WO2024118381A1

WO2024118381A1 - Methods and composition to target microglia and macrophages

Info

Publication number: WO2024118381A1
Application number: PCT/US2023/080574
Authority: WO
Inventors: Chun-li ZHANG; Carolina Ivonne SERRANO GARCIA; Sergio Giuseppe CANANZI
Original assignee: University of Texas System; University of Texas at Austin
Current assignee: University of Texas System; University of Texas at Austin
Priority date: 2022-11-28
Filing date: 2023-11-20
Publication date: 2024-06-06
Anticipated expiration: 2025-05-28

Abstract

Provided here are compositions and methods for targeting microglial and/or macrophage cells using promoter sequences derived from IBA1. Both therapeutic and non-therapeutic applications are disclosed wherein a suitable heterologous gene can be expressed with high efficiency and specificity in the microglial and/or macrophage cells.

Description

TITLE

METHODS AND COMPOSITION TO TARGET MICROGLIA AND MACROPHAGES

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/385,136 filed November 28, 2022 and titled “METHODS AND COMPOSITION TO TARGET MICROGLIA AND MACROPHAGES,” the content of which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] The present application contains a Sequence Listing that which has been submitted in .XML format via Patent Center and is hereby incorporated by reference in its entirety. The said WIPO Sequence Listing was created on November 6, 2023, XML copy is named 106546-779181 PCT 3989. xml, and is 19 kilobytes in size.

BACKGROUND

[0003] 1. Field

[0004] The present invention relates to compositions and methods facilitating targeted gene expression in microglial and macrophage cells and applications thereof.

[0005] 2. Background

[0006] Microglia/macrophages are immune cells, with the former residing in the nervous system and the latter being in the peripheral tissues/organs. Microglia account for about 15% of all cells in the brain. Like peripheral macrophages, microglia are the primary immune cells of the central nervous system. Microglia not only act as scavengers for pathogens and damaged cells but also secrete cytokines, chemokines, prostaglandins, and reactive oxygen species, which help to regulate and direct the immune response. Microglial dysregulation has been implicated in several neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Multiple sclerosis, as well as cardiac diseases, glaucoma, and viral and bacterial infections. Additionally, macrophage dysregulation may contribute to the pathophysiology of obsessive-compulsive disorder (OCD), and Tourette syndrome.

[0007] Like microglia, peripheral macrophage are key players of the innate immune system that engulf pathogens and damaged cells. Additionally, they secrete key cytokines that help regulate the immune system. Macrophage dysregulation has also been implicated in the number of disease conditions including atherosclerosis, myocardia infarction, infections, cancer, obesity, and fibrosis.

[0008] Despite their extreme relevance, gene therapy tools that specifically target microphage and microglial cells are not available or are suboptimal. This is particularly true in the case of viral vector-based gene therapy tools. There is therefore a need in the field for targeted gene therapy tools for these cells.

SUMMARY OF THE INVENTION

[0009] In some aspects, the current disclosure encompasses a recombinant polynucleotide sequence comprising: a heterologous gene; and a promoter operably linked to the heterologous gene, wherein the promoter has a nucleic acid sequence at least 70% identical to one or more of SEQ ID. NOS: 1-5. In some aspects of the recombinant polynucleotide sequence provided herein, the nucleic acid sequence of the promoter is less than 1 kb in length. In some aspects, the nucleic acid sequence of the promoter is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least a 100% identical to one or more of SEQ ID NOS: 1-5. In some aspects, the promoter is capable of inducing expression of the heterologous gene in a microglial cell. In some aspects, the promoter is capable of inducing expression of the heterologous gene in a macrophage.

[0010] In some aspects, the recombinant polynucleotide further comprises one or more additional transcriptional and/or post-transcriptional regulatory elements. In some aspects, the heterologous gene encodes a functional protein or an RNA. In some aspects, non-limiting examples of the heterologous gene could be a cytotoxic gene, a reporter gene, a recombinase, a nuclease, a cytokine, a chemokine, a transcriptional regulator, a translational regulator, a transporter, a regulator of endocytosis or exocytosis or a therapeutic gene.

[0011] In some aspects, the current disclosure also encompasses a viral vector comprising a polynucleotide sequence comprising: a heterologous gene; and a promoter operably linked to the heterologous gene, wherein the promoter has a nucleic acid sequence at least 70% identical to any one of SEQ ID NOS: 1-5. Non-limiting examples of viral vectors include adenovirus, adeno- associated virus, a retrograde virus, retrovirus, herpesvirus, lentivirus, poxvirus, or papilloma virus expression vector. In some aspects, the viral vector is adapted to induce expression of the heterologous gene in microglia and/or macrophages.

[0012] In some aspects, the current disclosure encompasses a therapeutic composition comprising the viral vectors disclosed herein. In some aspects, the current disclosure also encompasses therapeutic composition comprising the recombinant polynucleotide sequence disclosed herein. In some aspects, the therapeutic compositions may comprise a pharmaceutically acceptable buffer, diluent, or excipient. In some aspects, the therapeutic compositions disclosed herein may be used in treatment of diseases, non-limiting examples of which include treatment of Alzheimer's disease, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis, autism, brain lupus (NP-SLE), chemo-induced peripheral neuropathy (CIPN), postherapeutic neuralgia, chronic inflammatory demyelinating polyneuropathy (Cl DP), epilepsy, Guillain-Barre Syndrom (GBS), inclusion body myositis, lysosomal storage diseases, sphingomyelinlipidose (Niemann-Pick C), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), mucopolysaccharidose ll/IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, Myasthenia Gravis, NeuroBehcet's Disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett's Syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, bacterial meningitis, cancer, autoimmune disease, macrophage activation syndrome, atherosclerosis, liver fibrosis, liver cirrhosis, diabetes mellitus, Kawasaki disease, asthma, hemophagocytic lymphohistiocytosis, sarcoidosis, periodontitis, Whipple's disease, pulmonary alveolar proteinosis, autoimmune diseases, macrophage related pulmonary disease, Leishmaniasis, obesity complications, hemodialysis related inflammation, microbial infection, viral infection, inflammation, and complications thereof. In some exemplary aspects the therapeutic composition is used for treatment of liver fibrosis.

[0013] In some aspects, the current disclosure also encompasses a method of inducing expression in microglia and/or macrophage comprising contacting the microglia and/or macrophage cell with a polynucleotide sequence comprising: a heterologous gene; and a promoter operably linked to the heterologous gene, wherein the promoter has a nucleic acid sequence at least 70% identical to any one or more of SEQ ID. NOS: 1-5. In some aspects, the nucleic acid sequence of the promoter is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least a 100% identical to any one or more of SEQ ID. NOS: 1-5. In some aspects, the method of the current disclosure may also encompass incorporating the polynucleotide sequence into a viral vector and contacting said viral vector with a microglia and/or macrophage cell. Non-limiting examples of viral vector include adenovirus, adeno- associated virus, a retrograde virus, retrovirus, herpesvirus, lentivirus, poxvirus or papiloma virus expression vector.

[0014] In some aspects, the current disclosure encompasses a method of inducing expression in microglia and/or macrophage in a subject in need thereof, comprising administrating into the subject, a composition comprising a recombinant polynucleotide sequence comprising a heterologous gene and a promoter operably linked to a heterologous gene, wherein the promoter has a nucleic acid sequence of less than 1 kb and has a nucleic acid sequence at least 70% identical to one or more of SEQ ID NOS: 1-5. In some aspects, the method comprises contacting a liver macrophage cell with a polynucleotide sequence comprising: a heterologous gene; and a promoter operably linked to the heterologous gene, wherein the promoter has a nucleic acid sequence at least 70% identical to any one of SEQ ID. NOS 1-5. In some aspects, the heterologous gene is any one of trem2, or nfe2L2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Aspects of the present inventive concept are illustrated by way of example in which like reference numerals indicate similar elements and in which:

[0016] FIG 1A provides confocal immunofluorescence images showing results from an in vivo screen for microglia-targeting promoters using lentivirus vectors in a mouse with cerebral ischemia. Eight different promoters were tested (CD68, F4/80, IBA1 , CX3CR1 , C1Qa, C1Qb, TMEM119, and SP). Lentivirus injection was performed in the striatum of adult mice. Brains were analyzed one week post virus injection by immunofluorescence. Microglia targeting efficiency was analyzed by GFP expression in IBA1+ cells. Brain sections were stained with Hoechst DNA staining to indicate cells and IBA1 expression was used as a marker for microglial cells.

[0017] FIG. 1B provides quantification from the promoter screen of overlap between GFP expression and 1 BA1 (used as a marker for microglial cells) as indicated by the ratio of % overlapping cells (GFP+IBA1)/ total GFP labelled cells. Quantification of the number of GFP⁺ and IBA1⁺ cells shows that IBA1 promotor is specific to microglial cells.

[0018] FIG. 2A provides confocal immunofluorescence images showing that pseudotyped lentiviral vectors packaged in either LCMV (Lymphocytic Choriomeningitis virus) or VSV-G vesicular stomatitis virus GP) envelopes and comprising a full length IBA1 promoter linked to GFP reporter can effectively target microglial cells. Two different envelope plasmids were used for lentivirus packaging. VSV-G and LCMV were used to express GFP driven by IBA1 promoter in striatal microglia of adult mice after brain ischemia by MCAO. IBA1 expression was used as a marker for microglial cells.

[0019] FIG. 2B provides quantification of overlap between GFP expression and IBA1 (used as a marker for microglial cells) as indicated by the ratio of % overlapping cells (GFP+IBA1)/ total GFP labelled cells for LCMV and VSV-G lentiviruses. Quantification of GFP and IBA1+ cells showed no difference in GFP expression in microglia between the two envelopes.

[0020] FIG. 3A provides confocal immunofluorescence images of mouse brain striatal sections showing effective microglial targeting of GFP expression using full length IBA1 promoter sequence in scAAV (self-complementary adeno-associated virus) packaged with different AAV serotypes. Eight AAV serotypes were used (AAV1, AAV2, AAV5, AAV6, AAV6M, AAV8, AAV9, AAVPHP.eB) to induce GFP expression in microglia. scAAV vectors were injected into the striatum of mice with local ischemic injury by L-NIO.

[0021] FIG. 3B provides quantification of targeting specificity of scAAVs with different serotypes. The bars represent the % ratio of overlap between GFP and IBA1 (used as a marker for microglial cells)/ total GFP labelled cells. Analysis of GFP expression in microglia show high expression of GFP in microglia in the site of injection for all the serotypes, but with varying degrees of specificity.

[0022] FIG. 3C provides quantification of targeting efficiency of scAAVs with different serotypes. The bars represent the % ratio of overlap between GFP and IBA1 (used as a marker for microglial cells)/ total IBA1 labelled cells. scAAV5 and scAAV8 exhibited the highest efficiency of targeting.

[0023] FIG. 4A provides confocal immunofluorescence images of mouse brain striatal and cortical sections showing long-term microglial targeted GFP expression using full length IBA1 promoter sequence when using scAAV5 or scAAV8. Virus was injected into the striatum of adult mice with local ischemia induced by L-NIO injection. Two different titers of scAAV5 and scAAV8 were used for the experiment. Different areas around the site of virus injection were analyzed.

[0024] FIG. 4B provides quantification of targeting efficiency of scAAV5 and scAAV8 as seen from brain sections 4-week post injection in FIG. 4A (upper panels: merged image channel from the boxed regions; lower panels: separate and merged image channels from the injection sites). The bars represent the % ratio of overlap between GFP and IBA1 (used as a marker for microglial cells)/ total GFP labelled cells. Quantification of GFP+ and IBA1+ cells showed similar long-term specificity (upper bar graph) and efficiency (lower bar graph) between serotypes.

[0025] FIG. 5A provides a schematic representation of IBA1 promoter lengths used to determine the minimal effective promoter region. Shorter promoters derived from original IBA1 were tested in control and ischemic injured mice. AAV5 was injected into mouse striatum to check GFP expression in microglia after one week post virus injection. [0026] FIG. 5B provides confocal immunofluorescence images of mouse brain striatal sections showing microglial targeting of GFP expression using indicated promoter sequences in control and mouse model for stroke (MCAO).

[0027] FIG. 5C provides quantification of targeting efficiency of indicated promoters in control (white bars) and stroke models (black bars) using IBA1a promoter. The bars represent the % ratio of overlap between GFP and IBA1 (used as a marker for microglial cells)/ total GFP labelled cells. scAAV5 and scAAV8 exhibited the highest efficiency of targeting. Quantification of GFP⁺ and IBA1⁺ cells show that microglia-targeting efficiency and specificity of the promoters decreased proportionally to the length of the promoter. IBA1a (460 size) is the minimal promoter with efficient microglia-targeting.

[0028] FIG. 5D provides quantification of targeting specificity of indicated promoters in control (white bars) and stroke models (black bars) using IBA1a mini-promoter. The bars represent the % ratio of overlap between GFP and IBA1 (used as a marker for microglial cells)/total IBA1 labelled cells.

[0029] FIG. 6A shows a schematic of a new AAV vector designed to reduce GFP expression in neurons using the minimal IBA1 a promoter, by inclusion of a targeting sequences of the neuron- expressed miR124.

[0030] FIG. 6B shows confocal immunofluorescence images of mouse brain injected with the new vector. AAV5 packaged virus was injected into the striatum of adult mice. GFP-expressing cells were analyzed four weeks post virus injection.

[0031] FIG. 6C shows quantification of GFP+ and IBA1+ cells showed that this new vector exhibited superior specificity to microglia and no expression in neurons.

[0032] FIG 7A provides confocal immunofluorescence images of liver sections showing targeting of GFP to liver macrophages. scAAV5, scAAV8 and scAAVPHP.eB were administered in adult wild type mice by retro-orbital injection. One week after virus injection GFP expression in liver was analyzed by immunofluorescence.

[0033] FIG. 7B provides quantification of targeting efficiency of AAV serotypes to liver macrophages using the IBA1a mini-promoter. The bars represent the % ratio of overlap between GFP and IBA1 (used as a marker for macrophage cells)/ total GFP labelled cells. scAAV8 exhibited the highest efficiency of targeting.

[0034] FIG. 7C provides quantification of targeting specificity of AAV serotypes to liver macrophages using the IBA1a mini-promoter. The bars represent the % ratio of overlap between GFP and IBA1 (used as a marker for microglial cells)/ total IBA1 labelled cells.

[0035] The drawing figures do not limit the present inventive concept to the specific aspects disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain aspects of the present inventive concept.

DETAILED DESCRIPTION

[0036] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0037] In an aspect, the current disclosure overcomes limitations in the field of gene therapy by providing methods and compositions that may be used to specifically induce expression in microglial and macrophage cell populations of a mammal. The compositions and methods provided herein may be used, for example, in cell cultures, in the generation of genetically modified animals for research, or they may be used as a therapeutic to drive expression in microglial and/or macrophage cell populations in a mammalian subject, such as a human and non-human animal. In some aspects, minimal promoters are provided that can be incorporated into vectors and used to drive expression of heterologous genes in microglia and/or macrophages. In some aspects, both clinical and non-clinical use of the disclosed promoters is envisaged.

[0038] The disclosure is a result of extensive screening and testing of promoter sequences to identify minimal promoters for targeting microglial and/or macrophage cells. In some exemplary aspects, the identified promoters were incorporated into viral vectors and tested for their ability to drive heterologous gene expression in various mice disease models including stroke. It was not expected that minimal promoter sequences would be able to achieve cell specific gene expression in the microglial cells. The high specificity and efficiency of targeting that can be achieved using these minimal promoters make them excellent candidates for myriad of therapeutic and non- therapeutic applications.

I. Terminology [0039] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.

[0040] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0041] The terms “comprising,” “including” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described.

[0042] Lastly, the terms “or” and “and/or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and/or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0043] The terms "nucleic acid”, "nucleic acid molecule”, and "polynucleotide” are used interchangeably herein. The terms “nucleic acid encoding . . .”, or “nucleic acid molecule encoding . . . “ should be understood as referring to the sequence of nucleotides which encodes a polypeptide.

[0044] A polynucleotide described herein may comprise one or more nucleic acids each encoding a polypeptide, operably linked to a promoter (i.e. , in a functional relationship with) and one or more regulatory sequences. Such a polynucleotide may alternatively be referred to herein as a "nucleic acid construct” or "construct”. As used herein, the term “operably linked” refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene), such that the promoter, etc., operates to initiate, assist, affect, cause, and/or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in certain tissue(s), developmental stage(s) and/or condition(s).

[0045] The term “recombinant” as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.

[0046] As used herein, “regulatory elements” refer to any sequence elements that regulate, positively or negatively, the expression of an operably linked sequence. “Regulatory elements” include, without being limiting, a promoter, an enhancer, a leader, a transcription start site (TSS), a linker, 5' and 3' untranslated regions (UTRs), an intron, a polyadenylation signal, and a termination region or sequence, etc., that are suitable, necessary, or preferred for regulating or allowing expression of the gene or transcribable DNA sequence in a cell. Such additional regulatory element(s) can be optional and used to enhance or optimize expression of the gene or transcribable DNA sequence. A regulatory sequence can, for example, be inducible, noninducible, constitutive, cell-cycle regulated, metabolically regulated, and the like. A regulatory sequence may be a promoter. As used herein, the term “promoter” refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and/or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). A promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter sequence or other promoter sequence. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. A promoter of the present application can thus include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. In some exemplary aspects the promoter sequence is adapted to enable expression of a polynucleotide in microglial and/or macrophage cells.

[0047] As used herein, the term “operably linked” refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene), such that the promoter, etc., operates to initiate, assist, affect, cause, and/or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in certain tissue(s), developmental stage(s) and/or condition(s).

[0048] The term “microglial cell” or “microglia”, as used herein, refers to a class of glial cells involved in the mediation of an immune response within the central nervous system by acting as macrophages. Microglial cells are capable of producing exosomes, cytokines, chemokines, and neurotrophic factors, and further include different forms of microglial cells, including amoeboid microglial cells, ramified microglial cells and reactive microglial cells. Microglial cells include reactive microglia, which are defined as quiescent ramified microglia that transform into a reactive, macrophage-like state and accumulate at sites of brain injury and inflammation to assist in tissue repair and neural regeneration. It is known in the art that hematopoietic stem cells can migrate to the brain and differentiate into macrophages having many characteristics of microglia. Since the promoters of the invention have been demonstrated to be active in macrophages and microglia, it is at least plausible that these promoters will also be active in hematopoietic stem cell (HSC)- derived microglia-like cells.

[0049] The term “heterologous” when used in reference to a nucleic acid molecule (such as a coding sequence) or a polypeptide (such as an enzyme) refers to a nucleic acid molecule or a protein that is not natively found in the host organism or cell. “Heterologous” also includes a native coding region, or portion thereof, that is removed from the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism's genome. The heterologous nucleic acid molecule is deliberately introduced into the host cell. A “heterologous” nucleic acid molecule or protein may be derived from any source, e.g., eukaryotes, prokaryotes, viruses, etc. In an aspect, the heterologous nucleic acid molecule may be derived from a eukaryote (such as, for example, another yeast) or a prokaryote (such as, for example, a bacteria). The term “heterologous” as used herein also refers to an element (nucleic acid or protein) that is derived from a source other than the endogenous source. Thus, for example, a heterologous element could be derived from a different strain of host cell, or from an organism of a different taxonomic group (e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications). The term “heterologous” is also used synonymously herein with the term “exogenous”.

[0050] The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.

[0051] As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain aspects, the patient or subject is a primate. Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.

[0052] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[0053] “Prevention” or “preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0054] “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[0055] A “therapeutic polypeptide” is a polypeptide that may alleviate or reduce symptoms that result from an absence or defect in a protein in a cell or subject. Alternatively, a “therapeutic polypeptide” is one that otherwise confers a benefit to a subject, e.g., anti-cancer effects or improvement in transplant survivability. As used herein, the term “therapeutic polypeptide” also encompasses proteins useful as vaccines, therapeutics, and diagnostics.

II. Compositions

[0056] The current disclosure encompasses various compositions which may comprise, in the alternative or in any combination: recombinant polynucleotides; vectors; cells (or populations of cells) comprising the polynucleotides and/or vectors; cells expressing the polypeptides; and therapeutic compositions comprising any one or more polynucleotides, vectors, and/or cells as described herein, for use in microglial and/or macrophage targeting for laboratory, commercial or therapeutic purpose.

Recombinant Polynucleotides

[0057] In some aspects, the current disclosure encompasses recombinant polynucleotide sequences comprising a promoter sequence capable of inducing expression of a heterologous gene to which it is operably linked in microglial and/or peripheral macrophages. In some aspects, the current disclosure encompasses a recombinant polynucleotide sequence comprising: a heterologous gene; and a promoter operably linked to the heterologous gene, wherein the promoter is an IBA1 promoter or a variant or fragment thereof. In some aspects of the current disclosure, the promoter is a fragment (or variant thereof) of the wild-type I BA1 promoter, wherein the nucleic acid sequence of the promoter is less than 1kb in length. In some aspects, the nucleic acid sequence of the promoter is less than 750 bp in length. In some aspects, the promoter is less than about 950 bp, or 900 bp, or 850 bp, or 800 bp, or 750 bp, or 700 bp, or 650 bp, or 600 bp, 550 bp, 500 bp, or 450 bp, or 400 bp, or 350 bp, or 300 bp, or 250 bp, or 200 bp, or 150 bp, or 100 bp. In some aspects, the promoter is a minimal promoter derived from IBA1.

[0058] In some exemplary aspects, the promoter comprises a polynucleotide sequence with a nucleic sequence at least about 70% identical to any one of SEQ ID NOS: 1-5 as provided in Table 1 or variants, derivative or fragments thereof. In some aspect, the identity or similarity is of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some aspects, the promoter comprises a polynucleotide sequence at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a 950 bp, or 900 bp, or 850 bp, or 800 bp, or 750 bp, or 700 bp, or 650 bp, or 600 bp, 550 bp, 500 bp, or 450 bp, or 400 bp, or 350 bp, or 300 bp, or 250 bp, 200 bp, 150 bp, or 100 bp fragment of the wild type IBA1 promoter as provided in SEQ ID NO. 1.

Table 1 : Exemplary promoter sequences

[0059] In some aspects, the promoter is capable of inducing expression of a heterologous gene to which it is operably linked, to the microglia and/or peripheral macrophages with high specificity and efficiency. In some aspects, the promoter exhibits a specificity of at least about 60% to about 65%, or about 65% to about 70%, or about 70% to about 75%, or about 75% to about 80%, or about 80% to about 90%, or about 90% to about 95%, or about 95% to about 100% for microglial expression. In some aspects, the promoter exhibits high efficiency of gene expression of a heterologous gene to which it is operably linked, in the microglial cells comprising the recombinant polynucleotide sequence. In some aspects, the promoter exhibits at least over 50% efficiency of heterologous gene expression to which it is operably linked, in the microglial cells comprising the recombinant polynucleotide sequence.

[0060] In some aspects, the current disclosure also encompasses recombinant polynucleotides comprising a promoter sequence as disclosed herein, operably linked to a heterologous gene or cDNA. In some aspects, the heterologous gene or cDNA can be any gene of interest. In some aspects, the heterologous gene may comprise a polynucleotide sequence identical to or a variant, derivative or a fragment of a naturally occurring gene or corresponding cDNA sequence. In some aspects, the heterologous gene or cDNA may comprise a synthetic gene sequence. In some aspects, the heterologous gene or cDNA may comprise a polynucleotide sequence identical to or a variant, derivative or a fragment of a prokaryotic gene or corresponding cDNA. In some aspects, the heterologous gene or cDNA may comprise a polynucleotide sequence identical to or a variant, derivative, or a fragment of a eukaryotic gene or corresponding cDNA. In some aspect the heterologous gene or cDNA is a variant, derivative, or a fragment of a non-human mammalian gene (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species). In some aspects, the heterologous gene or cDNA is a variant, derivative, or a fragment of a human gene.

[0061] In some aspects, the heterologous gene or cDNA encodes a polypeptide that functions, for example, as a therapeutic, a reporter, a bioactive protein, or an antibody.

[0062] In some aspects, the heterologous gene or cDNA encodes a therapeutic polypeptide. In some aspect the heterologous gene or cDNA encodes a bioactive polypeptide. In some exemplary aspects, the heterologous gene or cDNA encodes one or more of a cytotoxic protein, anti-cancer, anti-inflammatory, immunomodulatory, anti-viral, anti-microbial, anti-fungal, anti- helminthic, hypocholestrolemic, anti-diabetic, anti-fibrotic, analgesics, anti-depressants, neuromodulatory, anti-pruritic, cardiovascular, and/or hormonal protein.

[0063] In some aspects, the heterologous gene or cDNA encodes a polypeptide that functions, for example as an antibody, a Chimeric Antigen Receptor (CAR), a vaccine, a recombinase, a transporter, translational regulator, transcriptional regulator, post-transcriptional regulator, endocytosis or exocytosis regulatory protein. In some aspects, the heterologous gene or cDNA encodes a polypeptide that functions as a diagnostic polypeptide.

[0064] In some aspects, the heterologous gene or cDNA encodes a polypeptide that is not a therapeutic polypeptide. In some aspects, the heterologous gene or cDNA encodes a polypeptide that is a reporter polypeptide, for example a fluorescent protein like GFP (green fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), mCherry, LacZ (b-galactosidase), CAT (chloramphenicol acetyltransferase), luciferase.

[0065] In some aspects, the heterologous gene may encode a non-coding RNA molecule. Nonlimiting examples of non-coding RNA include, but are not restricted to tRNA, rRNA, microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs, bifunctional RNA, IncRNA.

[0066] In some aspects, the heterologous gene or cDNA encodes a therapeutic polypeptide or ncRNA effective in treatment of any one of Alzheimer's disease, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis, autism, brain lupus (NP- SLE), chemo-induced peripheral neuropathy (CIPN), postherapeutic neuralgia, chronic inflammatory demyelinating polyneuropathy (Cl DP), epilepsy, Guillain-Barre Syndrom (GBS), inclusion body myositis, lysosomal storage diseases, sphingomyelinlipidose (Niemann-Pick C), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), mucopolysaccharidose ll/IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, Myasthenia Gravis, Neuro-Behcet's Disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett's Syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, bacterial meningitis, liver cirrhosis, autoimmune hepatitis, cholangitis, sclerosing cholangitis, or cancers.

[0067] In some exemplary aspects, the heterologous gene encodes a polypeptide comprising an amino acid sequence at least about 60% identical to SEQ ID NOS: 6-10 (GFP, TREM2, NFE2L2, GRN, CSF1 R).

Table 2: Exemplary polypeptide sequences

[0068] In some aspects, the recombinant polynucleotide sequence may further comprise other transcriptional and translational regulatory sequences. In some aspects, the transcriptional regulatory element constitutes a binding site for a transcriptional activator or repressor. A transcriptional activator is a protein that activates expression of the transgene when bound to the transcriptional regulatory element. A transcriptional repressor is a protein that prevents expression of the transgene when bound to the transcriptional regulatory element. Non-limiting examples of regulatory elements include transcription initiation, termination, enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; tissue specific regulatory sequences and when desired, sequences that enhance secretion of the encoded product. In some aspects, the regulatory signal is an enhancer sequence. By “enhancer” is meant a nucleic acid sequence that, when positioned proximate to a promoter, confers increased transcription activity relative to the transcription activity resulting from the promoter in the absence of the enhancer domain. Non-limiting examples of enhancers include CMV enhancer, MIE enhancer, GADD45G, HACNS1. In some aspects, the regulatory sequence is a viral posttranscriptional regulatory element for example woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), hepatitis B virus posttranscriptional regulatory element (HBVPRE), RNA transport element (RTE), or any variant thereof. In some aspects, the regulatory sequence is a transcription termination sequence, for example a SV40 late poly(A) sequence, a rabbit betaglobin poly(A) sequence, a bovine growth hormone poly(A) sequence, or any variant thereof. In some aspects, depending on the regulatory sequence, the sequence may be located anywhere on the recombinant polynucleotide sequence, for example before, or after the promoter sequence, between the promoter and the heterologous gene or cDNA, at the end of the gene or cDNA or after the start of the gene sequences. In some aspects, the regulatory sequence is a F2A, E2A, P2A, T2A Picornavirus IRES, Apthovirus IRES, Hepatitis A IRES, Pestivirus IRES, Hepesvirus IRES. In some aspects, the recombinant polynucleotide may comprise an untranslated regions (UTRs). In mRNA, the 5'UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. In some aspects, any suitable naturally occurring or synthetic UTR sequence can be incorporated into the polynucleotides disclosed herein. Other non-UTR sequences may also be used as regions or subregions within the polynucleotides. For example, introns or portions of introns sequences may be incorporated into regions of the polynucleotides of the invention. Incorporation of intronic sequences may increase protein production as well as polynucleotide levels. Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5'UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes.

Vectors and delivery systems

[0069] In some aspects, the compositions of the current disclosure encompass vectors comprising any recombinant polynucleotide disclosed herein. A vector can be any genetic element, e.g., a plasmid, chromosome, virus, transposon, behaving either as an autonomous unit of polynucleotide replication within a cell, (i.e., capable of replication under its own control) or being rendered capable of replication by insertion into a cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Vectors can contain polynucleotide sequences which are necessary to effect ligation or insertion of the vector into a desired host cell and to affect the expression of the attached segment. Such sequences differ depending on the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences and transcription and translation termination sequences. Alternatively, expression vectors can be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences. A vector can comprise a selectable marker gene. In some aspects, the vector is an “episomal expression vector” or “episome,” which is able to replicate in a host cell and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure.

[0070] In some aspects, the vector is a viral vector. In some aspects, the suitable delivery system may be a viral vector. In some aspects, the viral vector is an RNA viral vector. In some aspects, the viral vector is a DNA viral vector. Non-limiting examples of suitable viral vectors include adenovirus, adeno associated virus (AAV), retrovirus, herpesvirus, lentivirus, poxvirus, or papilloma virus vector. In some aspects, vector is a Lentiviral vector. Lentiviral vectors have the ability to infect and to stably integrate into the genome of dividing and non-dividing cells (Amado and Chen, 1999 Science 285: 674-6). Methods for the construction and use of lentiviral based expression constructs are described in U.S. Patent No.'s 6,165,782, 6,207,455, 6,218,181 , 6,277,633 and 6,323,031 the entirety of which are hereby incorporated by reference. Polynucleotides encoding envelope protein(s) of VSV (VSV-G), LCMV (Lymphocytic choriomeningitis Virus), or RRV (Ross River Virus) may be used to prepare lentivral vectors suitable to target microglial and/or macrophage cells. In some aspects, the vector is a AAV vector comprising the recombinant polynucleotide. In some aspects, the AAV vector is a self- complementary AAV vector. In some aspects, the AAV vector is composed of, at a minimum, a polynucleotide as disclosed herein, and 5' and 3' AAV inverted terminal repeats (ITRs). In some aspects, the AAV vector may be of any of the available serotypes. In some exemplary aspects, the serotype of the AAV vector is suitable for targeting microglia and/or macrophages. Examples of suitable serotypes include AAV1, 2, 5, 8, 9, PHP.eB.

[0071] In some aspects, the current disclosure also encompasses non-viral vectors or delivery systems. Non-limiting examples of non-viral vectors and delivery systems include transposons, plasmids, polynucleotides formulated with delivery systems like polymers, polyplexes, lipids, lipidoids, lipoplexes, liposomes, polymer nanoparticles, nanoparticles, lipid nanoparticles (LNPs), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembled nucleic acid nanoparticles, hyaluronidase, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer-RNA chimeras, RNA-fusion protein complexes and any combination thereof. All vector systems can be used in vitro, ex vivo or in vivo in cell cultures, tissue culture, ex vivo cell, tissue or organ samples or live animals.

Therapeutic compositions

[0072] In some aspects, the compositions disclosed herein may be a therapeutic composition and may further comprise one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients used in the manufacture of therapeutic compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, suspension aids, isotonic agents, thickening agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, preservatives, and/or oils. Such excipients may optionally be included in therapeutic formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator. Various excipients for formulating therapeutic compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006 ). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure.

[0073] In some aspects, the compositions disclosed herein are formulated for administration into a subject via one or more routes for example oral, intraadiposal, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatical, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, local, mucosal, parenteral, rectal, subconjunctival, subcutaneously, sublingual, topically, trans buccal, transdermal, vaginal, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via localized perfusion. In some aspects, the therapeutic compositions are formulated for administration via injection or transcranial delivery. In some aspects, the therapeutic compositions are formulated as a unit dose. In some aspects, the formulation may further comprise excipients suitable for administration via the routes provided herein.

[0074] In some aspects, the compositions disclosed herein may further comprise excipients suitable for one or more suitable administration means. In some aspects, the compositions may be formulated as injectables, liquids, emulsions, suspensions, syrups, pills, caplets, creams, ointments, lotions, patches, solutions, suspensions, suppositories, lyophilizates, gels and capsules. Methods of making therapeutic compositions are well known in the art (See, e.g., Remington, The Science and Practice of Pharmacy, Alfonso R. Gennaro (Ed.) Lippincott, Williams & Wilkins (pub)). The therapeutic composition may also be formulated so as to facilitate timed, sustained, pulsed, or continuous release. The therapeutic composition may also be administered in a device, such as a timed, sustained, pulsed, or continuous release device.

III. Methods

[0075] In some aspects, the current disclosure also encompasses methods of inducing heterologous gene expression in microglial and/or macrophages using the compositions disclosed herein. In some aspects, the method comprises contacting a microglial or macrophage cell with a recombinant polynucleotide sequence or vector disclosed herein.

[0076] In some aspects, any of the compositions disclosed herein can be used in the method. In some aspects, a polynucleotide comprising a heterologous gene operably linked to a microglial/macrophage promoter comprising a nucleic acid sequence at least 70% identical to any one or more of SEQ ID NOS: 1-5 can be used in the method, wherein the expression of the heterologous gene is at least about 60% specific to microglial and/or macrophage cells.

[0077] In some aspects, the method can be implemented in vitro for example in microglial or macrophage cell or tissue cultures. In some aspects, the method can be implemented ex vivo using tissue samples from a subject. In some aspects, the method can be implemented in vivo in a subject in need thereof. Methods to introduce gene editing components into a cell in vitro or ex vivo include, but are not limited to, electroporation, sonoporation, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viral vectors. In some aspects, the polynucleotide can be incorporated in any suitable vector sequence as disclosed herein and known in the art. In some aspects, the polynucleotides can be used with any gene editing system known in the art. These gene-editing components may comprise one or more of DNA, cDNA or RNA, viral vectors, CRISPR, TALEN, zinc finger nuclease (ZFN), meganuclease, Mega-TAL, and transposon-based systems. Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Examples of viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated viral vectors, poxvirus vectors; herpesvirus vectors, helper-dependent adenovirus vectors, hybrid adenovirus vectors, Epstein-Bar virus vectors, herpes simplex virus vectors, hemagglutinating virus of Japan (HVJ) vectors, and Moloney murine leukemia virus vectors.

[0078] In some aspects, the method can be implemented for therapeutic or non-therapeutic applications. Non limiting examples on non-therapeutic use include laboratory use, use for research purposes and/or industrial use. In some exemplary aspects, the method disclosed herein may be used to express one or more of a cytotoxic gene, a reporter gene, a recombinase, a nuclease, a cytokine, a chemokine, a transcriptional regulator, a translational regulator, a transporter, a regulator of endocytosis or exocytosis in a microglial and or macrophage cell. In some exemplary aspects, the method disclosed herein may be used to express one or more of a cytotoxic gene, a reporter gene, a recombinase, a nuclease, a cytokine, a chemokine, a transcriptional regulator, a translational regulator, a transporter, a regulator of endocytosis or exocytosis in a microglial and or macrophage cell in a cell culture, tissue culture, ex vivo tissue sample. In some exemplary aspects, the compositions disclosed herein can be used in vivo in mouse models to study macrophages and or microglial cells or for differential or cell specific gene expression in macrophages or microglial cells in suitable mouse models. In some aspects, the compositions can be used to express a therapeutic gene of interest in a subject. In some exemplary aspects the heterologous gene encodes a polypeptide comprising an amino acid sequence at least about 60% identical to SEQ. ID. NOS: 6-10.

[0079] In some aspects, the current disclosure also encompasses method of treating diseases by inducing expression of a heterologous therapeutic gene in macrophages or microglial cells, the method comprising administering a therapeutically effective amount of a compositions as disclosed herein into a subject in need thereof. In some aspects, the disease could be any disease wherein the subject in need thereof would benefit from microglial specific expression of a therapeutic. Non limiting examples include, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis, autism, brain lupus (NP- SLE), chemo-induced peripheral neuropathy (CIPN), postherapeutic neuralgia, chronic inflammatory demyelinating polyneuropathy (Cl DP), epilepsy, Guillain-Barre Syndrom (GBS), inclusion body myositis, lysosomal storage diseases, sphingomyelinlipidose (Niemann-Pick C), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), mucopolysaccharidose ll/IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, Myasthenia Gravis, Neuro-Behcet's Disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett's Syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis.

[0080] In one aspect the compositions can be used for the treatment of peripheral macrophage related disorders in a subject in need thereof. Non limiting examples include cancer, autoimmune disease, macrophage activation syndrome, atherosclerosis, liver fibrosis, liver cirrhosis, diabetes mellitus, Kawasaki disease, asthma, hemophagocytic lymphohistiocytosis, sarcoidosis, periodontitis, Whipple's disease, pulmonary alveolar proteinosis, autoimmune diseases, macrophage related pulmonary disease, Leishmaniasis, obesity complications, hemodialysis related inflammation, microbial infection, viral infection, inflammation, and complications thereof.

[0081] In some aspects, any suitable mode of administration can be used depending on the disease condition and the formulation. Suitable modes of administration are known in the art and further provided herein including but not limited to intravenous, intracranial, intrathecal, subcutaneous, intranasal route, cranial, transmucosal, transnasal, transcranial, intracerebroventricular, intestinal, and/or parenteral delivery.

[0082] In some aspects, a subject in need includes a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In some aspects, the human includes man, woman, children, elderly, adults, and teens. In some other aspects, the human is an adult human patient, or a pediatric human patient. In some aspects, the subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc. In some aspects, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In some aspects, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In some aspects, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a specific aspect, the animal is a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non-human primates. In certain aspects, the animal is a rodent. Non-limiting examples of rodents may include mice, rats, guinea pigs, etc. In some exemplary aspects the subject is a human.

[0083] For any formulation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0084] In some aspects, toxicity and therapeutic efficacy of the active ingredients disclosed herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In some aspects, data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in a human subject. In some aspects, a dosage for use herein may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1).

EXAMPLES

[0085] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0086] The current disclosure provides minimal promoter sequences capable of targeting heterologous gene expression into microglial and macrophage cells. In some aspects, the heterologous gene linked to the minimal promoter is incorporated into a viral vector for delivery. Effective targeting of gene expression into microglial and macrophage cells has immense therapeutic potential. The disclosed examples provide screening, development and testing of minimal promoters for therapeutic and non-therapeutic applications.

Example 1 : Experimental Procedures

Animals:

[0087] Wild-type C57/BL6J mice were purchased from Jackson Laboratories. Adult male and female mice at 2-3 months of age were used unless otherwise stated. All mice were housed under a 12 hrs light/dark cycle and had ad libitum access to food and water in the UT Southwestern animal facility. The experimental animals were randomized, and the experimenters were not blinded to the allocation of animals during experiments and outcome assessment. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern. Sample sizes were determined based on prior experiences with immunohistochemical analyses of cell reprogramming.

Virus preparation and intracranial injections:

Lentivirus preparation

[0088] The lentiviral vector SP-GFP was generated by sub-cloning the macrophage synthetic promoter (SP) into the CS-CDF-CG-PRE vector. The human CD68 promoter and enhancer was purchased from Addgene and PCR-based subcloned into hNG2-GFP vector at the Xbal and Agel sites. The mF4-80 promoter was PCR-subcloned into CD68-GFP at the EcoRI and Agel sites using template mouse DNA. hlBA1-GFP, hTmem119-GFP, hC1Qa-GFP, and hC1Qb-GFP were constructed by PCR-subcloning the promoters from template human DNA into hCD68-GFP vector at EcoRI/Agel sites. hCX3CR1-GFP was constructed by PCR-subcloning the promoters from template human DNA into hCD68-GFP vector at Xbal/Agel sites. The IBA1-Empty plasmid was subcloned by blunt self-ligation at the Agel and Xhol sites of the hIBAI-GFP vector. Replicationdeficient virus was produced in HEK293T cells by transient transfection with lentiviral vectors and packaging plasmids (pMDL, pREV, and VSV-G or pHCMV-LCMV-WE envelopes). Lentivirus was collected by PEG precipitation.

AAV vectors and virus production

[0089] The AAV vectors driven by human IBA1 promoter were constructed through PCR-based amplification of human genomic DNA. The hlBA1 promoter was then subcloned into the lentiviral hlBA1-GFP vector or into scAAV or ssAAV vector. All vectors were verified through restriction enzyme digestions and DNA sequencing. AAV viruses were packaged with pAd-deltaF6 (Addgene #112867) and the helper pAAV2/1 , pAAV2/2 (Addgene #104963), pAAV2/5 (Addgene #104964), pAAV2/6, pAAV2/6M, pAAV2/8 (Addgene #112864), pAAV2/9 (Addgene #112865), pUCmini-iCAP-PHP.eB (Addgene #103005) in HEK293T cells. Briefly, HEK293T cells were transfected with the packaging plasmids and a vector plasmid. Three days later, virus was collected from the cell lysates and culture media. Virus was purified through iodixanol gradient ultracentrifugation, washed with PBS, and concentrated with 100K PES concentrator (Pierce™, Thermo Scientific). Viral titers were determined by quantitative PCR with ITR primers (forward: 5- GGAACCCCTAGTGATGGAGTT-3; reverse: 5-CGGCCTCAGTGAGCGA-3). Based on publications, virus with a titer range from 5x10¹² - 8x10¹³ GC/mL was used for experiments.

Viral Stereotactic Injections

[0090] Two weeks after MCAO, striatum of adult mice was stereotactically injected with lentivirus or AAVs. Mice were placed on a stereotactic frame (Kopf, USA) under isoflurane anesthesia (5% induction, 1% maintenance at a flow of 100 ml/min). A vertical skin incision exposed the Bregma on the skull used for guiding the location of the Burr hole. Injection coordinates were as follows: anterior/posterior, +1.0 mm; medial/lateral, -2.0 mm; and dorsal/ventral from skull, -3.0 mm. 2 pL of lentivirus with an original titer of 2 x 10⁹ colony forming units per ml_ or 2 pL of AAV with an original titer of 0.5-8 x 10¹³ GC/mL was injected using a 10- pL Hamilton syringe with 33 G beveled tip metal needle. The mix was injected at a rate of 0.5 pL/min until the total volume was delivered. The needle was left in place for 5 minutes before it was slowly removed from the brain.

Immunohistochemistry and quantification

[0091] Mice were euthanized and transcardially perfused with ice-cold 1 x PBS, followed by 4% paraformaldehyde (PFA). Brains were then isolated, post-fixed overnight with 4% PFA at 4 °C, and cryoprotected with 30% sucrose solution for at least 24 hr at 4 °C. Coronal brain sections were collected at 40-pm thickness with a sliding microtome (Leica) and were stored in antifreezing solution at -20 °C. Immunostainings were conducted essentially as previously described (2). Primary antibodies are listed below in Table 1. Alexa Fluor 488-, 555- or 647-conjugated corresponding secondary antibodies from Jackson ImmunoResearch were used for indirect fluorescence. Nuclei were counterstained with Hoechst 33342 (HST). Images were taken with a Zeiss LSM 700 confocal microscope. For quantifying, 3 of 20x images were taken for each slice, with 3 slices per animal. (Data were obtained from one-sixth of the sections spanning the virus- injected striatal region in each mouse.) Animals with failed or mistargeted viral injections were excluded. A representative image was shown from at least three similar images. ImageJ and GraphPad Prism 9 software were used for imaging and statistical analysis.

Middle Cerebral Artery Occlusion

[0092] To model ischemic stroke, cerebral ischemia was induced in mice by performing middle cerebral artery occlusion (MCAO). Mice were anesthetized with isoflurane (1-5% at flow rate of 100ml/min). The left common and external carotid arteries were exposed and ligated. Occlusion of the middle cerebral artery (MCA) was accomplished through the insertion of a filament from the basal part of the external carotid artery and advancing it in the internal carotid artery toward the location of MCA branching from the circle of Willis. The occlusion lasted 20 min and reperfusion was initiated by removing the suture from the internal carotid artery. Once the suture was removed, the internal carotid artery was ligated.

L-NIO Induced Ischemia

[0093] L-NIO was used to specifically model ischemic stroke in the striatum. Briefly, a craniotomy was performed overlying the injection sites of the cortex, whereas the mice were anesthetized and securely mounted onto a stereotaxic apparatus. A Hamilton syringe was filled with L-NIO (27 pg/pL in sterile physiological saline; Calbiochem), secured onto the stereotaxic arm, and connected to a pressure pump. Three injections (each of 0.3 pl of L-NIO solution) were made in the following coordinates: anterior-posterior (AP) +1, medio-lateral (ML) +2, dorsoventral (DV) -3. Injections were made at a rate of 3 pL/min, targeting the striatum. Localized vasoconstriction leads to focal ischemia in the striatum.

Intravenous injections to eye

[0094] Mice were positioned on their side after anesthetization. Loose skin over the shoulders and behind the ears was pulled back to let the eye protrude slightly. The injection needle was inserted with bevel down at an angle of 45° behind the globe of the eye in the retro-bulbar sinus. Virus was slowly deposited. The needle was then slowly removed, and the eyelid was closed by applying little pressure.

Example 2: In vivo screen for microglia-targeting promoters

[0095] As a first step to developing an effective minimal promoter to target heterologous gene expression to microglia and macrophage, sequences were screened for effective targeting of green fluorescence protein (GFP) expression into the microglial cells of the striatum. Promoter sequences SP, CD28, F4/80, IBA1 , CX3CR1 , C1Qa, C1Qb, or Trem119 were cloned upstream of the GFP gene and the sequences were incorporated into lentiviral vectors. Equal amounts of lentiviruses were stereotactically injected with a Hamilton syringe and a 33-gauge needle into the striatum of each mice. When possible, mice were bilaterally injected with different viruses for reduced animal usages.

[0096] Mice were euthanized one week post injection and perfused. Cryosections were obtained and immunostained essentially as described using antibodies against GFP and IBA1. IBA1 was used as a biomarker for microglial cells. Nuclei were counterstained with Hoechst 33342 (HST). Confocal images were used to determine co-localization of GFP and IBA1. Targeting specificity was calculated as percentage of (GFP⁺IBA1⁺ double positive cells)/(GFP⁺ cells).

[0097] Results show that of the tested promoters IBA1 showed the highest targeting efficiency (see FIGs. 1A and 1B). IBA1 promoter was used for subsequent development and characterization.

Example 3: Testing lentiviral pseudotypes with IBA1 promoter for microglial targeting [0098] Pseudotyping is a glycoprotein optimization method to improve the transduction efficiency and stability of lentiviral vector by combining the foreign viral envelope proteins with the lentiviral vector. Pseudotyped lentiviral vectors packaged in either LCMV (Lymphocytic Choriomeningitis virus) or VSV-G (vesicular stomatitis virus GP) envelopes were tested for their ability to target GFP expression under IBA1 promoter to microglial cells. LCMV or VSVG lentiviral vectors comprising a IBA1-GFP sequence were stereotaxically injected with a Hamilton syringe and a 33-gauge needle into the striatum of mice. The mice were euthanized 4 weeks post injection and perfused. Cryosections were obtained and immunostained essentially as described using antibodies against GFP and IBA1. IBA1 was used as a biomarker for microglial cells. Nuclei were counterstained with Hoechst 33342 (HST). Confocal images were used to determine colocalization of GFP and IBA1 (see FIG. 2A). Targeting specificity was calculated as percentage of (GFP⁺IBA1⁺ double positive cells)/(GFP⁺ cells).

[0099] As seen in FIG. 2B both pseudotypes were equally effective in targeting GFP expression to microglial cells with greater than 80% specificity.

Example 4: Testing AAV vectors with IBA1 promoter for microglial targeting

[0100] Next AAV viral vectors were tested to determine if the vectors could specifically target microglial cells when used with IBA1 promoter. Self-complementary adeno-associated virus (scAAV) contains a dimeric inverted repeat genome able to fold into double stranded DNA (dsDNA) and provide several advantages including increased and prolonged expression and a broader range of targets. Self-complementary AAV (scAAV) of serotype 1 , 2, 5, 8, 9 and PHP.eB comprising a IBA1-GFP sequence were stereotactically injected with a Hamilton syringe and a 33-gauge needle into the striatum of mice. The mice were euthanized one week post injection and perfused. Microtome sections were obtained and immunostained essentially as described using antibodies against GFP and IBA1. IBA1 was used as a biomarker for microglial cells. Confocal images were used to determine co-localization of GFP and IBA1 (see FIG. 3A). Targeting specificity was calculated as percentage of (GFP⁺IBA1⁺ double positive cells)/(GFP⁺ cells). Targeting efficiency was calculated as percentage of (GFP⁺IBA1⁺ double positive cells)/(IBA1⁺ cells).

[0101] As seen in FIG. 3B serotypes 2/2, 2/5 and 2/8 exhibited greater than 75% targeting specificity to microglial cells. Serotypes 2/5 and 2/8 also exhibited greater than 75% targeting efficiency (see FIG. 3C). Example 5: Testing long-term microglial expression under the IBA1 promoter

[0102] Next scAAV viral vectors were tested to determine if sustained expression of a heterologous gene in microglial cells could be achieved with I BA1 promoter. Self-complementary AAV (scAAV) of serotype 5, 8 comprising a IBA1-GFP sequence were stereotactically injected with a Hamilton syringe and a 33-gauge needle into the striatum of mice. The mice were euthanized 4 weeks post injection and perfused. Cryosections were obtained and immunostained essentially as described using antibodies against GFP and IBA1. IBA1 was used as a biomarker for microglial cells. Nuclei were counterstained with Hoechst 33342 (HST). Confocal images were used to determine co-localization of GFP and IBA1 (see FIG. 4A). Targeting specificity was calculated as percentage of (GFP⁺IBA1⁺ double positive cells)/(GFP⁺ cells). Targeting efficiency was calculated as percentage of (GFP⁺IBA1⁺ double positive cells)/(l BA1 ⁺ cells).

[0103] As seen in FIG. 4B targeting specificity of scAAV5 and scAAV8 as seen from brain sections 4 weeks post injection was greater than 75% suggesting sustained expression of the heterologous gene. Both scAAV5 and scAAV8 also showed high sustained expression with scAAV5 exhibiting close to 80% efficiency at 4 weeks.

Example 6: Determining the minimal promoter sequence of IBA1 needed to target microglial cells

[0104] One aspect of the current disclosure is the determination of the minimal promoter sequence that can be used for targeting microglial and macrophages. Viral vectors have limitations in the length of sequence that can be packaged into a virion. As such finding a minimal promoter sequence is not only desirable but can become essential when the heterologous gene is large. To this end, promoter sequences derived from IBA1 of different lengths were tested (see FIG. 5A). Sequence 1 corresponds to a 756 bp core promoter sequence, IBA1a, IBA1 b, IBA1c and IBA1d correspond to truncated versions of the promoter of 466 bp, 313 bp, 179 bp and 118 bp respectively. AAV5 vector comprising the promoter sequences linked to GFP were stereotaxically injected into the striatum of control and mouse model for stroke. The mice were euthanized one week post injection and perfused. Cryosections were obtained and immunostained essentially as described using antibodies against GFP and IBA1. Confocal images were used to determine co-localization of GFP and IBA1 (see FIG. 5B). Targeting specificity was calculated as percentage of (GFP⁺IBA1⁺ double positive cells)/(GFP⁺ cells) (FIG. 5C). Targeting efficiency was calculated as percentage of (GFP⁺IBA1⁺ double positive cells)/(IBA1⁺ cells) (FIG. 5D). [0105] As seen in FIG. 5C and FIG. 5D the 460 bp fragment showed robust efficiency and specificity for expression in microglial cells. Fragments of about 310 bp and smaller showed a pattern of decreasing efficiency and specificity.

Example 7: Design of improved specificity AAV for brain microglial targeting

[0106] The 460-bp minimal promoter was redesigned for improved specificity for brain microglia by including the targeting sequences of miR124 (see FIG. 6A). The resulting AAV vector was stereotactically injected with a Hamilton syringe and a 33-gauge needle into the striatum of mice. The mice were euthanized 4 weeks post injection and perfused. Cryosections were obtained and immunostained essentially as described using antibodies against GFP and IBA1. The resulting promoter showed nearly 100% microglia targeting specificity. No neurons were targeted (FIG. 6B and FIG. 6C) . The packaging capacity of the vector with the minimal promoter is ~3.0 kb, which could be further increased if the WPRE sequence is removed, thus making this a highly desirable promoter.

Example 8: Targeting liver macrophages by AAV8 under IBA1 promoter post intravenous injection.

[0107] To target peripheral macrophages, AAV-hlBA1-GFP packaged with serotype 5, 8, or PHP.eB was injected intravenously through the retroorbital route. Expression of GFP was analyzed 4 weeks post injection. Liver macrophages were detected with staining of IBA1. Representative confocal images are shown (FIG. 7A). Quantification results show that AAV- hlBA1-GFP virus packaged with serotype 8 is the most efficient and specific in delivering gene expression in liver macrophages (FIG. 7B-C).

Claims

What is claimed is:

1 . A recombinant polynucleotide sequence comprising:

(a) a heterologous gene; and

(b) a promoter operably linked to the heterologous gene, wherein the promoter has a nucleic acid sequence at least 70% identical to one or more of SEQ ID NOS: 1-5.

2. The recombinant polynucleotide sequence of claim 1 , wherein the nucleic acid sequence of the promoter is less than 1kb in length.

3. The recombinant polynucleotide sequence of claim 2, wherein the nucleic acid sequence of the promoter is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least a 100% identical to one or more of SEQ ID NOS: 1-5.

4. The recombinant polynucleotide sequence of any one of claims 1 or 2, wherein the promoter is capable of inducing expression of the heterologous gene in a microglial cell.

5. The recombinant polynucleotide sequence of any one of claims 1 or 2, wherein the promoter is capable of inducing expression of the heterologous gene in a macrophage.

6. The recombinant polynucleotide sequence of any one of claims 1-5, wherein the recombinant polynucleotide sequence further comprises one or more additional transcriptional and/or post-transcriptional regulatory elements (such as miR124T).

7. The recombinant polynucleotide sequence of claim 1 , wherein the heterologous gene encodes a functional protein or an RNA. The recombinant polynucleotide sequence of claim 1 , wherein the heterologous gene is a cytotoxic gene, a reporter gene, a recombinase, a nuclease, a receptor, a cytokine, a chemokine, a transcriptional regulator, a translational regulator, a transporter, a regulator of endocytosis or exocytosis or a therapeutic gene. A viral vector comprising a polynucleotide sequence comprising:

(a) a heterologous gene; and

(b) a promoter operably linked to the heterologous gene, wherein the promoter has a nucleic acid sequence at least 70% identical to one or more of SEQ ID NOS: 1-5. The viral vector of claim 9, wherein the viral vector is selected from adenovirus, adeno- associated virus, a retrograde virus, retrovirus, herpesvirus, lentivirus, poxvirus, or papilloma virus expression vector. The viral vector of any one of claims 9 or 10, adapted to inducing expression of the heterologous gene in microglia and/or macrophages. A therapeutic composition comprising the viral vector of any one of claims 9-11. A therapeutic composition comprising the recombinant polynucleotide sequence of any one of claim 1-8. A therapeutic composition of any one of claims 12 or 13 further comprising pharmaceutically acceptable buffer, diluent, or excipient. Use of the therapeutic composition of any one of claims 12-14, for treatment of Alzheimer's disease, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis, autism, brain lupus (NP-SLE), chemo-induced peripheral neuropathy (CIPN), postherapeutic neuralgia, chronic inflammatory demyelinating polyneuropathy (Cl DP), epilepsy, Guillain-Barre Syndrom (GBS), inclusion body myositis, lysosomal storage diseases, sphingomyelinlipidose (Niemann-Pick C), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), mucopolysaccharidose ll/IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, Myasthenia Gravis, Neuro-Behcet's Disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett's Syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, bacterial meningitis, cancer, autoimmune disease, macrophage activation syndrome, atherosclerosis, liver fibrosis, liver cirrhosis, diabetes mellitus, Kawasaki disease, asthma, hemophagocytic lymphohistiocytosis, sarcoidosis, periodontitis, Whipple's disease, pulmonary alveolar proteinosis, autoimmune diseases, macrophage related pulmonary disease, liver disease, Leishmaniasis, obesity complications, hemodialysis related inflammation, microbial infection, viral infection, inflammation, and complications thereof.

A method of inducing expression in microglia and/or macrophage comprising contacting the microglia and/or macrophage cell with a polynucleotide sequence comprising:

(a) a heterologous gene; and

The method of inducing expression in microglia and/or macrophages of claim 16, wherein the nucleic acid sequence of the promoter is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least a 100% identical to one or more of SEQ ID NOS: 1-5. The method of inducing expression in microglia and/or macrophages of any one of claims 16 or 17, further comprising incorporating the polynucleotide sequence into a viral vector and contacting said viral vector with said microglia and/or macrophage cell. The method of inducing expression in microglia and/or macrophages of claim 18, wherein the viral vector is selected from adenovirus, adeno- associated virus, a retrograde virus, retrovirus, herpesvirus, lentivirus, poxvirus or papiloma virus expression vector. The method of any one of claims 17-19, wherein the heterologous gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 6-10. A method of inducing expression in microglia and/or macrophage in a subject in need thereof, comprising administrating into the subject, a composition comprising a recombinant polynucleotide sequence comprising a heterologous gene and a promoter operably linked to a heterologous gene, wherein the promoter has a nucleic acid sequence of less than 1kb and has a nucleic acid sequence at least 70% identical to one or more of SEQ ID NOS: 1-5. A method of modulating liver macrophage activity, the method comprising contacting a liver macrophage cell with a polynucleotide sequence comprising:

(a) a heterologous gene; and

(b) a promoter operably linked to the heterologous gene, wherein the promoter has a nucleic acid sequence at least 70% identical to any one of SEQ ID NOS: 1-5. The method of claim 22, wherein the heterologous gene is any one of trem2, nfe2L2, GRN, CSF1R.

24. The method of claim 22, wherein the heterologous gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 6-10.