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WO2024243361A1 - Compositions and methods to treat muscle injury and disease - Google Patents

Compositions and methods to treat muscle injury and disease Download PDF

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Publication number
WO2024243361A1
WO2024243361A1 PCT/US2024/030648 US2024030648W WO2024243361A1 WO 2024243361 A1 WO2024243361 A1 WO 2024243361A1 US 2024030648 W US2024030648 W US 2024030648W WO 2024243361 A1 WO2024243361 A1 WO 2024243361A1
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ifn
cell
subject
cells
injury
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PCT/US2024/030648
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French (fr)
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Kevin Robert KING
Van NINH
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The Regents Of The University Of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the field of the treatment and prevention of the damage to muscle tissues for example cardiomyocytes, injury such as myocardial injury, myocardial infarction, ischemia, pressure overload, and sharp needle trauma.
  • cardiomyocytes injury such as myocardial injury, myocardial infarction, ischemia, pressure overload, and sharp needle trauma.
  • BACKGROUND Innate immune responses to sterile tissue injury are often credited to professional immune cells (e.g., myeloid cells) sensing damage molecules released by dying cells 1,2 .
  • myeloid cells e.g., myeloid cells
  • MI myocardial infarction
  • IFN antiviral type I interferon
  • cGAS double stranded DNA
  • cGAS cyclic GMP-AMP Synthase
  • cGAMP cyclic di-GMP-AMP
  • STING adaptor Stimulator of Interferon Genes
  • Irf3 Interferon Regulatory Factor 3
  • ISGs interferon stimulated genes 6-9 . Since Ifnar-dependent ISG-expression can occur in any cell type, Applicant refer to cells expressing ISGs as interferon-induced cells, or IFNICs. It has been reported that globally inhibiting the MI-induced IFN response was protective against pathologic remodeling, ventricular dilation, and rupture; however, the reason for protection and its relevance to humans was unknown 6 . Single-cell transcriptomics has rapidly advanced the understanding of cellular heterogeneity within infarcted hearts.
  • IFNICs small subpopulations of IFNICs in multiple immune and non-immune cell subtypes. It is unknown how small fractions of so many cell types become activated by IFNs while the majority avoid activation 6,10 . Moreover, it is unknown which cell type secretes IFN to initiate the response. Sterile inflammation after myocardial infarction (MI) is classically credited to myeloid cells interacting with damage associated molecules dispersed throughout the infarct.
  • MI myocardial infarction
  • cardiomyocytes are essential initiators of the type I interferon (IFN) response at the infarct borderzone (BZ).
  • IFN response to MI is spatially clustered in focal colonies of interferon induced cells (IFNICs) expressing interferon stimulated genes (ISGs) that decorate the BZ where cardiomyocytes undergo mechanical destabilization and “loss-of- neighbor” stress.
  • IFNICs interferon induced cells
  • ISGs interferon stimulated genes
  • Cardiomyocyte-selective deletion of interferon regulatory factor 3 (Irf3) abrogated IFNIC colonies and phenocopied globally Irf3-deficient mice after MI.
  • IFNIC colonies remained at the BZs of mice lacking Irf3 in fibroblasts, endothelial cells, or neutrophils; of Ccr2-deficient mice, or of plasmacytoid dendritic cell-depleted mice.
  • IFN and ISG expression blunted the protective matricellular programs of BZ fibroblasts and left them vulnerable to pathologic remodeling and ventricular rupture.
  • mice that died within the first week after MI Applicant consistently found IFNIC Atty. Dkt. No.: 114198-5210 colonies at sites of ventricular rupture. Taken together, these results reveal an unexpected local pathologic niche initiated by mechanically stressed BZ cardiomyocytes that has a global impact on survival.
  • Applicant suggests that selective inhibition of Irf3 activation in cardiomyocytes could limit ischemic cardiomyopathy while avoiding broad immunosuppression.
  • Applicant also discovered that heart attacks initiate the interferon response, not in immune cells, but unexpectedly in cardiomyocytes. They do so via the cGAS-STING-IRF3 pathway. Thus, this pathway was targeted so that one can block the pathway and improve outcomes after myocardial infarction by targeting this more selective signaling pathway (upstream of IFNAR and thus causing less broad immunosuppression) and in a specific cell type not responsible for immune surveillance.
  • therapies targeting the cGAS-STING-IRF3 pathway in cardiomyocytes have a better risk-benefit profile with retained efficacy but reduced immunosuppression and infections while on therapy.
  • Applicant also reports herein that when cells are mechanically distorted, stretched, or locally separated from each other, whether by an external force or because they are contractile cells that hyper contract or contract against a sudden imbalance of forces (e.g., heart muscle cells contracting at the edge of a “heart attack” where neighbor cells have died of ischemia), they induce a type I interferon (IFN) response.
  • the type I IFN response is a fundamental multi-step innate immune response that can be inhibited at multiple steps using small molecules, antibodies, and nucleic acids.
  • This pathway involves molecules such as cGAS, cGAMP, STING or MDA5, RIG-I, MAVS, or TLRs, TRIF, and TBK1, IRF3, type I IFNs, which are secreted and spread to neighboring cells where they bind to the IFN alpha receptor (IFNAR) on neighboring cells, signal via Jak/Stat, activate the transcriptional regulator ISGF3, and induce hundreds of effector interferon stimulated genes (ISGs) with proinflammatory and anti-viral functions.
  • IFNAR IFN alpha receptor
  • Applicant exploits this response and pathway in new disease contexts and therapeutic settings where the IFN response is induced by mechanical distortion. Inhibition of this response provides new methods to treat cardiac and muscular injury, including myocardial infarction. Atty. Dkt. No.: 114198-5210 Thus, Applicant provides herein targeted inhibitors of the pathway with better side effect profiles (comparable efficacy with fewer infections). While conventional wisdom would say the pathway is initiated by immune cells, Applicant cast a broader net and created cell-specific knockouts for cardiomyocytes, fibroblasts, endothelial cells, neutrophils, and macrophages.
  • Applicant’s evidence derives from construction of a cardiomyocyte-specific Irf3- deficient mouse, which was exposed to experimental myocardial infarction and analyzed by single cell and spatial transcriptomics. It was compared to other cell type specific mice, wild- type mice, and globally Irf3-deficient mice. As disclosed herein, it is shown that Irf3 is activated via the cGAS-STING- IRF3 pathway which comes from cGAS-deficient mice, STING-deficient mice, compared to MAVS- deficient and TRIF-deficient mice.
  • reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina) due to ischemia and a heart attack each method comprising, or consisting essentially of, or yet further consisting of, administering a cardiomyocyte cGAS-STING-IRF3 inhibitor thereby reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack
  • the inhibitor is a small molecule or a gene therapy which inhibits the activity of Irf3.
  • a non-limiting example of gene therapy is the administration of an inhibitory RNA molecule to reduce the activity of Irf3 and/or its upstream activators.
  • the subject is a mammal such as a human patient.
  • the administration of the inhibitor treats and prevents ischemic heart disease as well. Modes of administration are known in the art and described herein.
  • the inhibitors and methods can be administered directly in vitro or in a composition, in vitro or in vivo.
  • the methods can be used for personalized therapies or to test for new drugs or new drug combinations or compositions.
  • they can be used for veterinary applications, or as an animal model for pre-clinical testing, personalized therapies or new formulations or combination therapies.
  • FIGS. 1A – 1P Myocardial infarction induces focal colonies of IFNICs at borderzones in mice.
  • FIGS. 1A – 1O Spatial transcriptomics analysis of short axis sections from infarcted mouse hearts on post-MI Day 3 (D3).
  • FIGS. 1A – 1B Borderzone gene scores (data not shown) for (FIG. 1A) WT - 2 representative biological replicates, and (FIG. 1A) Irf3 -/- mice.
  • FIG. 1C – 1D Ischemic zone gene scores (data not shown) for (FIG. 1C) WT and (FIG. 1D) Irf3 -/- hearts.
  • FIGS. 1E – 1F Ifit1 interferon stimulated gene (ISG) expression for (FIG. 1E) WT and (FIG. 1F) Irf3 -/- hearts.
  • FIGGS.1G – 1L Magnified view of selected ISGs in IFNIC colonies of D3 WT hearts – (FIG. 1G) Ifit1, (FIG. 1I) Rsad2, and (FIG. 1K) the ISG score (FIG. 7B). Quantification of (FIG. 1H) Ifit1+ and (FIG.
  • FIG. 1H, FIG. 1J, FIG. 1L, FIG. 1O, FIG. 1P Data are presented as mean values ⁇ s.e.m. *P ⁇ 0.05, **P ⁇ .001, ***P ⁇ .0001.
  • FIGS. 2A – 2N Myocardial infarction induces focal colonies of IFNICs in humans. Spatial transcriptomics analysis of tissue sections from infarcted human hearts or controls. (FIGS. 2A – 2B) Borderzone gene scores for representative (FIG. 2A) 2 infarcted hearts and (FIG.
  • FIG. 2B a control heart.
  • FIGS. 2C – 2D Ischemic zone gene scores for the same (FIG. 2C) infarcted and (FIG.2D) control hearts.
  • FIGGS. 2E – 2F Ifit1 interferon stimulated gene (ISG) expression (data not shown) for the same (FIG. 2E) infarcted and (FIG. 2F) control hearts.
  • FIGGS. 2G – 2L Close-up view of IFNIC gene expression in the same infarcted hearts – (FIG. 2G) MX1, (FIG. 2I) IFIT3, and (FIG. 2K) the ISG score with quantification of (FIG. 2H) MX1+ and (FIG.
  • FIGS. 2M – 2N Ranked Moran’s I test statistics of the top 2,000 differentially expressed genes with annotated ISGs in the same (FIG. 2M) infarcted and (FIG. 2N) control human hearts.
  • FIG. 2H, FIG. 2J, FIG. 2L Data are presented as mean values ⁇ s.e.m. *P ⁇ 0.05, **P ⁇ .01.
  • FIG. 2H, FIG. 2J, FIG. 2L Unpaired student’s t-test.
  • FIGS. 3A – 3J Cardiomyocytes are dominant initiators of the type I IFN response in MI. (FIGS.
  • Irf3 fl/fl mice were crossed with mice expressing Cre recombinase under the regulation of cell-type selective promoters: cardiomyocytes (Irf3 fl/fl X Myh6 cre/+ , Irf3 CM ), fibroblasts (Irf3 fl/fl X Col1 ⁇ 1 creERT2/+ , Irf3 FB ), macrophages (Irf3 fl/fl X Cx3cr1 creERT2/+ , Irf3 Mac ), neutrophils (Irf3 fl/fl X S100a8 cre/+ , Irf3 Neut ), and endothelium (Irf3 fl/fl X Tie2 cre/+ , Irf3 EC ).
  • cardiomyocytes Irf3 fl/fl X Myh6 cre/+ , Irf3 CM
  • fibroblasts Irf3 fl
  • FIG. 3A Borderzone gene scores and ISG scores at representative BZs for each conditional knockout mouse.
  • FIG. 3B Moran’s I test statistic with annotated ISGs for the top 2,000 variably expressed genes for each conditional knockout line.
  • FIG. 3C Comparison of spatial autocorrelation metrics between WT and conditional Irf3 knockout mice on day 3 after MI. Spatially enriched features were ordered and the ranked lists were converted to a percentile.
  • FIG. 3F Representative image of cell marker gene probes used for RNA MERFISH and identification of cell types and zones in the infarcted heart.
  • FIG. 3G RNA MERFISH-labeled type I IFN transcript, Ifna2 (red), localized primarily to BZ cardiomyocyte gene Ankrd1 (blue) and fibroblast gene Col6 ⁇ 3 (cyan) with closeup view in panel (FIG. 3H) below.
  • FIG. 3I Ifna2 transcripts assigned to individual cells and represented by red circles as Ifna2+ cells.
  • FIG. 3J Relative expression of Ifna2 in each cell type in WT and Irf3 -/- mice.
  • FIG. 3D Data are presented as mean values ⁇ s.e.m. and ** P ⁇ 0.005, ****P ⁇ 0.0001.
  • FIGS. 4A – 4F Nuclear rupture and extranuclear DNA are found in load- bearing cells of the infarct borderzone in vivo.
  • FIG. 4A Representative image of short axis cross section from a cardiomyocyte-specific nuclear reporter mouse harvested at post-MI D3. created by crossing fl-STOP-fl tdTomNLS x Myh6 cre/+ .
  • CM-tdTom-NLS Upon cre-mediated recombination, the flanked STOP codons are removed and tdTomato fluorescence reporter with a nuclear localization sequence are expressed specifically by cardiomyocytes (CM-tdTom-NLS).
  • FIG. 4B Representative images of CM-tdTom-NLS in nuclei of the remote zone and borderzone. Nuclear rupture was visualized as fluorescent reporter diffused throughout the cytoplasm of outlined cardiomyocytes whereas tdTom fluorescence was confined within the nuclear membrane in non-ruptured nuclei.
  • FIG. 4C Quantification of the rate of ruptured nuclei in the remote zone vs.
  • FIG. 4D Sequence-specific DNA probes were designed and synthesized to detect nuclear and extranuclear DNA in mouse hearts post-MI D1 and D3. Representative images of Atty. Dkt. No.: 114198-5210 DNA MERFISH encoding probes for 260 gene loci in 21 mouse chromosomes and rounds of hybridization of fluorescently labeled readout probes. The last image in the panel represents computationally decoded gene loci performed after data collection.
  • FIGS. 5A – 5K MI-induced IFNIC colonies co-localize at sites of ventricular rupture and antagonize protective fibroblast function.
  • FIG. 5A Relative difference in survival post-MI in previously published studies that inhibit the type I IFN signaling (Irf3 -/- , Ifnar -/- , anti-IFNAR Ab) or fibroblast activation and the matricellular response (anti-TGFbR Ab, Sparc -/- , Postn -/- ) 3,44-50 .
  • FIG. 5B Representative sample after fatal ventricular rupture - H&E staining and spatial transcriptomic BZ gene score and ISG score. Inset shows magnified view of the rupture site.
  • Panel below shows magnified view of IFNIC colony #7 from the RNA MERFISH sample shown in (FIG. 5C).
  • RNA MERFISH fluorescent probes within the IFNIC colony (DAPI in grey, cardiomyocytes (blue - Flnc, Nppa), fibroblasts (cyan – Col1a1, Col6a3), and macrophages.
  • FIG. 5E Neighborhood based clustering analysis using the centroid of each IFNIC colony as the reference point and chosen as the highest ISG expressing capture spots. Adjacent spots were designated as primary, secondary, and tertiary degree neighbors to the ISG centroid(s) for each cluster. Differential gene expression testing was performed for each neighbor against the centroid of ISG clusters. Atty. Dkt. No.: 114198-5210 (FIG.
  • FIG. 5F Violin plots of Ifit3 and Irf7 from the centroid towards spots labeled as tertiary neighbors.
  • FIG. 5G Violin plot of fibroblast-expressed, matricellular genes selected from differential expression testing between neighbors and centroid of IFNIC colonies. Postn and Col1 ⁇ 1 display a reciprocal relationship to ISGs in both expression level and distance.
  • FIG. 5K Proposed model in which borderzone cardiomyocytes are dominant initiators of IFNIC colonies leading to pathogenic Irf3- dependent responses to MI. Effector cells represented below and relative contributions of cell types to adverse remodeling, dilation, and ventricular rupture remain to be fully elucidated.
  • FIG. 5J Data represented as ⁇ s.e.m. and *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG. 5C, FIG. 5H, FIG. 5I, FIG. 5J One-way ANOVA with multiple comparisons using Dunnett’s multiple comparison post-hoc analysis.
  • FIG. 5F, FIG. 5G Wilcoxon rank-sum and FDR P-value adjusted.
  • FIG. 6 Summary table of biological replicates in imaging- and sequencing-based spatial transcriptomics. Summary table of mouse and human biological samples and replicates. Total cells single cells are quantified for imaging-based RNA and DNA MERFISH. Total spatial transcriptomes are quantified for capture-based Visium spatial transcriptomics.
  • FIG. 7A – 7D Gene scoring method using integrated sc/snRNA-seq data to map transcriptional zones of infarcted myocardium in space.
  • FIG. 7A Experimental workflow. Gene scores are derived from differentially expressed genes (DEG) after clustering integrated sc/snRNA-seq data from infarcted mouse hearts on day 3 after MI or using previously developed scores for BZ and IZ, as published 18 .
  • FIG. 7B Table of gene scores are summed within the SCT assay of the integrated spatial object for analysis of mouse and human spatial transcriptomes and were normalized to minimum UMI counts in integrated samples.
  • FIG. 7C Replicate sequencing-based spatial transcriptomic datasets were Atty. Dkt.
  • FIG. 8A Comparison of transcriptionally defined BZ and underlying H&E tissue histology.
  • FIG. 8A H&E tissue histology underlying the transcriptionally defined anterior and posterior BZs for representative spatial transcriptomics samples.
  • FIGS. 9A – 9I The type I IFN response produces spatially clustered ISG expression.
  • FIG. 9B Spatially variable features were calculated by Seurat’s FindVariableFeatures() using vst as the selection method. Standard variance plotted against log normalized average expression of highly variable features. ISGs were among the 2000 topmost variable features are labeled on the VariableFeaturesPlot.
  • FIG. 9C Interferon stimulated genes projected onto space in a D3 post-MI cardiac section.
  • FIGS. 10A – 10C Methods for identifying and qualitatively characterizing IFNIC colonies and spatial correlation.
  • FIGS. 11A – 11D Biological replicates of the clustered type I IFN response after MI in mice and humans and corresponding negative controls.
  • FIG. 11A Table representing all biological replicates and samples used for spatial transcriptomic analysis in this study. Column (FIG.
  • FIGS. 11A contains representative images of sample conditions that were negative for IFNIC colonies including the validation of using knockouts of Cgas-Sting-IRF3 pathway as negative controls for the study.
  • FIG. 11B Positive IFNIC colonies represented in biological replicates for conditions used to determine the presence of ISG+ conditions.
  • FIGS. 11D Data represented as mean values ⁇ s.e.m. and ****P ⁇ 0.0001, Two-sided Mann- Whitney U-test.
  • FIGS. 11D Data represented as mean values ⁇ s.e.m. and ****P ⁇ 0.0001, Two-sided Mann- Whitney U-test.
  • FIG. 12A Representative images of 4 individual ISGs in cardiomyocyte-specific Irf3 deletion display minimal ISG cluster formation compared to ISG expression in cardiomyocyte- (FIG. 12B) fibroblast- (FIG. 12C) macrophage- (FIG. 12D) neutrophil-, and (FIG. 12E) endothelial-specific deletions of Irf3.
  • FIG. 12A Representative images of 4 individual ISGs in cardiomyocyte-specific Irf3 deletion display minimal ISG cluster formation compared to ISG expression in cardiomyocyte- (FIG. 12B) fibroblast- (FIG. 12C) macrophage- (FIG. 12D) neutrophil-, and (FIG. 12E) endothelial-specific deletions of Irf3.
  • FIG. 13A – 13H Infiltrating myeloid cells are dispensable for IFNIC colony formation in the infarcted murine heart.
  • FIG. 13A Representative Ifit1+ IFNIC colony found at the BZ in Ccr2-defiicient hearts at D3 post-infarct.
  • FIG. 13C Timeline of antibody-mediated plasmacytoid dendritic cell (pDC) depletion.
  • FIG. 13D Gating strategy to determine pDCs depletion in isotype control-treated or anti-BST2-ab treated, infarcted mouse hearts. Cd45+B220+ positive myeloid cells were gated by MHCII expression and BST2 to identify pDCs.
  • FIG. 13E Feature plots of Ifit1 expression in neutrophils and macrophages isolated from mice treated with either isotype control-antibody or anti-Bst2-Ab treated mice.
  • FIG. 13H Gene expression analysis by qPCR in D4 infarcted hearts between WT-> WT transfer and IRF3KO -> WT transfer conditions.
  • FIGS. 14A – 14F Validation of RNA MERFISH encoding probe library by comparison of log-normalized of transcripts detected in WT and Irf3-deficient mice.
  • FIG. 14A Number of total probes designed for each transcript in the encoding library used in this study.
  • FIG. 14C Quality control metrics of the raw data and total counts of each transcript detected in WT D3 MI heart and (FIG. 14D) in Irf3-deficient D3 MI heart.
  • FIG. 14E Comparison of the total mean counts in the raw data and (FIG. 14F) log-normalized counts of detected transcripts for BZ genes such as Nppa, Flnc, Ankrd1 between WT and Irf3-deficient mice. Atty. Dkt.
  • FIGS. 15A – 15I DNA MERFISH enables sequence-specific detection of extranuclear DNA.
  • FIG. 15A Time course of needle pass injury. The appearance of Ifit1 expression occur at D3 post-injury at the side of needle insertion, similar to the time course of ISG expression in infarcted hearts.
  • FIG. 15B Close-up view of scored ISGs,
  • FIG.15C mechanically-induced Piezo1,
  • FIG.15D scored BZ genes, and
  • FIG. 15E H&E at the site of day 3 post-injury.
  • FIG. 15F Schematic of experimental traumatic injury induced by in vivo needle-insertion in healthy hearts.
  • FIG. 15G BZ gene score and ISG score spatial transcriptomics and
  • FIG. 15I Regions of interest used to quantify extranuclear probes in WT D1 and WT D3 infarcted hearts were chosen based on underlying histology to demarcate the RZ and BZ. The regions designated as RZ are shown in blue dashed boxes, and the regions designated as the BZ are shown in red dashed boxes for each sample.
  • FIGS. 16A – 16E IFNIC colonies and ISG expression appear directly adjacent to the site of ventricular rupture and have high calculated spatial autocorrelation.
  • FIG. 16A Representative images of 7 collected rupture samples in WT mice during monitoring on D3-D7 post-MI show IFNIC colonies directly adjacent to the site of rupture.
  • FIG. 16B In situ hybridization of ruptured cardiac cross section with representative Flnc (blue), Postn (green), and Ifit3 (magenta).
  • FIG. 16C Table of rupture rates and successful spatial transcriptomics (sectioning and identification of rupture site), and qualitative assessment of ISG expression proximal to rupture site.
  • FIG. 16D Spatial autocorrelation determined for each biological rupture sample as measured by Moran’s I test statistic.
  • FIG. 16E Moran’s I test statistic was significantly higher across all biological replicates of ruptured WT hearts compared to non-ruptured Irf3 -/- hearts.
  • Data are presented as mean values ⁇ s.e.m.
  • FIGS. 17A – 17F Characterizing IFNICs and the inverse spatial relationship between ISG and Postn expression.
  • FIG. 17A Representative IFNIC cell types showing minority contribution by neutrophils and endothelial cells.
  • FIG. 17B Line scans performed in WT D3 MI sample and
  • FIG. 17C biological replicate, and
  • FIG. 17D ruptured sample to power the observation of inversely correlated Postn and ISG scores.
  • FIGS. 18A – 18H Analysis of IFNAR-deficiency in single-cells.
  • FIG. 18B UMAP of clustered fibroblasts from scRNA-seq integrated data of WT and Ifnar -/- cells from D3 infarcted hearts.
  • FIG. 18C Heatmap of marker genes for each fibroblast subcluster.
  • FIG. 18D Integrated fibroblast subset from WT and IFNAR-KO infarcted hearts were split by biological condition and by activated vs.
  • FIG. 18E IFNAR-KO mouse hearts contain a higher percentage of activated fibroblasts compared to those captured in WT infarcted hearts.
  • FIG. 18F Log normalized expression of gene markers in activated fibroblast populations between Ifnar -/- and WT mice.
  • FIG. 18G UMAP of captured macrophages in the scRNA-seq dataset divided into macrophages from Ifnar -/- mice in blue and macrophages from WT mice in magenta.
  • FIGS. 19A – 19E Irf3-deficient hearts have greater fibroblast activation and protective matricellular responses.
  • FIG. 19A Nuclei isolation from WT and Irf3- deficient hearts are processed for snRNA-seq. Nuclei data sets were integrated to enable Atty. Dkt. No.: 114198-5210 direct comparison of cell types.
  • FIG. 19B Heatmap displaying averaged values of DEGs in each fibroblast subpopulation were stratified into activated and not activated fibroblasts Variable expression level of Postn underlie fibroblasts clustered into the activated subtype.
  • FIG. 19C UMAP of clustered fibroblasts from snRNA-seq integrated data of WT and Irf3 -/- nuclei from D3 infarcted hearts on the left panel and separation of activated vs. non-activated fibroblast populations in the right panel (Wilcoxon-signed rank test, Bonferroni-adjusted P ⁇ 0.01).
  • FIG. 19C UMAP of clustered fibroblasts from snRNA-seq integrated data of WT and Irf3 -/- nuclei from D3 infarcted hearts on the left panel and separation of activated vs. non-activated fibroblast populations in the right panel (Wilcoxon-signed rank test, Bonferroni-adjust
  • FIG. 19E Log normalized average expression of fibroblast activation markers between those captured from Irf3-deficient and WT and D3 MI hearts.
  • FIG. 19D Data represented as mean values ⁇ s.e.m.
  • FIGS. 20A – 20C Type I IFN antagonizes mouse fibroblast activation and function.
  • FIG. 20A Collagen Gel contraction assays performed with L929 fibroblasts treated with TGF ⁇ , IFN ⁇ , or combination treatment. IFN ⁇ blocked the TGF ⁇ -mediated response of fibroblast activation as determined by the lack of gel contraction.
  • FIG. 20B Treatment with anti-IFNAR-ab abrogated the IFN ⁇ -mediated blockade of the fibroblast contractile phenotype.
  • FIG. 20C Top panel: L929 fibroblast gene expression data of matricellular genes after treatment with TGF ⁇ , IFN ⁇ , or combination treatment, and corresponding inverse expression of ISG’s in the bottom panel.
  • FIG. 20A, FIG. 20B, FIG. 20C Data represented as mean values ⁇ s.e.m. *P > 0.05, **P > 0.005, ***P > 0.0005, ****P > 0.00005.
  • One-way ANOVA with Dunnett’s multiple comparisons test (n 2-3 per condition).
  • FIG. 21 The 2 IFNIC sources are compared and contrasted. Table summarizing intracardiac and extracardiac sources of IFNICs.
  • FIGS. 22A – 22D Quality control metrics of sequencing-based spatial transcriptomics.
  • FIG.22A Total number of Visium spots detected over tissue for each sample.
  • FIG. 22B Total number of genes detected (nCount) per sample.
  • FIG. 22C Feature scatter plot of unique molecular identifiers and total genes detected in integrated Atty. Dkt. No.: 114198-5210 dataset consisting of all the labeled samples from (FIG. 22B).
  • FIG. 22D List of the highest expressed features detected in WT samples.
  • compositions and methods are intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others.
  • Consisting essentially of when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically Atty. Dkt. No.: 114198-5210 acceptable carriers, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.
  • “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • the term “mammal” includes both human and non-human mammals.
  • the term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non- Atty. Dkt.
  • a mammal is a human.
  • a mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero).
  • a mammal can be male or female.
  • a subject is a human.
  • composition refers to an active agent, such as a compound as disclosed herein and a carrier, inert or active.
  • the carrier can be, without limitation, solid such as a bead or resin, or liquid, such as phosphate buffered saline.
  • Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra- oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein.
  • Pharmaceutically acceptable carriers Atty. Dkt.
  • No.: 114198-5210 include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate,
  • Suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
  • the compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the result and/or protection desired.
  • Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.
  • An “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the Atty. Dkt. No.: 114198-5210 therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration.
  • a “type I IFN response inhibitors or inhibitory IFN agents” intends any agent that inhibits the type I IFN response.
  • Non-limiting examples include anti-IFNAR antibodies, such as an anti-IFNAR antibody, e.g., Anifrolumab TM ( Astrazeneca, an FDA-approved biologic approved for chronic systemic delivery for lupus based on the TULIP-2 Trial (https://www.saphnelohcp.com/how-saphnelo-works.html); and Ventus Therapeutics’ Phase 1 small molecular cGAS-inhibitor, VENT-003 (https://www.ventustx.com/pipeline/cgas/).
  • an anti-IFNAR antibody e.g., Anifrolumab TM ( Astrazeneca, an FDA-approved biologic approved for chronic systemic delivery for lupus based on the TULIP-2 Trial (https://www.saphnelohcp.com/how-saphnelo-works.html); and Ventus Therapeutics’ Phase 1 small molecular cGAS-inhibitor,
  • anti-IFNAR antibodies do not bind IFN or its upstream activators but instead blocks its downstream receptor, IFNAR on secondary-responding cells.
  • this antibody is useful to treat local injury such as surgery, needle trauma, injection of viral gene therapy, cell therapy, cannula placement, etc.
  • Additional agents include agents that inhibit other molecules in this pathway (cGAS inhibitors, STING inhibitors, TBK1 inhibitors, IRF3 inhibitors, anti-IFN therapies, anti-IFNAR therapies, Jak/Stat inhibitors, anti-ISG therapies, etc.), e.g., an inhibitory antibody or inhibitory nucleic acid such as antisense oligonucleotides (ASO), RNAi or siRNA.
  • cGAS inhibitors e.g., STING inhibitors, TBK1 inhibitors, IRF3 inhibitors, anti-IFN therapies, anti-IFNAR therapies, Jak/Stat inhibitors, anti-ISG therapies, etc.
  • an inhibitory antibody or inhibitory nucleic acid such as antisense oligonucleotides (ASO), RNAi or siRNA.
  • Yet further examples include IFN-kinoid, AGS- 009 JNJ-55920839 Rontalizumab Sifalimumab BMS-986165 Baricitinib Filgotinib Solcitinib Tofacitinib, for example as shown in Fig 1 of Felten R, et al. (2019) Drug Des Devel Ther., 13:1535-1543, incorporated herein by reference.
  • the acronym “cGAS” intends. cyclic Guanosine monophosphate- Adenosine monophosphate (cGMP-AMP) Synthase.
  • cGAMP intends icyclic Guanosine monophosphate- Adenosine monophosphate (cGMP-AMP). Atty. Dkt. No.: 114198-5210
  • STING intends STimulator of InterferoN Genes.
  • TK1 intends TANK binding kinase 1.
  • IRF3 intends Interferon Regulatory Factor 3 (IRF3).
  • IFNs intends Type I interferons.
  • IFNAR intends IFN Alpha Receptor.
  • ISG intends interferon stimulating genes, which are genes that are expressed in response to stimulation by interferons. Interferons bind to receptors on the surface of a cell, initiating protein signaling pathways within the cell.
  • IFIT1 interferon induced protein with tetratricopeptide 1
  • IFIT2 interferon induced protein 44 like
  • IFI44L interferon induced protein 44 like
  • RSD2 radical S-adenosyl methionine domain containing
  • ISG15 cytidine/uridine monophosphate kinase 2 (CMPK2); circulating inflammatory marker elevated in advanced heart failure (CXCL10); and MX dynamin like GTPase 1 (MX1) and 2 (MX2).
  • mechanically induced type I IFN response intends mechanical tissue injury causes cGAS activation, which catalyzes production of the small molecule cGAMP, which can signal within the cell and be transferred between cells via gap junctions or the extracellular space to agonize STING, which activates TBK1, which phosphorylates the transcription factor IRF3, which leads to dimerization, nuclear translocation, and upregulation of IRF3-dependent genes such as the type I IFNs.
  • Type I IFNs are secreted cytokines that can signal via the cell surface receptor IFNAR to induce expression of 100s of interferon stimulated genes (ISGs) in neighboring cells.
  • ISGs are the dominant effector molecules so blocking any step in this pathway has potential to protect from IFN- and ISG- mediated pathology, particularly in patients who show evidence of ISG expression by imaging nuclear or contrast-associated ISGs -labeled with tissue that shows evidence of ISG expression, for example by immunostaining, in situ hybridization, or spatial transcriptomics.
  • cardiac regeneration intends reversing or repairing damaged heart cells or tissue. Atty. Dkt.
  • treating or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • treatment excludes prophylaxis.
  • oligonucleotide or “polynucleotide” or “portion,” or “segment” thereof refer to a stretch of polynucleotide residues which is long enough to use in PCR or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules.
  • the polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • pendent moieties e.
  • contacting means direct or indirect binding or interaction between two or more.
  • direct interaction is binding.
  • Atty. Dkt. No.: 114198-5210 indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity.
  • Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo.
  • an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence.
  • Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST.
  • equivalent polynucleotides, proteins and corresponding sequences can be determined using BLAST (accessible at blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on August 1, 2021).
  • an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity, or at least about 85 % homology or identity, or alternatively at least about 90 % homology or identity, or alternatively at least about 95 % homology or identity, or alternatively at least about 96 % homology or identity, or alternatively at least about 97 % homology or identity, or alternatively at least about 98 % homology or identity, or alternatively at least about 99 % homology or identity (in one aspect, as determined using the Clustal Omega alignment program) and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
  • an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complementary sequence.
  • a first sequence nucleic acid sequence or amino acid
  • the identity percentage between the two sequences can Atty. Dkt. No.: 114198-5210 be calculated.
  • the first sequence can be referred to herein as an equivalent and the second sequence can be referred to herein as a reference sequence.
  • the identity percentage is calculated based on the full-length sequence of the first sequence. In other embodiments, the identity percentage is calculated based on the full-length sequence of the second sequence.
  • CM cardiomyocyte
  • a “cardiomyocyte” or “cardiac myocyte” is a specialized muscle cell which primarily forms the myocardium of the heart. Cardiomyocytes have five major components: 1. cell membrane (sarcolemma) and T-tubules, for impulse conduction, 2. sarcoplasmic reticulum, a calcium reservoir needed for contraction, 3. contractile elements, 4. mitochondria, and 5. a nucleus.
  • Cardiomyocytes can be subdivided into subtypes including, but not limited to, atrial cardiomyocyte, ventricular cardiomyocyte, SA nodal cardiomyocyte, peripheral SA nodal cardiomyocyte, or central SA nodal cardiomyocyte.
  • Stem cells can be propagated to mimic the physiological functions of cardiomyocytes or alternatively, differentiate into cardiomyocytes. This differentiation can be detected by the use of markers selected from, but not limited to, myosin heavy chain, myosin light chain, actinin, troponin, tropomyosin, GATA4, Mef2c, and Nkx2.5.
  • the cardiomyocyte marker “myosin heavy chain” and “myosin light chain” are part of a large family of motor proteins found in muscle cells responsible for producing contractile force.
  • the cardiomyocyte marker “actinin” is a microfilament protein which are the thinnest filaments of the cytoskeleton found in the cytoplasm of all eukaryotic cells. Actin polymers also play a role in actomyosin-driven contractile processes and serve as platforms for myosin’s ATP hydrolysis-dependent pulling action in muscle contraction.
  • This protein has been sequenced and characterized, see for example GenBank Accession Nos. NP_001093, NP_001095, NP_001094, NP_004915, P35609, NP_598917, NP_112267, AAI07534, and NP_001029807. The gene for this protein has also been sequenced and characterized, see for example GenBank Accession Nos.
  • the cardiomyocyte marker “troponin” is a complex of three proteins that is integral to muscle contraction in skeletal and cardiac muscle. Troponin is attached to the protein “tropomyosin” and lies within the groove between actin filaments in muscle tissue. Tropomyosin can be used as a cardiomyocyte marker. These proteins have been sequenced and characterized, see for example GenBank Accession Nos. NP_000354, NP_003272, P19429, NP_001001430, AAB59509, AAA36771, and NP_001018007. The gene for this protein has also been sequenced and characterized, see for example GenBank Accession Nos.
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus, adeno-associated virus, and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • Gene delivery are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction.
  • exogenous polynucleotide sometimes referred to as a “transgene”
  • transgene an exogenous polynucleotide
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection, sometimes called transduction), transfection, transformation or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • transfected, transduced or transformed may be used interchangeably herein to indicate the presence of exogenous polynucleotides or the expressed polypeptide therefrom in a cell.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
  • the term “express” refers to the production of a gene product.
  • the gene product is a polypeptide or protein.
  • the gene product is an mRNA, a tRNA, an rRNA, a miRNA, a dsRNA, or a siRNA, e.g., inhibitory RNA molecules.
  • a cell that “stably expresses” an exogenous polypeptide is one that continues to express a polypeptide encoded by an exogenous gene introduced into the cell either after replication if the cell is dividing or for longer than a day, up to about a week, up to about two weeks, up to three weeks, up to four weeks, for several weeks, up to a month, up to two months, up to three months, for several months, up to a year or more.
  • a “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors examples include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med.5(7):823-827. Atty. Dkt.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
  • the integrated DNA form is called a provirus.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • a “lentiviral vector” is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene.
  • Ads adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos.
  • AAV is a standard abbreviation for adeno-associated virus.
  • Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus.
  • co-infecting helper virus There are currently thirteen serotypes of AAV that have been characterized.
  • AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2.
  • the degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self- annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs).
  • ITRs inverted terminal repeat sequences
  • An “AAV expression cassette” as used herein refers to a nucleotide sequence comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV expression cassette can be replicated and packaged into infectious viral particles (e.g., AAV vectors) when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.
  • infectious viral particles e.g., AAV vectors
  • An “AAV virion” or “AAV vector” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV expression cassette.
  • AAV vector particle a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector”.
  • production of AAV vector particle necessarily includes production of AAV expression cassette, as such a plasmid is contained within an AAV vector particle.
  • Adeno-associated virus is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV.
  • nucleotide sequences of the genomes of the AAV serotypes are known.
  • the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983) as corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994).
  • Atty. Dkt. No.: 114198-5210 the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No.
  • AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004).
  • Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs.
  • Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes.
  • the two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene.
  • Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.
  • the cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins.
  • a single consensus polyadenylation site is located at map position 95 of the AAV genome.
  • AAV genomes for use in the methods of this disclosure comprise a polynucleotide to modulate type I IFN expression and one or more AAV ITRs flanking a nucleic acid molecule.
  • AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13.
  • Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692.
  • Other types of rAAV Atty. Dkt. No.: 114198-5210 variants, for example rAAV with capsid mutations, are also contemplated.
  • AAV1 AAV6, AAV8 or AAVrh.74 is used to promote skeletal muscle specific expression.
  • the polynucleotide that modulates type I IFN expression can be under the control of a tissue specific promoter, e.g., a cardiomyocyte- specific promoter, e.g., the promoter of the gene encoding the contractile protein TroponinT, or myosin light chain 2 promoter (MLC-2V), or the promoter of alpha myosin heavy chain (Myh6) (see Griscelli et al. (1997) CR Acad. Sci. 329(2):103-12, or Lin Z et al. (2014) Circ Res 115:354-363, or Breckenridge R.
  • a tissue specific promoter e.g., a cardiomyocyte- specific promoter, e.g., the promoter of the gene encoding the contractile protein TroponinT, or myosin light chain 2 promoter (MLC-2V), or the promoter of alpha myosin heavy chain (Myh6) (see Griscelli et al. (1997) CR
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI).
  • Under transcriptional control is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription.
  • “Operably linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.
  • a “composition” is intended to mean a combination of polynucleotides, active agent or another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant. Atty. Dkt. No.: 114198-5210
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein.
  • Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, microspheres, microparticles, or nanoparticles (comprising e.g., biode
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
  • “Liposomes” are microscopic vesicles consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer.
  • bipolar neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other types of bipolar lipids including but not limited to dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C ⁇ C bonds.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PI phosphatidylinositol
  • SM sphingomyelin
  • DOPE dioleoylphosphatidylethanolamine
  • lipids capable of producing a stable liposome are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic Atty. Dkt.
  • HSPC hydrogenated soy phosphatidylcholine
  • lecithin phosphatidylethanolamine
  • lysolecithin lysophosphatidylethanol-amine
  • phosphatidylserine phosphatidylinositol
  • sphingomyelin cephalin
  • cardiolipin phosphatidic Atty. Dkt.
  • 114198-5210 acid cerebrosides, distearoylphosphatidylethan-olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloteoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1- carboxylate (DOPE-mal).
  • DSPE distearoylphosphatidylethan-olamine
  • DOPC dipalmitoylphosphatidylcholine
  • POPC palmitoyloteoylphosphatidylcholine
  • POPE palmitoyloleoylphosphatidylethanolamine
  • DOPE-mal dioleoylphosphatidylethanolamine 4-(N-maleimi
  • Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol).
  • DDAB DODAC
  • DMRIE DMTAP
  • DOGS DOGS
  • DOTAP DOTMA
  • DOSPA DPTAP
  • DSTAP DC-Chol
  • Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioteoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles.
  • liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure. The three classifications, as developed by the New York Academy Sciences Meeting, “Liposomes and Their Use in Biology and Medicine,” December 1977, are multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles (LUVs).
  • MLVs multi-lamellar vesicles
  • SUVs small uni-lamellar vesicles
  • LUVs large uni-lamellar vesicles
  • the polynucleotides can be encapsulated in such for administration in accordance with the methods described herein.
  • a “micelle” is an aggregate of surfactant molecules dispersed in a liquid colloid.
  • a typical micelle in aqueous solution forms an aggregate with the hydrophilic “head” regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center.
  • This type of micelle is known as a normal phase micelle (oil-in-water micelle).
  • Inverse micelles have the head groups at the center with the tails extending out (water-in-oil micelle).
  • Micelles can be used to attach a polynucleotide, polypeptide, antibody or composition described herein to facilitate efficient delivery to the target cell or tissue.
  • lipid nanoparticles are also included as a micelles.
  • Gene delivery vehicles also include several non-viral vectors, including DNA/liposome complexes, and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
  • the nucleic acid or proteins of this invention can be Atty. Dkt. No.: 114198-5210 conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., a cell surface marker found on stem cells.
  • RNA interference refers to sequence-specific or gene specific suppression of gene expression (protein synthesis) that is mediated by short interfering RNA (siRNA).
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA molecules
  • RNAi RNA interference
  • 11 nucleotides in length 12 nucleotides in length
  • 13 nucleotides in length 14 nucleotides in length
  • 15 nucleotides in length 16 nucleotides in length
  • 17 nucleotides in length 18 nucleotides in length
  • 19 nucleotides in length 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length
  • 24 nucleotides in length 25 nucleotides in length
  • 26 nucleotides in length 27 nucleotides in length
  • 28 nucleotides in length or 29 nucleotides in length.
  • siRNA includes short hairpin RNAs (shRNAs).
  • “Double stranded RNA” refers to double stranded RNA molecules that may be of any length and may be cleaved intracellularly into smaller RNA molecules, such as siRNA. In cells that have a competent interferon response, longer dsRNA, such as those longer than about 30 base pair in length, may trigger the interferon response. In other cells that do not have a competent interferon response, dsRNA may be used to trigger specific RNAi.
  • siRNA includes short hairpin RNAs (shRNAs).
  • shRNAs comprise a single strand of RNA that forms a stem-loop structure, where the stem consists of the complementary sense and antisense strands that comprise a double-stranded siRNA, and the loop is a linker of varying size.
  • the stem structure of shRNAs generally is from about 10 to about 30 nucleotides in length.
  • the stem can be 10-30 nucleotides in length, or alternatively, 12-28 nucleotides in length, or alternatively, 15-25 nucleotides in length, or alternatively, 19-23 nucleotides in length, or alternatively, 21-23 nucleotides in length.
  • Tools to assist siRNA design are readily available to the public.
  • siRNA design tool is available on the internet at www.dharmacon.com, Ambion-www.ambion.com/jp/techlib/misc/siRNA_finder.html; Thermo Scientific- Dharmacon-www.dharmacon.com/DesignCenter/DesignCenterPage.aspx; Bioinformatics Atty. Dkt. No.: 114198-5210 Research Center-sysbio.kribb.re.kr:8080/AsiDesigner/menuDesigner.jsf; and Invitrogen- rnaidesigner.invitrogen.com/rnaiexpress/.
  • shRNA that targets and downregulates type I IFN can be prepared using methods known in the art (see, e.g., Cattaneo et al. (2016) Cell Death and Differentiation, 23:555-564 or are commercially available.
  • Antisense oligonucleotides (ASOs) are small pieces of DNA or RNA that can bind to specific RNA molecules, preventing the RNA from making proteins or functioning in other ways. Methods to administer ASOs to cardiac tissue are described in the literature, e.g., Prakash, et al. (2019) Nucl. Acids Res. Vol. 47(12):6029-6044.
  • CRISPR systems can also be used to downregulate or abrogate type I IFN expression by inhibiting transcription (also designated CRISPRi) using dCas9 (a mutant version of the Cas9 enzyme that lacks endonuclease activity) fused to a repressor domain (for example the Krüppel Associated Box – KRAB – domain) that is guided to the type I IFN gene by a single guide RNA designed to specifically target the promoter or exons of this gene, thereby repressing its transcription.
  • a repressor domain for example the Krüppel Associated Box – KRAB – domain
  • Crispr regulation of translation employs a catalytically dead dCas13 that targets RNA molecules (unlike Cas9 that targets DNA).
  • KRAB domain is a category of transcriptional repression domains present in approximately 400 human zinc finger protein-based transcription factors (KRAB zinc finger proteins).
  • the KRAB domain typically consists of about 75 amino acid residues, while the minimal repression module is approximately 45 amino acid residues. It is predicted to function through protein-protein interactions via two amphipathic helices.
  • administer intends to mean delivery of a substance to a subject such as an animal or human. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Atty. Dkt. No.: 114198-5210 Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, as well as the age, health or gender of the subject being treated.
  • Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of pets and animals, treating veterinarian.
  • Suitable dosage formulations and methods of administering the agents are known in the art.
  • Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and the target cell or tissue.
  • Non-limiting examples of route of administration include intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.
  • An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the optimal route will vary with the condition and age of the recipient, and the disease being treated. These strategies can be administered acutely (in a scenario of myocardial infarction) or chronically in any setting of heart failure. They can be administered systemically or locally.
  • Non-limiting examples of locally delivery includes intracardiac injection or delivery during reperfusion of a coronary artery.
  • Cardiac injury intends any injury to the heart muscle or cardiac cells.
  • Non-limiting examples include heart failure, blunt cardiac injury, coronary artery disease, ischemic heart disease, or myocardial infarction.
  • the injury can be acute or chronic.
  • Heart failure also known as congestive heart failure, is a condition that develops when the heart does not pump enough blood to meet the body needs.
  • “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent that is an amount sufficient to obtain a pharmacological response such as passive immunity; or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the Atty. Dkt. No.: 114198-5210 specified disorder or disease in the patient.
  • a therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.
  • the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from a control or reference sample.
  • the expression level of a gene from one sample may be directly compared to the expression level of that gene from the same sample following administration of a compound.
  • Modes For Carrying Out the Disclosure Applicant’s discovery differs from conventional wisdom in two ways that leads to the disclosed methods. Applicant provides herein methods to treat type I IFN responses that result from mechanical injury rather than the classic systemic autoimmune disease or infections. Applicant also provides methods to treat type I IFN responses that are initiated by non-immune cells rather than immune cells. Conventional wisdom attributes initiation of the type I IFN response to professional innate immune cells such as dendritic cells and myeloid cells. This is primarily viewed as a systemic response.
  • Applicant found it is activated in spatially localized clusters within mechanically injured tissue rather than a consequence of systemic autoimmune diseases or local infection.
  • Applicant shows herein that staining for ISGs (the consequence of type I IFN response activation) after traumatic needle injury, which mimics the effects of needles or cannulas used for therapeutic delivery or injection showed staining in clusters at the site of sharp needle trauma.
  • Applicant also stained for ISGs after myocardial infarction or ischemia reperfusion injury (models of human heart attacks) and observed clustered staining at the borderzone in the infarct and in the stretched atria.
  • Applicant stained for ISGs after transaortic constriction and pressure overload which mimics hypertensive heart disease and aortic stenosis and observed it in clusters in the ventricle and in the stretched atria. Based on these observations, provided herein are methods Atty. Dkt. No.: 114198-5210 to inhibit the type I IFN response in new clinical contexts that are caused by mechanical injury. Methods are also provided to inhibit the type I IFN response selectively in immune and non-immune immune cells rather than immune cells to achieve efficacy without broad immunosuppression (professional innate immune cells use the IFN pathway to defend against pathogenic viruses and other pathogens so inhibiting it in all cells would impair this protective antiviral response).
  • Applicant provides a method to inhibit the type I interferon (IFN) response systemically or locally in a subject that has experienced, is experiencing, or is anticipated to experience an endogenous or exogenous mechanical tissue injury, the method comprising, or alternatively consisting essentially of, or yet further consisting of systemically or locally administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting IFN response in the subject.
  • IFN type I interferon
  • Non-limiting examples of endogenous mechanical tissue injury include injury to any tissue caused by mechanical compression, tension, bending, shearing, torsion, stress, stretch distortion, hypercontraction, imbalanced contraction; sharp or blunt trauma, abrasion, or concussion; or mechanical destabilization caused by loss of tissue such as due to resection or cell death or caused by addition of acellular or cellular material such as amyloid, calcium, atherosclerotic plaque, thrombus, or tumor.
  • tissues that may have experienced, or are experiencing, or are anticipated to experience include cardiac, vascular, skeletal muscle, and other.
  • cardiac diseases or conditions involving endogenous mechanical tissue injury include exercise; ischemia or infarction; cardiomyopathies such as dilated, hypertrophic, infiltrative, inflammatory, arrhythmogenic, drug-induced, toxin-induced, or takosubo; valvular heart disease; acute or chronic arterial or venous systemic or pulmonary hypertension; atrial fibrillation, stretch, or remodeling; myocarditis, pericarditis, or sarcoid; tumor.
  • vascular diseases or conditions involving endogenous mechanical tissue injury include aortic or arterial atherosclerosis or calcific disease, aneurysms or dissection, acute or chronic arterial or venous systemic or pulmonary hypertension, microvascular dysfunction.
  • Examples of skeletal muscle diseases or conditions involving endogenous mechanical tissue injury include exercise or muscle injury due to exercise.
  • Examples of other endogenous mechanical injury are those caused by space-occupying cellular growths or acellular deposits Atty. Dkt. No.: 114198-5210 such as tumors, calcium, or amyloid; swelling or compression such as due to inflammation or infection, concussive injury (e.g., traumatic brain injury).
  • the cells or tissue that are treated are mechanically loaded immune or non-immune cells, such as for example, such as cardiomyocytes, skeletal muscle cells, smooth muscle cells, fibroblasts, vascular tissue, and endothelial cells.
  • Administration can be local or systemic as determined by the condition to be treated as well as the subject being treated.
  • Applicant also provides a method to treat or prevent a disease or condition caused by an endogenous or exogenous mechanical tissue injury in a subject in need thereof, the method comprising, or alternatively consisting essentially of, or yet further consisting of administering to the subject an effective amount of an inhibitory IFN agent, thereby treat or prevent a disease or condition caused by an endogenous or exogenous mechanical tissue injury in the subject.
  • the endogenous or exogeneous mechanical injury is associated with systemic or local type I interferon (IFN) response.
  • IFN systemic or local type I interferon
  • Non-limiting examples of conditions related to endogenous mechanical tissue injury include injury to any tissue caused by mechanical compression, tension, bending, shearing, torsion, stress, stretch distortion, hypercontraction, imbalanced contraction; sharp or blunt trauma, abrasion, or concussion; or mechanical destabilization caused by loss of tissue such as due to resection or cell death or caused by addition of acellular or cellular material such as amyloid, calcium, atherosclerotic plaque, thrombus, or tumor.
  • tissues that may have experienced, or are experiencing, or are anticipated to experience include cardiac, vascular, skeletal muscle, and other.
  • cardiac diseases or conditions involving endogenous mechanical tissue injury include exercise; ischemia or infarction; cardiomyopathies such as dilated, hypertrophic, infiltrative, inflammatory, arrhythmogenic, drug-induced, toxin-induced, or takosubo; valvular heart disease; acute or chronic arterial or venous systemic or pulmonary hypertension; atrial fibrillation, stretch, or remodeling; myocarditis, pericarditis, or sarcoid; tumor.
  • vascular diseases or conditions involving endogenous mechanical tissue injury include aortic or arterial atherosclerosis or calcific disease, aneurysms or dissection, acute or chronic arterial or venous systemic or pulmonary hypertension; microvascular dysfunction.
  • Examples of skeletal muscle diseases or conditions involving endogenous mechanical tissue injury include exercise or muscle injury due to exercise.
  • Examples of other Atty. Dkt. No.: 114198-5210 endogenous mechanical injury are those caused by space-occupying cellular growths or acellular deposits such as tumors, calcium, or amyloid; swelling or compression such as due to inflammation or infection, concussive injury (e.g., traumatic brain injury).
  • the cells or tissue that are treated are mechanically loaded immune or non-immune cells, such as for example, such as cardiomyocytes, skeletal muscle cells, smooth muscle cells, fibroblasts, vascular cells and endothelial cells.
  • Administration can be local or systemic as determined by the condition to be treated as well as the subject being treated.
  • the disease or condition is selected from mechanical stretch, hypercontraction, inflammatory heart disease, myocarditis, myopericarditis, sarcoid, inflammatory cardiomyopathies, acute myocardial ischemia/infarction or chronic ischemic heart disease, localized cardiac injury, vascular stretch or mechanical deformation due to systemic arterial hypertension, pressure overload due to chronic hypertension, and valvular heart disease, atrial stretch and atrial fibrillation, aortic or arterial aneurysms, pulmonary arterial hypertension, pulmonary venous hypertension due to chronically elevated filling pressures, atherosclerosis, skeletal muscle injury, suture sites, cannula insertion sites, biopsy sites, or therapeutic injection sites. Additional diseases and conditions are described herein.
  • exogeneous mechanical tissue injury are disclosed herein.
  • Non-limiting examples include the use of sutures, a cannula, a medical device, a needle, a guidewire, a catheter, an electrode, a bioptome, a stent, or a staple, e.g., a surgical staple.
  • administration of the device such as the suture or staple is coated or incorporated into the agent for delivery to the subject.
  • a method to inhibit the type I IFN response systemically or locally in a subject prior to, concurrently or subsequent to local delivery of a therapy comprising, or Atty. Dkt.
  • No.: 114198-5210 alternatively consisting essentially of, or yet further consisting of administering to the subject an effective amount of an inhibitory IFN agent prior to or concurrently to the local delivery of the therapy, thereby inhibiting the type I IFN response.
  • therapies include administration of one or more of; drugs such as biologics, small molecules, or nucleic acids; viral-based therapies, biomaterials; therapeutic cells, engineered cells, and stem cells; or devices such as deep brain stimulators.
  • a method is provided to inhibit the type I IFN response in and/or to treat mechanical pathologies in a subject in need thereof comprising, or alternatively consisting essentially of, or yet further consisting of administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the IFN response in mechanical pathologies in and/or treat mechanical pathologies.
  • Non-limiting examples of mechanical pathologies are selected from mechanical stretch, hypercontraction, inflammatory heart disease, myocarditis, myopericarditis, sarcoid, inflammatory cardiomyopathies, acute myocardial ischemia/infarction or chronic ischemic heart disease, localized cardiac injury, vascular stretch or mechanical deformation due to systemic arterial hypertension, pressure overload due to chronic hypertension, and valvular heart disease, atrial stretch and atrial fibrillation, aortic or arterial aneurysms, pulmonary arterial hypertension, pulmonary venous hypertension due to chronically elevated filling pressures, atherosclerosis, skeletal muscle injury, suture sites, cannula insertion sites, biopsy sites, or therapeutic injection sites.
  • the methods can further comprise detecting one or more of the quantity, location, distribution, and amount of type I IFN in the subject.
  • the methods can be practiced on subjects such as mammals and human patients. Any appropriate means of administration can be used, e.g., by a method comprising intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.
  • a method for one or more of: reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack each method comprising, or consisting essentially of, or yet further consisting of, by Atty. Dkt. No.: 114198-5210 administering a cardiomyocyte cGAS-STING-IRF3 inhibitor thereby reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack.
  • the inhibitor is a small molecule or a gene therapy which inhibits the activity of Irf3, cGAS, STING and TBK1.
  • Administration can be in vitro or in vivo.
  • the methods can be practiced on subjects such as mammals and human patients.
  • Non-limiting examples of administration include intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub- retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.
  • the subject to be treated is a mammal, or a human patient.
  • the methods can be used in” animal models such as murines, simians, canines, ovines, to test for new therapies or combination therapies.
  • the subject can be a human patient under the supervision of a treating physician. Reducing or treating cardiac injury, promoting, or supporting cardiac regeneration.
  • reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack each method comprising, or consisting essentially of, or yet further consisting of, by administering a type I IFN inhibitory agent or a cardiomyocyte cGAS-STING-IRF3 inhibitor thereby reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack.
  • the inhibitor is a small molecule or a gene therapy which inhibits the activity of Irf3.
  • Administration can be local or systemic and can be in vitro in a tissue system or in vivo in a subject.
  • Subjects can be a mammal, such as a human patient.
  • Inhibiting the type I IFN response systemically or locally before, during, or shortly after surgery. Atty. Dkt. No.: 114198-5210
  • the insertion of needles during suturing of tissue induces mechanical disruption of cells and locally activates the type I IFN response. This can induce local inflammation and promote surgical site complications, e.g., short surgical tissue trauma caused by staples or cautery. Inhibiting the type I IFN response can reduce complications.
  • this disclosure provides a method to inhibit the type I IFN response systemically or locally in a subject who will receive or has received a suture, by administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the IFN response.
  • the subject is a mammal such as a human patient.
  • Administration can be accomplished by administration of a pharmaceutical composition comprising the inhibitory IFN agent, or by coating the suture or other device used for insertions into the subject during surgery. Administration can be prior to, concurrently, or after surgery.
  • the method further comprises surgery prior to, after or concurrently with the administration of the inhibitory IFN agent.
  • Applicant inserted a needle into the hearts of mice as if performing surgery and 3 days later performed in situ hybridization for ISGs. Applicant observed spatially clustered staining for ISGs at the site of needle insertion, indicating that the type I IFN response was locally activated.
  • systemic anti-IFNAR Ab the mouse equivalent of Astrazeneca’s Anifrolumab TM
  • the local needle site ISG response was eliminated.
  • Applicant delivered the anti-IFNAR antibody therapy locally through the inserted needle without systemic inhibition and again the ISG response was eliminated. This could also be accomplished by coating the needle or suture with anti- IFN therapy.
  • the proinflammatory type I IFN response is induced by surgery and can be inhibited systemically or locally to reduce surgical site complications. Inhibit the type I IFN response systemically or locally before, during, or shortly after local delivery of therapy.
  • Applicant also provides a method to inhibit the type I IFN response systemically or locally in a subject prior to, concurrently or subsequent to local delivery of therapy by administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the type I IFN response.
  • the subject is a mammal such as a human Atty. Dkt. No.: 114198-5210 patient.
  • Administration can be accomplished by administration of a pharmaceutical composition comprising the inhibitory IFN agent.
  • the method further comprises administration of the local delivery of a therapy prior to, after or concurrently with the administration of the inhibitory IFN agent.
  • the subject is a mammal such as a human patient.
  • Therapies are often delivered locally by direct injection of tissue or by inserting a cannula and delivering therapy via the cannula. Examples of therapies delivered this way include drugs such as biologics, small molecules, or nucleic acids; viral-based therapies, biomaterials; therapeutic cells, engineered cells, and stem cells; or devices such as deep brain stimulators. Both needles and cannulas mechanically disrupt tissue when they are inserted and thus induce the type I IFN response which can be a barrier to therapy. Applicant has reduced this to practice as detailed above with the needle experiments.
  • Applicant has shown that this response can be inhibited with systemic or local type I IFN inhibitory agent.
  • CLPT Clearpoint Neuro
  • This is used for introduction of device therapies (deep brain stimulation), and other therapeutic modalities (drug, viral, cell, etc.).
  • a secondary benefit of inhibiting the type I IFN response in the context of local delivery of gene therapy is that it would locally reduce the anti-viral response, which is a tissue response that impedes infection by therapeutic viruses.
  • an ISG-binding compounds can serve as systemically delivered companion diagnostic imaging agents (nuclear, PET, MRI, CT, ultrasound) by localizing to sites of ISG induction to quantify location, distribution, and amount of type I IFN response and predict which patients are most likely to benefit from type I IFN inhibition. Atty. Dkt.
  • cGAS is part of an IFN-inducing pathways, but it is itself an inducible ISG. Therefore, the cGAS-binding compounds being developed as therapeutics (e.g., VENT-003) could be radiolabeled or similarly modified and repurposed as imaging agents to serve as a companion diagnostic and identify which patients have the greatest type I IFN responses and are most likely to respond to therapy and to localize the response to a specific organ or tissue. More generally, any selective ISG-binding compound, if modified to create modality-specific contrast, could serve as an imaging agent for a companion diagnostic to quantify which patients are candidates for type I IFN inhibition.
  • tissue whether obtained by biopsy or excision, can be stained for ISG proteins or probed for interferon stimulating genes (ISG) RNA using in situ hybridization to quantify the amount and distribution of type I IFN response activation and serve as a companion diagnostic to predict which patients are most likely to benefit from type I IFN inhibition.
  • ISG interferon stimulating genes
  • Applicant performed in situ hybridization for ISGs such as Ifit1 and Cxcl10 or full spatial transcriptomic profiling using 10x Visium or MERFISH, and by each method, Applicant observed spatially clustered ISG responses. Therefore, staining or probing for ISGs can serve as a companion diagnostic to identify which patients have type I IFN responses and are most likely to respond to type I IFN response inhibition. Inhibit the type I IFN response in mechanical pathologies. Applicant also provides a method to inhibit the type I IFN response in mechanical pathologies and/or treat mechanical pathologies.
  • this disclosure provides a method to inhibit the type I IFN response in mechanical pathologies in a subject in need thereof by administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the IFN response in mechanical pathologies and/or treat mechanical pathologies.
  • the subject is a mammal such as a human patient.
  • Administration can be accomplished by administration of a pharmaceutical composition comprising the inhibitory IFN agent.
  • Atty. Dkt. No.: 114198-5210 The method can be used to treat the following mechanical injuries: Mechanical stretch – due to volume overload or dysrhythmia including atrial stretch associated with atrial fibrillation, ventricular stretch associated with diverse causes of cardiomyopathy or valvular heart disease leading to atrial or ventricular wall stretch.
  • valvular heart disease As both can cause pressure overload seen in the ventricle and atria, atrial stretch and atrial fibrillation since focal interferon responses in stretched atria (stretched atria can be caused by many disorders including chronic hypertension, diastolic dysfunction, and valvular heart disease (e.g., aortic stenosis, mitral regurgitation, mitral stenosis).
  • Ischemic heart disease Acute myocardial ischemia/infarction or chronic ischemic heart disease occurs due to insufficient delivery of oxygenated blood through one or more obstructed branches of the coronary artery tree. This causes localized cardiac injury and induces the type I IFN response in the infarct and at the borderzone.
  • Atherosclerosis stretch of vascular smooth muscle cell, endothelial cells, or fibroblasts in or surrounding atherosclerotic lesions.
  • Skeletal muscle injury due to exercise, trauma, or disease
  • Cannula insertion sites for example in the brain as performed by all Clearpoint Neuro (CLPT) procedures (see above).
  • Biopsy sites explicit mechanical disruption of tissue similar to sutures/cannulas above (brain, heart, liver, kidney, etc.) Atty. Dkt. No.: 114198-5210
  • Therapeutic injection sites – drugs such as biologics, small molecules, or nucleic acids; viral-based therapies, biomaterials; therapeutic cells, engineered cells, and stem cells are being injected into many different tissues for therapy.
  • Such injections will induce the type I IFN response which can make these therapies less effective.
  • Applicant propose inhibiting the IFN response to improve the therapeutic efficacy of these injected therapies.
  • Selective inhibit of the type I IFN response in non-immune cells Conventional wisdom attributes initiation of the type I IFN response to professional innate immune cells (e.g., dendritic cells and myeloid cells).
  • Applicant discovered that mechanical pathologies initiate the type I IFN response in non-immune cells.
  • Applicant also provides a method to inhibit the type I IFN in non-immune cells in a subject in need thereof, by administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the type I IFN response in non-immune cells in the subject.
  • the subject is a mammal such as a human patient.
  • Administration can be accomplished by administration of a pharmaceutical composition comprising the inhibitory IFN agent.
  • a pharmaceutical composition comprising the inhibitory IFN agent.
  • an effective amount of an anti-senso oligo (ASO) therapy to cardiomyocytes can be delivered to cardiomyocytes to inhibit activation of the type I IFN response by targeting one or more molecules in the activation of the type I IFN response as detailed above.
  • compositions and modes of Administration are compositions comprising the inhibitory agents for use in the methods as described herein.
  • the compositions, including pharmaceutical compositions comprising, consisting essentially of, or consisting of the inhibitory agent can be in combination of other therapeutic agents can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes.
  • the pharmaceutical formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral, subcutaneous, oral, buccal, rectal, sublingual, or transdermal administration routes.
  • parenteral administration comprises, or consists essentially of, or yet further consists of, intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra- arterial, intra-articular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration.
  • the pharmaceutical composition is formulated for local administration.
  • the pharmaceutical composition is formulated for systemic administration. In some aspects, they are administered by coating a device or suture for example, that will be mechanically injuring the tissue of the subject.
  • the pharmaceutical formulations include, but are not limited to, lyophilized formulations, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.
  • the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form.
  • Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like.
  • Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like.
  • the pharmaceutical formulations further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane, and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids
  • bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane
  • buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.
  • the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range
  • Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
  • the pharmaceutical formulations include, but are not limited to, sugars like trehalose, sucrose, mannitol, maltose, glucose, or salts like potassium phosphate, Atty. Dkt.
  • the pharmaceutical formulations further include diluent which are used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling.
  • Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as AVICEL ® , dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar, such as Di- PAC ® (Amstar), mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner’s sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like.
  • the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance.
  • disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or AMIJEL ® , or sodium starch glycolate such as PROMOGEL ® or EXPLOTAB ® , a cellulose such as a wood product, methylcrystalline cellulose, e.g., AVICEL ® , AVICEL ® PH101, AVICEL ® PH102, AVICEL ® PH105, ELCEMA ® P100, EMCOCEL ® , VIVACEL ® , MING TIA ® , and SOLKA-FLOC ® , methylcellulose, croscarmellose,
  • the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.
  • Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials.
  • Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (STEROTEX ® ), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, STEAROWET ® , boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as CARBOWAXTM, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as SYLOIDTM, CAB-O-SIL ® ,
  • Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents.
  • Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N- methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, Atty. Dkt.
  • Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.
  • Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium biphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, poly sorb ate-20 or TWEEN® 20, or trometamol.
  • Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g
  • Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., PLURONIC ® (BASF), and the like.
  • compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., PLURONIC ® (BASF), and the like.
  • BASF PLURONIC ®
  • Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants is included to enhance physical stability or for other purposes. Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, Atty. Dkt.
  • wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.
  • compositions for the administration can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy.
  • the pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the compounds provided herein into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • each compound of the combination provided herein is included in an amount sufficient to produce the desired therapeutic effect.
  • compositions of the present technology may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal, and vaginal, or a form suitable for administration by inhalation or insufflation.
  • topical administration the combination of compounds can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well-known in the art.
  • Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection) as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.
  • Useful injectable preparations include sterile suspensions, solutions, or emulsions of the compounds provided herein in aqueous or oily vehicles.
  • the compositions may also contain formulating agents, such as suspending, stabilizing, and/or dispersing agents.
  • the formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.
  • the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, Atty. Dkt. No.: 114198-5210 buffer, and dextrose solution, before use.
  • the combination of compounds provided herein can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.
  • the pharmaceutical compositions may take the form of, for example, lozenges, tablets, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc, or silica
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the combination of compounds provided herein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., corn starch or alginic acid); binding agents (e.g. starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc).
  • the tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques well known to the skilled artisan.
  • the pharmaceutical compositions of the present technology may also be in the form of oil-in-water emulsions. Atty. Dkt. No.: 114198-5210
  • Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore TM , or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.
  • one or more compositions disclosed herein are contained in a kit.
  • kits comprising, consisting essentially of, or consisting of one or more compositions disclosed herein and instructions for their use.
  • Dosage and Dosage Formulations the combinations or compositions comprising the inhibitory agent are administered to a subject suffering from a condition as disclosed herein, such as a mammal or a human, either alone or as part of a pharmaceutically acceptable formulation, once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently.
  • Administration of the composition or combination alone or in combination with the additional therapeutic agent and compositions containing same can be accomplished by any method that enables delivery to the site of action.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient can also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient.
  • dosage values can vary with the type and severity of the condition to be alleviated and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values.
  • one or more of the methods described herein further comprise, or consists essentially of, or yet further consists of, a diagnostic step.
  • a sample is first obtained from a subject in need of the therapy or suspected of having a disease or condition described above.
  • Exemplary samples include, but are not limited to, cell sample, tissue sample, tumor biopsy, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord
  • the sample is a tumor biopsy.
  • the sample is a liquid sample, e.g., a blood sample.
  • the sample is a cell-free DNA sample.
  • Various methods known in the art can be utilized to determine the presence of a type I IFN in a cell or tissue. Expression levels or abundance can be determined by direct measurement of expression at the protein or mRNA level, for example by microarray analysis, quantitative PCR analysis, or RNA sequencing analysis. Alternatively, labeled antibody systems may be used to quantify target protein abundance in the cells, followed by immunofluorescence analysis, such as FISH analysis.
  • the diagnostic steps can be performed prior to or after administration and may be performed throughout the course of treatment to monitor the subject’s response.
  • the diagnostic method is performed to detect one or more of the quantity, location, distribution, and amount of type I IFN in the subject or tissue.
  • Atty. Dkt. No.: 114198-5210 Experimental Description Applicant hypothesized that the distribution of ISG expression could be explained by focal initiation of the type I IFN response followed by spread and amplification of the secondary response in neighboring cells. To test this hypothesis, Applicant analyzed infarcted mouse and human hearts using spatial transcriptomics and single molecule RNA fluorescence in situ hybridization RNA (smFISH). Experimental Methods Animals Adult C57BL/6J mice were purchased from the Jackson Laboratory at 10 weeks of age used as wildtype controls. Irf3 -/- were bred inhouse from existing colonies.
  • Ccr2 -/- (strain 004999), Ifnar 1- (028288), Cgas -/- (026554), and STING gt/gt (017537) mice were purchased from Jackson Laboratory.
  • Nuclear reporter mice (tdTom-NLS) were purchased from Jackson Laboratory (025106).
  • Cardiomyocyte-specific nuclear rupture reporter mice were generated by crossing floxed tdTom-NLS with Myh6 cre/+ mice. Irf3 fl/fl were a generous gift donated from Dr. Tadatsugu Taniguchi.
  • Cell type-specific transgenics were generated by crossing Irf3 fl/fl with transgenic mice expressing cre-recombinase under the control of the following promoters purchased from Jackson Laboratory: Myh6 cre/+ (011038), Col1 ⁇ 1 creERT2/+ (016241), Cx3cr1 creERT2/+ (025524), S100a8 cre/+ (0216141), Tie2 cre/+ (008863).
  • Inducible deletion was accomplished by intraperitoneal injection of tamoxifen 20 mg/mL in corn oil or vehicle injection for 5 consecutive days in Col1 ⁇ 1 creERT2/+ and Cx3cr1 creERT2/+ mice. Animals were allowed 7 days to recover after the last tamoxifen or vehicle injection before conducting any surgical procedures.
  • mice were maintained in a pathogen-free environment of the University of California San Diego (UCSD) facilities and all animal experiments were approved by the subcommittee on Animal Research Care at UCSD (Institutional Animal Care and Use Committee, S17144).
  • Permanent ligation (MI) surgery and cardioprotective therapy For permanent ligation, mice were intubated and ventilated with 2% isoflurane. Thoracotomy was performed at the fourth left intercostal space was performed to expose the Atty. Dkt. No.: 114198-5210 heart and visualize the left anterior descending artery (LAD).
  • LAD left anterior descending artery
  • the LAD was permanently ligated with an 8-0 nylon suture in mice with MI, and hearts were collected at various time points as hours (hr) or days (D) post-surgery (1hr, 4hr, D1, D3, D7, or D28). Intercostal space and skin were sutured closed using 6-0 prolene sutures. Cardioprotective therapy of anti- IFNAR-Ab was performed as previously described. Mice were treated with two intraperitoneal doses of 500 ⁇ g of MAR1-5A3 IFNAR neutralizing antibody (anti-IFNAR- ab) at 8 hours and 48 hours post-MI surgery (BioXCell catalog no. BE04241)3,14. Hearts were harvested at D3 post-infarct.
  • NP Needle Pass
  • mice were anesthetized under 2% isoflurane and partial thoracotomy was performed above the fourth left intercostal space; a chest retractor was inserted and placed between the third and fourth intercostal spaces.
  • a 28G beveled needle was inserted into the lateral LV free-wall directly to the right of the LAD at the position in which Applicant normally perform permanent ligation in MI; the needle did not penetrate through to the endocardium.
  • the 28G beveled needle was held in position in the midventricular wall for 3 seconds before being withdrawn.
  • the chest retractor was then removed, and the intercostal space was closed using 6-0 prolene sutures.
  • a sterile 20G flexible angiocatheter was placed within the pleural space prior to removal of chest retractor.
  • the intercostal space and dermis were closed using 6-0 prolene sutures.
  • a syringe was attached to the angiocatheter and negative pressure was manually applied simultaneously as the catheter was withdrawn. Surgical glue was then applied to the remainder of the skin incision.
  • H9 embryonic stem cells Human H9 embryonic stem cells (ESCs) were commercially acquired and cultured on Matrigel-coated 6-well plates (WiCell, Madison, WI). H9 ESCs were maintained in mTeSR1 Media (StemCell Technologies #85851) until 90% confluency. Once confluent, cells were split with mTeSR1 media and 5 ⁇ M ROCK inhibitor Y27632, counted, and plated at a confluency of .5 million cells/mL on Matrigel-coated 12-well plates (Tocris #1524). Cells Atty. Dkt.
  • No.: 114198-5210 were fed daily for 3 days, and on the following day, cells were fed with RPMI basal medium and 4 ⁇ L of 36 mM of GSK3 inhibitor CHIR99021 (Sigma R7388; Tocris #4423) to begin differentiation.
  • RPMI basal medium 4 ⁇ L of 36 mM of GSK3 inhibitor CHIR99021 (Sigma R7388; Tocris #4423) to begin differentiation.
  • media was removed, and cells were fed with only RPMI basal medium.
  • RPMI media After 2 days, and after another 2 days, cells were detached with ACCUTASE and inactivated by 20% fetal bovine serum (FBS) in RPMI medium (RPMI20) (Stemcell Technologies, 07922). Cells were resuspended in LaSR basal medium containing 5 ⁇ M Y27632 ROCK inhibitor and seeded onto gelatin-coated 12-well plates at a density of 5,000 cells per well. For the following 5 days, cells were fed with 12 mL of LaSR medium (advanced DMEM/F12 medium; 6.5 mL Glutamax, 500 ⁇ L of antioxidant 100 mg/mL ascorbic acid solution) + 3 ⁇ M CHIR99021 GSK3 inhibitor.
  • LaSR medium advanced DMEM/F12 medium
  • Glutamax 500 ⁇ L of antioxidant 100 mg/mL ascorbic acid solution
  • Epicardial cells were removed from plates with ACCUTASE and quenched with RPMI20. Cells were resuspended in LaSR medium supplemented with 5 ⁇ M Y27632 ROCK inhibitor. Following overnight cell attachment, epicardial cells were differentiated into fibroblasts and maintained in LaSR medium supplemented with 10 ng/mL bFGF (RnD Systems, #233-FB). After differentiation, hiPSC-Fibroblasts were then replated and expanded on gelatin-coated 6 well plates and maintained on Human Cardiac Fibroblast Media (catalog no. MSDS 315-500, Cell Application, INC).
  • Applicant used recombinant human TGF ⁇ (10 ng/mL, Peprotech 100-21) and/or human IFN ⁇ (10 ng/mL, Peprotech 300-02) suspended in culture medium for 24 hours before harvesting for downstream analysis.
  • Atty. Dkt. No.: 114198-5210 Fibroblast functional assessment with collagen gel contraction assay Human iPSC-derived cardiac fibroblasts or L929 fibroblasts were enzymatically dissociated from confluent culture plates, pelleted by centrifugation, and quantified with hemocytometer.
  • RNA isolation and qPCR RNA was isolated from myocytes using RNeasy Mini Kit (Qiagen catalog no.74536) and reverse transcribed with high-efficiency enzymes (Applied Biosystems catalog no. 438813).
  • POSTN Hs01566750_m1
  • SPARC Hs00234160_m1
  • COL1 ⁇ 1 Hs00164004_m1
  • IRF7 Hs00164004_m1
  • IFI27 Hs01086373_g1
  • IFIT3 Hs01922752_s1
  • GAPDH Hs02786624_g1
  • earsnip samples were digested in NaOH and neutralized by Tris HCL. DNA was extracted and amplicons were amplified for gel electrophoresis. Primer sequences used for each transgenic line are included in Tables 1 and 2. Atty. Dkt.
  • mice were closely monitored for 12 consecutive hours each day starting from day 3 to day 14 post- MI, which is designated as the window of susceptibility for a rupture event to occur in mice. When an acute mortality was observed, the chest cavity was immediately opened, and hearts were immediately perfused with cold PBS and harvested.
  • Plasmacytoid dendritic cell depletion Mice were pre-treated with intraperitoneal doses of 500 ⁇ g of anti-mouse CD317 (BST2) antibody or isotype control antibody once a day for 3 days prior to MI surgery (BioXCell #BE0311). Hearts were harvested at D3 post-infarct, flow sorted, and processed for single cell RNA sequencing.
  • BMDC bone marrow derived cells
  • cytosolic NLS signal was visualized and imaged using the Nikon AXR point scanning confocal microscope. For each field of view taken at 60X magnification, 15-20 optical slices were obtained and used for maximal intensity projections. Ruptured cardiomyocyte nuclei were normalized to total number of nuclei analyzed in each field of view acquired from the remote zone or borderzone adjacent to infarct. Sequencing-based spatial transcriptomics OCT-embedded cardiac tissue blocks were cryosectioned in short axis orientation at approximately 10 ⁇ m in thickness at cryostat temperature set to -22 ⁇ C. Sections were stained with H&E, and images were obtained using 20X magnification on a Nikon Eclipse Ti2-E widefield microscope.
  • Sections were then processed for spatially resolved gene expression using the Visium Spatial Transcriptomics Kit according to the manufacturer’s protocol (10X Genomics). Permeabilization time of infarcted murine hearts was previously optimized and determined at 30 minutes. Quality control for cDNA and libraries were performed on Agilent TapeStation before sequencing on the Illumina NovaSeq6000 instrument. The resulting sequencing data was processed, and images were aligned using the SpaceRanger v.1.3.1 pipeline (10X Genomics). Quality control, normalization, and integration for sequencing-based spatial transcriptomics Sequencing-based spatial transcriptomic assays were initially preprocessed to assess variance in feature counts/spot and the biological differences in cell density across heterogenous tissue morphology of the infarcted heart.
  • the stimulated group was similarly normalized using SCTransform() and PCA analysis.
  • FindIntegrationAnchors() was performed followed by PrepSCTIntegration() on a merged list of the 2 objects to anchor and integrate the datasets together.
  • Graph-based clustering using K-nearest neighbors (KNN) function FindNeighbors() was performed and shared nearest neighbors were identified using FindClusters() at a resolution of .60 on the entire integrated dataset.
  • Moran I test statistic
  • Sepal score and IFNIC colony size quantification
  • Moran I test statistic is a spatial autocorrelation coefficient used to quantify and measure spatial enrichment and distribution of individual genes.
  • Moran’s I was chosen as it is independent from differential gene expression and unrelated to clustering information. This metric scale ranges from 1 (significant spatial enrichment) to 0 (homogenously distributed throughout biological sample).
  • Moran’s I revealed ISGs are spatially enriched and formed colonies with high autocorrelation test statistic indicating numerous focal IFN-responses.
  • Applicant used the Sepal method, which quantifies spatially clustered features using a diffusion-based simulation.
  • K-means nearest neighbors clustering of chosen ISG transcript (ex. Ifit1) was then performed, and this approach yielded 4-8 colonies that were significantly absent in Irf3 -/- mice.
  • IFNIC colony area was calculated based on the 55 ⁇ m diameter of an individual Visium spot, and the distance between the centers of each adjacent spot measures 100 ⁇ m.
  • Activated fibroblasts, IZ, and BZ mapping strategy Applicant mapped BZ or IZ labels to spatial transcriptomic clusters using a list of differentially expressed genes compiled from sn/scRNA-seq data of cardiomyocytes, innate immune cells, and fibroblasts based on previously described methods 18 . .
  • CM-rich spatial clusters To classify CM-rich spatial clusters, Applicant evaluated the gene-set scores found uniquely elevated in post-MI samples specific to the borderzone. Mapping of gene-set scores from CM snRNA-seq data to space was performed using area under the receiver operating characteristic (AUROC) analysis. Clusters with an AUROC > 0.7 were positively classified. For IZ clusters, Applicant performed subclustering to determine immune cell niches to map to space. To map activated fibroblasts to space, Applicant performed subclustering of our integrated snRNA-seq dataset to determine subclusters of fibroblasts designated as activated (Postn+) or non-activated (Postn-).
  • AUROC receiver operating characteristic
  • Activated Postn+ fibroblasts were used in downstream analysis and average expression of genes within this subset when comparing D3 post-MI snRNA-seq data from WT samples vs. Irf3-/- or Ifnar-/- samples. Zones were quantified and normalized to total UMI and scaled by 10,000. This analysis was performed with all spots from representative day 3 post-MI sample and with IZ pixels (defined by clustering) to further explore heterogeneity at a timepoint predetermined to yield the highest expression of ISGs by snRNA-seq and spatial transcriptomic data. Correlation tests were also performed with gene- set scores to confirm colocalization patterns inferred from clustering analyses. Atty. Dkt.
  • the coordinates of Visium spots with overlying tissue were extracted in which a random coordinate or location was chosen for each simulation.
  • the randomly chosen coordinate was assigned a neighboring spot (N+1) and assessed for any overlap or adjacency with BZ HI pixels.
  • N+1 a neighboring spot
  • Probabilities generated from the simulations were transformed using Chi square contingency analysis and P-values were assessed using Fisher’s Exact test to compare probability versus outcome i.e., the percentage of clusters that overlapped with any BZ HI pixels.
  • IZ and BZ neighbor analysis Applicant quantified the fraction of BZ and ISG pixels in second-order neighbors (defined by clustering analyses). Results were binned based on the reference pixel classification; IZ contained primarily transcripts designated as innate Atty. Dkt. No.: 114198-5210 immune process or cells whereas BZ contained primarily transcripts designated from BZ myocytes and activated fibroblasts. From this, Applicant quantified both scores as a function of distance (0–400 ⁇ m) relative to reference pixels.
  • IFNIC colonies using infarcted human tissue Spatial transcriptomic analysis of infarcted human samples was determined using deposited datasets from a recently published study using the same genome-wide Visium platform and from our previously published samples. Patient samples were integrated using SCTransform described above, and metrics of spatial autocorrelation and size of IFNIC colonies were performed using methods described above and in the same manner as murine spatial transcriptomic analysis.
  • RNA MERFISH Imaging-based spatial transcriptomics
  • MEFISH error-robust fluorescent in situ hybridization
  • encoding probes were designed to target specifically 40-nt sub-regions of selected transcripts 37,63 .
  • RNA MERFISH Single mRNA molecules were computationally decoded, and the total transcript signal and DAPI nuclear stain were used to perform cell segmentation with machine learning algorithm Cellpose 64 .
  • Quality control, normalization, and integration for RNA MERFISH Data analysis of RNA MERFISH data was performed with single-cell sequencing analysis tools like Scanpy and Squidpy 65 . Quality control metrics were determined by visualizing distribution plots of the raw data for total transcript counts and number of genes with more than one counts per cell. These metrics were used to determine covariates that may affect the quality of the dataset and filtered the data accordingly. QC metrics are displayed in FIGS. 14B – 14F). The data is then normalized using Scanpy and log normalization of the total number of transcripts.
  • RNA MERFISH experiments with multiple cardiac tissue sections and experimental conditions to control for batch effects. Data integration across sections from different animals was performed using Scanpy’s principal component-based method ingest. Applicant used the WT D3 MI mouse cardiac section as our reference dataset and performed Leiden clustering with subsets of marker genes from our encoding probe gene panel as follows: borderzone cardiomyocytes [Nppa, Flnc, Ankrd1], fibroblasts [Col1 ⁇ 1, Col6 ⁇ 3], macrophages [Cd68, Adgre1], neutrophils [Cxcr2, Csf3r], and endothelial cells [Pecam1, Flt1].
  • DNA MERFISH Genome-scale in situ chromatin imaging DNA MERFISH was performed as previously described 37 . Briefly, the mouse genome was partitioned into 260 distinct DNA loci that spanned all 21 murine chromosomes. Each genomic loci spanned ⁇ 30-kb and was targeted by 200-300 specific DNA encoding probes. These primary probes were synthesized from an oligonucleotide pool from Twist Biosciences and each contained a 40-nt target sequence specific to a region of DNA loci.
  • This method considers whether a set of features are densely grouped or homogenously distributed with a certain radius ⁇ .
  • Epsilon was unbiasedly selected using k-nearest neighbor analysis. This algorithm identified 8 groups of densely clustered ISG transcripts (Ifit1, Ifit2, Ifit3, Cxcl10) as ISG expression in low density regions that were designated as outliers or “scattered” ISG expression.
  • Nuclear Morphology In cardiac sections that had undergone RNA MERFISH, cardiomyocyte nuclei were identified by the colocalization of hybridized Tnnt2 probes and DAPI fluorescence present in both the borderzone and remote zone. Approximately 200 nuclei were quantified in each region by performing nuclear segmentation and creating a binary segmentation mask.
  • hearts were enzymatically digested for 45 minutes in continuous agitation at 37oC in 450 U/mL collagenase, 125 U/mL collagenase XI, 60 U/mL DNAse, and 60 U/mL hyaluronidase.
  • minced ventricles were suspended in .6 mL nucleus lysis buffer supplemented with .2 U/ ⁇ L RNase inhibitor (Sigma product no. NUC101, Enzymatics part no. Y9240L).
  • Minced tissue was further homogenized with 2 mL dounce grinder for approximately 10 strokes with A-sized pestle and 20 strokes with B-sized pestle (Sigma catalogue no. D8938).
  • the lysates were treated with an additional 1 mL of lysis buffer and incubated for 2 additional minutes; lysates were filtered through consecutive 100, 50, and 20- ⁇ m strainers (CellTrics 04-004-2318, 04-004-2317 and 04-0042- 2315).
  • Nuclei were pelleted by centrifugation at 1,000g for 5 minutes at 4oC. Subsequent washes were performed until final suspension of nuclei was made using 2% BSA in PBS supplemented with RNase inhibitor. Nuclei suspension was treated with 10 ⁇ g ml ⁇ 1 4′,6- diamidino-2-phenylindole (DAPI), counted on hemocytometer, and volume adjusted to produce a final suspension of 1000 nuclei/ ⁇ L. Atty. Dkt.
  • DAPI diamidino-2-phenylindole
  • sn/scRNA-seq Normalization Quality control, normalization, and integration for sn/scRNA-seq Normalization was performed to account for variability in depth of sequencing reads per cell or nuclei as previously described.
  • the total transcript count for each nucleus was scaled to 10,000 molecules, and raw counts for each gene were normalized to the total count of captured transcripts associated with the barcoded cells or nucleus and natural log transformed.
  • Nuclei that contain at least 200 uniquely expressed genes represented in at least 3 nuclei were retained for further analysis. Ribosomal and hemoglobin transcripts were excluded to avoid incorporation of transcriptional artifacts or technical variables accrued during nuclei isolation.
  • MI causes ischemic cell death in the IZ that leads to mechanical stress in the BZ resulting in nuclear rupture and loss of compartmentalization of genomic DNA primarily in BZ cardiomyocytes and secondarily in BZ fibroblasts.
  • This enables activation of the cGAS-STING-IRF3 DNA sensing pathway, and production of secreted IFNs, which diffusively spread to IFNAR-expressing neighbor cells that respond by expressing ISGs and forming the observed IFNIC colonies.
  • IFN-exposed fibroblasts exhibit impaired activation, contractile function, and expression of protective matricellular proteins, which when localized at sites of high mechanical stress (e.g., the junction of the ventricular free wall and septum), increases vulnerability to pathologic remodeling and catastrophic rupture (FIG. 5K).
  • the newly described BZ IFNIC colonies represent a novel IFN response of intracardiac origin initiated by non-immune cells that is distinct from previously described extra cardiac sources (FIG. 21).
  • multi-omics technologies are poised to reveal new microenvironment niches in healthy and injured tissues that shape biological form and function.
  • Applicant uncover a new pathologic niche within the borderzone of the infarcted heart that has global effects on pathologic remodeling and survival.
  • Applicant shows in one aspect that selectively inhibiting the cGAS-STING-IRF3 pathway in non- Atty. Dkt. No.: 114198-5210 immune BZ cardiomyocytes and fibroblasts provides therapeutic benefit while avoiding broader immunosuppression associated with inhibiting the type I IFN response across all innate immune cells 61,62 .
  • Equivalents It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention.
  • Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786- 791 (2013). https://doi.org/10.1126/science.1232458 9 Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826-830 (2013). https://doi.org/10.1126/science.1229963 Atty. Dkt. No.: 114198-5210 Patel, S. J., King, K. R., Casali, M. & Yarmush, M. L. DNA-triggered innate immune responses are propagated by gap junction communication.
  • STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674-678 (2008). https://doi.org/10.1038/nature07317 Sheehan, K. C. et al. Blocking monoclonal antibodies specific for mouse IFN- alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo hydrodynamic transfection. J Interferon Cytokine Res 26, 804-819 (2006). https://doi.org/10.1089/jir.2006.26.804 Schneider, W. M., Chevillotte, M. D. & Rice, C. M. Interferon-stimulated genes: a complex web of host defenses.
  • IFNAR-1 mouse IFN- alpha/beta receptor subunit 1
  • IFN-beta Plays Both Pro- and Anti-inflammatory Roles in the Rat Cardiac Fibroblast Through Differential STAT Protein Activation. Frontiers in pharmacology 9, 1368 (2018). https://doi.org/10.3389/fphar.2018.01368 Oka, T. et al. Genetic manipulation of periostin expression reveals a role in cardiac hypertrophy and ventricular remodeling. Circ Res 101, 313-321 (2007). https://doi.org/10.1161/CIRCRESAHA.107.149047 Atty. Dkt. No.: 114198-5210 Humeres, C. et al.
  • Anifrolumab an Anti-Interferon-alpha Receptor Monoclonal Antibody, in Moderate-to-Severe Systemic Lupus Erythematosus. Arthritis Rheumatol 69, 376-386 (2017). https://doi.org/10.1002/art.39962 Huang, R. J. et al. A spatially mapped gene expression signature for intestinal stem- like cells identifies high-risk precursors of gastric cancer. bioRxiv (2023). https://doi.org/10.1101/2023.09.20.558462 Atty. Dkt. No.: 114198-5210 Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M.

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Abstract

Provided are methods to inhibit the type I interferon (IFN) response in mechanically injured immune or non-immune cells and tissue and to treat or prevent associated disorders in a subject in need thereof by administering to the subject an effective amount of an inhibitory IFN agent.

Description

Atty. Dkt. No.: 114198-5210 COMPOSITIONS AND METHODS TO TREAT MUSCLE INJURY AND DISEASE CROSS-REFERENCE TO RELATED APPLICATION This application claims priority under 35 U.S.C. § 119(e) of U.S. Application Serial No.: 63/468,507, filed May 23, 2023, the contents of which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under DP2AR075321 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. TECHNICAL FIELD The present invention relates generally to the field of the treatment and prevention of the damage to muscle tissues for example cardiomyocytes, injury such as myocardial injury, myocardial infarction, ischemia, pressure overload, and sharp needle trauma. BACKGROUND Innate immune responses to sterile tissue injury are often credited to professional immune cells (e.g., myeloid cells) sensing damage molecules released by dying cells1,2. Though inflammation is necessary for wound healing, excessive inflammation can exacerbate tissue injury and dysfunction3. Nowhere is this more evident than in the heart, where ischemic injury provokes exuberant inflammation after myocardial infarction (MI) followed by infarct expansion, pathologic remodeling, and heart failure, making it the most common cause of death in the world4,5. Thus a need exists in the art to treat MI and associated disorder of cardiac and other muscles and muscle organs. This disclosure satisfies this need and provides related advantages as well. SUMMARY OF THE DISCLOSURE In addition to the many proinflammatory innate immune pathways activated after MI, Applicant recently discovered that MI induces the antiviral type I interferon (IFN) response Atty. Dkt. No.: 114198-5210 via the double stranded DNA (dsDNA) sensor cyclic GMP-AMP Synthase (cGAS). Upon sensing dsDNA, cGAS catalyzes production of cyclic di-GMP-AMP (cGAMP), a gap junction permeable second messenger that signals via the adaptor Stimulator of Interferon Genes (STING) and activates Interferon Regulatory Factor 3 (Irf3), which upregulates expression of secreted type I IFN (IFNα and IFNβ). The diffusible IFN cytokine then signals via the interferon α receptor (Ifnar) on the surface of cells in the local microenvironment and upregulates expression of hundreds of effector molecules known as interferon stimulated genes (ISGs)6-9. Since Ifnar-dependent ISG-expression can occur in any cell type, Applicant refer to cells expressing ISGs as interferon-induced cells, or IFNICs. It has been reported that globally inhibiting the MI-induced IFN response was protective against pathologic remodeling, ventricular dilation, and rupture; however, the reason for protection and its relevance to humans was unknown6. Single-cell transcriptomics has rapidly advanced the understanding of cellular heterogeneity within infarcted hearts. Curiously, Applicant and others have noticed small subpopulations of IFNICs in multiple immune and non-immune cell subtypes. It is unknown how small fractions of so many cell types become activated by IFNs while the majority avoid activation6,10. Moreover, it is unknown which cell type secretes IFN to initiate the response. Sterile inflammation after myocardial infarction (MI) is classically credited to myeloid cells interacting with damage associated molecules dispersed throughout the infarct. Here, Applicant show that in contrast, cardiomyocytes are essential initiators of the type I interferon (IFN) response at the infarct borderzone (BZ). Using spatial transcriptomic analysis of MI in mice and humans, Applicant find that the IFN response to MI is spatially clustered in focal colonies of interferon induced cells (IFNICs) expressing interferon stimulated genes (ISGs) that decorate the BZ where cardiomyocytes undergo mechanical destabilization and “loss-of- neighbor” stress. Cardiomyocyte-selective deletion of interferon regulatory factor 3 (Irf3) abrogated IFNIC colonies and phenocopied globally Irf3-deficient mice after MI. In contrast, IFNIC colonies remained at the BZs of mice lacking Irf3 in fibroblasts, endothelial cells, or neutrophils; of Ccr2-deficient mice, or of plasmacytoid dendritic cell-depleted mice. IFN and ISG expression blunted the protective matricellular programs of BZ fibroblasts and left them vulnerable to pathologic remodeling and ventricular rupture. In mice that died within the first week after MI, Applicant consistently found IFNIC Atty. Dkt. No.: 114198-5210 colonies at sites of ventricular rupture. Taken together, these results reveal an unexpected local pathologic niche initiated by mechanically stressed BZ cardiomyocytes that has a global impact on survival. Applicant suggests that selective inhibition of Irf3 activation in cardiomyocytes could limit ischemic cardiomyopathy while avoiding broad immunosuppression. Applicant also discovered that heart attacks initiate the interferon response, not in immune cells, but unexpectedly in cardiomyocytes. They do so via the cGAS-STING-IRF3 pathway. Thus, this pathway was targeted so that one can block the pathway and improve outcomes after myocardial infarction by targeting this more selective signaling pathway (upstream of IFNAR and thus causing less broad immunosuppression) and in a specific cell type not responsible for immune surveillance. Without being bound by theory, therapies targeting the cGAS-STING-IRF3 pathway in cardiomyocytes have a better risk-benefit profile with retained efficacy but reduced immunosuppression and infections while on therapy. Applicant also reports herein that when cells are mechanically distorted, stretched, or locally separated from each other, whether by an external force or because they are contractile cells that hyper contract or contract against a sudden imbalance of forces (e.g., heart muscle cells contracting at the edge of a “heart attack” where neighbor cells have died of ischemia), they induce a type I interferon (IFN) response. The type I IFN response is a fundamental multi-step innate immune response that can be inhibited at multiple steps using small molecules, antibodies, and nucleic acids. This pathway involves molecules such as cGAS, cGAMP, STING or MDA5, RIG-I, MAVS, or TLRs, TRIF, and TBK1, IRF3, type I IFNs, which are secreted and spread to neighboring cells where they bind to the IFN alpha receptor (IFNAR) on neighboring cells, signal via Jak/Stat, activate the transcriptional regulator ISGF3, and induce hundreds of effector interferon stimulated genes (ISGs) with proinflammatory and anti-viral functions. These ISGs can induce local inflammation and antiviral responses that can drive tissue pathology and inhibit the effectiveness of locally any virally-delivered gene therapies. Applicant exploits this response and pathway in new disease contexts and therapeutic settings where the IFN response is induced by mechanical distortion. Inhibition of this response provides new methods to treat cardiac and muscular injury, including myocardial infarction. Atty. Dkt. No.: 114198-5210 Thus, Applicant provides herein targeted inhibitors of the pathway with better side effect profiles (comparable efficacy with fewer infections). While conventional wisdom would say the pathway is initiated by immune cells, Applicant cast a broader net and created cell-specific knockouts for cardiomyocytes, fibroblasts, endothelial cells, neutrophils, and macrophages. Applicant’s evidence derives from construction of a cardiomyocyte-specific Irf3- deficient mouse, which was exposed to experimental myocardial infarction and analyzed by single cell and spatial transcriptomics. It was compared to other cell type specific mice, wild- type mice, and globally Irf3-deficient mice. As disclosed herein, it is shown that Irf3 is activated via the cGAS-STING- IRF3 pathway which comes from cGAS-deficient mice, STING-deficient mice, compared to MAVS- deficient and TRIF-deficient mice. Thus, in one aspect, provided herein are methods for one or more of: reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina) due to ischemia and a heart attack, each method comprising, or consisting essentially of, or yet further consisting of, administering a cardiomyocyte cGAS-STING-IRF3 inhibitor thereby reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack, In one aspect, the inhibitor is a small molecule or a gene therapy which inhibits the activity of Irf3. A non-limiting example of gene therapy is the administration of an inhibitory RNA molecule to reduce the activity of Irf3 and/or its upstream activators. Non-limiting examples of such are known in the art, e.g., C6orf106 (see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036214/#:~:text=Unlike%20canonical%20inh ibitors%20of%20antiviral,%2C%20cellular%20expression%2C%20or%20degradation, last accessed on May 12, 2024). In one aspect, the subject is a mammal such as a human patient. Without being bound by theory, because the central process by which patients develop ischemic heart disease, the most common cause of death in the US and the world, the administration of the inhibitor treats and prevents ischemic heart disease as well. Modes of administration are known in the art and described herein. The inhibitors and methods can be administered directly in vitro or in a composition, in vitro or in vivo. When Atty. Dkt. No.: 114198-5210 administered in vitro, the methods can be used for personalized therapies or to test for new drugs or new drug combinations or compositions. When administered to a mammal, they can be used for veterinary applications, or as an animal model for pre-clinical testing, personalized therapies or new formulations or combination therapies. As apparent to the skilled artisan, an effective amount should be administered for the desired treatment and will vary with the disease and treatment. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A – 1P: Myocardial infarction induces focal colonies of IFNICs at borderzones in mice. (FIGS. 1A – 1O) Spatial transcriptomics analysis of short axis sections from infarcted mouse hearts on post-MI Day 3 (D3). (FIGS. 1A – 1B) Borderzone gene scores (data not shown) for (FIG. 1A) WT - 2 representative biological replicates, and (FIG. 1A) Irf3-/- mice. (FIGS. 1C – 1D) Ischemic zone gene scores (data not shown) for (FIG. 1C) WT and (FIG. 1D) Irf3-/- hearts. (FIGS. 1E – 1F) Ifit1 interferon stimulated gene (ISG) expression for (FIG. 1E) WT and (FIG. 1F) Irf3-/- hearts. (FIGS.1G – 1L) Magnified view of selected ISGs in IFNIC colonies of D3 WT hearts – (FIG. 1G) Ifit1, (FIG. 1I) Rsad2, and (FIG. 1K) the ISG score (FIG. 7B). Quantification of (FIG. 1H) Ifit1+ and (FIG. 1I) Rsad2+ pixels per section and (FIG. 1L) IFNIC colony size in D3 WT hearts (n = 5 biologically independent samples) compared to D3 Irf3-/- hearts (n = 3 biologically independent samples). (FIGS. 1M – 1N) Ranked Moran’s I test statistics of the top 2,000 differentially expressed genes with annotated ISGs in the same (FIG. 1M) WT hearts and (FIG. 1N) Irf3-/- hearts. (FIG. 1O) ISG cluster localization to the infarct BZ quantified by permutation testing using Monte-Carlo simulation of randomly placed ISG clusters of different sizes in each WT D3 sample (n = 5). Each cluster size underwent 500 simulation and was statistically tested using Fisher’s Exact test. (FIG. 1P) Time course of cardiac spatial transcriptomic patterns (left) and quantification (right) of the BZ gene score (top) and Ifit1 (bottom) for Sham, 1hr, 4hr, day 1 (D1), day 3 (D3), day 7 (D7), and day 28 (D28) after MI hearts (n = 1-5). Quantification is shown at right. (FIG. 1H, FIG. 1J, FIG. 1L, FIG. 1O, FIG. 1P) Data are presented as mean values േs.e.m. *P < 0.05, **P < .001, ***P < .0001. (FIG. 2H, FIG. 1J, FIG. 1L) Unpaired student’s t-test. (FIG. 1M, FIG. 1N) Moran’s I test statistic was computed using Benjamini-Hochberg false discovery rate-adjusted (FDR) P- Atty. Dkt. No.: 114198-5210 values. (FIG. 1O) Spatial correlation coefficients were transformed using Chi Squared and P- values computed with Fisher’s Exact Test. FIGS. 2A – 2N: Myocardial infarction induces focal colonies of IFNICs in humans. Spatial transcriptomics analysis of tissue sections from infarcted human hearts or controls. (FIGS. 2A – 2B) Borderzone gene scores for representative (FIG. 2A) 2 infarcted hearts and (FIG. 2B) a control heart. (FIGS. 2C – 2D) Ischemic zone gene scores for the same (FIG. 2C) infarcted and (FIG.2D) control hearts. (FIGS. 2E – 2F) Ifit1 interferon stimulated gene (ISG) expression (data not shown) for the same (FIG. 2E) infarcted and (FIG. 2F) control hearts. (FIGS. 2G – 2L) Close-up view of IFNIC gene expression in the same infarcted hearts – (FIG. 2G) MX1, (FIG. 2I) IFIT3, and (FIG. 2K) the ISG score with quantification of (FIG. 2H) MX1+ and (FIG. 2I) IFIT3+ pixels per section and (FIG. 2L) IFNIC colony size (n = 7 infarcted hearts and n = 5 control hearts). (FIGS. 2M – 2N) Ranked Moran’s I test statistics of the top 2,000 differentially expressed genes with annotated ISGs in the same (FIG. 2M) infarcted and (FIG. 2N) control human hearts. (FIG. 2H, FIG. 2J, FIG. 2L) Data are presented as mean values േs.e.m. *P < 0.05, **P < .01. (FIG. 2H, FIG. 2J, FIG. 2L) Unpaired student’s t-test. (FIG. 2M, FIG. 2N) Moran’s I test statistic was computed using Benjamini-Hochberg FDR-adjusted P-values. FIGS. 3A – 3J: Cardiomyocytes are dominant initiators of the type I IFN response in MI. (FIGS. 3A – 3C) Cell-type specific deletion of Irf3 was achieved by crossing Irf3fl/fl mice with mice expressing Cre recombinase under the regulation of cell-type selective promoters: cardiomyocytes (Irf3fl/fl X Myh6cre/+, Irf3CM), fibroblasts (Irf3fl/fl X Col1α1creERT2/+, Irf3FB), macrophages (Irf3fl/fl X Cx3cr1creERT2/+, Irf3Mac), neutrophils (Irf3fl/fl X S100a8cre/+, Irf3Neut), and endothelium (Irf3fl/fl X Tie2cre/+, Irf3EC). Spatial transcriptomics data from infarcted hearts of each cell specific Irf3KO were collected on post-MI day 3, integrated, and analyzed. (n = 2 each transgenic line) (FIG. 3A) Borderzone gene scores and ISG scores at representative BZs for each conditional knockout mouse. (FIG. 3B) Moran’s I test statistic with annotated ISGs for the top 2,000 variably expressed genes for each conditional knockout line. (FIG. 3C) Comparison of spatial autocorrelation metrics between WT and conditional Irf3 knockout mice on day 3 after MI. Spatially enriched features were ordered and the ranked lists were converted to a percentile. Sepal scores for several ISGs quantify the degree of spatially clustering (n = 4 WT, n = 2 per transgenic line). (FIG. 3D) Atty. Dkt. No.: 114198-5210 Gene counts of ISGs designated in ISG score were summed in each spatial capture spot in WT and transgenic samples (n = 2 WT, n = 2 per transgenic line). All spots were thresholded for counts above 10 determined against the lower 25th percentile of ISG counts score in Irf3-/- post-MI D3. (FIG. 3E) RNA MERFISH clustering of cells was annotated in a WT D3 MI mouse heart to use as the reference for Scanpy ingestion of cells from Irf3-/- D3 MI heart; the resulting uniform manifold approximation and projection (UMAP) plot contained 41,953 cells fitted between the 2 conditions (n = 86,559 total cells). (FIG. 3F) Representative image of cell marker gene probes used for RNA MERFISH and identification of cell types and zones in the infarcted heart. (FIG. 3G) RNA MERFISH-labeled type I IFN transcript, Ifna2 (red), localized primarily to BZ cardiomyocyte gene Ankrd1 (blue) and fibroblast gene Col6α3 (cyan) with closeup view in panel (FIG. 3H) below. (FIG. 3I) Ifna2 transcripts assigned to individual cells and represented by red circles as Ifna2+ cells. (FIG. 3J) Relative expression of Ifna2 in each cell type in WT and Irf3-/- mice. (FIG. 3D) Data are presented as mean values ±s.e.m. and ** P < 0.005, ****P < 0.0001. (FIG. 3B) Moran’s I test statistic was computed using Benjamini-Hochberg FDR-adjusted P-values. (FIG. 3C, FIG. 3D) Ranked percentiles for sepal scores and ISG counts per spot were compared using one-way ANOVA with Dunnett’s multiple comparisons test to determine adjusted P-values. FIGS. 4A – 4F: Nuclear rupture and extranuclear DNA are found in load- bearing cells of the infarct borderzone in vivo. (FIG. 4A) Representative image of short axis cross section from a cardiomyocyte-specific nuclear reporter mouse harvested at post-MI D3. created by crossing fl-STOP-fl tdTomNLS x Myh6cre/+. Upon cre-mediated recombination, the flanked STOP codons are removed and tdTomato fluorescence reporter with a nuclear localization sequence are expressed specifically by cardiomyocytes (CM-tdTom-NLS). (FIG. 4B) Representative images of CM-tdTom-NLS in nuclei of the remote zone and borderzone. Nuclear rupture was visualized as fluorescent reporter diffused throughout the cytoplasm of outlined cardiomyocytes whereas tdTom fluorescence was confined within the nuclear membrane in non-ruptured nuclei. (FIG. 4C) Quantification of the rate of ruptured nuclei in the remote zone vs. borderzone of infarcted CM-tdTom-NLS mice measured as a ratio between ruptured nuclei observed over total nuclei in each field of view (n = 8-13 fields of view). (FIG. 4D) Sequence-specific DNA probes were designed and synthesized to detect nuclear and extranuclear DNA in mouse hearts post-MI D1 and D3. Representative images of Atty. Dkt. No.: 114198-5210 DNA MERFISH encoding probes for 260 gene loci in 21 mouse chromosomes and rounds of hybridization of fluorescently labeled readout probes. The last image in the panel represents computationally decoded gene loci performed after data collection. (FIG. 4E) Quantification of neighborhood-based clustering and classification of DNA probes as intranuclear or extranuclear in the RZ and BZ on days 1 and 3 after MI (n = 1,535 cells). (FIG. 4F) Relative amounts of extranuclear DNA probes for each cell type. (FIG. 4C, FIG. 4F) Data are presented as mean values ±s.e.m. and *P <0.01, ****P < 0.0001. (FIG. 4C) Unpaired student’s t-test. (FIG. 4D) Wilcoxon rank sum test. FIGS. 5A – 5K: MI-induced IFNIC colonies co-localize at sites of ventricular rupture and antagonize protective fibroblast function. (FIG. 5A) Relative difference in survival post-MI in previously published studies that inhibit the type I IFN signaling (Irf3-/-, Ifnar-/-, anti-IFNAR Ab) or fibroblast activation and the matricellular response (anti-TGFbR Ab, Sparc-/-, Postn-/-)3,44-50. (FIG. 5B) Representative sample after fatal ventricular rupture - H&E staining and spatial transcriptomic BZ gene score and ISG score. Inset shows magnified view of the rupture site. (FIG. 5C) IFNIC colonies were algorithmically identified using a density-based clustering algorithm (DBSCAN) to group neighboring (distance-based eps=70) densely packed fluorescent Ifit1 transcripts. Clustered and scattered expression of Ifit1 is indicated by color and labels. Quantification appears in graph below showing the relative proportion of Ifit1+ expression in each cell type within the IFNIC colonies shown in c (n = 1250 cells, 8 clusters). (FIG. 5D) Representative overlay of RNA MERFISH fluorescent oligonucleotide probes targeting Ifit1 (magenta) to designate IFNIC colonies, Adgre1 (yellow) to designate the macrophages, and Nppa (blue) to designate BZ cardiomyocytes with visual representation of cell-type contributions of IFNIC clusters in following panels. Panel below shows magnified view of IFNIC colony #7 from the RNA MERFISH sample shown in (FIG. 5C). Overlay of Ifit1 and cell-type specific RNA MERFISH fluorescent probes within the IFNIC colony (DAPI in grey, cardiomyocytes (blue - Flnc, Nppa), fibroblasts (cyan – Col1a1, Col6a3), and macrophages. (FIG. 5E) Neighborhood based clustering analysis using the centroid of each IFNIC colony as the reference point and chosen as the highest ISG expressing capture spots. Adjacent spots were designated as primary, secondary, and tertiary degree neighbors to the ISG centroid(s) for each cluster. Differential gene expression testing was performed for each neighbor against the centroid of ISG clusters. Atty. Dkt. No.: 114198-5210 (FIG. 5F) Violin plots of Ifit3 and Irf7 from the centroid towards spots labeled as tertiary neighbors. (FIG. 5G) Violin plot of fibroblast-expressed, matricellular genes selected from differential expression testing between neighbors and centroid of IFNIC colonies. Postn and Col1α1 display a reciprocal relationship to ISGs in both expression level and distance. (FIG. 5H) Collagen gel contraction assay. Human iPSC-differentiated fibroblasts were seeded into 3D collagen hydrogel matrix and treated with various combinations of 10 ng/mL of TGFβ, IFNβ and anti-IFNAR neutralizing antibody (n = 7-9 each condition). Representative images of gels are shown at bottom. (FIGS. 5I – 5J) Expression of (FIG. 5I) matricellular genes and (FIG. 5J) ISGs by human iPSC fibroblasts treated with combinations of 10 ng/mL TGFβ and/or 10 ng/mL IFNβ, measured by qPCR, and expressed as fold change increase compared to control (n = 3-4 each condition). (FIG. 5K) Proposed model in which borderzone cardiomyocytes are dominant initiators of IFNIC colonies leading to pathogenic Irf3- dependent responses to MI. Effector cells represented below and relative contributions of cell types to adverse remodeling, dilation, and ventricular rupture remain to be fully elucidated. (FIG.5C, FIG. 5H, FIG. 5I, FIG. 5J) Data represented as േ s.e.m. and *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (FIG. 5C, FIG. 5H, FIG. 5I, FIG. 5J) One-way ANOVA with multiple comparisons using Dunnett’s multiple comparison post-hoc analysis. (FIG. 5F, FIG. 5G) Wilcoxon rank-sum and FDR P-value adjusted. FIG. 6: Summary table of biological replicates in imaging- and sequencing-based spatial transcriptomics. Summary table of mouse and human biological samples and replicates. Total cells single cells are quantified for imaging-based RNA and DNA MERFISH. Total spatial transcriptomes are quantified for capture-based Visium spatial transcriptomics. FIGS. 7A – 7D: Gene scoring method using integrated sc/snRNA-seq data to map transcriptional zones of infarcted myocardium in space. (FIG. 7A) Experimental workflow. Gene scores are derived from differentially expressed genes (DEG) after clustering integrated sc/snRNA-seq data from infarcted mouse hearts on day 3 after MI or using previously developed scores for BZ and IZ, as published18. (FIG. 7B) Table of gene scores are summed within the SCT assay of the integrated spatial object for analysis of mouse and human spatial transcriptomes and were normalized to minimum UMI counts in integrated samples. (FIG. 7C) Replicate sequencing-based spatial transcriptomic datasets were Atty. Dkt. No.: 114198-5210 integrated using reciprocal principal component analysis (RPCA) and normalized using Seurat’s SCTransform method. Uniform manifold approximation and projection (UMAP) of Louvain clustering of spatial transcriptomic spots (n = 37). (FIG. 7D) Averaged heat map of top marker genes selected from Wilcoxon signed-rank method for each spatial cluster. FIG. 8A: Comparison of transcriptionally defined BZ and underlying H&E tissue histology. (FIG. 8A) H&E tissue histology underlying the transcriptionally defined anterior and posterior BZs for representative spatial transcriptomics samples. FIGS. 9A – 9I: The type I IFN response produces spatially clustered ISG expression. (FIG. 9A) Representative depiction of the type I IFN pathway. The primary response involves cGAS sensing of decompartmentalized dsDNA in the cytosol and resulting 2nd messenger production of cGAMP. This activates the STING adaptor resulting in phosphorylation and activation of the master transcriptional regulator IRF3. Translocation of IRF3 into the nucleus induces expression and secretion of type I IFN cytokines which signal to bystander cells via binding to the IFNAR receptor in an autocrine or paracrine manner. IFNAR-binding results in the expression of hundreds of interferon stimulated genes (ISGs) as a robust readout of the secondary response and delineation of IFNICs. (FIG. 9B) Spatially variable features were calculated by Seurat’s FindVariableFeatures() using vst as the selection method. Standard variance plotted against log normalized average expression of highly variable features. ISGs were among the 2000 topmost variable features are labeled on the VariableFeaturesPlot. (FIG. 9C) Interferon stimulated genes projected onto space in a D3 post-MI cardiac section. (FIG. 9D) Genetic knockout mice of signaling components in the cGAS-STING-IRF3 axis were infarcted and harvested D3 post-MI for spatial transcriptomic analysis. Representative Ifit1 expression in infarcted (FIG. 9D) Irf3-/- (n = 2), (FIG. 9E) Ifnar-/- (n = 2), (FIG. 9F) anti-IFNAR-ab treatment (n = 1), (FIG. 9G) Cgas-/- (n = 1) and (FIG. 9H) STING-/- (Tmem173) hearts (n = 1). (FIG. 9I) Number of positive spots (above zero) for Ifit1 expression for infarcted WT and genetic knockout mice of the cGAS-STING- IRF3 signaling axis. (FIG. 9I) Data represented as േs.e.m. FIGS. 10A – 10C: Methods for identifying and qualitatively characterizing IFNIC colonies and spatial correlation. (FIG. 10A) Thresholds for individual interferon stimulated genes or ISG score was determined for Ifit1, Rsad2, and ISG score expression by using max expression level in Irf3-/- negative control mice and using the maximum value. Atty. Dkt. No.: 114198-5210 Each Ifit1, Rsad2, or ISG spot above threshold (Ifit1+, Rsad2+, ISG+) was assigned a value of 0-6 corresponding to the number of ISG+ neighbors (N). (FIG. 10B) Spots that retained positive expression levels after threshold were clustered using k-means. (FIG. 10C) Computation of IFNIC colony localization to the transcriptional BZ or IZ in integrated spatial dataset. Thresholds for ISG scores were determined as the maximum expression level in infarcted Irf3-/- mice (n = 3). Visium spatial coordinates were extracted for random coordinate selection and cluster localization. From these coordinates, the probability of incrementally increasing ISG cluster sizes (N nearest neighbors) landing in a specific region (BZ or IZ) was computed and compared against the actual occurrence of ISG clusters overlapping with BZHI pixels. FIGS. 11A – 11D: Biological replicates of the clustered type I IFN response after MI in mice and humans and corresponding negative controls. (FIG. 11A) Table representing all biological replicates and samples used for spatial transcriptomic analysis in this study. Column (FIG. 11A) contains representative images of sample conditions that were negative for IFNIC colonies including the validation of using knockouts of Cgas-Sting-IRF3 pathway as negative controls for the study. (FIG. 11B) Positive IFNIC colonies represented in biological replicates for conditions used to determine the presence of ISG+ conditions. (FIG. 11C) Representative IFNIC colonies in cell-specific knockouts (n = 2 each transgenic line). (d) Percent of pixels that were positive for ISG scores out of total pixels overlying tissue were calculated for each sample in the negative controls vs. positive datasets (n = 40). (FIG. 11D) Data represented as mean values േs.e.m. and ****P < 0.0001, Two-sided Mann- Whitney U-test. FIGS. 12A – 12F: IFNIC colonies are initiated by BZ cardiomyocytes. (FIG. 12A) Representative images of 4 individual ISGs in cardiomyocyte-specific Irf3 deletion display minimal ISG cluster formation compared to ISG expression in cardiomyocyte- (FIG. 12B) fibroblast- (FIG. 12C) macrophage- (FIG. 12D) neutrophil-, and (FIG. 12E) endothelial-specific deletions of Irf3. (FIG. 12F) Ranked percentile of Moran’s I test statistic for ISGs Ifit1, Ifit3, Rsad2, Irf7, and Isg15 computed for each biological replicate of WT D3 infarcted mice and cell-type deletions of Irf3-/- mice (n = 3 biological replicates per WT and n=2 transgenic conditions). (FIG. 12F) Data are presented as mean values േs.e.m. and **P < 0.005, One-way ANOVA and Dunnett’s post-hoc analysis for multiple comparisons. Atty. Dkt. No.: 114198-5210 FIGS. 13A – 13H: Infiltrating myeloid cells are dispensable for IFNIC colony formation in the infarcted murine heart. (FIG. 13A) Representative Ifit1+ IFNIC colony found at the BZ in Ccr2-defiicient hearts at D3 post-infarct. (FIG. 13B) Recruited macrophages are significantly decreased in Ccr2-deficient D3 infarcted hearts compared to WT infarcted samples and no differences in Ifit1 expression (n=2 per group). (FIG. 13C) Timeline of antibody-mediated plasmacytoid dendritic cell (pDC) depletion. Anti-Bst2 antibody or isotype control antibody was administered for 3 days prior to MI surgery and hearts were harvested D3 post-MI and processed for scRNA-seq. (FIG. 13D) Gating strategy to determine pDCs depletion in isotype control-treated or anti-BST2-ab treated, infarcted mouse hearts. Cd45+B220+ positive myeloid cells were gated by MHCII expression and BST2 to identify pDCs. (FIG. 13E) Feature plots of Ifit1 expression in neutrophils and macrophages isolated from mice treated with either isotype control-antibody or anti-Bst2-Ab treated mice. (FIG. 13F) Plasmacytoid dendritic cell depletion does not affect the percentage of ISG+ neutrophils or monocytes at post-MI D3 (n = 2 mice per condition or 8,500 isolated cells per mouse). (FIG. 13G) Chimeric bone marrow transplant experiment and qPCR analysis of D4 infarcted hearts. Nlrp1a and Irf3 gene expression analysis measured by qPCR in the control WT-> WT condition compared to IRF3KO-> WT condition (n = 5 per condition). (FIG. 13H) Gene expression analysis by qPCR in D4 infarcted hearts between WT-> WT transfer and IRF3KO -> WT transfer conditions. (FIG. 13B, FIG. 13G, FIG. 13H) Data are presented as mean values േs.e.m. and **P < 0.01, ****P < 0.0001. (FIG. 13B, FIG. 13G) Unpaired student’s t-test. FIGS. 14A – 14F: Validation of RNA MERFISH encoding probe library by comparison of log-normalized of transcripts detected in WT and Irf3-deficient mice. (FIG. 14A) Number of total probes designed for each transcript in the encoding library used in this study. (FIG. 14B) Representation of Ifna2 (red), Ifit1 (magenta), and Nppa (blue) in WT and Irf3-/- D3 MI mice (n = 1 per condition, 85,995 total cells). (FIG. 14C) Quality control metrics of the raw data and total counts of each transcript detected in WT D3 MI heart and (FIG. 14D) in Irf3-deficient D3 MI heart. (FIG. 14E) Comparison of the total mean counts in the raw data and (FIG. 14F) log-normalized counts of detected transcripts for BZ genes such as Nppa, Flnc, Ankrd1 between WT and Irf3-deficient mice. Atty. Dkt. No.: 114198-5210 FIGS. 15A – 15I: DNA MERFISH enables sequence-specific detection of extranuclear DNA. (FIG. 15A) Time course of needle pass injury. The appearance of Ifit1 expression occur at D3 post-injury at the side of needle insertion, similar to the time course of ISG expression in infarcted hearts. (FIG. 15B) Close-up view of scored ISGs, (FIG.15C) mechanically-induced Piezo1, (FIG.15D) scored BZ genes, and (FIG. 15E) H&E at the site of day 3 post-injury. (FIG. 15F) Schematic of experimental traumatic injury induced by in vivo needle-insertion in healthy hearts. BZ gene score and ISG score spatial transcriptomics and (FIG. 15G) quantitative comparison of ISG scores at BZ vs. RZ at the site of mechanical injury (n = 4). (FIG. 15H) Nuclear solidity of RZ vs. BZ cardiomyocytes identified by Tnnt2 expression (n = 200 nuclei per region of interest). (FIG. 15I) Regions of interest used to quantify extranuclear probes in WT D1 and WT D3 infarcted hearts were chosen based on underlying histology to demarcate the RZ and BZ. The regions designated as RZ are shown in blue dashed boxes, and the regions designated as the BZ are shown in red dashed boxes for each sample. The ingested cells are found in borderzone cardiomyocytes using WT D3 cells as the reference sample. (FIG. 15G, FIG. 15H) Data are presented as mean values േs.e.m. and ****P < 0.0001, unpaired student t-test. FIGS. 16A – 16E: IFNIC colonies and ISG expression appear directly adjacent to the site of ventricular rupture and have high calculated spatial autocorrelation. (FIG. 16A) Representative images of 7 collected rupture samples in WT mice during monitoring on D3-D7 post-MI show IFNIC colonies directly adjacent to the site of rupture. (FIG. 16B) In situ hybridization of ruptured cardiac cross section with representative Flnc (blue), Postn (green), and Ifit3 (magenta). Clustering of Ifit3 to the site of rupture is seen and demarcated by white dotted line. (FIG. 16C) Table of rupture rates and successful spatial transcriptomics (sectioning and identification of rupture site), and qualitative assessment of ISG expression proximal to rupture site. (FIG. 16D) Spatial autocorrelation determined for each biological rupture sample as measured by Moran’s I test statistic. (FIG. 16E) Moran’s I test statistic was significantly higher across all biological replicates of ruptured WT hearts compared to non-ruptured Irf3-/- hearts. (FIG. 16E) Data are presented as mean values േs.e.m. and *P < 0.05, spatial correlation coefficients were transformed with Fisher’s Z and compared with multiple comparisons unpaired t-test with False Discovery Rate (FDR). Atty. Dkt. No.: 114198-5210 FIGS. 17A – 17F: Characterizing IFNICs and the inverse spatial relationship between ISG and Postn expression. (FIG. 17A) Representative IFNIC cell types showing minority contribution by neutrophils and endothelial cells. (FIG. 17B) Line scans performed in WT D3 MI sample and (FIG. 17C) biological replicate, and (FIG. 17D) ruptured sample to power the observation of inversely correlated Postn and ISG scores. (FIG. 17E) Integrated scRNA-seq dataset from post-MI D1 and D3 mouse hearts. UMAP depicting captured immune and stroma cell types. (FIG.17F) Feature scatterplot of an integrated snRNA-seq dataset of WT fibroblasts from D3 MI hearts showing an inverse scatter profile between PostnHI and ISG scores. Approximately 5% of fibroblasts isolated from WT infarcted hearts are IFNICs and express low counts of Postn (n = 3 mice). (FIG. 17B, FIG. 17C, FIG. 17D) ANCOVA regression with LOESS moving average and regression analysis, *P > 0.05. FIGS. 18A – 18H: Analysis of IFNAR-deficiency in single-cells. (FIG. 18A) Whole cell isolation from WT and IFNAR-deficient hearts processed for scRNA-seq. Single cell data sets were integrated and log normalized to enable direct comparison of cell types between samples and replicates (n = 3 WT, 2 Ifnar-/- mice). (FIG. 18B) UMAP of clustered fibroblasts from scRNA-seq integrated data of WT and Ifnar-/- cells from D3 infarcted hearts. (FIG. 18C) Heatmap of marker genes for each fibroblast subcluster. (FIG. 18D) Integrated fibroblast subset from WT and IFNAR-KO infarcted hearts were split by biological condition and by activated vs. nonactivated fibroblast based on differential expression analysis Wilcoxon-signed rank test, Bonferroni-adjusted P < 0.01). (FIG. 18E) IFNAR-KO mouse hearts contain a higher percentage of activated fibroblasts compared to those captured in WT infarcted hearts. (FIG. 18F) Log normalized expression of gene markers in activated fibroblast populations between Ifnar-/- and WT mice. (FIG. 18G) UMAP of captured macrophages in the scRNA-seq dataset divided into macrophages from Ifnar-/- mice in blue and macrophages from WT mice in magenta. (FIG. 18H) Volcano plot of differentially expressed genes between WT and Ifnar-/- macrophages corrected for FDR rate-adjusted p- values. (FIG. 18E, FIG. 18F) Data represented as mean values ±s.e.m. *P > 0.05, ****P > 0.00005 Mann-Whitney U test. FIGS. 19A – 19E: Irf3-deficient hearts have greater fibroblast activation and protective matricellular responses. (FIG. 19A) Nuclei isolation from WT and Irf3- deficient hearts are processed for snRNA-seq. Nuclei data sets were integrated to enable Atty. Dkt. No.: 114198-5210 direct comparison of cell types. (FIG. 19B) Heatmap displaying averaged values of DEGs in each fibroblast subpopulation were stratified into activated and not activated fibroblasts Variable expression level of Postn underlie fibroblasts clustered into the activated subtype. (FIG. 19C) UMAP of clustered fibroblasts from snRNA-seq integrated data of WT and Irf3-/- nuclei from D3 infarcted hearts on the left panel and separation of activated vs. non-activated fibroblast populations in the right panel (Wilcoxon-signed rank test, Bonferroni-adjusted P < 0.01). (FIG. 19D) Percent of activated fibroblasts out of total captured fibroblasts for each replicate sample (n = 3 per condition). (FIG. 19E) Log normalized average expression of fibroblast activation markers between those captured from Irf3-deficient and WT and D3 MI hearts. (FIG. 19E) Volcano plot with selected gene annotation of differentially expressed genes between PostnHI fibroblasts in Irf3-/- and WT mice on day 3 after MI. Colored points represent genes significantly increased (magenta, right) or decreased (blue, left) in WT fibroblasts compared to Irf3-/- (n = 3 mice per condition). (FIG. 19D) Data represented as mean values േs.e.m. *P > 0.05, Mann-Whitney U test. FIGS. 20A – 20C: Type I IFN antagonizes mouse fibroblast activation and function. (FIG. 20A) Collagen Gel contraction assays performed with L929 fibroblasts treated with TGFβ, IFNβ, or combination treatment. IFNβ blocked the TGFβ-mediated response of fibroblast activation as determined by the lack of gel contraction. (FIG. 20B) Treatment with anti-IFNAR-ab abrogated the IFNβ-mediated blockade of the fibroblast contractile phenotype. (FIG. 20C) Top panel: L929 fibroblast gene expression data of matricellular genes after treatment with TGFβ, IFNβ, or combination treatment, and corresponding inverse expression of ISG’s in the bottom panel. (FIG. 20A, FIG. 20B, FIG. 20C) Data represented as mean values േs.e.m. *P > 0.05, **P > 0.005, ***P > 0.0005, ****P > 0.00005. One-way ANOVA with Dunnett’s multiple comparisons test (n = 2-3 per condition). FIG. 21: The 2 IFNIC sources are compared and contrasted. Table summarizing intracardiac and extracardiac sources of IFNICs. FIGS. 22A – 22D: Quality control metrics of sequencing-based spatial transcriptomics. (FIG.22A) Total number of Visium spots detected over tissue for each sample. (FIG. 22B) Total number of genes detected (nCount) per sample. (FIG. 22C) Feature scatter plot of unique molecular identifiers and total genes detected in integrated Atty. Dkt. No.: 114198-5210 dataset consisting of all the labeled samples from (FIG. 22B). (FIG. 22D) List of the highest expressed features detected in WT samples. DETAILED DESCRIPTION Definitions As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting or separating or both limiting and separating the subject matter described. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entireties to more fully describe the state of the art to which this invention pertains. The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)). As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of” when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically Atty. Dkt. No.: 114198-5210 acceptable carriers, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology. “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (–) 15%, 10%, 5%, 3%, 2%, or 1 %. “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals. The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non- Atty. Dkt. No.: 114198-5210 human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. A “composition” as used herein, refers to an active agent, such as a compound as disclosed herein and a carrier, inert or active. The carrier can be, without limitation, solid such as a bead or resin, or liquid, such as phosphate buffered saline. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra- oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers Atty. Dkt. No.: 114198-5210 include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein. An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the Atty. Dkt. No.: 114198-5210 therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. A “type I IFN response inhibitors or inhibitory IFN agents” intends any agent that inhibits the type I IFN response. Non-limiting examples include anti-IFNAR antibodies, such as an anti-IFNAR antibody, e.g., AnifrolumabTM (Astrazeneca, an FDA-approved biologic approved for chronic systemic delivery for lupus based on the TULIP-2 Trial (https://www.saphnelohcp.com/how-saphnelo-works.html); and Ventus Therapeutics’ Phase 1 small molecular cGAS-inhibitor, VENT-003 (https://www.ventustx.com/pipeline/cgas/). Some anti-IFNAR antibodies (e.g., Anifrolumab), do not bind IFN or its upstream activators but instead blocks its downstream receptor, IFNAR on secondary-responding cells. In one aspect, this antibody is useful to treat local injury such as surgery, needle trauma, injection of viral gene therapy, cell therapy, cannula placement, etc. Additional agents include agents that inhibit other molecules in this pathway (cGAS inhibitors, STING inhibitors, TBK1 inhibitors, IRF3 inhibitors, anti-IFN therapies, anti-IFNAR therapies, Jak/Stat inhibitors, anti-ISG therapies, etc.), e.g., an inhibitory antibody or inhibitory nucleic acid such as antisense oligonucleotides (ASO), RNAi or siRNA. Yet further examples include IFN-kinoid, AGS- 009 JNJ-55920839 Rontalizumab Sifalimumab BMS-986165 Baricitinib Filgotinib Solcitinib Tofacitinib, for example as shown in Fig 1 of Felten R, et al. (2019) Drug Des Devel Ther., 13:1535-1543, incorporated herein by reference. As used herein, the acronym “cGAS” intends. cyclic Guanosine monophosphate- Adenosine monophosphate (cGMP-AMP) Synthase. As used herein, the acronym “cGAMP” intends icyclic Guanosine monophosphate- Adenosine monophosphate (cGMP-AMP). Atty. Dkt. No.: 114198-5210 As used herein, the acronym “STING” intends STimulator of InterferoN Genes. As used herein, the acronym “TBK1” intends TANK binding kinase 1. As used herein, the acronym “IRF3” intends Interferon Regulatory Factor 3 (IRF3). As used herein, the acronym “IFNs” intends Type I interferons. As used herein, the acronym “IFNAR” intends IFN Alpha Receptor. As used herein, the acronym “ISG” intends interferon stimulating genes, which are genes that are expressed in response to stimulation by interferons. Interferons bind to receptors on the surface of a cell, initiating protein signaling pathways within the cell. Non- limiting examples of such include: interferon induced protein with tetratricopeptide 1 (IFIT1), 2 (IFIT2), and 3 (IFIT3); interferon induced protein 44 like (IFI44L); radical S-adenosyl methionine domain containing (RSAD2); interferon-induced ubiquitin-like modifier (ISG15); cytidine/uridine monophosphate kinase 2 (CMPK2); circulating inflammatory marker elevated in advanced heart failure (CXCL10); and MX dynamin like GTPase 1 (MX1) and 2 (MX2). As used herein, “mechanically induced type I IFN response” intends mechanical tissue injury causes cGAS activation, which catalyzes production of the small molecule cGAMP, which can signal within the cell and be transferred between cells via gap junctions or the extracellular space to agonize STING, which activates TBK1, which phosphorylates the transcription factor IRF3, which leads to dimerization, nuclear translocation, and upregulation of IRF3-dependent genes such as the type I IFNs. Type I IFNs are secreted cytokines that can signal via the cell surface receptor IFNAR to induce expression of 100s of interferon stimulated genes (ISGs) in neighboring cells. ISGs are the dominant effector molecules so blocking any step in this pathway has potential to protect from IFN- and ISG- mediated pathology, particularly in patients who show evidence of ISG expression by imaging nuclear or contrast-associated ISGs -labeled with tissue that shows evidence of ISG expression, for example by immunostaining, in situ hybridization, or spatial transcriptomics. As used herein, the term “cardiac regeneration” intends reversing or repairing damaged heart cells or tissue. Atty. Dkt. No.: 114198-5210 As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. In one aspect, treatment excludes prophylaxis. The terms “oligonucleotide” or “polynucleotide” or “portion,” or “segment” thereof refer to a stretch of polynucleotide residues which is long enough to use in PCR or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules. The polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an Atty. Dkt. No.: 114198-5210 indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.” As used herein, an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence. Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST. In one aspect, equivalent polynucleotides, proteins and corresponding sequences can be determined using BLAST (accessible at blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on August 1, 2021). It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, amino acid sequence, protein, polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity, or at least about 85 % homology or identity, or alternatively at least about 90 % homology or identity, or alternatively at least about 95 % homology or identity, or alternatively at least about 96 % homology or identity, or alternatively at least about 97 % homology or identity, or alternatively at least about 98 % homology or identity, or alternatively at least about 99 % homology or identity (in one aspect, as determined using the Clustal Omega alignment program) and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complementary sequence. In some embodiments, a first sequence (nucleic acid sequence or amino acid) is compared to a second sequence, and the identity percentage between the two sequences can Atty. Dkt. No.: 114198-5210 be calculated. In further embodiments, the first sequence can be referred to herein as an equivalent and the second sequence can be referred to herein as a reference sequence. In yet further embodiments, the identity percentage is calculated based on the full-length sequence of the first sequence. In other embodiments, the identity percentage is calculated based on the full-length sequence of the second sequence. “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals. A cardiomyocyte (CM) is a muscle cell that forms the chambers of the heart. They are usually divided into two types of cells, the pacemaker cells and the force-producing ventricular and atrial CMs. See, Talman and Kivela, Front. Cardiovasc. Med. (2018) July 26, 2018, https://www.frontiersin.org/article/10.3389/fcvm.2018.00101. In some aspects, a “cardiomyocyte” or “cardiac myocyte” is a specialized muscle cell which primarily forms the myocardium of the heart. Cardiomyocytes have five major components: 1. cell membrane (sarcolemma) and T-tubules, for impulse conduction, 2. sarcoplasmic reticulum, a calcium reservoir needed for contraction, 3. contractile elements, 4. mitochondria, and 5. a nucleus. Cardiomyocytes can be subdivided into subtypes including, but not limited to, atrial cardiomyocyte, ventricular cardiomyocyte, SA nodal cardiomyocyte, peripheral SA nodal cardiomyocyte, or central SA nodal cardiomyocyte. Stem cells can be propagated to mimic the physiological functions of cardiomyocytes or alternatively, differentiate into cardiomyocytes. This differentiation can be detected by the use of markers selected from, but not limited to, myosin heavy chain, myosin light chain, actinin, troponin, tropomyosin, GATA4, Mef2c, and Nkx2.5. The cardiomyocyte marker “myosin heavy chain” and “myosin light chain” are part of a large family of motor proteins found in muscle cells responsible for producing contractile force. These proteins have been sequenced and characterized, see for example GenBank Accession Nos. AAD29948, CAC70714, CAC70712, CAA29119, P12883, NP_000248, Atty. Dkt. No.: 114198-5210 P13533, CAA37068, ABR18779, AAA59895, AAA59891, AAA59855, AAB91993, AAH31006, NP_000423, and ABC84220. The genes for these proteins have also been sequenced and characterized, see for example GenBank Accession Nos. NM_002472 and NM_000432. The cardiomyocyte marker “actinin” is a microfilament protein which are the thinnest filaments of the cytoskeleton found in the cytoplasm of all eukaryotic cells. Actin polymers also play a role in actomyosin-driven contractile processes and serve as platforms for myosin’s ATP hydrolysis-dependent pulling action in muscle contraction. This protein has been sequenced and characterized, see for example GenBank Accession Nos. NP_001093, NP_001095, NP_001094, NP_004915, P35609, NP_598917, NP_112267, AAI07534, and NP_001029807. The gene for this protein has also been sequenced and characterized, see for example GenBank Accession Nos. NM_001102, NM_004924, and NM_001103. The cardiomyocyte marker “troponin” is a complex of three proteins that is integral to muscle contraction in skeletal and cardiac muscle. Troponin is attached to the protein “tropomyosin” and lies within the groove between actin filaments in muscle tissue. Tropomyosin can be used as a cardiomyocyte marker. These proteins have been sequenced and characterized, see for example GenBank Accession Nos. NP_000354, NP_003272, P19429, NP_001001430, AAB59509, AAA36771, and NP_001018007. The gene for this protein has also been sequenced and characterized, see for example GenBank Accession Nos. NM_000363, NM_152263, and NM_001018007. A “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus, adeno-associated virus, and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. Atty. Dkt. No.: 114198-5210 “Gene delivery,” “gene transfer,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection, sometimes called transduction), transfection, transformation or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). Unless otherwise specified, the term transfected, transduced or transformed may be used interchangeably herein to indicate the presence of exogenous polynucleotides or the expressed polypeptide therefrom in a cell. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein. The term “express” refers to the production of a gene product. In some embodiments, the gene product is a polypeptide or protein. In some embodiments, the gene product is an mRNA, a tRNA, an rRNA, a miRNA, a dsRNA, or a siRNA, e.g., inhibitory RNA molecules. A cell that “stably expresses” an exogenous polypeptide is one that continues to express a polypeptide encoded by an exogenous gene introduced into the cell either after replication if the cell is dividing or for longer than a day, up to about a week, up to about two weeks, up to three weeks, up to four weeks, for several weeks, up to a month, up to two months, up to three months, for several months, up to a year or more. A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med.5(7):823-827. Atty. Dkt. No.: 114198-5210 In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. As used herein, “retroviral mediated gene transfer” or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. A “lentiviral vector” is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg. In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/11984. Wild-type AAV has high infectivity and specificity integrating into the host cell’s genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski, et al. (1988) Mol. Cell. Biol. 8:3988-3996. As used herein, the term “AAV” is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are currently thirteen serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, Handbook of Parvoviruses 1:169-228, 1989, and Berns, Atty. Dkt. No.: 114198-5210 Virology 1743-1764, 1999. However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, Parvoviruses and Human Disease 165-174, 1988, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61, 1974). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self- annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. An “AAV expression cassette” as used herein refers to a nucleotide sequence comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV expression cassette can be replicated and packaged into infectious viral particles (e.g., AAV vectors) when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. An “AAV virion” or “AAV vector” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV expression cassette. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector”. Thus, production of AAV vector particle necessarily includes production of AAV expression cassette, as such a plasmid is contained within an AAV vector particle. Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983) as corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). As other examples, Atty. Dkt. No.: 114198-5210 the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). Cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007). Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). Recombinant AAV genomes for use in the methods of this disclosure comprise a polynucleotide to modulate type I IFN expression and one or more AAV ITRs flanking a nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV Atty. Dkt. No.: 114198-5210 variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. In some embodiments, to promote skeletal muscle specific expression, AAV1, AAV6, AAV8 or AAVrh.74 is used. The polynucleotide that modulates type I IFN expression (e.g., a shRNA targeting type I IFN) can be under the control of a tissue specific promoter, e.g., a cardiomyocyte- specific promoter, e.g., the promoter of the gene encoding the contractile protein TroponinT, or myosin light chain 2 promoter (MLC-2V), or the promoter of alpha myosin heavy chain (Myh6) (see Griscelli et al. (1997) CR Acad. Sci. 329(2):103-12, or Lin Z et al. (2014) Circ Res 115:354-363, or Breckenridge R. et al (2007) Genesis 45(3):135-44, or Sohal DS et al. (2001) Circ Res 89 (1):20-5) for additional examples of tissue specific promoters. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5’ and/or 3’ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5’ of the start codon to enhance expression. “Under transcriptional control” is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. “Operably linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell. A “composition” is intended to mean a combination of polynucleotides, active agent or another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant. Atty. Dkt. No.: 114198-5210 A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, microspheres, microparticles, or nanoparticles (comprising e.g., biodegradable polymers such as Poly(Lactic Acid-co-Glycolic Acid)), and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. “Liposomes” are microscopic vesicles consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer. These are neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other types of bipolar lipids including but not limited to dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C═C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic Atty. Dkt. No.: 114198-5210 acid, cerebrosides, distearoylphosphatidylethan-olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloteoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1- carboxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioteoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles. Typically, liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure. The three classifications, as developed by the New York Academy Sciences Meeting, “Liposomes and Their Use in Biology and Medicine,” December 1977, are multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles (LUVs). The polynucleotides can be encapsulated in such for administration in accordance with the methods described herein. A “micelle” is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic “head” regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center. This type of micelle is known as a normal phase micelle (oil-in-water micelle). Inverse micelles have the head groups at the center with the tails extending out (water-in-oil micelle). Micelles can be used to attach a polynucleotide, polypeptide, antibody or composition described herein to facilitate efficient delivery to the target cell or tissue. Also included as a micelles are lipid nanoparticles. Gene delivery vehicles also include several non-viral vectors, including DNA/liposome complexes, and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention. To enhance delivery to a cell, the nucleic acid or proteins of this invention can be Atty. Dkt. No.: 114198-5210 conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., a cell surface marker found on stem cells. “RNA interference” (RNAi) refers to sequence-specific or gene specific suppression of gene expression (protein synthesis) that is mediated by short interfering RNA (siRNA). “Short interfering RNA” (siRNA) refers to double-stranded RNA molecules (dsRNA), generally, from about 10 to about 30 nucleotides in length that are capable of mediating RNA interference (RNAi), or 11 nucleotides in length, 12 nucleotides in length, 13 nucleotides in length, 14 nucleotides in length, 15 nucleotides in length, 16 nucleotides in length, 17 nucleotides in length, 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, or 29 nucleotides in length. As used herein, the term siRNA includes short hairpin RNAs (shRNAs). “Double stranded RNA” (dsRNA) refers to double stranded RNA molecules that may be of any length and may be cleaved intracellularly into smaller RNA molecules, such as siRNA. In cells that have a competent interferon response, longer dsRNA, such as those longer than about 30 base pair in length, may trigger the interferon response. In other cells that do not have a competent interferon response, dsRNA may be used to trigger specific RNAi. The term siRNA includes short hairpin RNAs (shRNAs). shRNAs comprise a single strand of RNA that forms a stem-loop structure, where the stem consists of the complementary sense and antisense strands that comprise a double-stranded siRNA, and the loop is a linker of varying size. The stem structure of shRNAs generally is from about 10 to about 30 nucleotides in length. For example, the stem can be 10-30 nucleotides in length, or alternatively, 12-28 nucleotides in length, or alternatively, 15-25 nucleotides in length, or alternatively, 19-23 nucleotides in length, or alternatively, 21-23 nucleotides in length. Tools to assist siRNA design are readily available to the public. For example, a computer-based siRNA design tool is available on the internet at www.dharmacon.com, Ambion-www.ambion.com/jp/techlib/misc/siRNA_finder.html; Thermo Scientific- Dharmacon-www.dharmacon.com/DesignCenter/DesignCenterPage.aspx; Bioinformatics Atty. Dkt. No.: 114198-5210 Research Center-sysbio.kribb.re.kr:8080/AsiDesigner/menuDesigner.jsf; and Invitrogen- rnaidesigner.invitrogen.com/rnaiexpress/. shRNA that targets and downregulates type I IFN can be prepared using methods known in the art (see, e.g., Cattaneo et al. (2016) Cell Death and Differentiation, 23:555-564 or are commercially available. Antisense oligonucleotides (ASOs) are small pieces of DNA or RNA that can bind to specific RNA molecules, preventing the RNA from making proteins or functioning in other ways. Methods to administer ASOs to cardiac tissue are described in the literature, e.g., Prakash, et al. (2019) Nucl. Acids Res. Vol. 47(12):6029-6044. CRISPR systems can also be used to downregulate or abrogate type I IFN expression by inhibiting transcription (also designated CRISPRi) using dCas9 (a mutant version of the Cas9 enzyme that lacks endonuclease activity) fused to a repressor domain (for example the Krüppel Associated Box – KRAB – domain) that is guided to the type I IFN gene by a single guide RNA designed to specifically target the promoter or exons of this gene, thereby repressing its transcription. Crispr regulation of translation, on the other hand, employs a catalytically dead dCas13 that targets RNA molecules (unlike Cas9 that targets DNA). In this case, a guide RNA targeting the translation start site of the type I IFN mRNA is used to ensure that dCas13 selectively represses production of type I IFN protein, without affecting translation of mRNAs encoding other proteins. The Krüppel associated box (KRAB) domain is a category of transcriptional repression domains present in approximately 400 human zinc finger protein-based transcription factors (KRAB zinc finger proteins). The KRAB domain typically consists of about 75 amino acid residues, while the minimal repression module is approximately 45 amino acid residues. It is predicted to function through protein-protein interactions via two amphipathic helices. The most prominent interacting protein is called TRIM28 initially visualized as SMP1, cloned as KAP1 and TIF1-beta. Substitutions for the conserved residues abolish repression. As used herein, the term “administer” or “administration” or “administering” intends to mean delivery of a substance to a subject such as an animal or human. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Atty. Dkt. No.: 114198-5210 Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, as well as the age, health or gender of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of pets and animals, treating veterinarian. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and the target cell or tissue. Non-limiting examples of route of administration include intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation. An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the optimal route will vary with the condition and age of the recipient, and the disease being treated. These strategies can be administered acutely (in a scenario of myocardial infarction) or chronically in any setting of heart failure. They can be administered systemically or locally. Non-limiting examples of locally delivery includes intracardiac injection or delivery during reperfusion of a coronary artery. Cardiac injury intends any injury to the heart muscle or cardiac cells. Non-limiting examples include heart failure, blunt cardiac injury, coronary artery disease, ischemic heart disease, or myocardial infarction. The injury can be acute or chronic. Heart failure, also known as congestive heart failure, is a condition that develops when the heart does not pump enough blood to meet the body needs. “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent that is an amount sufficient to obtain a pharmacological response such as passive immunity; or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the Atty. Dkt. No.: 114198-5210 specified disorder or disease in the patient. A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from the same sample following administration of a compound. Modes For Carrying Out the Disclosure Applicant’s discovery differs from conventional wisdom in two ways that leads to the disclosed methods. Applicant provides herein methods to treat type I IFN responses that result from mechanical injury rather than the classic systemic autoimmune disease or infections. Applicant also provides methods to treat type I IFN responses that are initiated by non-immune cells rather than immune cells. Conventional wisdom attributes initiation of the type I IFN response to professional innate immune cells such as dendritic cells and myeloid cells. This is primarily viewed as a systemic response. However, Applicant found it is activated in spatially localized clusters within mechanically injured tissue rather than a consequence of systemic autoimmune diseases or local infection. Applicant shows herein that staining for ISGs (the consequence of type I IFN response activation) after traumatic needle injury, which mimics the effects of needles or cannulas used for therapeutic delivery or injection showed staining in clusters at the site of sharp needle trauma. Applicant also stained for ISGs after myocardial infarction or ischemia reperfusion injury (models of human heart attacks) and observed clustered staining at the borderzone in the infarct and in the stretched atria. Applicant stained for ISGs after transaortic constriction and pressure overload, which mimics hypertensive heart disease and aortic stenosis and observed it in clusters in the ventricle and in the stretched atria. Based on these observations, provided herein are methods Atty. Dkt. No.: 114198-5210 to inhibit the type I IFN response in new clinical contexts that are caused by mechanical injury. Methods are also provided to inhibit the type I IFN response selectively in immune and non-immune immune cells rather than immune cells to achieve efficacy without broad immunosuppression (professional innate immune cells use the IFN pathway to defend against pathogenic viruses and other pathogens so inhibiting it in all cells would impair this protective antiviral response). Therapeutic Methods Thus, in one aspect, Applicant provides a method to inhibit the type I interferon (IFN) response systemically or locally in a subject that has experienced, is experiencing, or is anticipated to experience an endogenous or exogenous mechanical tissue injury, the method comprising, or alternatively consisting essentially of, or yet further consisting of systemically or locally administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting IFN response in the subject. Non-limiting examples of endogenous mechanical tissue injury include injury to any tissue caused by mechanical compression, tension, bending, shearing, torsion, stress, stretch distortion, hypercontraction, imbalanced contraction; sharp or blunt trauma, abrasion, or concussion; or mechanical destabilization caused by loss of tissue such as due to resection or cell death or caused by addition of acellular or cellular material such as amyloid, calcium, atherosclerotic plaque, thrombus, or tumor. Examples of tissues that may have experienced, or are experiencing, or are anticipated to experience such include cardiac, vascular, skeletal muscle, and other. Examples of cardiac diseases or conditions involving endogenous mechanical tissue injury include exercise; ischemia or infarction; cardiomyopathies such as dilated, hypertrophic, infiltrative, inflammatory, arrhythmogenic, drug-induced, toxin-induced, or takosubo; valvular heart disease; acute or chronic arterial or venous systemic or pulmonary hypertension; atrial fibrillation, stretch, or remodeling; myocarditis, pericarditis, or sarcoid; tumor. Examples of vascular diseases or conditions involving endogenous mechanical tissue injury include aortic or arterial atherosclerosis or calcific disease, aneurysms or dissection, acute or chronic arterial or venous systemic or pulmonary hypertension, microvascular dysfunction. Examples of skeletal muscle diseases or conditions involving endogenous mechanical tissue injury include exercise or muscle injury due to exercise. Examples of other endogenous mechanical injury are those caused by space-occupying cellular growths or acellular deposits Atty. Dkt. No.: 114198-5210 such as tumors, calcium, or amyloid; swelling or compression such as due to inflammation or infection, concussive injury (e.g., traumatic brain injury). In some aspects, the cells or tissue that are treated are mechanically loaded immune or non-immune cells, such as for example, such as cardiomyocytes, skeletal muscle cells, smooth muscle cells, fibroblasts, vascular tissue, and endothelial cells. Administration can be local or systemic as determined by the condition to be treated as well as the subject being treated. Applicant also provides a method to treat or prevent a disease or condition caused by an endogenous or exogenous mechanical tissue injury in a subject in need thereof, the method comprising, or alternatively consisting essentially of, or yet further consisting of administering to the subject an effective amount of an inhibitory IFN agent, thereby treat or prevent a disease or condition caused by an endogenous or exogenous mechanical tissue injury in the subject. In one aspect, the endogenous or exogeneous mechanical injury is associated with systemic or local type I interferon (IFN) response. Non-limiting examples of conditions related to endogenous mechanical tissue injury include injury to any tissue caused by mechanical compression, tension, bending, shearing, torsion, stress, stretch distortion, hypercontraction, imbalanced contraction; sharp or blunt trauma, abrasion, or concussion; or mechanical destabilization caused by loss of tissue such as due to resection or cell death or caused by addition of acellular or cellular material such as amyloid, calcium, atherosclerotic plaque, thrombus, or tumor. Examples of tissues that may have experienced, or are experiencing, or are anticipated to experience such include cardiac, vascular, skeletal muscle, and other. Examples of cardiac diseases or conditions involving endogenous mechanical tissue injury include exercise; ischemia or infarction; cardiomyopathies such as dilated, hypertrophic, infiltrative, inflammatory, arrhythmogenic, drug-induced, toxin-induced, or takosubo; valvular heart disease; acute or chronic arterial or venous systemic or pulmonary hypertension; atrial fibrillation, stretch, or remodeling; myocarditis, pericarditis, or sarcoid; tumor. Examples of vascular diseases or conditions involving endogenous mechanical tissue injury include aortic or arterial atherosclerosis or calcific disease, aneurysms or dissection, acute or chronic arterial or venous systemic or pulmonary hypertension; microvascular dysfunction. Examples of skeletal muscle diseases or conditions involving endogenous mechanical tissue injury include exercise or muscle injury due to exercise. Examples of other Atty. Dkt. No.: 114198-5210 endogenous mechanical injury are those caused by space-occupying cellular growths or acellular deposits such as tumors, calcium, or amyloid; swelling or compression such as due to inflammation or infection, concussive injury (e.g., traumatic brain injury). In some aspects, the cells or tissue that are treated are mechanically loaded immune or non-immune cells, such as for example, such as cardiomyocytes, skeletal muscle cells, smooth muscle cells, fibroblasts, vascular cells and endothelial cells. Administration can be local or systemic as determined by the condition to be treated as well as the subject being treated. In one aspect, the disease or condition is selected from mechanical stretch, hypercontraction, inflammatory heart disease, myocarditis, myopericarditis, sarcoid, inflammatory cardiomyopathies, acute myocardial ischemia/infarction or chronic ischemic heart disease, localized cardiac injury, vascular stretch or mechanical deformation due to systemic arterial hypertension, pressure overload due to chronic hypertension, and valvular heart disease, atrial stretch and atrial fibrillation, aortic or arterial aneurysms, pulmonary arterial hypertension, pulmonary venous hypertension due to chronically elevated filling pressures, atherosclerosis, skeletal muscle injury, suture sites, cannula insertion sites, biopsy sites, or therapeutic injection sites. Additional diseases and conditions are described herein. Also provided is a method to inhibit the type I IFN response, or to treat or prevent mechanical tissue injury (systemically or locally) in a subject who will receive or has received an exogenous mechanical tissue injury, the method comprising, or alternatively consisting essentially of, or yet further consisting of administering to the subject an effective amount of an inhibitory IFN agent prior to or subsequent to receiving the mechanical tissue injury, thereby inhibiting the type I IFN response, or treating or preventing mechanical tissue injury (systemically or locally) in the subject. Examples of exogeneous mechanical tissue injury are disclosed herein. Non-limiting examples include the use of sutures, a cannula, a medical device, a needle, a guidewire, a catheter, an electrode, a bioptome, a stent, or a staple, e.g., a surgical staple. In one aspect, administration of the device such as the suture or staple is coated or incorporated into the agent for delivery to the subject. Further provided is a method to inhibit the type I IFN response systemically or locally in a subject prior to, concurrently or subsequent to local delivery of a therapy comprising, or Atty. Dkt. No.: 114198-5210 alternatively consisting essentially of, or yet further consisting of administering to the subject an effective amount of an inhibitory IFN agent prior to or concurrently to the local delivery of the therapy, thereby inhibiting the type I IFN response. Non-limiting examples of therapies include administration of one or more of; drugs such as biologics, small molecules, or nucleic acids; viral-based therapies, biomaterials; therapeutic cells, engineered cells, and stem cells; or devices such as deep brain stimulators. In another aspect, a method is provided to inhibit the type I IFN response in and/or to treat mechanical pathologies in a subject in need thereof comprising, or alternatively consisting essentially of, or yet further consisting of administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the IFN response in mechanical pathologies in and/or treat mechanical pathologies. Non-limiting examples of mechanical pathologies are selected from mechanical stretch, hypercontraction, inflammatory heart disease, myocarditis, myopericarditis, sarcoid, inflammatory cardiomyopathies, acute myocardial ischemia/infarction or chronic ischemic heart disease, localized cardiac injury, vascular stretch or mechanical deformation due to systemic arterial hypertension, pressure overload due to chronic hypertension, and valvular heart disease, atrial stretch and atrial fibrillation, aortic or arterial aneurysms, pulmonary arterial hypertension, pulmonary venous hypertension due to chronically elevated filling pressures, atherosclerosis, skeletal muscle injury, suture sites, cannula insertion sites, biopsy sites, or therapeutic injection sites. In each of the above methods, the methods can further comprise detecting one or more of the quantity, location, distribution, and amount of type I IFN in the subject. The methods can be practiced on subjects such as mammals and human patients. Any appropriate means of administration can be used, e.g., by a method comprising intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation. Further provided is a method for one or more of: reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack, each method comprising, or consisting essentially of, or yet further consisting of, by Atty. Dkt. No.: 114198-5210 administering a cardiomyocyte cGAS-STING-IRF3 inhibitor thereby reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack. In one aspect, the inhibitor is a small molecule or a gene therapy which inhibits the activity of Irf3, cGAS, STING and TBK1. Administration can be in vitro or in vivo. The methods can be practiced on subjects such as mammals and human patients. Non-limiting examples of administration include intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub- retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation. In each of the above methods, the subject to be treated is a mammal, or a human patient. When performed on a mammal, it can be used for the treatment of sport animals, livestock or pets. Alternatively, the methods can be used in” animal models such as murines, simians, canines, ovines, to test for new therapies or combination therapies. In addition, the subject can be a human patient under the supervision of a treating physician. Reducing or treating cardiac injury, promoting, or supporting cardiac regeneration. In one aspect, provided herein is method for one or more of: reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack, each method comprising, or consisting essentially of, or yet further consisting of, by administering a type I IFN inhibitory agent or a cardiomyocyte cGAS-STING-IRF3 inhibitor thereby reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack. In one aspect, the inhibitor is a small molecule or a gene therapy which inhibits the activity of Irf3. Administration can be local or systemic and can be in vitro in a tissue system or in vivo in a subject. Subjects can be a mammal, such as a human patient. Inhibiting the type I IFN response systemically or locally before, during, or shortly after surgery. Atty. Dkt. No.: 114198-5210 The insertion of needles during suturing of tissue induces mechanical disruption of cells and locally activates the type I IFN response. This can induce local inflammation and promote surgical site complications, e.g., short surgical tissue trauma caused by staples or cautery. Inhibiting the type I IFN response can reduce complications. Thus, this disclosure provides a method to inhibit the type I IFN response systemically or locally in a subject who will receive or has received a suture, by administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the IFN response. In one aspect, the subject is a mammal such as a human patient. Administration can be accomplished by administration of a pharmaceutical composition comprising the inhibitory IFN agent, or by coating the suture or other device used for insertions into the subject during surgery. Administration can be prior to, concurrently, or after surgery. Thus, in another embodiment, the method further comprises surgery prior to, after or concurrently with the administration of the inhibitory IFN agent. Applicant inserted a needle into the hearts of mice as if performing surgery and 3 days later performed in situ hybridization for ISGs. Applicant observed spatially clustered staining for ISGs at the site of needle insertion, indicating that the type I IFN response was locally activated. When Applicant did the same experiment but first pretreated with a single dose of systemic anti-IFNAR Ab (the mouse equivalent of Astrazeneca’s AnifrolumabTM) the local needle site ISG response was eliminated. In a separate experiment, Applicant delivered the anti-IFNAR antibody therapy locally through the inserted needle without systemic inhibition and again the ISG response was eliminated. This could also be accomplished by coating the needle or suture with anti- IFN therapy. Without being bound by theory, these results show that the proinflammatory type I IFN response is induced by surgery and can be inhibited systemically or locally to reduce surgical site complications. Inhibit the type I IFN response systemically or locally before, during, or shortly after local delivery of therapy. Applicant also provides a method to inhibit the type I IFN response systemically or locally in a subject prior to, concurrently or subsequent to local delivery of therapy by administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the type I IFN response. In one aspect, the subject is a mammal such as a human Atty. Dkt. No.: 114198-5210 patient. Administration can be accomplished by administration of a pharmaceutical composition comprising the inhibitory IFN agent. Thus, in another embodiment, the method further comprises administration of the local delivery of a therapy prior to, after or concurrently with the administration of the inhibitory IFN agent. In one aspect, the subject is a mammal such as a human patient. Therapies are often delivered locally by direct injection of tissue or by inserting a cannula and delivering therapy via the cannula. Examples of therapies delivered this way include drugs such as biologics, small molecules, or nucleic acids; viral-based therapies, biomaterials; therapeutic cells, engineered cells, and stem cells; or devices such as deep brain stimulators. Both needles and cannulas mechanically disrupt tissue when they are inserted and thus induce the type I IFN response which can be a barrier to therapy. Applicant has reduced this to practice as detailed above with the needle experiments. Applicant has shown that this response can be inhibited with systemic or local type I IFN inhibitory agent. In one aspect, at the site of cannula insertion in the brain, as is performed commercially via FDA-cleared delivery systems made by Clearpoint Neuro (CLPT). This is used for introduction of device therapies (deep brain stimulation), and other therapeutic modalities (drug, viral, cell, etc.). A secondary benefit of inhibiting the type I IFN response in the context of local delivery of gene therapy is that it would locally reduce the anti-viral response, which is a tissue response that impedes infection by therapeutic viruses. Moreover, it limits the ability of the tissue to mount an IFN response induced by the therapeutic virus (viruses induce the type I IFN response In one aspect, intramyocardial injection of stem cells or gene therapy at the cardiac infarct or borderzone to treat ischemic heart failure (https://www.jacc.org/doi/10.1016/j.jacc.2022.11.061). The method can be further combined with a companion diagnostic. In one aspect, an ISG-binding compounds can serve as systemically delivered companion diagnostic imaging agents (nuclear, PET, MRI, CT, ultrasound) by localizing to sites of ISG induction to quantify location, distribution, and amount of type I IFN response and predict which patients are most likely to benefit from type I IFN inhibition. Atty. Dkt. No.: 114198-5210 cGAS is part of an IFN-inducing pathways, but it is itself an inducible ISG. Therefore, the cGAS-binding compounds being developed as therapeutics (e.g., VENT-003) could be radiolabeled or similarly modified and repurposed as imaging agents to serve as a companion diagnostic and identify which patients have the greatest type I IFN responses and are most likely to respond to therapy and to localize the response to a specific organ or tissue. More generally, any selective ISG-binding compound, if modified to create modality-specific contrast, could serve as an imaging agent for a companion diagnostic to quantify which patients are candidates for type I IFN inhibition. Alternatively, tissue, whether obtained by biopsy or excision, can be stained for ISG proteins or probed for interferon stimulating genes (ISG) RNA using in situ hybridization to quantify the amount and distribution of type I IFN response activation and serve as a companion diagnostic to predict which patients are most likely to benefit from type I IFN inhibition. Applicant observed clustered ISG expression in the ventricles of ischemic, infarcted, or pressure overloaded ventricles, sharp needle injured ventricles, and in mechanically stretched atria consequent to pressure overloaded ventricles. Specifically, Applicant performed in situ hybridization for ISGs such as Ifit1 and Cxcl10 or full spatial transcriptomic profiling using 10x Visium or MERFISH, and by each method, Applicant observed spatially clustered ISG responses. Therefore, staining or probing for ISGs can serve as a companion diagnostic to identify which patients have type I IFN responses and are most likely to respond to type I IFN response inhibition. Inhibit the type I IFN response in mechanical pathologies. Applicant also provides a method to inhibit the type I IFN response in mechanical pathologies and/or treat mechanical pathologies. Thus, this disclosure provides a method to inhibit the type I IFN response in mechanical pathologies in a subject in need thereof by administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the IFN response in mechanical pathologies and/or treat mechanical pathologies. In one aspect, the subject is a mammal such as a human patient. Administration can be accomplished by administration of a pharmaceutical composition comprising the inhibitory IFN agent. Atty. Dkt. No.: 114198-5210 The method can be used to treat the following mechanical injuries: Mechanical stretch – due to volume overload or dysrhythmia including atrial stretch associated with atrial fibrillation, ventricular stretch associated with diverse causes of cardiomyopathy or valvular heart disease leading to atrial or ventricular wall stretch. Also included are pressure overload due to chronic hypertension, valvular heart disease as both can cause pressure overload seen in the ventricle and atria, atrial stretch and atrial fibrillation since focal interferon responses in stretched atria (stretched atria can be caused by many disorders including chronic hypertension, diastolic dysfunction, and valvular heart disease (e.g., aortic stenosis, mitral regurgitation, mitral stenosis). Hypercontraction – stress takotsubo cardiomyopathy, hypertrophic cardiomyopathy, hypertensive heart disease, infiltrative cardiomyopathy (e.g., cardiac amyloid). Inflammatory heart disease - myocarditis, myopericarditis, sarcoid, or other inflammatory cardiomyopathies. Ischemic heart disease - Acute myocardial ischemia/infarction or chronic ischemic heart disease occurs due to insufficient delivery of oxygenated blood through one or more obstructed branches of the coronary artery tree. This causes localized cardiac injury and induces the type I IFN response in the infarct and at the borderzone. Vascular stretch or mechanical
Figure imgf000046_0001
due to systemic arterial hypertension, aortic or arterial aneurysms, pulmonary arterial hypertension, pulmonary venous hypertension due to chronically elevated filling pressures. Atherosclerosis – stretch of vascular smooth muscle cell, endothelial cells, or fibroblasts in or surrounding atherosclerotic lesions. Skeletal muscle injury – due to exercise, trauma, or disease Suture sites – (see above) Cannula insertion sites – for example in the brain as performed by all Clearpoint Neuro (CLPT) procedures (see above). Biopsy sites – explicit mechanical disruption of tissue similar to sutures/cannulas above (brain, heart, liver, kidney, etc.) Atty. Dkt. No.: 114198-5210 Therapeutic injection sites – drugs such as biologics, small molecules, or nucleic acids; viral-based therapies, biomaterials; therapeutic cells, engineered cells, and stem cells are being injected into many different tissues for therapy. Such injections will induce the type I IFN response which can make these therapies less effective. Applicant propose inhibiting the IFN response to improve the therapeutic efficacy of these injected therapies. Selective inhibit of the type I IFN response in non-immune cells. Conventional wisdom attributes initiation of the type I IFN response to professional innate immune cells (e.g., dendritic cells and myeloid cells). Applicant discovered that mechanical pathologies initiate the type I IFN response in non-immune cells. Applicant also provides a method to inhibit the type I IFN in non-immune cells in a subject in need thereof, by administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the type I IFN response in non-immune cells in the subject. In one aspect, the subject is a mammal such as a human patient. Administration can be accomplished by administration of a pharmaceutical composition comprising the inhibitory IFN agent. There are 2 ways to target non-immune cells. Targeted or biased delivery of molecules that inhibit the type I IFN response or reduce expression of key molecules in the type I IFN response pathway preferentially in non- immune cells. In one aspect, an effective amount of an anti-senso oligo (ASO) therapy to cardiomyocytes. Without being bound by theory, ASOs can be delivered to cardiomyocytes to inhibit activation of the type I IFN response by targeting one or more molecules in the activation of the type I IFN response as detailed above. This would achieve protection of the ventricle from development of heart failure while minimizing immunosuppression by inhibiting the pathway in professional innate immune cells that fight pathogenic viruses. Selective expression (or inhibition) of molecules involved in the type I IFN response to limit its initiation in non-immune cells. This can be accomplished using nucleic acid or gene therapy with regulatory sequences that cause it to selectively expressed in non-immune cells such as cardiomyocytes or fibroblasts (non-immune cells) rather than dendritic cells or macrophages (professional innate immune cells). Non-limiting examples of such include Atty. Dkt. No.: 114198-5210 agents and molecules that inhibit expression of cGAS or STING, or IRF3, or IFN alpha or IFN beta, or over expression of the DNAse TREX1. Compositions and Modes of Administration Also provided herein are compositions comprising the inhibitory agents for use in the methods as described herein. The compositions, including pharmaceutical compositions comprising, consisting essentially of, or consisting of the inhibitory agent can be in combination of other therapeutic agents can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes. These can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, micelles, gene delivery vehicles, lipid nanoparticles, vectors, or auxiliaries which facilitate processing of the combinations of compounds provided herein into preparations which can be used pharmaceutically. In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral, subcutaneous, oral, buccal, rectal, sublingual, or transdermal administration routes. In some cases, parenteral administration comprises, or consists essentially of, or yet further consists of, intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra- arterial, intra-articular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration. In some instances, the pharmaceutical composition is formulated for local administration. In other instances, the pharmaceutical composition is formulated for systemic administration. In some aspects, they are administered by coating a device or suture for example, that will be mechanically injuring the tissue of the subject. In some embodiments, the pharmaceutical formulations include, but are not limited to, lyophilized formulations, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations. Atty. Dkt. No.: 114198-5210 In some embodiments, the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington’s Pharmaceutical Sciences (Mack Publishing Co., Easton, Pennsylvania 1975), Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, (Marcel Decker, New York, N.Y., 1980,) and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins l999). In some instances, the pharmaceutical formulations further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane, and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range. In some instances, the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate. In some embodiments, the pharmaceutical formulations include, but are not limited to, sugars like trehalose, sucrose, mannitol, maltose, glucose, or salts like potassium phosphate, Atty. Dkt. No.: 114198-5210 sodium citrate, ammonium sulfate and/or other agents such as heparin to increase the solubility and in vivo stability of polypeptides. In some instances, the pharmaceutical formulations further include diluent which are used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as AVICEL®, dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar, such as Di- PAC® (Amstar), mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner’s sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like. In some cases, the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance. The term “disintegrate” include both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. Examples of disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or AMIJEL®, or sodium starch glycolate such as PROMOGEL® or EXPLOTAB®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., AVICEL®, AVICEL® PH101, AVICEL®PH102, AVICEL® PH105, ELCEMA® P100, EMCOCEL®, VIVACEL®, MING TIA®, and SOLKA-FLOC®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (AC-DI-SOL®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross- linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as VEEGUM® HV (magnesium aluminum silicate), a gum such as agar, Atty. Dkt. No.: 114198-5210 guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like. In some instances, the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like. Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (STEROTEX®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, STEAROWET®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as CARBOWAX™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as SYLOID™, CAB-O-SIL®, a starch such as corn starch, silicone oil, a surfactant, and the like. Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents. Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N- methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, Atty. Dkt. No.: 114198-5210 bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like. Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like. Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium biphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, poly sorb ate-20 or TWEEN® 20, or trometamol. Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like. Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., PLURONIC® (BASF), and the like. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants is included to enhance physical stability or for other purposes. Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, Atty. Dkt. No.: 114198-5210 hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof. Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like. The pharmaceutical compositions for the administration can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy. The pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the compounds provided herein into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition, each compound of the combination provided herein is included in an amount sufficient to produce the desired therapeutic effect. For example, pharmaceutical compositions of the present technology may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal, and vaginal, or a form suitable for administration by inhalation or insufflation. For topical administration, the combination of compounds can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well-known in the art. Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection) as well as those designed for transdermal, transmucosal, oral, or pulmonary administration. Useful injectable preparations include sterile suspensions, solutions, or emulsions of the compounds provided herein in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing, and/or dispersing agents. The formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, Atty. Dkt. No.: 114198-5210 buffer, and dextrose solution, before use. To this end, the combination of compounds provided herein can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art. For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art with, for example, sugars, films, or enteric coatings. Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the combination of compounds provided herein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., corn starch or alginic acid); binding agents (e.g. starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). The tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques well known to the skilled artisan. The pharmaceutical compositions of the present technology may also be in the form of oil-in-water emulsions. Atty. Dkt. No.: 114198-5210 Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophoreTM, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate. In some embodiments, one or more compositions disclosed herein are contained in a kit. Accordingly, in some embodiments, provided herein is a kit comprising, consisting essentially of, or consisting of one or more compositions disclosed herein and instructions for their use. Dosage and Dosage Formulations In some embodiments, the combinations or compositions comprising the inhibitory agent are administered to a subject suffering from a condition as disclosed herein, such as a mammal or a human, either alone or as part of a pharmaceutically acceptable formulation, once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently. Administration of the composition or combination alone or in combination with the additional therapeutic agent and compositions containing same can be accomplished by any method that enables delivery to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration. Bolus doses can be used, or infusions over a period of 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, 90, 120 or more minutes, or any intermediate time period can also be used, as can infusions lasting 3, 4, 5, 6, 7, 8, 9, 10, 12, 1416, 20, 24 or more hours or lasting for 1-7 days or more. Infusions can be administered by drip, continuous infusion, infusion pump, metering pump, depot formulation, or any other suitable means. Atty. Dkt. No.: 114198-5210 Dosage regimens can be adjusted to provide the optimum desired response. For example, a single bolus can be administered, several divided doses can be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient can also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that can be provided to a patient in practicing the present disclosure. It is to be noted that dosage values can vary with the type and severity of the condition to be alleviated and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present disclosure encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens for Atty. Dkt. No.: 114198-5210 administration are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein. Diagnostic Methods In some embodiments, one or more of the methods described herein further comprise, or consists essentially of, or yet further consists of, a diagnostic step. In some instances, a sample is first obtained from a subject in need of the therapy or suspected of having a disease or condition described above. Exemplary samples include, but are not limited to, cell sample, tissue sample, tumor biopsy, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In some instances, the sample is a tumor biopsy. In some cases, the sample is a liquid sample, e.g., a blood sample. In some cases, the sample is a cell-free DNA sample. Various methods known in the art can be utilized to determine the presence of a type I IFN in a cell or tissue. Expression levels or abundance can be determined by direct measurement of expression at the protein or mRNA level, for example by microarray analysis, quantitative PCR analysis, or RNA sequencing analysis. Alternatively, labeled antibody systems may be used to quantify target protein abundance in the cells, followed by immunofluorescence analysis, such as FISH analysis. The diagnostic steps can be performed prior to or after administration and may be performed throughout the course of treatment to monitor the subject’s response. In one aspect, the diagnostic method is performed to detect one or more of the quantity, location, distribution, and amount of type I IFN in the subject or tissue. Atty. Dkt. No.: 114198-5210 Experimental Description Applicant hypothesized that the distribution of ISG expression could be explained by focal initiation of the type I IFN response followed by spread and amplification of the secondary response in neighboring cells. To test this hypothesis, Applicant analyzed infarcted mouse and human hearts using spatial transcriptomics and single molecule RNA fluorescence in situ hybridization RNA (smFISH). Experimental Methods Animals Adult C57BL/6J mice were purchased from the Jackson Laboratory at 10 weeks of age used as wildtype controls. Irf3-/- were bred inhouse from existing colonies. Ccr2-/- (strain 004999), Ifnar1- (028288), Cgas-/- (026554), and STINGgt/gt (017537) mice were purchased from Jackson Laboratory. Nuclear reporter mice (tdTom-NLS) were purchased from Jackson Laboratory (025106). Cardiomyocyte-specific nuclear rupture reporter mice were generated by crossing floxed tdTom-NLS with Myh6cre/+ mice. Irf3fl/fl were a generous gift donated from Dr. Tadatsugu Taniguchi. Cell type-specific transgenics were generated by crossing Irf3fl/fl with transgenic mice expressing cre-recombinase under the control of the following promoters purchased from Jackson Laboratory: Myh6cre/+ (011038), Col1α1creERT2/+ (016241), Cx3cr1creERT2/+ (025524), S100a8cre/+ (0216141), Tie2cre/+ (008863). Inducible deletion was accomplished by intraperitoneal injection of tamoxifen 20 mg/mL in corn oil or vehicle injection for 5 consecutive days in Col1α1creERT2/+ and Cx3cr1creERT2/+ mice. Animals were allowed 7 days to recover after the last tamoxifen or vehicle injection before conducting any surgical procedures. All experiments were performed with 12–14-week- old animals and were carried out using age- and sex-matched groups without using randomization. All experiments involving mice were maintained in a pathogen-free environment of the University of California San Diego (UCSD) facilities and all animal experiments were approved by the subcommittee on Animal Research Care at UCSD (Institutional Animal Care and Use Committee, S17144). Permanent ligation (MI) surgery and cardioprotective therapy For permanent ligation, mice were intubated and ventilated with 2% isoflurane. Thoracotomy was performed at the fourth left intercostal space was performed to expose the Atty. Dkt. No.: 114198-5210 heart and visualize the left anterior descending artery (LAD). The LAD was permanently ligated with an 8-0 nylon suture in mice with MI, and hearts were collected at various time points as hours (hr) or days (D) post-surgery (1hr, 4hr, D1, D3, D7, or D28). Intercostal space and skin were sutured closed using 6-0 prolene sutures. Cardioprotective therapy of anti- IFNAR-Ab was performed as previously described. Mice were treated with two intraperitoneal doses of 500 μg of MAR1-5A3 IFNAR neutralizing antibody (anti-IFNAR- ab) at 8 hours and 48 hours post-MI surgery (BioXCell catalog no. BE04241)3,14. Hearts were harvested at D3 post-infarct. Cardiac needle trauma For in vivo trauma of the myocardium, Applicant used Needle Pass (NP) injury previously described18. Briefly, mice were anesthetized under 2% isoflurane and partial thoracotomy was performed above the fourth left intercostal space; a chest retractor was inserted and placed between the third and fourth intercostal spaces. After visualization of the heart and LAD, a 28G beveled needle was inserted into the lateral LV free-wall directly to the right of the LAD at the position in which Applicant normally perform permanent ligation in MI; the needle did not penetrate through to the endocardium. The 28G beveled needle was held in position in the midventricular wall for 3 seconds before being withdrawn. The chest retractor was then removed, and the intercostal space was closed using 6-0 prolene sutures. To reduce complications due to pneumothorax, a sterile 20G flexible angiocatheter was placed within the pleural space prior to removal of chest retractor. The intercostal space and dermis were closed using 6-0 prolene sutures. After the skin was sutured and closed, a syringe was attached to the angiocatheter and negative pressure was manually applied simultaneously as the catheter was withdrawn. Surgical glue was then applied to the remainder of the skin incision. Culturing and Differentiation of Human iPSC-derived Fibroblasts Human H9 embryonic stem cells (ESCs) were commercially acquired and cultured on Matrigel-coated 6-well plates (WiCell, Madison, WI). H9 ESCs were maintained in mTeSR1 Media (StemCell Technologies #85851) until 90% confluency. Once confluent, cells were split with mTeSR1 media and 5 μM ROCK inhibitor Y27632, counted, and plated at a confluency of .5 million cells/mL on Matrigel-coated 12-well plates (Tocris #1524). Cells Atty. Dkt. No.: 114198-5210 were fed daily for 3 days, and on the following day, cells were fed with RPMI basal medium and 4 μL of 36 mM of GSK3 inhibitor CHIR99021 (Sigma R7388; Tocris #4423) to begin differentiation. One day after differentiation with GSK3 inhibitor, media was removed, and cells were fed with only RPMI basal medium. Two days later, combined medium was prepared as follows: 1 mL RPMI medium, 1 μL of 5mM IWP2 (2.5 μM final concentration). Cells were fed with RPMI media after 2 days, and after another 2 days, cells were detached with ACCUTASE and inactivated by 20% fetal bovine serum (FBS) in RPMI medium (RPMI20) (Stemcell Technologies, 07922). Cells were resuspended in LaSR basal medium containing 5 μM Y27632 ROCK inhibitor and seeded onto gelatin-coated 12-well plates at a density of 5,000 cells per well. For the following 5 days, cells were fed with 12 mL of LaSR medium (advanced DMEM/F12 medium; 6.5 mL Glutamax, 500 μL of antioxidant 100 mg/mL ascorbic acid solution) + 3 μM CHIR99021 GSK3 inhibitor. Epicardial cells were removed from plates with ACCUTASE and quenched with RPMI20. Cells were resuspended in LaSR medium supplemented with 5 μM Y27632 ROCK inhibitor. Following overnight cell attachment, epicardial cells were differentiated into fibroblasts and maintained in LaSR medium supplemented with 10 ng/mL bFGF (RnD Systems, #233-FB). After differentiation, hiPSC-Fibroblasts were then replated and expanded on gelatin-coated 6 well plates and maintained on Human Cardiac Fibroblast Media (catalog no. MSDS 315-500, Cell Application, INC). In vitro fibroblast treatment with TGFβ and IFNβ Human iPSC-derived fibroblasts or mouse L929 fibroblasts originally derived from connective tissue were used for in vitro cell culture experiments (ATCC CCL-1). Fibroblasts were seeded onto 10 mm gelatin-coated, treated culture plates and maintained with 10% FBS/1% penicillin-supplemented DMEM until reaching confluency. Fibroblasts were subsequently subcultured and/or seeded onto 6-well gelatin coated plates for experimental treatments and designated as passage 3 (P3) cells. For in vitro treatment of cells, Applicant used recombinant human TGFβ (10 ng/mL, Peprotech 100-21) and/or human IFNβ (10 ng/mL, Peprotech 300-02) suspended in culture medium for 24 hours before harvesting for downstream analysis. Atty. Dkt. No.: 114198-5210 Fibroblast functional assessment with collagen gel contraction assay Human iPSC-derived cardiac fibroblasts or L929 fibroblasts were enzymatically dissociated from confluent culture plates, pelleted by centrifugation, and quantified with hemocytometer. Approximately 250,000 cells were seeded suspended into 2 mg/mL collagen hydrogel solution and adjusted to 1 mL with 10% FBS-supplemented clear DMEM without phenol red (Advanced Biomatrix catalog no. 5074, Gibco catalog #31053028). The 1 mL solution is added to 12-well culture plate and placed in 37oC incubator for 30 minutes to allow solidification of hydrogel. Following solidification, gels were released from the sides of the wells by careful separation using a pipette tip traced along the perimeter of gel. Gels were supplemented with 1 mL of clear culture media and photographed to determine the gel area pre-contraction. After 24 hours, gels were treated with TGFβ (10 ng/mL) as the positive control for gel contraction and/or IFNβ (10 ng/mL). For relevant experiments, anti-IFNAR-ab was administered immediately after TGFβ/IFNβ cotreatment with 1 μg/mL suspended in full media and imaged at 24 hours and 3 days post-treatment. Gel area was determined using scaled images in Fiji ImageJ. RNA isolation and qPCR RNA was isolated from myocytes using RNeasy Mini Kit (Qiagen catalog no.74536) and reverse transcribed with high-efficiency enzymes (Applied Biosystems catalog no. 438813). Quantitative, real-time qPCR was then performed using TaqMan primers for the following murine transcripts Col1α1 (Mm00801666_g1), Bgn (Mm001191753_m1), Postn (Mm01284919_m1), Sparc (Mm05915229_s1), Irf7 (Mm00516793_g1), Oasl1 (Mm00455081_m1), Ifnb1 (Mm00439552_s1), Irf3 (Mm00516784_m1), Cxcl10 (Mm00445235_m1), Ifit1 (Mm07295796_m1), Isg15 (Mm01705338_s1), and Gapdh (Mm99999915_g1). In the studies done on human iPSC-derived cells, the following human transcripts were used POSTN (Hs01566750_m1), SPARC (Hs00234160_m1), COL1α1 (Hs00164004_m1), IRF7 (Hs00164004_m1), IFI27 (Hs01086373_g1), IFIT3 (Hs01922752_s1), and GAPDH (Hs02786624_g1). For genotyping of transgenic animals, earsnip samples were digested in NaOH and neutralized by Tris HCL. DNA was extracted and amplicons were amplified for gel electrophoresis. Primer sequences used for each transgenic line are included in Tables 1 and 2. Atty. Dkt. No.: 114198-5210 Table 1 Primers Genotyping
Figure imgf000062_0001
Table 2
Figure imgf000062_0002
Atty. Dkt. No.: 114198-5210 Collection of ruptured ventricles Since permanent LAD ligation in mice produce the mechanical defect of ventricular rupture comparable to clinical observations of myocardial rupture, Applicant performed MI surgery in a cohort of 40 WT mice aged between 12-15 weeks purchased from Jackson Laboratory (strain 000664). To preserve the integrity of RNA of ruptured ventricles, mice were closely monitored for 12 consecutive hours each day starting from day 3 to day 14 post- MI, which is designated as the window of susceptibility for a rupture event to occur in mice. When an acute mortality was observed, the chest cavity was immediately opened, and hearts were immediately perfused with cold PBS and harvested. Tissue was quickly observed under surgical microscope and then flash-frozen by embedding in OCT. Each acute mortality event was observed for 1) presence of blood in the chest cavity and 2) visualization of rupture site under the microscope. These 2 criteria qualified a harvested sample as a ruptured ventricle. Plasmacytoid dendritic cell depletion Mice were pre-treated with intraperitoneal doses of 500 μg of anti-mouse CD317 (BST2) antibody or isotype control antibody once a day for 3 days prior to MI surgery (BioXCell #BE0311). Hearts were harvested at D3 post-infarct, flow sorted, and processed for single cell RNA sequencing. Chimeric bone marrow transfer For transfer of bone marrow derived cells (BMDC) from WT mice into Irf3-/- (WT -> KO) experiments, recipient mice were irradiated for 12 minutes using 10Gy of ionizing radiation dose. Bone marrow cells were isolated from the femurs of WT or Irf3-/- donor mice, counted, and resuspended in 1 mL of 5% bovine serum albumin/PBS solution. Approximately half a million cells were suspended per 100 μL and donated via retroorbital injection into irradiated recipients. Immunohistochemistry and Nuclear Rupture Imaging All hearts harvested for downstream analysis were perfused with first 10 mL of cold phosphate buffered saline (PBS) contained within a syringe attached to a 28G needle to remove contaminating blood. Tissue was harvested and embedded in optimal cutting temperature (OCT) compound and flash-frozen in isopentane bath cooled by dry ice. OCT- Atty. Dkt. No.: 114198-5210 embedded hearts were sectioned into 10 μm thick, short-axis sections for use with H&E staining, immunofluorescence, and spatial transcriptomics assays (Visium and MERFISH). H&E staining was performed according to manufacturer’s suggested protocol. The 3D distribution of cytosolic NLS signal was visualized and imaged using the Nikon AXR point scanning confocal microscope. For each field of view taken at 60X magnification, 15-20 optical slices were obtained and used for maximal intensity projections. Ruptured cardiomyocyte nuclei were normalized to total number of nuclei analyzed in each field of view acquired from the remote zone or borderzone adjacent to infarct. Sequencing-based spatial transcriptomics OCT-embedded cardiac tissue blocks were cryosectioned in short axis orientation at approximately 10 μm in thickness at cryostat temperature set to -22οC. Sections were stained with H&E, and images were obtained using 20X magnification on a Nikon Eclipse Ti2-E widefield microscope. Sections were then processed for spatially resolved gene expression using the Visium Spatial Transcriptomics Kit according to the manufacturer’s protocol (10X Genomics). Permeabilization time of infarcted murine hearts was previously optimized and determined at 30 minutes. Quality control for cDNA and libraries were performed on Agilent TapeStation before sequencing on the Illumina NovaSeq6000 instrument. The resulting sequencing data was processed, and images were aligned using the SpaceRanger v.1.3.1 pipeline (10X Genomics). Quality control, normalization, and integration for sequencing-based spatial transcriptomics Sequencing-based spatial transcriptomic assays were initially preprocessed to assess variance in feature counts/spot and the biological differences in cell density across heterogenous tissue morphology of the infarcted heart. Visual assessment of the underlying H&E confirmed lower molecular counts in tissue regions of dead and necrotic tissue of the infarct zone whereas high molecular counts were consistently seen in borderzone areas. Normalization was performed using Seurat’s SCTransform v2 method based on negative binomial models that account for technical artifacts such as sequencing depth variations but detects and preserves highly variant biological features. This is performed by placing a lower bound on the standard deviation of lowly expressed genes when using Pearson residuals to Atty. Dkt. No.: 114198-5210 estimate highly variable features. Replicate data of experimental conditions were split into two Seurat objects labeled as the control condition and the stimulated condition; our control condition consisted of all samples that served as our negative control i.e., infarcted hearts that lacked a type I interferon response (Irf3-/-, Ifnar-/-, Cgas-/-, STINGgt/gt). All wildtype post- MI day 3 hearts, all tissue-specific Irf3-/- mice, and Ccr2-/- mice were assigned to the stimulated Seurat object. The control dataset was normalized using SCTransform and dimensional reduction performed using PCA. Visium spots were clustered using Seurat’s FindClusters function at a resolution of 0.9. The stimulated group was similarly normalized using SCTransform() and PCA analysis. To perform integration of the two categorized Seurat objects using Pearson residuals, FindIntegrationAnchors() was performed followed by PrepSCTIntegration() on a merged list of the 2 objects to anchor and integrate the datasets together. Graph-based clustering using K-nearest neighbors (KNN) function FindNeighbors() was performed and shared nearest neighbors were identified using FindClusters() at a resolution of .60 on the entire integrated dataset. Moran I’s test statistic, Sepal score, and IFNIC colony size quantification Spatial distribution of genes was examined using Moran’s I test statistic, which is a spatial autocorrelation coefficient used to quantify and measure spatial enrichment and distribution of individual genes. Moran’s I was chosen as it is independent from differential gene expression and unrelated to clustering information. This metric scale ranges from 1 (significant spatial enrichment) to 0 (homogenously distributed throughout biological sample). Moran’s I revealed ISGs are spatially enriched and formed colonies with high autocorrelation test statistic indicating numerous focal IFN-responses. As orthogonal validation of identifying spatially variable genes, Applicant used the Sepal method, which quantifies spatially clustered features using a diffusion-based simulation. Highly expressed, clustered gene expression are assigned with a high sepal score since it would take a longer diffusion time (dt) to reach homogenous distribution across space. To define and quantify ISG-expressing colonies, Applicant considered first-order, contiguous neighbors as a requisite to be designated as an IFNIC colony (see FIG. 10). Applicant first compared log normalized expression of Ifit1, Rsad2, and scored ISG transcripts in -/-
Figure imgf000065_0001
infarcted tissue to determine that Irf3 mice are appropriate to use as a negative control for ISG expression. Applicant then compared the relative frequency Atty. Dkt. No.: 114198-5210 of Ifit1 and Rsad2 in all our biological replicate samples between WT and Irf3-/- D3 MI spatial transcriptomic samples to determine a threshold or cutoff limit for expression levels of each ISG. Once the threshold was determined, Applicant created a binomial “neighborhood matrix” that consist of ISG+ pixels and neighboring pixels that were also ISG+ above the designated threshold expression value of .70, .75, and 3.0, respectively. This was performed by assigning each pixel with a value of 0-6 corresponding to the number of ISG+ pixels (N) and quantified by taking the intersect of a binary neighborhood matrix (positive ISG neighbors) with binary ISG classification matrix and summing by each column in the generated matrix. K-means nearest neighbors clustering of chosen ISG transcript (ex. Ifit1) was then performed, and this approach yielded 4-8 colonies that were significantly absent in Irf3-/- mice. IFNIC colony area was calculated based on the 55 μm diameter of an individual Visium spot, and the distance between the centers of each adjacent spot measures 100 μm. Activated fibroblasts, IZ, and BZ mapping strategy Applicant mapped BZ or IZ labels to spatial transcriptomic clusters using a list of differentially expressed genes compiled from sn/scRNA-seq data of cardiomyocytes, innate immune cells, and fibroblasts based on previously described methods18. . To classify CM-rich spatial clusters, Applicant evaluated the gene-set scores found uniquely elevated in post-MI samples specific to the borderzone. Mapping of gene-set scores from CM snRNA-seq data to space was performed using area under the receiver operating characteristic (AUROC) analysis. Clusters with an AUROC > 0.7 were positively classified. For IZ clusters, Applicant performed subclustering to determine immune cell niches to map to space. To map activated fibroblasts to space, Applicant performed subclustering of our integrated snRNA-seq dataset to determine subclusters of fibroblasts designated as activated (Postn+) or non-activated (Postn-). Activated Postn+ fibroblasts were used in downstream analysis and average expression of genes within this subset when comparing D3 post-MI snRNA-seq data from WT samples vs. Irf3-/- or Ifnar-/- samples. Zones were quantified and normalized to total UMI and scaled by 10,000. This analysis was performed with all spots from representative day 3 post-MI sample and with IZ pixels (defined by clustering) to further explore heterogeneity at a timepoint predetermined to yield the highest expression of ISGs by snRNA-seq and spatial transcriptomic data. Correlation tests were also performed with gene- set scores to confirm colocalization patterns inferred from clustering analyses. Atty. Dkt. No.: 114198-5210 Applicant then curated subset-specific gene lists using Seurat’s FindMarkers() function (logfc.threshold = .5, min. pct = 0.25, assay = “SCT”) in comparing respective clusters to relevant transcriptional neighbors. Gene lists were filtered to remove genes with adjusted P values > 0.0001 and sorted by log fold change and gene scores were generated from the top 10 genes of each cluster, which were then summed in each spatial assay. Determination of interferon-induced cell (IFNIC) colony localization To assess whether observed IFNIC colonies localized to the infarct borderzone at a rate greater than chance occurrence, the Monte Carlo simulation method was employed to approximate random sampling of our dataset. The coordinates of Visium spots with overlying tissue were extracted in which a random coordinate or location was chosen for each simulation. During a simulation, the randomly chosen coordinate was assigned a neighboring spot (N+1) and assessed for any overlap or adjacency with BZHI pixels. Each simulation was performed 500 times until N+1=12 for each sample. Probabilities generated from the simulations were transformed using Chi square contingency analysis and P-values were assessed using Fisher’s Exact test to compare probability versus outcome i.e., the percentage of clusters that overlapped with any BZHI pixels. Inverse spatial patterning of activated fibroblast and IFNIC signatures Centroids were selected from each IFNIC colony in analyzed samples (n=3 WT males) and primary, secondary, and tertiary neighbors were then assigned. Differential gene expression analysis was performed using Wilcoxon rank sum and each neighbor and centroids of ISG colonies were compared. Line scans were performed by measuring gene scores along a vector drawn from the IZ through the BZ. Applicant previously determined gene scores by spatial clustering of transcripts that characterized the BZ and IZ described in detail above. Here Applicant used an ISG gene score determined from our previously generated datasets and spatially clustered genes. Gene scores were reported as a function of distance from a reference vector line (orthogonal to image analysis line scans). Scores from pixels with similar distances were averaged across 3 biological samples. For IZ and BZ neighbor analysis, Applicant quantified the fraction of BZ and ISG pixels in second-order neighbors (defined by clustering analyses). Results were binned based on the reference pixel classification; IZ contained primarily transcripts designated as innate Atty. Dkt. No.: 114198-5210 immune process or cells whereas BZ contained primarily transcripts designated from BZ myocytes and activated fibroblasts. From this, Applicant quantified both scores as a function of distance (0–400 μm) relative to reference pixels. Analysis of IFNIC colonies using infarcted human tissue Spatial transcriptomic analysis of infarcted human samples was determined using deposited datasets from a recently published study using the same genome-wide Visium platform and from our previously published samples. Patient samples were integrated using SCTransform described above, and metrics of spatial autocorrelation and size of IFNIC colonies were performed using methods described above and in the same manner as murine spatial transcriptomic analysis. Borderzone and ischemic zone genes were transcriptionally determined using snRNA-seq data of “CM2” from the referenced multiomic study (data not shown) RNA MERFISH: Imaging-based spatial transcriptomics To perform RNA multiplexed error-robust fluorescent in situ hybridization (MERFISH) and sequential imaging in the infarcted murine heart, a 33-gene encoding probe library targeting Tnnt2, Ttn, Ankrd1, Nppa, Shroom3, Nppb, Xirp2, Flnc, Col1α1, Col6α3, Postn, Cxcl5, Adgre1, Cd68, Ccr2, Chil3, S100a4, Ly6c2, Timd4, Lyve1, Cxcr2, Csf3r, Ly6g, Retnlg, S100a8, Pecam1, Flt1, Ifna2, Ifnb1, Ifit1, Ifit2, Ifit3, and Cxcl10 was designed and constructed to include cell-type specific marker genes and genes covering the type I IFN signaling pathway. Approximately 20-60 barcoded encoding probes were designed to target specifically 40-nt sub-regions of selected transcripts37,63. Ventricular short-axis tissue sections were cut on a cryostat at 16 μm in thickness onto silanized coverslips and were fixed in 4% PFA at room temperature for 10 minutes=. The samples were then permeabilized with 5% SDS in PBS for minutes followed by 80% ethanol in water for a few hours. Tissue hybridization of encoding probes was performed for approximately 16 hrs in a humidified oven at 47^C. Following overnight hybridization and washing for 30 minutes with 40% formamide in 2xSSC with .1% TWEEN-20, samples were cast in Bis/Acrylamide to crosslink and stabilize acrydite-modified encoding probes, which were acrydite-modified29. Tissue clearing with ProteinaseK digestion or photobleaching of samples were performed to quench background autofluorescence in tissue samples. Sequential hybridization and stripping of Atty. Dkt. No.: 114198-5210 fluorescently labeled readout probes and subsequent was performed by an automated, custom built fluidics system. Imaging was performed on a custom-built system with 60x objective lens as previously described37. After data collection, raw images underwent fitting analysis and image registration to correct for drift that occurred during image acquisition. Single mRNA molecules were computationally decoded, and the total transcript signal and DAPI nuclear stain were used to perform cell segmentation with machine learning algorithm Cellpose64. Quality control, normalization, and integration for RNA MERFISH Data analysis of RNA MERFISH data was performed with single-cell sequencing analysis tools like Scanpy and Squidpy65. Quality control metrics were determined by visualizing distribution plots of the raw data for total transcript counts and number of genes with more than one counts per cell. These metrics were used to determine covariates that may affect the quality of the dataset and filtered the data accordingly. QC metrics are displayed in FIGS. 14B – 14F). The data is then normalized using Scanpy and log normalization of the total number of transcripts. Applicant performed RNA MERFISH experiments with multiple cardiac tissue sections and experimental conditions to control for batch effects. Data integration across sections from different animals was performed using Scanpy’s principal component-based method ingest. Applicant used the WT D3 MI mouse cardiac section as our reference dataset and performed Leiden clustering with subsets of marker genes from our encoding probe gene panel as follows: borderzone cardiomyocytes [Nppa, Flnc, Ankrd1], fibroblasts [Col1α1, Col6α3], macrophages [Cd68, Adgre1], neutrophils [Cxcr2, Csf3r], and endothelial cells [Pecam1, Flt1]. Cells from infarcted Irf3-/- mice were ingested or embedded into the UMAP space of the annotated cells in WT mice Applicant used as reference. DNA MERFISH: Genome-scale in situ chromatin imaging DNA MERFISH was performed as previously described37. Briefly, the mouse genome was partitioned into 260 distinct DNA loci that spanned all 21 murine chromosomes. Each genomic loci spanned ~30-kb and was targeted by 200-300 specific DNA encoding probes. These primary probes were synthesized from an oligonucleotide pool from Twist Biosciences and each contained a 40-nt target sequence specific to a region of DNA loci. Sample preparation was performed as previously described and hybridization of probes contained Atty. Dkt. No.: 114198-5210 pools of both acrydite-modified RNA and DNA encoding libraries to facilitate downstream analysis of cell identity and DNA localization. Fluorescently labeled readout probes were combinatorially hybridized and stripped using a custom-built fluidics and microscopy system (see the RNA-MERFISH section above). After data collection, raw images underwent fitting analysis and image registration to correct for drift. DNA loci were computationally decoded using signal intensity between FOV’s in multiple fluorescent channels to filter noise from signal. Nuclear-localized DNA probes were determined using neighborhood-based clustering with minimally 10-decoded spots per 5 μm radius. Sparse, decoded DNA probes that met the criteria for positive signal but were outliers to the neighborhood distance were designated as extranuclear DNA. Nuclear shearing artifacts from cryosectioning were controlled and excluded from analysis by removing data in the first and last 5 μm regions of tissue sections. Cell segmentation was performed with Cellpose on DAPI staining and RNA molecules. Cells with extranuclear DNA underwent quality control analysis as described above, normalized, and ingested with cells from our WT D3 MI cells from the RNA MERFISH experiment. Density-based clustering of applications with noise (DBSCAN) analysis Analysis of RNA MERFISH IFNIC colonies was performed using the density-based clustering algorithm (DBSCAN). This method considers whether a set of features are densely grouped or homogenously distributed with a certain radius ε. Epsilon was unbiasedly selected using k-nearest neighbor analysis. This algorithm identified 8 groups of densely clustered ISG transcripts (Ifit1, Ifit2, Ifit3, Cxcl10) as ISG expression in low density regions that were designated as outliers or “scattered” ISG expression. Nuclear Morphology In cardiac sections that had undergone RNA MERFISH, cardiomyocyte nuclei were identified by the colocalization of hybridized Tnnt2 probes and DAPI fluorescence present in both the borderzone and remote zone. Approximately 200 nuclei were quantified in each region by performing nuclear segmentation and creating a binary segmentation mask. These masks were analyzed by Fiji ImageJ Particle Analysis to measure cardiomyocyte-specific nuclear solidity. The solidity of a nucleus is calculated as the area of the DAPI-stained nuclei divided by the area of the convex hull; large deviations below solidity ratio of 1.0 indicate irregularity of the nuclear contour36. Atty. Dkt. No.: 114198-5210 Single cell isolation, flow cytometry, and cell sorting Whole cell suspensions were isolated from freshly harvested hearts as previously described. Briefly, hearts were enzymatically digested for 45 minutes in continuous agitation at 37ºC in 450 U/mL collagenase, 125 U/mL collagenase XI, 60 U/mL DNAse, and 60 U/mL hyaluronidase. Cell suspensions were then filtered through 40 μm nylon mesh cell strainer containing flow cytometry staining buffer (FACS) and stained at 4ºC with DAPI to exclude permeabilized cells and anti-mouse cocktail directed against major hematopoietic lineage markers Terr119 (BioLegend, clone TER119), CDB220 (BioLegend, clone RA3-6B), CD49b (BioLegend, clone DX5), and CD90.2 (BioLegend clone 53-2.1). Secondary staining of myeloid and stromal cell subsets were performed using anti-mouse antibody cocktail against CD11b (BioLegend, clone M1/70), and CD 45.2 (BioLegend, clone 104), Ly6G (BioLegend, clone A1A8), and F4/80 (BioLegend, clone BM8). Primary and secondary master mixes were suspended in FACS. Flow cytometry was performed on SONY MA900 multi-application cell sorter. Single nuclei isolation Single nuclei suspensions were isolated from frozen ventricular tissue as previously described. Briefly, murine ventricles were harvested, weighed, and minced before flash- freezing by immersion in liquid nitrogen. For isolation of nuclei, minced ventricles were suspended in .6 mL nucleus lysis buffer supplemented with .2 U/μL RNase inhibitor (Sigma product no. NUC101, Enzymatics part no. Y9240L). Minced tissue was further homogenized with 2 mL dounce grinder for approximately 10 strokes with A-sized pestle and 20 strokes with B-sized pestle (Sigma catalogue no. D8938). The lysates were treated with an additional 1 mL of lysis buffer and incubated for 2 additional minutes; lysates were filtered through consecutive 100, 50, and 20-μm strainers (CellTrics 04-004-2318, 04-004-2317 and 04-0042- 2315). Nuclei were pelleted by centrifugation at 1,000g for 5 minutes at 4oC. Subsequent washes were performed until final suspension of nuclei was made using 2% BSA in PBS supplemented with RNase inhibitor. Nuclei suspension was treated with 10 μg ml−14′,6- diamidino-2-phenylindole (DAPI), counted on hemocytometer, and volume adjusted to produce a final suspension of 1000 nuclei/μL. Atty. Dkt. No.: 114198-5210 Single cell and single nuclei RNA sequencing (sn/scRNA-seq) Microfluidic droplet-based separation of single cells or individual nuclei were performed in which each were encapsulated with reagents for reverse transcription of mRNA, barcodes, and unique molecular identifiers (UMIs) (10X Genomics Chromium). Paired-end sequencing was performed using Illumina dye sequencing performed on NovaSeq6000 instrument. Demultiplexing of pooled samples and low-level analysis were performed using the Cell Ranger 6.1.1 pipeline from 10X Genomics in which the sequenced samples were mapped to a murine reference transcriptome (refdata-gex-mm10-2020-A, which includes introns), and redundant UMIs were eliminated. Quality control, normalization, and integration for sn/scRNA-seq Normalization was performed to account for variability in depth of sequencing reads per cell or nuclei as previously described. The total transcript count for each nucleus was scaled to 10,000 molecules, and raw counts for each gene were normalized to the total count of captured transcripts associated with the barcoded cells or nucleus and natural log transformed. Nuclei that contain at least 200 uniquely expressed genes represented in at least 3 nuclei were retained for further analysis. Ribosomal and hemoglobin transcripts were excluded to avoid incorporation of transcriptional artifacts or technical variables accrued during nuclei isolation. Additionally, quality control was performed to assess the quantity of low-quality/dying cells by assessing the percentage of mitochondrial transcripts present using the PercentageFeatureSet function and excluded nuclei containing more than 5% mitochondrial content. Highly variable genes across individual datasets were identified with FindVariableFeatures function using Seurat R package v.4.9, which performs variance- stabilizing transformation with subsequent selection of 4,000 genes with the highest feature variance. Doublets and aggregated nuclei were determined by assessing non-endogenous gene markers (for example, presence of CM genes such as Myh6 in the fibroblast subset) and ambient RNA were removed using SoupX, which displayed subsets after filtering and removing doublets/multiples. Integration of multiple scRNA-seq and snRNA-seq datasets was performed in Seurat using canonical correlation analysis (CCA) to identify anchors between datasets and anchored using mutual nearest neighbor method using Seurat FindIntegrationAnchors function. Atty. Dkt. No.: 114198-5210 Statistics Statistical analysis was performed using Prism 9 (GraphPad), and all data are represented as mean values ± s.e.m. unless otherwise indicated. Unpaired Mann–Whitney U- tests, Wilcoxon rank-sum tests, Pearson correlation, one-way analysis of variance (ANOVA) with post-hoc analysis using Dunnett’s multiple comparisons tests, or two-way ANOVA with Tukey’s post-hoc analysis used to determine statistically significant data between experimental conditions where P < 0.05 was considered significant. P values are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. To account for multiple hypotheses testing in data generated via Seurat differential gene expression analysis, Seurat- derived P values were rate-adjusted to determine false discovery results using Benjamini- Hochberg or Fisher’s Z. Conclusion In conclusion, Applicant shows herein that MI causes ischemic cell death in the IZ that leads to mechanical stress in the BZ resulting in nuclear rupture and loss of compartmentalization of genomic DNA primarily in BZ cardiomyocytes and secondarily in BZ fibroblasts. This enables activation of the cGAS-STING-IRF3 DNA sensing pathway, and production of secreted IFNs, which diffusively spread to IFNAR-expressing neighbor cells that respond by expressing ISGs and forming the observed IFNIC colonies. Within the IFNIC niche, IFN-exposed fibroblasts exhibit impaired activation, contractile function, and expression of protective matricellular proteins, which when localized at sites of high mechanical stress (e.g., the junction of the ventricular free wall and septum), increases vulnerability to pathologic remodeling and catastrophic rupture (FIG. 5K). The newly described BZ IFNIC colonies represent a novel IFN response of intracardiac origin initiated by non-immune cells that is distinct from previously described extra cardiac sources (FIG. 21). As partially-resolved, multi-omics technologies are poised to reveal new microenvironment niches in healthy and injured tissues that shape biological form and function. In this work, Applicant uncover a new pathologic niche within the borderzone of the infarcted heart that has global effects on pathologic remodeling and survival. Applicant shows in one aspect that selectively inhibiting the cGAS-STING-IRF3 pathway in non- Atty. Dkt. No.: 114198-5210 immune BZ cardiomyocytes and fibroblasts provides therapeutic benefit while avoiding broader immunosuppression associated with inhibiting the type I IFN response across all innate immune cells61,62. Equivalents It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All nucleotide sequences provided herein are presented in the 5′ to 3′ direction. The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or Atty. Dkt. No.: 114198-5210 negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
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Claims

Atty. Dkt. No.: 114198-5210 WHAT IS CLAIMED IS: 1. A method to inhibit the type I interferon (IFN) response systemically or locally in a subject that has experienced, is experiencing, or is anticipated to experience endogenous or exogenous mechanical cell or tissue injury, the method comprising systemically or locally administering to the subject an effective amount of an inhibitory IFN agent, thereby inhibiting the type I IFN response in the subject. 2. A method to treat or prevent a disease or condition caused by an endogenous or exogenous mechanical cell or tissue injury in a subject in need thereof, the method comprising systemically or locally administering to the subject an effective amount of an inhibitory IFN agent, thereby treating or preventing a disease or condition caused by an endogenous or exogenous mechanical tissue injury in the subject. 3. The method of claim 1 or 2, wherein the cell or tissue is selected from are mechanically loaded immune or non-immune tissue or cell. 4. The method of claim 3, wherein the mechanically loaded non-immune cell is selected from a cardiomyocyte, a skeletal muscle cell, a smooth muscle cells, a fibroblast, a vascular cell, an epithelial cell, or an endothelial cell. 5. The method of any of claims 2-4, wherein the disease or condition is selected from mechanical stretch, hypercontraction, inflammatory heart disease, myocarditis, myopericarditis, sarcoid, inflammatory cardiomyopathies, acute myocardial ischemia/infarction or chronic ischemic heart disease, localized cardiac injury, vascular stretch or mechanical deformation due to systemic arterial hypertension, pressure overload due to chronic hypertension, and valvular heart disease, atrial stretch and atrial fibrillation, aortic or arterial aneurysms, pulmonary arterial hypertension, pulmonary venous hypertension due to chronically elevated filling pressures, atherosclerosis, skeletal muscle injury, suture sites, cannula insertion sites, biopsy sites, or therapeutic injection sites. 6. A method to inhibit the type I IFN response or to treat or prevent mechanical tissue injury systemically or locally in a subject who will receive or has received an exogenous mechanical tissue injury, comprising administering to the subject an effective amount of an inhibitory IFN agent prior to or subsequent to receiving the exogenous mechanical tissue Atty. Dkt. No.: 114198-5210 injury, thereby inhibiting the type I IFN response or treating or preventing mechanical tissue injury. 7. A method to inhibit the type I IFN response systemically or locally in a subject prior to, concurrently or subsequent to local delivery of a therapy comprising administering to the subject an effective amount of an inhibitory IFN agent prior to or concurrently with the local delivery of the therapy, thereby inhibiting the type I IFN response. 8. The method of claim 7, wherein the therapy comprises administration of one or more of: biologics, small molecules, nucleic acids; viral-based therapies, biomaterials; therapeutic cells, engineered cells, stem cells; a device, or a deep brain stimulator. 9. The method of any of claims 1-8, wherein the type I IFN inhibitory agent is coated on or incorporated into the device causing the mechanical tissue injury. 10. The method of claim 9, wherein the mechanical injury device is selected from a needle, a suture, a guidewire, a cannula, a catheter, an electrode, a bioptome, a stent, or a staple. 11. The method of any one of claims 1-10, further comprising detecting one or more of the quantity, location, distribution, and amount of type I IFN in the subject. 12. The method of any one of claims 1-11, wherein the subject is a mammal. 13. The method of claim 12, wherein the mammal is a human patient. 14. The method of any of claims 1-8 or 11-13, wherein administration is by a method comprising intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation. 15. A method for one or more of: reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack, each method comprising, or consisting essentially of, or yet further consisting of, by administering a cardiomyocyte cGAS-STING-IRF3 inhibitor thereby reducing or treating cardiac injury, promoting or supporting cardiac regeneration, reducing or inhibiting the Atty. Dkt. No.: 114198-5210 progression to heart failure, or treating a subject that presents with chest pain (angina), due to ischemia and a heart attack. 16. The method of claim 15, wherein the inhibitor is a small molecule or a gene therapy which inhibits the activity of Irf3. 17. The method of claim 15 or 16, wherein the administration is in vitro or in vivo. 18. The method of claim 15 or 16, wherein the subject is a mammal. 19. The method of claim 18, wherein the mammal is a human patient. 20. The method of any of claims 15-19, wherein administration is by a method comprising intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.
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