+

WO2025005990A1 - Formulations de nanoparticules lipidiques pour thérapie cellulaire et procédés associés - Google Patents

Formulations de nanoparticules lipidiques pour thérapie cellulaire et procédés associés Download PDF

Info

Publication number
WO2025005990A1
WO2025005990A1 PCT/US2023/085851 US2023085851W WO2025005990A1 WO 2025005990 A1 WO2025005990 A1 WO 2025005990A1 US 2023085851 W US2023085851 W US 2023085851W WO 2025005990 A1 WO2025005990 A1 WO 2025005990A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipid
mol
cells
pni
formulation
Prior art date
Application number
PCT/US2023/085851
Other languages
English (en)
Inventor
Pierrot Harvie
Lloyd Brian JEFFS
Ruo Yu ZHANG
Mohammadreza KAZEMIAN
Harish CHAKRAPANI
Nikita Jain
Anitha THOMAS
Andrew KONDRATOWICZ
Original Assignee
Global Life Sciences Solutions Canada Ulc
Global Life Sciences Solutions Usa Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2023/069303 external-priority patent/WO2024006863A1/fr
Application filed by Global Life Sciences Solutions Canada Ulc, Global Life Sciences Solutions Usa Llc filed Critical Global Life Sciences Solutions Canada Ulc
Publication of WO2025005990A1 publication Critical patent/WO2025005990A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Nucleic-acid-based cell therapy reagents offer advantages over electroporation and traditional oncological treatments in terms of safety and efficacy.
  • RNA cell therapies are subject to degradation by exonucleases and endonucleases in vivo without a delivery system, so they need a carrier.
  • Lipid nanoparticles generally consist of different lipids, each serving distinct functions.
  • LNP can have a lipidic or aqueous core and may contain bilayer structures depending on the abundance of each type of lipids.
  • the components of LNP formulations include: ionizable cationic lipids, which spontaneously encapsulate negatively-charged nucleic acids by a combination of attractive electrostatic interactions with and hydrophobic interactions; neutral phospholipids to reduce charge-related toxicity and to maintain structure of the LNP; and cholesterol to stabilize the LNP and help with cell entry, and a lipid-conjugated polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the disclosure provides, e.g., lipid formulations and related methods suitable for forming nucleic acid-based cell therapy reagents in lipid nanoparticles.
  • the disclosure provides a lipid formulation for forming a lipid-based nanoparticle suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage comprising an ionizable lipid, a phospholipid, and optionally a stabilizer.
  • the disclosure provides a lipid formulation comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, for forming a lipid-based nanoparticle suitable for transfecting a nucleic acid cargo into T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells.
  • the disclosure provides a lipid formulation for forming a lipid-based nanoparticle suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, wherein the formulation is substantially free of cholesterol.
  • the disclosure provides a lipid formulation comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, for forming a lipid-based nanoparticle suitable for transfecting a nucleic acid cargo into T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells, wherein the formulation is substantially free of cholesterol.
  • the disclosure provides a method for screening lipid composition of lipid nanoparticles for preferential delivery of RNA to T cells, comprising: (a) preparing a plurality of lipid nanoparticles with different lipid compositions and a reporter RNA, (b) administering the lipid nanoparticles to Jurkat cells, (c) measuring the relative abundance of reporter RNA, (d) comparing the relative abundance of reporter RNA to a threshold, and (e) identifying lipid compositions above threshold as candidates for preferential delivery of RNA to T cells.
  • FIG. 1 A is graphical representation of transfection efficacy percentage for V46
  • FIG. IB is a graphical representation of GFP expression for V46 PNI 516, V47 PNI 516, V57 PNI 516, V57 PNI 659, V22 PNI 550, and V40 PNI 550, as discussed in Example 3.
  • FIG. 2 is a line graph of the percent TCR knockout normalized to untreated cells with V46 PNI 516, V47 PNI 516, V57 PNI 516, V57 PNI 659, and V22 PNI 550 LNP as delivery system, as discussed in Example 5.
  • FIG. 3 A is a graphical representation of transfection efficiency for V57-PNI-516, V57-PNI-659 and V22-PNI-550 LNP compared to electroporation, as discussed in Example 6.
  • FIG. 3B is a graphical representation of payload expression of GFP mRNA efficiency for V57-PNI-516, V57-PNI-659 and V22-PNI-550 LNP compared to electroporation, as discussed in Example 6.
  • FIG. 3C is a graphical representation of cell viability post treatment for efficiency for V57-PNI-516, V57-PNI-659 and V22-PNI-550 LNP compared to electroporation, as discussed in Example 6.
  • FIG. 4A is a graphical representation of cytometry post 24 hours for LNP comprising GFP mRNA payload in V57-PNI-516, V57-PNI-659 and V22-PNI-550 LNP and electroporation for 0.5 and 2 pg of mRNA per million cells on cell count at 24 H post treatment, as discussed in Example 6.
  • FIG. 4B is a graphical representation of flow cytometry for LNP comprising GFP mRNA payload in V57-PNI-516, V57-PNI-659 and V22-PNI-550 LNP and electroporation for 0.5 and 2 pg of mRNA per million cells on percent viability at 24 H post treatment, as discussed in Example 6.
  • FIG. 5 A is a graphical representation of cytometry post 24 hours for LNP comprising GFP mRNA payload in V57-PNI-516, V57-PNI-659 and V22-PNI-550 LNP for 0.5 and 2 pg of mRNA per million cells on cell count at 24 H post treatment, as discussed in Example 6.
  • FIG. 5B is a graphical representation of flow cytometry for LNP comprising GFP mRNA payload in V57-PNI-516, V57-PNI-659 and V22-PNI-550 LNP for 0.5 and 2 pg of mRNA per million cells on percent viability at 24 H post treatment, as discussed in Example 6.
  • Embodiments of the present disclosure provide lipid formulations comprising an ionizable lipid, a phospholipid, and optionally a stabilizer.
  • the lipid formulations are useful for forming a lipid-based nanoparticle suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage.
  • the lipid formulations as provided herein can exclude cholesterol in some embodiments, which has conventionally been believed to be an important component.
  • the lipid formulations in accordance with embodiments of the present disclosure demonstrate effective transfection, even without the presence of cholesterol.
  • the lipid nanoparticles disclosed herein can further comprise therapeutic agents such as nucleic acid cargos, which can comprise chimeric antigen receptor (CAR) encoded mRNA, a gene editing nuclease and a guide RNA to perform permanent gene knockout and/or insertion, or an mRNA encoding a protein to correct a genetic deficiency.
  • therapeutic agents can be used for cell therapy.
  • Some of the lipid formulations disclosed herein lack cholesterol, known to provide stability and assist in cell entry, but surprisingly the lipid nanoparticles they form are able to effectively transfect target cells.
  • the lipid nanoparticles disclosed herein beneficially exhibit increased transfection efficiency and cell viability compared to other methods of transfection, such as electroporation and other lipid particles.
  • the lipid nanoparticles described herein also demonstrate this increased transfection efficiency and cell viability when targeting T cells, which are known in the art to be difficult to transfect without reducing cell viability.
  • the lipid formulations are useful for forming a lipid-based nanoparticle, e.g., suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage.
  • lipid mix formulations are provided for use in generating more effective lipid- based formulations of nucleic acid cargo (including, e.g., vaccine, gene editing tools, and protein replacement mRNA) and other oligomers such as peptides, and methods for using these lipid mixes and resulting formulations to prepare vaccines.
  • nucleic acid cargo including, e.g., vaccine, gene editing tools, and protein replacement mRNA
  • other oligomers such as peptides
  • the disclosure provides a lipid formulation for forming a lipid-based nanoparticle suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, wherein the formulation is substantially free of cholesterol.
  • the disclosure provides a lipid formulation comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, for forming a lipid-based nanoparticle suitable for, e.g., transfecting a nucleic acid cargo into T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells.
  • the disclosure provides a lipid formulation comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, for forming a lipid-based nanoparticle suitable for transfecting a nucleic acid cargo into T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells, wherein the formulation is substantially free of cholesterol.
  • the lipid formulation forms part of a composition for cell therapy, which include a nucleic acid cargo.
  • the lipid formulation can further comprise a nucleic acid cargo.
  • the term “cargo,” or “payload,” refers to a substance intended to have a direct effect in the mitigation or prevention of disease, i.e., to act as a therapeutic agent, or to act as a research reagent.
  • the term “therapeutic agents” includes nucleic acids, as herein described, or nucleic acid therapeutics (“NAT”), oligopeptides, polypeptides, and small molecules.
  • the therapeutic agent in accordance with some embodiments, is a nucleic acid therapeutic, such as an RNA polynucleotide.
  • the therapeutic agent is messenger RNA (mRNA) or self-amplifying RNA (saRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • the therapeutic agent is double stranded small interfering RNA (siRNA), circular DNA (plasmid), linearized plasmid DNA, minicircles, or msDNA (multicopy single stranded DNA).
  • nucleic acid cargoes includes deoxyribonucleic acid, complementary deoxyribonucleic acid, complete genes, ribonucleic acid, oligonucleotides and ribozymes, acting as a nucleic acid therapeutic (NAT) for gene therapies targeting a variety of diseases, such as cancer, infectious diseases, genetic disorders and neurodegenerative diseases.
  • the nucleic acid cargo in accordance with some embodiments, comprises chimeric antigen receptor (CAR) encoded mRNA.
  • the nucleic acid cargo comprises a gene editing nuclease and a guide RNA to perform permanent gene knockout and/or insertion.
  • the nucleic acid cargo comprises, in accordance with further embodiments, an mRNA encoding a protein to correct a genetic deficiency.
  • the nucleic acid cargo comprises an mRNA encoding a protein to treat disease and wherein the nucleic acid cargo comprises two or more nucleic acids.
  • the nucleic acid cargo comprises at least two nucleic acids, such as 3 nucleic acids, 4 nucleic acids, 5 nucleic acids, 6 nucleic acids, 7 nucleic acids, 8 nucleic acids, 9 nucleic acids, 10 nucleic acids, etc.
  • the nucleic acid cargo comprises two or more nucleic acids
  • the two or more nucleic acids are packaged in separate nanoparticles.
  • the NAT is incorporated into lipid particle during its formation with compounds of the invention. More than one nucleic acid therapeutic may be incorporated in this way. They may be derived from natural sources, or more commonly, synthesized or grown in culture. Examples of nucleic acid cargo include, but are not limited to, antisense oligonucleotides, ribozymes, microRNA, mRNA, ribozyme, tRNA, tracrRNA, sgRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, pre-condensed DNA, plasmid or pDNA, or an aptamer.
  • nucleic acid cargo include, but are not limited to, antisense oligonucleotides, ribozymes, microRNA, mRNA, ribozyme, tRNA, tracrRNA, sgRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, pre-condensed DNA, plasm
  • Nucleic acid reagents are used to silence genes (with for example siRNA), express genes (with for example mRNA), edit genomes (with for example CRISPR/Cas9), and reprogram cells for return to the originating organism (for example ex vivo cell therapy to reprogram immune cells for cancer therapy; autologous transfer or allogenic transfer).
  • genes with for example siRNA
  • express genes with for example mRNA
  • edit genomes with for example CRISPR/Cas9
  • reprogram cells for return to the originating organism for example ex vivo cell therapy to reprogram immune cells for cancer therapy; autologous transfer or allogenic transfer.
  • nucleic acid refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single stranded, double stranded, or contain portions of both double stranded and single stranded sequence, as appropriate. Nucleic acids are meant to include any oligonucleotide or polynucleotide whose delivery into a cell causes a desirable effect.
  • nucleic acids can be modified or unmodified, base modified, and may include different type of capping structures, such as Capl.
  • nucleic acids includes diagnostic agents and research reagents which follow the same physical principles afforded by aspects of the invention.
  • nucleic acid refers to self-amplifying RNA (“saRNA”).
  • nucleic acid in further embodiments, refers to a plasmid including self-amplifying RNA.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to single-stranded and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, e.g., 3'-5' and 2'-5', inverted linkages, e.g., 3'- 3' and 5'-5', branched structures, or internucleotide analogs.
  • DNA 2'-deoxyribonucleotides
  • RNA ribonucleotides linked by internucleotide phosphodiester bond linkages, e.g., 3'-5' and 2'-5', inverted linkages, e.g., 3'- 3' and 5'-5', branched structures, or internucleotide analogs.
  • Polynucleotides have associated counter ions, such as H+, NH4+, trialkylammonium, Mg2+, Na+, and the like.
  • a polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof.
  • Polynucleotides may include intemucleotide, nucleobase and/or sugar analogs. Fragments containing up to 50 nucleotides are generally termed oligonucleotides, and longer RNA are called polynucleotides. In embodiments, oligonucleotides of the present invention are 20-50 nucleotides in length.
  • polynucleotides are 996 to 4500 nucleotides in length, as in the case of messenger RNA. Further, polynucleotides of the invention can include up to 14,000 nucleotides, in accordance with embodiments of the present disclosure.
  • the nucleic acid that is present in a lipid particle according to the present disclosure includes any form of nucleic acid that is known.
  • the nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA.
  • double-stranded RNA examples include siRNA and other RNA interference reagents.
  • Single-stranded nucleic acids include antisense oligonucleotides, guide RNA, including CRISPR-Cas9 gRNA, ribozymes, microRNA, mRNA, and triplex-forming oligonucleotides. More than one nucleic acid may be incorporated into the lipid particle, for example mRNA and guide RNA together, or different types of each, or in combination with protein.
  • a nucleic acid encodes a genetically engineered receptor that specifically binds to a ligand, such as a recombinant receptor, and a molecule involved in a metabolic pathway, or functional portion thereof.
  • a ligand such as a recombinant receptor
  • the molecule involved in a metabolic pathway is a recombinant molecule, including an exogenous entity.
  • a genetically engineered receptor and the molecule involved in a metabolic pathway may be encoded by one nucleic acid or two or more different nucleic acids.
  • a first nucleic acid might encode a genetically engineered receptor that specifically binds to a ligand and a second nucleic acid might encode the molecule involved in a metabolic pathway.
  • polypeptides encompasses the terms “oligopeptides” and “proteins” as well as tertiary and quaternary structures thereof, that are therapeutic agents in some embodiments. Further, the term “polypeptide” refers to a single linear chain of many amino acids of any length held together by amide bonds and the term “oligopeptide” generally consists of from two to twenty amino acids, as used herein.
  • protein refers to one or more polypeptide and may include structural proteins, energy catalysts, albumin, hemoglobin, immunoglobulins, and enzymes.
  • cell therapy refers to transferring cells into the body to replace diseased or damaged cells.
  • the transferred cells may be a patient’s own cells, and by using cell therapy, one of ordinary skill in the art can rectify disease processes in these cells and restore them to the patient.
  • electroporation is considered the most feasible way to genetically modify T cells. Electroporation physically disrupts the cell membrane to force genetic material into cells, and results in some T cells being “irreversibly electroporated” or killed. Electroporation may cause some risk to the genetic materials being damaged before or during transfer.
  • T cells can take a long time to proliferate and a recent study showed that the viability of T Cells after electroporation was 31% as opposed to LNP mediated mRNA delivery.
  • T cells are well-known as difficult to transfect without impairing their survival, even with lipid nanoparticles as carriers.
  • CAR T cell therapy uses cells from the subject being treated, selects and enriches for T cells, and then engineers these cells using a viral vector to express a chimeric antigen receptor (CAR). The cells are returned to the subject, resulting in immunotherapy.
  • CAR treatment there are issues: a) not all the treated T cells have CAR, b) there is variability in the amount of CARs expressed on the T cells that are transfected, c) patients undergoing CAR have often had multiple rounds of chemotherapy which means less healthy T cells which are harder to enrich, and 4) there is a high incidence (46% or more) in patients of Cytokine Release Syndrome (CRS).
  • CRS Cytokine Release Syndrome
  • Viral based to T-cell transformation has been tried, but is labor intensive, expensive and pose manufacturing and regulatory challenges. Vector design and development takes time as suitable vectors determine the efficiency of transduction. Also, virus manufacturing methods are expensive because they are highly regulated, need a lot of equipment, and are labor intensive (one batch for each patient). Viral based transfection also poses the risk that viral genome may randomly insert into the human genome, and requires that the patient leave the hospital to have T cells harvested and treated at a specialized viral manufacturing facility.
  • the present disclosure provides methods for introducing a nucleic acid into a cell (i.e., transfection) in accordance with some embodiments.
  • the disclosure provides a method of preferentially transfecting cells of hematopoietic lineage using the lipid nanoparticles disclosed herein, the method comprising contacting the cells with lipid nanoparticles, wherein the cells maintain at least 50 percent immune cell viability post contact.
  • transfection refers to the transfer of nucleic acid into cells for the purpose of inducing the expression of a specific gene(s) of interest in both laboratory and clinical settings.
  • LIPOFECTINTM and LIPOFECT AMINETM are established commercial transfecting reagents sold by ThermoFisher Scientific. These research reagents contain permanently cationic lipid/s and are not suitable for clinical use.
  • the transfected cells of hematopoietic lineage are T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells.
  • Transfection is a technique commonly used in molecular biology for the introduction of nucleic acid cargo (or NATs) from the extracellular to the intracellular space for the purpose of transcription, translation, and expression of the delivered nucleic acid therapeutic (NAT)cargo for production of some gene product or for down regulating the expression of a disease-related gene.
  • nucleic acid cargo or NATs
  • NAT nucleic acid therapeutic
  • Transfection efficiency is commonly defined as either the i) percentage of cells in the total treated population showing positive expression of the delivered gene, as measured by live or fixed cell imaging (for detection of fluorescent protein), and flow cytometry or ii) the intensity or amount of protein expressed by treated cell(s) as analyzed by live or fixed cell imaging or flow cytometry or iii) using protein quantification techniques such as ELISA, or western blot.
  • These methods may be carried out by contacting the lipid particles or lipid mix formulations of the present disclosure with the cells for a period of time sufficient for intracellular delivery to occur.
  • Typical applications include using well-known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets in vitro and in vivo.
  • applications include delivery of DNA or mRNA sequences that code for therapeutically useful polypeptides. In this manner, therapy is provided for genetic diseases by supplying deficient or absent gene products.
  • lipid refers to a structurally diverse group of organic compounds that are fatty acid derivatives or sterols or could be lipid like materials as in lipidoids (example C 12-200) and are characterized by being insoluble in water but soluble in many organic solvents.
  • lipid mix formulations refer to the types of components, ratios of components, and the ratio of the total lipid components.
  • lipid mix formulations for the manufacture of lipid nanoparticles for nucleic acid delivery comprise cationic or ionizable lipid, phospholipid, cholesterol and a stabilizer such as polyethylene glycol conjugated lipids.
  • the lipid mix formulation comprises an ionizable lipid, a phospholipid, and a stabilizer.
  • the lipid mix formulations are substantially free of cholesterol. Lipid mix formulations are supplied to customers preparing their own LNP on instruments in their facilities.
  • lipid particles refers to generally spherical assemblies of lipids, which can include nucleic acid, cholesterol, and stabilizing agents.
  • the lipid particle represents the physical organization of the lipid mix formulation with the therapeutic agent among the components. Positive and negative charges, ratios, as well as hydrophilicity and hydrophobicity dictate the physical structure of the lipid particles in terms of size and orientation of components.
  • the structural organization of these lipid particles may lead to an aqueous interior with one or more bilayers as in liposomes or it may have a solid interior as in a solid nucleic acid lipid nanoparticle. There may be phospholipid monolayers or bilayers in single or multiple forms. Lipid particles are between 1 and 1000 microns in diameter.
  • the present disclosure provides lipid particles manufactured from the lipid mix formulations described herein.
  • lipid nanoparticle refers to a lipid particle about 300 nm or less in diameter, such as from about 200 nm or less, e.g., from about 15 nm to about 300 nm, from about 25 nm to about 300 nm, from about 50 nm to about 300 nm, from about 75 nm to about 300 nm, from about 100 nm to about 300 nm, from about 125 nm to about 300 nm, from about 150 nm to about 300 nm, from about 175 nm to about 300 nm, from about 200 nm to about 300 nm, from about 225 nm to about 300 nm, from about 250 nm to about 300 nm, from about 275 nm to about 300 nm, from about 15 nm to about 275 nm, from about 15 nm to about 250 nm, from about 15 nm to about 225 .
  • the present disclosure provides lipid nanoparticles manufactured from the lipid mix formulations described herein.
  • the term “viability” refers the ability to continue to grow, divide, and continue to grow and divide, as is normal for the cell type or tissue culture strain, when referring to cells in vitro. Cell viability is affected by harsh conditions or treatments. Cell viability is critical in ex vivo therapy or parenteral administration.
  • the term “ionizable lipid,” as described herein, refers to a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pKa, also referred to as the acid dissociation constant or acid ionization constant, of the ionizable group of the lipid but is more neutral at higher pH values.
  • the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides).
  • nucleic acids e.g., oligonucleotides
  • the term “ionizable lipid” includes lipids that assume a positive charge on pH decrease from physiological pH, and any of a number of lipid species that carry a net positive charge at a selective pH. Examples of suitable ionizable lipids are found in PCT Pub. Nos. WO20252589 and W021000041. In some embodiments the ionizable lipid has a jasmonic acid headgroup. In other embodiments, the ionizable lipid has a tetrahydrofuran head group.
  • the ionizable lipid is present in lipid formulations according to other embodiments of the present disclosure, preferably in a ratio of about 10 to about 50 Mol%, (“Mol%” means the percentage of the total moles that is of a particular component).
  • Mol% means the percentage of the total moles that is of a particular component.
  • the term “about” in this paragraph signifies a plus or minus range of 5 Mol% at increments of 0.1. For example, 28.7 Mol % would be in the claimed range of embodiments.
  • DODMA or l,2-dioleyloxy-3- dimethylaminopropane
  • MC3 is an alternative ionizable lipid, as is DLin-MC3-DMA or O- (Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (“MC3”).
  • the compositions of the present disclosure comprise ionizable lipids as a component.
  • the ionizable lipid comprises a mixture of ionizable lipids.
  • LNPs can be generated from the lipid formulations including the ionizable lipids of the present disclosure.
  • Exemplary ionizable lipids include, but are not limited to of (Z)-3-(2-((l , 17- bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl 4- (dimethylamino)butanoate (PNI 516), 3-(2-((l,17-bis(2-octylcyclopropyl)heptadecan-9- yl)oxy)-2-oxoethyl)cyclopentyl 4-(dimethylamino)butanoate (PNI 550), Z)-3-(2-((l, 17- bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl 1,4- dimethylpiperidine
  • phospholipids or “helper lipids” or “neutral lipids” refer to any one of several lipid species that exist in either in an anionic, uncharged or neutral zwitterionic form at physiological pH. Phospholipids are incorporated into lipid formulations and lipid particles in embodiments of the present disclosure.
  • the lipid formulations and lipid particles of embodiments of the present disclosure include one or more phospholipids at about 10 to about 85 Mol% of the composition, e.g., from about about 0.2 to about 85 Mol%, about 0.3 to about 85 Mol%, about 0.4 to about 85 Mol%, about 0.5 to about 85 Mol%, about 0.6 to about 85 Mol%, about 0.7 to about 85 Mol%, about 0.8 to about 85 Mol%, about 0.9 to about 85 Mol%, about 1 to about 85 Mol%, about 2 to about 85 Mol%, about 3 to about 85 Mol%, about 4 to about 85 Mol%, about 5 to about 85 Mol%, about 6 to about 85 Mol%, about 7 to about 85 Mol%, about 8 to about 85 Mol%, about 9 to about 85 Mol%, about 10 to about 85 Mol%, about 20 to about 85 Mol%, about 30 to about 85 Mol%, about 40 to about 85 Mol%, about 50 to about 85 Mol%, about 60 to about 85 Mol%, about 70 to about 85 Mol%, about 80 to about 85 Mol%, about
  • Suitable phospholipids support the formation of particles during manufacture.
  • Representative phospholipids include but are not limited to diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, and although not strictly “phospholipids” in a technical sense, is intended to include sphingomyelins (SM), dihydrosphingomyelins, cephalins, and cerebrosides.
  • SM sphingomyelins
  • dihydrosphingomyelins dihydrosphingomyelins
  • cephalins cephalins
  • cerebrosides cerebrosides
  • Exemplary phospholipids include but are not limited to zwitterionic lipids, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1
  • the phospholipid is any lipid that is negatively charged at physiological pH.
  • lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups joined to neutral lipids.
  • DOPG dioleoylphosphatidylglycerol
  • DPPG dipalmitoylphosphatidylglycerol
  • POPG palmitoyloleyolphosphatidylglycerol
  • cardiolipin phosphatidylinositol
  • diacylphosphatidylserine diacylphosphatidic acid
  • anionic modifying groups joined to neutral lipid
  • stabilizer refers to an agent that is added to the ionizable lipid, the phospholipid, and the optional sterol that form the lipid formulation according to embodiments of the present disclosure.
  • nonionic stabilizing agents include but are not limited to: Polyethyleneglycol (PEG), DMG- PEG2000 (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-200), Polysorbates (Tweens), DMPE-PGA-diol(30), DMPE-PSar(50)-H-acetyl,TPGS (Vitamin E polyethylene glycol succinate), BrijTM S20 (polyoxyethylene (20) stearyl ether), BrijTM35 (Polyoxyethylene lauryl ether, Polyethyleneglycol lauryl ether), BrijTMS10 (Polyethylene glycol octadecyl ether, Polyoxyethylene (10) stearyl ether), and MyijTM52 (polyoxyethylene (40) stearate), sucrose monolaurate, Kolliphor(r) HS 15.
  • PEG Polyethyleneglycol
  • DMG- PEG2000 l,2-dimyristoyl
  • the stabilizing agent includes PEGylated lipids including PEG-DMG 2000, also known as DMG PEG 2000 (“PEG-DMG”).
  • PEG-DMG DMG PEG 2000
  • Other polyethylene glycol conjugated lipids may also be used in some embodiments, such as DPE-PEG, DOPEPEG, DMPG-PEG, PEG-PE, PEG Ceramide, DPPG-PEG, four armed PEGs, and many more. Variations are sold by Broadpharm (San Diego, CA) and CreativePEGworks (Chapel Hill, NC).
  • the stabilizing agent may be used alone or in combinations with each other.
  • the stabilizing agent comprises about 0.1 to about 10 Mol% of the overall lipid mixture, such as from about 0.1 to about 10 Mol%, from about 0.5 to about 10 Mol%, from about 1 to about 10 Mol%, from about 1.5 to about 10 Mol%, from about 2 to about 10 Mol%, from about 2.5 to about 10 Mol%, from about 3 to about 10 Mol%, from about 3.5 to about 10 Mol%, from about 4 to about 10 Mol%, from about 4.5 to about 10 Mol%, from about 5 to about 10 Mol%, from about 5.5 to about 10 Mol%, from about 6 to about 10 Mol%, from about 6.5 to about 10 Mol%, from about 7 to about 10 Mol%, from about 7.5 to about 10 Mol%, from about 8 to about 10 Mol%, from about 8.5 to about 10 Mol%, from about 9 to about 10 Mol%, from about 9.5 to about 10 Mol%, from about 0.5 to about 9 Mol%, from about 0.5 to about 8.5 Mol%, from about 9.5 to about 10 Mol%, from about 0.5 to about 9 Mol%, from about
  • lipid formulations can be substantially cholesterol-free.
  • Lipid formulations that are considered “substantially free” of cholesterol are formulations that contain either (i) 0 wt.%, or no cholesterol, or (ii) an ineffective or (iii) an immaterial amount of cholesterol.
  • An example of an ineffective amount is an amount below the threshold amount to achieve the intended purpose of using cholesterol for stability and improve cell entry of the LNP, as one of ordinary skill in the art will appreciate.
  • An immaterial amount may be, e.g., below about 1 wt.%, e.g., from about 1 wt.% or less, such as from about 0.9 wt.% or less, from about 0.8 wt.% or less, from about 0.7 wt.% or less, from about 0.6 wt.% or less, from about 0.5 wt.% or less, from about 0.4 wt.% or less, from about 0.3 wt.% or less, from about 0.2 wt.% or less, from about 0.1 wt.% or less, etc.
  • cholesterol-free lipid formulations can use cholesteryl hemisuccinate as a substitute for cholesterol.
  • lipid formulations using cholesteryl hemisuccinate as a cholesterol substitute are found in commonly owned, co-assigned International Application No. , filed on December 22, 2023, titled “LIPID NANOPARTICLE FORMULATIONS FOR CELL THERAPY AND RELATED METHODS,” Attorney Docket No. 770158, herein incorporated by reference.
  • cholesterol can be included in the lipid formulations.
  • cholesterol includes about 10 to about 50 Mol% of the overall lipid mixture, e.g., such as from about 10 to about 45 Mol%, from about 10 to about 40 Mol%, from about 10 to about 35 Mol%, from about 10 to about 30 Mol%, from about 10 to about 25 Mol%, from about 10 to about 20 Mol%, from about 10 to about 15 Mol%, from about 15 to about 50 Mol%, from about 20 to about 50 Mol%, from about 25 to about 50 Mol%, from about 30 to about 50 Mol%, from about 35 to about 50 Mol%, from about 40 to about 50 Mol%, from about 45 to about 50 Mol%, etc.
  • Cholesterol can include, in some embodiments, about 10 Mol% of the overall lipid mixture. In some embodiments cholesterol includes about 50 Mol% of the overall lipid mixture.
  • the ionizable lipid is PNI 550 at from about 30 to about 50 %Mol (e.g. 40 %Mol), the phospholipid is DSPC at from about 10 to about 30 %Mol (e.g. 20 %Mol), the stabilizer is polyoxyethylene (10) stearyl ether at from about 0.1 to about 5 %Mol (e.g. 2.5 %Mol), and cholesterol is from about 30 to about 45 %Mol (e.g. 37.5 %Mol).
  • the ionizable lipid is PNI 550 at from about 35 to about 45 %Mol (e.g.
  • the phospholipid is DSPC at from about 15 to about 25 %Mol (e.g. 20 %Mol)
  • the stabilizer is polyoxyethylene (10) stearyl ether at from about 1.5 to about 3.5 %Mol (e.g. 2.5 %Mol)
  • cholesterol is from about 35 to about 40 %Mol (e.g. 37.5 %Mol).
  • the ionizable lipid is PNI 550 at from about 30 to about 50 %Mol (e.g. 40 %Mol)
  • the phospholipid is DSPC at from about 10 to about 30 %Mol (e.g.
  • the stabilizer is TPGS at from about 0.1 to about 3 %Mol (e.g. 0.7 %Mol), and cholesterol is from about 30 to about 50 %Mol (e.g. 39.3 %Mol).
  • the ionizable lipid is PNI 550 at from about 35 to about 45 %Mol (e.g. 40 %Mol)
  • the phospholipid is DSPC at from about 15 to about 25 %Mol (e.g. 20 %Mol)
  • the stabilizer is TPGS at from about 0.25 to about 1.5 %Mol (e.g.
  • the ionizable lipid is PNI 127 or PNI 516 at from about 20 to about 40 %Mol (e.g. 28.7 %Mol)
  • the phospholipid is DSPC at from about 40 to about 60 %Mol (e.g. 49.8 %Mol
  • the stabilizer is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-200 (DMG-PEG2000) at from about 0.1 to about 3 %Mol (e.g.
  • the ionizable lipid is PNI 127 or PNI 516 at from about 25 to about 35 %Mol (e.g. 28.7 %Mol)
  • the phospholipid is DSPC at from about 45 to about 55 %Mol (e.g. 49.8 %Mol
  • the stabilizer is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-200 (DMG-PEG2000) at from about 0.5 to about 2.5 %Mol (e.g.
  • the ionizable lipid is PNI 659 at from about 30 to about 50 %Mol (e.g. 42 %Mol), the phospholipid is DSPC at from about 45 to about 65 %Mol (e.g. 56.5 %Mol), and the stabilizer is DMG-PEG2000 at from about 0.1 to about 3 %Mol (e.g. 1.5 %Mol).
  • the ionizable lipid is PNI 659 at from about 35 to about 45 %Mol (e.g.
  • the phospholipid is DSPC at from about 50 to about 60 %Mol (e.g. 56.5 %Mol)
  • the stabilizer is DMG-PEG2000 at from about 0.5 to about 2.5 %Mol (e.g. 1.5 %Mol).
  • the particle size of the lipid particles can be increased and/or decreased, in accordance with embodiments of the present disclosure.
  • the change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the NAT delivered to mammals by changing biodistribution. Size can also be used to determine target tissue, with larger particles being cleared quickly and smaller one reaching different organ systems.
  • the lipid particles of the present disclosure can be assessed for size using devices that size particles in solution, such as the Wyatt Technology NanostarTM or the MalvernTM ZetasizerTM.
  • the particles generally have a mean particle diameter of from 15nm to lOOOnm.
  • a subgroup of lipid particles is “lipid nanoparticles” or “LNP” with a mean diameter of from about 15 to about 300 nm.
  • the mean particle diameter is greater than 300 nm.
  • the lipid particle has a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less.
  • the lipid particle has a diameter of from about 50 to about 150 nm. Smaller particles generally exhibit increased circulatory lifetime in vivo compared to larger particles and can have an increased ability to reach tumor sites than larger nanoparticles. In embodiments, the lipid particle has a diameter from about 15 to about 50 nm.
  • the lipid particles according to embodiments of the present disclosure can be prepared by standard T-tube mixing techniques, turbulent mixing, trituration mixing, agitation promoting orders self-assembly, or passive mixing of all the elements with selfassembly of elements into nanoparticles.
  • LNP lipid nanoparticles
  • Microfluidic two-phase droplet techniques have been applied to produce monodisperse polymeric microparticles for drug delivery or to produce large vesicles for the encapsulation of cells, proteins, or other biomolecules.
  • the use of hydrodynamic flow focusing to create monodisperse liposomes of controlled size has also been demonstrated.
  • Parameters such as the relative lipid and NAT concentrations at the time of mixing, as well as the mixing rates are difficult to control using current formulation procedures, resulting in variability in the characteristics of NAT produced, both within and between preparations.
  • the lipid mix formulation disclosed herein is unique in that the ratio of ionizable lipid to phospholipid is surprisingly low.
  • NanoAssemblr® instruments Precision NanoSystems ULC, Vancouver, Canada
  • NanoAssemblr® instruments enable the rapid and controlled manufacture of nanomedicines (liposomes, lipid nanoparticles, and polymeric nanoparticles).
  • NanoAssemblr® instruments accomplish controlled molecular self-assembly of nanoparticles via microfluidic mixing cartridges that allow millisecond mixing of nanoparticle components at the nanoliter, microliter, or larger scale with customization or parallelization. Rapid mixing on a small scale allows reproducible control over particle synthesis and quality that is not possible in larger instruments.
  • the lipid particles are prepared by a process by which from about 75 to about 100% of the nucleic acid used in the formation process is encapsulated in the particles.
  • U.S. Pat. Nos. 9,758,795 and 9,943,846 describe methods of using small volume mixing technology and novel formulations derived thereby.
  • U.S. Pat. No. 10,159,652 describes more advanced methods of using small volume mixing technology and products to formulate different materials.
  • U.S. Pat. No. 9,943,846 discloses microfluidic mixers with different paths and wells to elements to be mixed.
  • PCT Pub. No. WO 2017117647 discloses microfluidic mixers with disposable sterile paths.
  • U.S. Pat. No. 10,076,730 discloses bifurcating toroidal micro mixing geometries and their application to microfluidic mixing.
  • W02018006166 discloses a programmable automated micromixer and mixing chips therefore.
  • U.S. Design Nos. D771834, D771833, D772427, D803416, D800335, D800336 and D812242 disclose mixing cartridges having microchannels and mixing geometries for mixer instruments sold by Precision NanoSystems ULC.
  • devices for biological microfluidic mixing are used to prepare the lipid particles.
  • the devices include a first and second stream of reagents, which feed into the microfluidic mixer, and lipid particles are collected from the outlet, or emerge into a sterile environment.
  • the first stream includes a therapeutic agent in a first solvent.
  • Suitable first solvents include solvents in which the therapeutic agents are soluble and that are miscible with the second solvent.
  • Suitable first solvents include aqueous buffers.
  • Representative first solvents include citrate and acetate buffers, or optionally other low pH buffers.
  • the second stream includes lipid mix materials in a second solvent.
  • Suitable second solvents include solvents in which the ionizable lipids according to embodiments of the present disclosure are soluble, and that are miscible with the first solvent.
  • Suitable second solvents include 1,4-di oxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, and alcohols.
  • Representative second solvents include aqueous ethanol 90%, or anhydrous ethanol.
  • a suitable device includes one or more microchannels (i.e., a channel having its greatest dimension less than 2 millimeters).
  • the microchannel has a diameter from about 15 to about 300pm, e.g., from about 20 to about 300pm, from about 25 to about 300pm, from about 50 to about 300pm, from about 75 to about 300pm, from about 100 to about 300pm, from about 125 to about 300pm, from about 150 to about 300pm, from about 175 to about 300pm, from about 200 to about 300pm, from about 225 to about 300pm, from about 250 to about 300pm, from about 275 to about 300pm, from about 15 to about 275pm, from about 15 to about 250pm, from about 15 to about 225pm, from about 15 to about 200pm, from about 15 to about 175pm, from about 15 to about 150pm, from about 15 to about 125pm, from about 15 to about 100pm, from about 15 to about 75pm, from about 15 to about 50pm, from about
  • the microchannel has a diameter from about 300 to about 1000pm, e.g., from about 350 to about 1000pm, from about 400 to about 1000pm, from about 450 to about 1000pm, from about 500 to about 1000pm, from about 550 to about 1000pm, from about 600 to about 1000pm, from about 650 to about 1000pm, from about 700 to about 1000pm, from about 750 to about 1000pm, from about 800 to about 1000pm, from about 850 to about 1000pm, from about 900 to about 1000pm, from about 950 to about 1000pm, from about 300 to about 950pm, from about 300 to about 900pm, from about 300 to about 850pm, from about 300 to about 800pm, from about 300 to about 750pm, from about 300 to about 700pm, from about 300 to about 650pm, from about 300 to about 600pm, from about 300 to about 550pm, from about 300 to about 500pm, from about 300 to about 450pm, from about 300 to about 400pm, from about 300 to about 350pm, etc.
  • At least one region of the microchannel has a principal flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the principal direction (e.g., a staggered herringbone mixer), as described in U.S. Pat.
  • compositions comprise an effective amount of the lipid formulations described herein (e.g., LNP), as well as any other components, as needed.
  • the disclosure provides a pharmaceutical composition, comprising the lipid nanoparticle as described herein and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient may generally be equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage including, but not limited to, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1 percent and 99 percent (w/w) of the active ingredient.
  • compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • excipients for formulating pharmaceutical 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 and Wilkins, Baltimore, MD, 2006).
  • any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
  • the disclosure provides a method of treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of the lipid nanoparticle described herein or the pharmaceutical composition described herein to transfect cells of hematopoietic lineage.
  • the cells of hematopoietic lineage are T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells.
  • the transfected cells are in the liver of a subject.
  • the terms “treat,” “treating,” “treatment,” “therapeutically effective,” etc. used herein do not necessarily imply 100% or complete treatment/etc.
  • a lipid nanoparticle or pharmaceutical composition described herein can provide any amount of any level of treatment.
  • the treatment provided by the disclosed method can include the treatment of one or more conditions or symptoms of the disease or condition being treated.
  • the disclosed methods comprise using an effective amount of a lipid nanoparticle or pharmaceutical composition as described herein.
  • an “effective amount” means an amount sufficient to show a meaningful benefit.
  • a meaningful benefit includes, for example, detectably treating, relieving, or lessening one or more symptoms of a disease; inhibiting, arresting development, preventing, or halting further development of a diseases; reducing the severity of a disease; preventing a disease from occurring in a subject at risk thereof but yet to be diagnosed.
  • the meaningful benefit observed can be to any suitable degree.
  • the meaningful benefit can be observed at least 10%, such as from 10% to 95%, e.g., from 10% to 90%; from 15% to 90 from 10% to 90%; from 15% to 90%; from 20% to 90%; from 25% to 90%; from 30% to 90%; from 35% to 90%; from 40% to 90%; from 45% to 90%; from 50% to 90%; from 55% to 90%; from 60% to 90%; from 65% to 90%; from 70% to 90%; from 75% to 90%; from 80% to 90%; from 85% to 90%; from 10% to 85%; from 10% to 80%; from 10% to 75%; from 10% to 70%; from 10% to 65%; from 10% to 60%; from 10% to 55%; from 10% to 50%; from 10% to 45%; from 10% to 40%; from 10% to 35%; from 10% to 30%; from 10% to 25%; from 10% to 20%; from 10% to 15%; from 15% to 85%; from 20% to 80%; from 25% to 75%; from 30% to 65%; from 45% to 60%; from 50% to 75%; from 50% to 90%; etc.
  • 10% to 95% e.
  • an effective amount of a lipid nanoparticle is from about 0.1 to about 2.5 pg per million cells of hematopoietic lineage, e.g., such as from about 0.1 to about 2.5 pg, from about 0.2 to about 2.5 pg, from about 0.3 to about 2.5 pg, from about 0.4 to about 2.5 pg, from about 0.5 to about 2.5 pg, from about 0.6 to about 2.5 pg, from about 0.7 to about 2.5 pg, from about 0.8 to about 2.5 pg, from about 0.9 to about 2.5 pg, from about 1 to about 2.5 pg, from about 1.2 to about 2.5 pg, from about 1.4 to about 2.5 pg, from about 1.5 to about 2.5 pg, from about 1.6 to about 2.5 pg,
  • an effective amount of a lipid nanoparticle is from about 0.1 to about 2.5 mg/kg of a subject, e.g., such as from about 0.1 to about 2.5 mg/kg, from about 0.2 to about 2.5 mg/kg, from about 0.3 to about 2.5 mg/kg, from about 0.4 to about 2.5 mg/kg, from about 0.5 to about 2.5 mg/kg, from about 0.6 to about 2.5 mg/kg, from about 0.7 to about 2.5 mg/kg, from about 0.8 to about 2.5 mg/kg, from about 0.9 to about 2.5 mg/kg, from about 1 to about 2.5 mg/kg, from about 1.2 to about 2.5 mg/kg, from about 1.4 to about 2.5 mg/kg, from about 1.5 to about 2.5 mg/kg, from about 1.6 to about 2.5 mg/kg, from about 1.8 to about 2.5 mg/kg, from about 2 to about 2.5 mg/kg, from about 2.2 to about 2.5 mg/kg, from about 0.1 to about 2.2 mg/kg, from about 0.1 to about 2 mg/kg, from about 0.1 to about 2.5 mg/kg, from
  • dosage will depend upon a variety of factors, including the age, condition or disease state, predisposition to disease, genetic defect or defects, and body weight of the subject.
  • the size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular active agent and the desired effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.
  • the mammal may be any suitable mammal.
  • Mammals include, but are not limited to, the order Rodentia, such as mice, and the order Lagomorpha, such as rabbits.
  • the mammal can be from the order Carnivora, including Felines (cats) and Canines (dogs).
  • the mammal can be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perissodactyla, including Equines (horses).
  • the mammal can be of the order Primates, Cebids, or Simioids (monkeys) or of the order Anthropoids (humans and apes). In some embodiments, the mammal is human.
  • the administration is in vivo.
  • the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally).
  • the pharmaceutical compositions are administered intravenously, intramuscularly, intrathecally, or intraperitoneally by a bolus injection.
  • the administration routes include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.
  • the administration is intravenous, intramuscular, intrathecal, or intraperitoneal by bolus injection. In some embodiments, the administration is intramuscular. In other embodiments, the administration is intravenous.
  • the administration is ex vivo.
  • the cells of hematopoietic lineage are (i) removed from the subject, (ii) transfected, (iii) washed, and (iv) returned to the subject.
  • this method is used for cell reprogramming, genetic restoration, or immunotherapy, for example. Examples of current cell products available commercially for immuno-oncology applications are KymriahTM for B cell precursor acute lymphoblastic leukemia and YescartaTM for use in B cell lymphoma.
  • this ex vivo therapy is CAR- T therapy, wherein modified T cells with CD19-targeted chimeric antigen receptor attacks the CD 19 presenting cancer cells of a patient.
  • blood cells are drawn from the patient, isolated as to type (T cells, for example), activated, and expanded in sterile culture, and then mixed with LNP according to the present disclosure up to about four days later.
  • the LNP may have been prepared with gene editing nucleic acid products such as CRISPR (Cas9 mRNA and sgRNA), whereafter the treated cells are assessed and expanded over days four to eleven. On day thirteen, cells are treated with chimeric antigen receptor (CAR) LNP, and the newly-created CAR-expressing cells are returned to the patient.
  • CRISPR Cas9 mRNA and sgRNA
  • the disease is a genetic disease. In accordance with some embodiments of the present disclosure, the disease is a cancer.
  • the present disclosure provides, in some embodiments, a composition for modifying human T cells with chimeric antigen receptor (CAR) encoded mRNA to produce CAR-T cell product to be infused back into the patient, without any viral means of delivery of nucleic acid.
  • the present disclosure provides a composition for delivery of gene editing nuclease and guide RNA to perform permanent gene knockout and/or insertion (when a donor DNA is also included).
  • the present disclosure provides a composition for transfecting cells involved in the hematopoietic systems including hematopoietic stem cells, natural killer cells, and antigen presenting cells.
  • the present disclosure provides a method of modulating the T cell receptors to recognize and destroy neoantigens present on the surface of the tumor cells of the patient, in accordance with some embodiments.
  • the present disclosure provides, in some embodiments, a method of modulating the expression of a target polynucleotide or polypeptide.
  • These methods generally comprise contacting a cell with a lipid particle of the present disclosure that is associated with a nucleic acid capable of modulating the expression of a target polynucleotide or polypeptide.
  • the term “modulating” refers to altering the expression of a target polynucleotide or polypeptide. Modulating can mean increasing or enhancing, or it can mean decreasing or reducing.
  • a hematopoietic stem cell is capable of differentiating into different types of cells, and is capable of dividing, unlike most circulating cells in the peripheral blood stream.
  • the term “cells of a hematopoietic lineage’” encompass hematopoietic stem and progenitor cells and any cells differentiated from hematopoietic stem and progenitor cells.
  • Bone marrow (BM) is the major site of hematopoiesis in humans and usually there are small numbers of hematopoietic stem and progenitor cells (HSPCs) in the peripheral blood (PB).
  • cytokines in particular, granulocyte colonystimulating factor; G-CSF
  • G-CSF granulocyte colonystimulating factor
  • Jurkat cells are an immortalized acute T cell lymphoma cell line available from the ATCC via Cedarlane, Burlington, Ontario in Canada. Jurkat cells are a useful model for cancers and T cells.
  • a T cell or T lymphocyte, is the principal cell type involve in cell-mediated immunity.
  • T cells have a T cell receptor on the cell surface.
  • the main categories of T cells include Helper (CD4+), Cytotoxic (CD8+), Memory, and Regulatory T cells.
  • the log phase of growth with reference to T cell cultures means the time that the cells undergo a rapid expansion, around day 5 or day 6 post activation. Log phase can be observed through a sudden increase in cell count, this rapid expansion can be used as a time point to begin preparing LNPs for T cell treatment.
  • T cells may be activated in different ways. In some embodiments, the triple activation method using anti-CD3/CD28/CD2 antibodies is used.
  • T cells may be derived or differentiated from induced pluripotent stem cells (IPSC) or Embryonic Stem Cells (ESC).
  • IPC induced pluripotent stem cells
  • ESC Embryonic Stem Cells
  • Preparation of T cells for transformation by methods of the disclosure includes, in some embodiments, one or more culture and/or preparation steps.
  • the T cells are usually isolated from biological tissue (such as peripheral blood or arterial blood) derived from a mammalian subject.
  • the subject from which the cell is isolated has a disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the cells in some embodiments are primary cells, such as primary human cells.
  • the tissue sources include but are not limited to blood, tissue, lymph, and other tissue sources taken directly from the subject, and samples resulting from one or more processing steps, such as separation, centrifugation, washing, and/or incubation.
  • the tissue source from which the T cells are derived may be a blood or a blood-derived tissue source, or an apheresis or leukapheresis product.
  • tissue sources include but are not limited to whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, lymph node, spleen, or other lymphoid tissues.
  • PBMCs peripheral blood mononuclear cells
  • Isolation of the cells may include more preparation or non-affinity based cell separation.
  • cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove or enrich for certain components.
  • Cells from the circulating blood of a subject are obtained by apheresis or leukapheresis, in some embodiments.
  • the blood cells may be washed to remove the plasma fraction and an appropriate buffer or media is used for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • a washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions (Spectrum Krosflo, GE Akta Flux, for example) in accordance with embodiments of the present disclosure.
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ++ /Mg ++ free PBS.
  • Separating the T cells from tissue sources can involve density-based cell separation methods, including the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a PercollTM or FicollTM gradient.
  • methods include the separation of different cell types based on the expression or presence in the cell of one or more specific surface markers.
  • T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and/or CD45RO + T cells, can be isolated by positive or negative selection techniques in some embodiments.
  • CD3 + , CD28 + T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
  • a CD4 + or CD8 + selection step can be used to separate CD4 + helper and CD8 + cytotoxic T cells.
  • Memory T cells are present in both CD62L + and CD62L' subsets of CD8 + peripheral blood lymphocytes. Alternatively, a selection for CD4 + helper cells may be undertaken. In some cases, naive CD4 + T lymphocytes are CD45RO', CD45RA + , CD62L + , or CD4 + T cells. In others, central memory CD4 + cells are CD62L + and CD45RO + . In still other cases, effector CD4 + cells are CD62L' and CD45RO. [0107] Cell populations can also be isolated using affinity magnetic separation techniques.
  • the cells to be separated are incubated with magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., DynabeadsTM (Clontech) or MACSTM (Miltenyi) beads).
  • the magnetically responsive material is attached to a binding partner that specifically binds to a surface marker, present on the cell, cells, or population of cells that it is desired to separate.
  • T cells may be isolated by positive or negative selection processes from tissue sources depending on preference. Kits for both are available, for example, from StemCell Technologies in Vancouver, Canada.
  • isolation or separation is carried out using an apparatus that carries out one or more of the isolation, cell preparation, separation, processing, an incubation, required to transform the T cells in accordance with embodiments of the present disclosure.
  • the system is used to carry out each of these steps in a closed or sterile environment.
  • the system is a system as described in United States Patent Pub. No. 20110003380 Al . Separation and/or other steps may be accomplished using the CliniMACS system (Miltenyi Biotec). See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1 :72-82, and Wang et al.
  • a desired cell population can be collected and enriched via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream.
  • Other methods include FACS or microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140).
  • T cell incubation and treatment may be carried out in a culture vessel, such as a chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, tank or other container for culture or cultivating cells.
  • Stimulating conditions or agents include one or more agent, such as a ligand, capable of activating an intracellular signaling domain of a TCR complex.
  • incubation may be carried out as described in U.S. Pat. No. 6,040,177 to Riddell et al.
  • T cell cultures can be expanded by adding nondividing peripheral blood mononuclear cells (PBMC) and incubating the culture.
  • PBMC peripheral blood mononuclear cells
  • the resulting population of cells contains at least about 5 PBMC feeder cells for each T lymphocyte in the initial population to be expanded, such as from 5 to 50 PBMC feeder cells, e.g., from 5 to 45 PBMC feeder cells, from 5 to 40 PBMC feeder cells, from 5 to 35 PBMC feeder cells, from 5 to 30 PBMC feeder cells, from 5 to 25 PBMC feeder cells, from 5 to 20 PBMC feeder cells, from 5 to 15 PBMC feeder cells, from 5 to 10 PBMC feeder cells, from 10 to 45 PBMC feeder cells, from 15 to 45 PBMC feeder cells, from 20 to 45 PBMC feeder cells, from 25 to 45 PBMC feeder cells, from 30 to 45 PBMC feeder cells, from 35 to 45 PBMC feeder cells, from 40 to 45 PBMC feeder cells, etc.
  • 5 to 50 PBMC feeder cells e.g., from 5 to 45 PBMC feeder cells, from 5 to 40 PBMC feeder cells, from 5 to 35 PBMC feeder cells, from 5 to 30 PBMC feeder cells
  • T cell stimulating conditions include temperatures suitable for the growth of human T lymphocytes, for example, from 25 to 37 degrees Celsius.
  • the incubation may further include a supportive population of non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells, at a ratio to initial T cells of 10 to 1.
  • LCL lymphoblastoid cells
  • the present disclosure provides a method of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the present disclosure, wherein the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA includes a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.
  • the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide
  • the siRNA, microRNA, or antisense RNA includes a polynucleotide that specifically
  • the present disclosure provides, in embodiments, a method of treating a disease or disorder characterized by under-expression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the present disclosure, wherein the therapeutic agent is selected from an mRNA, a self-amplifying RNA (saRNA), or a plasmid, includes a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof. Examples include RNA vaccines, and more particularly self-amplifying mRNA vaccines.
  • formulation of the present disclosure is delivered intramuscularly, after which immune cells can infiltrate the delivery site and process delivered RNA and/or process encoded antigen produced by non-immune cells, such as muscle cells.
  • such immune cells can include macrophages (e.g., bone marrow derived macrophages), dendritic cells (e.g., bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myeloid dendritic cells), T cells, and monocytes (e.g., human peripheral blood monocytes), etc. (for example, see W02012/006372J.
  • macrophages e.g., bone marrow derived macrophages
  • dendritic cells e.g., bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myeloid dendritic cells
  • T cells e.g., T cells
  • monocytes e.g., human peripheral blood monocytes
  • RNA is delivered with a lipid formulation of an embodiment of the present disclosure (e.g., formulated as a liposome or LNP) in accordance with embodiments of the present disclosure.
  • the invention utilizes LNPs within which immunogenencoding RNA is encapsulated by LNPs that have an aqueous core or cores, and complexed with the LNPs that have a lipid core by noncovalent interactions (e.g, ionic interactions between negatively charged RNA and cationic lipid). Encapsulation and complexation within LNPs can protect RNA from RNase digestion. The encapsulation efficiency does not have to be 100%.
  • RNA molecules e.g., on the exterior surface of a liposome or LNP
  • naked RNA molecules RNA molecules not associated with a liposome or LNP
  • at least half of the RNA molecules e.g., at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the RNA molecules
  • Some lipid nanoparticles have multilamellar components such as phospholipid bilayers and aqueous pockets.
  • immunogen RNA molecules are used for immunization purposes.
  • immunogen RNA molecules encode a polypeptide immunogen.
  • the RNA is translated in vivo and the immunogen can elicit an immune response in the recipient.
  • the immunogen may elicit an immune response against a pathogen (e.g., a bacterium, a virus, a fungus or a parasite) but, in some embodiments, it elicits an immune response against an allergen or a tumor antigen.
  • the immune response may comprise an antibody response (usually including IgG) and/or a cell mediated immune response.
  • the polypeptide immunogen will typically elicit an immune response which recognizes the corresponding pathogen (or allergen or tumor) polypeptide, but in some embodiments the polypeptide may act as a mimotope to elicit an immune response which recognizes a saccharide.
  • the immunogen will typically be a surface polypeptide, e.g., an adhesin, a hemagglutinin, an envelope glycoprotein, a spike glycoprotein, etc.
  • the RNA molecule can encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides.
  • immunogens are expressed as separate polypeptides from a replicon, then one or more of these may be provided with an upstream IRES or an additional viral promoter element.
  • multiple immunogens may be expressed from a polyprotein that encodes individual immunogens fused to a short autocatalytic protease (e.g., foot-and-mouth disease virus 2A protein), or as inteins.
  • polypeptide immunogens e.g., I, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more immunogens
  • RNA molecule such as a selfreplicating RNA, encoding one or more immunogens (either the same or different as the polypeptide immunogens).
  • the immunogen elicits an immune response against Coronavirus spp., whose immunogens include, but are not limited to, those derived from a SARS CoV-1, SARS-CoV-2(12); human influenza virus, and Neisseria meningitidis for which useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding protein.
  • a combination of three useful polypeptides is disclosed in Giuliani et al. (Proc Natl Acad Sci U S A. 2006; 103(29): 10834-9. Epub 2006/07/06. doi: 10.1073/pnas.0603940103.
  • the immunogen elicits an immune response to Chikungunya virus; in other embodiments, the immunogen elicits an immune response to Zika virus.
  • the immunogen elicits an immune response against a virus which infects fish.
  • Fungal immunogens may be derived from Dermatophytres and other opportunistic organisms.
  • the immunogen elicits an immune response against a parasite from the Plasmodium genus, such as P. falciparum, P. vivax, P. malarias, or P. ovale.
  • the invention may be used for immunizing against malaria.
  • the immunogen elicits an immune response against a parasite from the Caligidae family, particularly those from the Lepeophtheirus and Caligus genera e.g., sea lice such as Lepeophtheirus salmonis or Caligus rogercresseyi.
  • the immunogen is an mRNA specific to neoantigens in cancer cells or solid tumors.
  • the immunogen is a tumor antigen selected from: (a) cancer-testis antigens such as NY-ESO-I, SSX2, SCPI as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE- 3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUMI (associated with, e.g., melanoma),
  • tumor immunogens include, but are not limited to, pl 5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn-23HI, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, pl6, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29&BCAA), CA 195, CA 242, CA-50, CAM43, CD68&KPI, CO-029, F
  • the range of LNP diameters is 60-180 nm. In other embodiments, the range of LNP diameters is 80-160 nm.
  • An LNP can be part of a composition comprising a population of LNPs, and the LNPs within the population can have a range of diameters.
  • a composition comprising a population of LNPs with different diameters
  • at least 80% by number of the LNP have diameters in the range of 60-180 nm, e.g., in the range of 80-160 nm
  • the average diameter (by intensity, e.g., Z-average) of the population is ideally in the range of 60-180 nm, e.g., in the range of 80-160 nm
  • the diameters within the plurality have a poly dispersity index ⁇ 0.2.
  • mixing can be performed using a process in which two feed streams of aqueous RNA solution are combined in a single mixing zone with one stream of an ethanolic lipid solution, all at the same flow rate e.g., in a microfluidic channel. See other description relating to NanoAssemblr® microfluidic mixers sold by Precision NanoSystems ULC, Vancouver, Canada.
  • the delivered RNA is released and is translated inside a cell to provide the immunogen in situ.
  • the RNA is plus (“+”) stranded, so it can be translated by cells without needing any intervening replication steps such as reverse transcription.
  • the RNA is a self-replicating RNA.
  • a self-replicating RNA molecule (replicon) can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy which it generates from itself).
  • a self-replicating RNA molecule is thus in some embodiments: a (+) strand molecule that can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs.
  • These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded immunogen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the immunogen.
  • the overall result of this sequence of transcriptions is an amplification in the number of the introduced replicon RNAs and so the encoded immunogen becomes a major polypeptide product of the host cells.
  • One suitable system for achieving self-replication is to use an alphavirus-based RNA replicon.
  • These (+) stranded replicons are translated after delivery to a cell to yield a replicase (or replicase- transcriptase).
  • the replicase is translated as a polyprotein which auto cleaves to provide a replication complex which creates genomic ( — ) strand copies of the (+) strand delivered RNA.
  • These ( — ) strand transcripts can themselves be transcribed to give further copies of the (+) stranded parent RNA, and also to give a subgenomic transcript which encodes the immunogen.
  • Suitable alphavirus replicons can use a replicase from a Sindbis virus, a Semliki Forest virus, an eastern equine encephalitis virus, or more preferably, a Venezuelan equine encephalitis virus, etc.
  • the system may be a hybrid or chimeric replicase in some embodiments.
  • the system uses a PNI-V101 replicon capable of self-amplifying in mammalian cells and expressing, through mRNA assembled, immunogenic proteins such as Sars-COV-2 spike proteins and as described in WO23057979 Al by Abraham, Suraj; Chemmannur, Sijo; Geall, Andy; et al.
  • RNA molecule in some embodiments, may have a 5' cap (e.g., a 7- methylguanosine). This cap can enhance in vivo translation of the RNA.
  • the 5' nucleotide of an RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA, this may be linked to a 7-m ethylguanosine via a 5'-to-5' bridge. A 5' triphosphate can enhance RIG-I binding and thus promote adjuvant effects.
  • An RNA molecule may have a 3' polyA tail. It may also include a polyA polymerase recognition sequence (e.g., AAUAAA) near its 3' end.
  • An RNA molecule useful with the invention for immunization purposes will typically be single-stranded. Single-stranded RNAs can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKR.
  • RNA molecules in accordance with embodiments of the present disclosure, can conveniently be prepared by in vitro transcription (IVT).
  • IVT can use a (cDNA) template created and propagated in plasmid form in bacteria, or created synthetically (for example by gene synthesis and/or polymerase chain-reaction (PCR) engineering methods).
  • the self-replicating RNA can include (in addition to any 5' cap structure) one or more nucleotides having a modified nucleobase.
  • a self-replicating RNA can include one or more modified pyrimidine nucleobases, such as pseudouridine and/or 5 methylcytosine residues.
  • the RNA includes no modified nucleobases, and may include no modified nucleotides, i.e., all of the nucleotides in the RNA are standard A, C, G and U ribonucleotides (except for any 5' cap structure, which may include a 7' methylguanosine).
  • the RNA may include a 5' cap comprising a 7' methylguanosine, and the first I, 2 or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.
  • an RNA used with the invention for immunization purposes ideally includes only phosphodiester linkages between nucleosides.
  • RNAs in other embodiments, it contains phosphoramidate, phosphorothioate, and/or methylphosphonate linkages.
  • multiple species of RNAs are formulated with a lipid formulation, such as two, three, four or more species of RNA, including different classes of RNA (such as mRNA, siRNA, selfreplicating RNAs, and combinations thereof).
  • the RNA is an mRNA specific to neoantigens in cancer cells or solid tumors.
  • the RNA is an mRNA to a tumor antigen selected from: (a) cancer-testis antigens such as NY-ESO-I, SSX2, SCPI as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK
  • cancer-testis antigens such as
  • tumor immunogens include, but are not limited to, pl 5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP- 180, pl85erbB2, pl80erbB-3, c-met, mn-23HI, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, pl6, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29&BCAA), CA 195, CA 242, CA-50, CAM43, CD68&KPI, CO-029, F
  • the disclosure provides a method for screening lipid composition of lipid nanoparticles for preferential delivery of RNA to T cells, comprising: (a) preparing a plurality of lipid nanoparticles with different lipid compositions and a reporter RNA, (b) administering the lipid nanoparticles to Jurkat cells, (c) measuring the relative abundance of reporter RNA, (d) comparing the relative abundance of reporter RNA to a threshold, and (e) identifying lipid compositions above threshold as candidates for preferential delivery of RNA to T cells.
  • the different lipid compositions of step (a) comprise an ionizable lipid and a phospholipid.
  • the different lipid compositions of step (a) comprise an ionizable lipid and a phospholipid and further comprise at least one of a sterol or a stabilizing agent.
  • the different lipid compositions of step (a) vary in at least one of (i) types of components, (ii) ratios of components, or (iii) the ratio of the total lipid components.
  • the term “reporter RNA” refers to any RNA that when delivered to a cell can have its relative abundance measured.
  • the reporter RNA is an mRNA encoding a bioluminescent or biofluorescent protein.
  • the reporter RNA is an mRNA encoding a gene editing nuclease and a guide RNA targeting a reporter gene.
  • the reporter RNA is an siRNA targeting a reporter gene.
  • the reporter RNA is labelled with a contrast agent.
  • the contrast agent is a radionuclide, an iodine agent, a luminescent dye, or a fluorescent dye.
  • the relative abundance of a reporter RNA can be measured by any appropriate method known in the art.
  • the relative abundance of reporter RNA in step (c) is measured by optical imaging.
  • the threshold can be determined using any appropriate control.
  • a threshold control be determined by measuring relative abundance of reporter RNA in a control group administered with LNPs without a reporter RNA, or any other appropriate control group. Any lipid composition that demonstrates a relative abundance of reporter RNA above the threshold is identified as a candidate for preferential delivery or RNA to T cells. Candidates can be further sorted based on a measurement of the relative abundance of reporter RNA above the threshold.
  • the word “comprising” is used in a non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It will be understood that in embodiments which comprise or may comprise a specified feature or variable or parameter, alternative embodiments may consist, or consist essentially of such features, or variables or parameters. A reference to an element by the indefinite article “a” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements.
  • a lipid formulation for forming a lipid-based nanoparticle suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage comprising an ionizable lipid, a phospholipid, and optionally a stabilizer.
  • a lipid formulation comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, for forming a lipid-based nanoparticle suitable for transfecting a nucleic acid cargo into T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells.
  • nucleic acid cargo comprises chimeric antigen receptor (CAR) encoded mRNA.
  • CAR chimeric antigen receptor
  • nucleic acid cargo comprises a gene editing nuclease and a guide RNA to perform permanent gene knockout and/or insertion.
  • nucleic acid cargo comprises an mRNA encoding a protein to correct a genetic deficiency.
  • nucleic acid cargo comprises an mRNA encoding a protein to treat disease.
  • PEGylated lipid [0151 ]
  • PNI 550 at from about 30 to about 50 %Mol (e.g. 40 %Mol), the phospholipid is DSPC at from about 10 to about 30 %Mol (e.g. 20 %Mol), the stabilizer is polyoxyethylene (10) stearyl ether at from about 0.1 to about 5 %Mol (e.g. 2.5 %Mol), and cholesterol is from about 30 to about 45 %Mol (e.g. 37.5 %Mol).
  • %Mol e.g. 40 %Mol
  • the phospholipid is DSPC at from about 10 to about 30 %Mol (e.g. 20 %Mol)
  • the stabilizer is polyoxyethylene (10) stearyl ether at from about 0.1 to about 5 %Mol (e.g. 2.5 %Mol)
  • cholesterol is from about 30 to about 45 %Mol (e.g. 37.5 %Mol).
  • PNI 550 at from about 35 to about 45 %Mol (e.g. 40 %Mol)
  • the phospholipid is DSPC at from about 15 to about 25 %Mol (e.g. 20 %Mol)
  • the stabilizer is polyoxyethylene (10) stearyl ether at from about 1.5 to about 3.5 %Mol (e.g. 2.5 %Mol)
  • cholesterol is from about 35 to about 40 %Mol (e.g. 37.5 %Mol).
  • 01651 The lipid formulation of aspect 1 or 2, wherein the ionizable lipid is
  • PNI 550 at from about 30 to about 50 %Mol (e.g. 40 %Mol)
  • the phospholipid is DSPC at from about 10 to about 30 %Mol (e.g. 20 %Mol)
  • the stabilizer is TPGS at from about 0.1 to about 3 %Mol (e.g. 0.7 %Mol)
  • cholesterol is from about 30 to about 50 %Mol (e.g. 39.3 %Mol).
  • PNI 550 at from about 35 to about 45 %Mol (e.g. 40 %Mol)
  • the phospholipid is DSPC at from about 15 to about 25 %Mol (e.g. 20 %Mol)
  • the stabilizer is TPGS at from about 0.25 to about 1.5 %Mol (e.g. 0.7 %Mol)
  • cholesterol is from about 35 to about 45 %Mol (e.g. 39.3 %Mol).
  • PNI 127 or PNI 516 at from about 20 to about 40 %Mol (e.g. 28.7 %Mol)
  • the phospholipid is DSPC at from about 40 to about 60 %Mol (e.g. 49.8 %Mol
  • the stabilizer is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-200 (DMG-PEG2000) at from about 0.1 to about 3 %Mol (e.g. 1.5 %Mol)
  • cholesterol is from about 10 to about 30 %Mol (e.g. 20 %Mol).
  • PNI 127 or PNI 516 at from about 25 to about 35 %Mol (e.g. 28.7 %Mol)
  • the phospholipid is DSPC at from about 45 to about 55 %Mol (e.g. 49.8 %Mol
  • the stabilizer is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-200 (DMG-PEG2000) at from about 0.5 to about 2.5 %Mol (e.g. 1.5 %Mol)
  • cholesterol is from about 15 to about 25 %Mol (e.g. 20 %Mol).
  • the phospholipid is DSPC at from about 45 to about 65 %Mol (e.g. 56.5 %Mol)
  • the stabilizer is DMG-PEG2000 at from about 0.1 to about 3 %Mol (e.g. 1.5 %Mol).
  • the phospholipid is DSPC at from about 50 to about 60 %Mol (e.g. 56.5 %Mol)
  • the stabilizer is DMG-PEG2000 at from about 0.5 to about 2.5 %Mol (e.g. 1.5 %Mol).
  • a lipid formulation for forming a lipid-based nanoparticle suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, wherein the formulation is substantially free of cholesterol.
  • a lipid formulation comprising an ionizable lipid, a phospholipid, and optionally a stabilizer, for forming a lipid-based nanoparticle suitable for transfecting a nucleic acid cargo into T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells, wherein the formulation is substantially free of cholesterol.
  • nucleic acid cargo comprises chimeric antigen receptor (CAR) encoded mRNA.
  • CAR chimeric antigen receptor
  • nucleic acid cargo comprises a gene editing nuclease and a guide RNA to perform permanent gene knockout and/or insertion.
  • nucleic acid cargo comprises an mRNA encoding a protein to correct a genetic deficiency.
  • nucleic acid cargo comprises an mRNA encoding a protein to treat disease.
  • stabilizer comprises from about 0.1 to about 10.0 %Mol.
  • lipid formulation of any of aspects 35-52, wherein the ionizable lipid is selected from the group consisting of (Z)-3-(2-((l,17-bis(2- octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl 4- (dimethylamino)butanoate (PNI 516), 3-(2-((l,17-bis(2-octylcyclopropyl)heptadecan-9- yl)oxy)-2-oxoethyl)cyclopentyl 4-(dimethylamino)butanoate (PNI 550), Z)-3-(2-((l, 17- bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl
  • a lipid nanoparticle comprising the lipid formulation of any of aspects
  • a lipid nanoparticle comprising the lipid formulation of aspect 1-2 or
  • the lipid nanoparticle of aspect 60 or 61 further comprising a peptide or polypeptide.
  • nucleic acid cargo comprises two or more nucleic acids.
  • a pharmaceutical composition comprising the lipid nanoparticle of any one of aspects 60-62 and a pharmaceutically acceptable excipient.
  • a vaccine comprising the lipid formulation of any of aspects 3-34 or
  • nucleic acid cargo is a nucleic acid vaccine element.
  • nucleic acid vaccine element encodes an antigen selected from coronavirus spike protein or influenza hemagglutinin protein.
  • nucleic acid vaccine element is derived from influenza virus.
  • nucleic acid vaccine element is derived from coronavirus.
  • nucleic acid vaccine element encodes an antigen to cancer cells or solid tumors.
  • a method of treating or preventing a disease in a subject comprising administering to the subject an effective amount of the lipid nanoparticle of any of aspects 60-62 or the pharmaceutical composition of aspect 69 or the vaccine of any of aspects 70-74 to transfect cells of hematopoietic lineage.
  • nucleic acid cargo is a
  • RNA encoding a chimeric antigen receptor [0221 ] (85) The method of any of aspects 82-84, wherein an effective amount of a lipid nanoparticle is from about 0.1 to about 2.5 pg per million cells of hematopoietic lineage.
  • a method for screening lipid composition of lipid nanoparticles for preferential delivery of RNA to T cells comprising: (a) preparing a plurality of lipid nanoparticles with different lipid compositions and a reporter RNA, (b) administering the lipid nanoparticles to Jurkat cells, (c) measuring the relative abundance of reporter RNA, (d) comparing the relative abundance of reporter RNA to a threshold, and (e) identifying lipid compositions above threshold as candidates for preferential delivery of RNA to T cells.
  • the different lipid compositions of step (a) comprise an ionizable lipid and a phospholipid.
  • step (a) The method of aspect 91, wherein the different lipid compositions of step (a) further comprise at least one of a sterol or a stabilizing agent.
  • step (a) The method of any of aspects 90-92, wherein the different lipid compositions of step (a) vary in at least one of (i) types of components, (ii) ratios of components, or (iii) the ratio of the total lipid components.
  • reporter RNA is an mRNA encoding a bioluminescent or biofluorescent protein.
  • reporter RNA is an mRNA encoding a gene editing nuclease and a guide RNA targeting a reporter gene.
  • the contrast agent is a radionuclide, an iodine agent, a luminescent dye, or a fluorescent dye.
  • GFP green fluorescent protein
  • MFI Median Fluorescence Intensity
  • PBS phosphate buffered saline
  • Gene of interest signifies a genetic element or elements intended for expression to achieve a therapeutic goal, including immunization.
  • A5 SARS Cov-2 antigenic coding elements and epidermal growth factor (EPO) are examples of a GOI to illustrate the present invention, but GOI is not limited to these demonstrated examples.
  • EPO epidermal growth factor
  • GOI might be a group of nucleic acid tools including Cas-9, sgRNA, and a replacement gene like PCSK9 and ANGPTL3 for familial hypercholesterolemia, TTR for transthyretin amyloidosis, DMD for Duchenne muscular dystrophy, KLKB1 for hereditary angioedema, for example.
  • Lipid Mixes include the ionizable lipid, phospholipid, and stabilizing agent.
  • Low pH buffers (3-6) may be used.
  • the pH of the buffer is typically below the pKa of the lipid.
  • iL ionizable lipid, a lipid that is cationic at higher pH, and converts to uncharged at lower pH.
  • Non-limiting examples include:
  • Ionizable lipids disclosed in PCT Publication WO20252589 by Jain et al. are:
  • PNI 516 is ionizable lipid (Z)-3-(2-((l,17-bis(2-octylcyclopropyl)heptadecan-9- yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl 4-(dimethylamino)butanoate;
  • PNI 560 is ionizable lipid (Z)-3-(2-((l,17-bis(2-octylcyclopropyl)heptadecan-9- yl)oxy)-2-oxoethyl)-2-(pent-2-en-l-yl)cyclopentyl l,4-dimethylpiperidine-4-carboxylate; and
  • PNI 550 is 3-(2-((l,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2- oxoethyl)cyclopentyl 4-(dimethylamino)butanoate.
  • Ionizable lipids disclosed in PCT Publication W02100041 by Thomas et al. are:
  • PNI 127 is (2R,3S,4S)-2-(((l,4-dimethylpiperidine-4- carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl (9Z,9'Z, 12Z, 12'Z)-bis(octadeca-9, 12- dienoate);
  • PNI 580 is (2R,3S,4S)-2-(((4-
  • PNI 660 is ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl l,4-dimethylpiperidine-4-carboxylate;
  • PNI 659 is ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl
  • PNI 721 is (2R,3S,4S)-2-(((2-
  • PNI 722 is 2-(((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2- yl)methoxy)-N,N-dimethylethan- 1 -amine;
  • PNI 723 is ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl
  • PNI 726 (2R,3S,4S)-2-((3-(dimethylamino)propoxy)methyl)tetrahydrofuran-3,4- diyl bis(2-hexyldecanoate);
  • PNI 728 is ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl
  • PNI 730 is (2R,3S,4S)-2-((2-(dimethylamino)ethoxy)methyl)tetrahydrofuran-3,4- diyl bis(2-hexyldecanoate).
  • SCR712 (contains both TagRFP RNA & B18R RNA) (Millipore Sigma Canada, Oakville Ontario).
  • eGFP mRNA made in house 5' Cap: m7GpppGm (vaccinia capping method), 5'UTR: Synthetic UTR, CDS: eGFP, 3’UTR: human alpha globin subunit 1 (HBA1), polyA tail: 45 nucleotides long sequence
  • AGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGA CCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAACCGCGGUGA UAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCA GCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUG
  • Cas9 mRNA was obtained from TriLink Biotechnologies, SKU: L-7606-100, and
  • TCR 1+3 sgRNA was obtained from Integrated DNA Technologies, Coralville, Iowa.
  • RNA or self-amplifying mRNA as described above was diluted using sodium acetate buffer to the required concentration.
  • Lipid nucleic acid particle (LNAP) samples were then prepared by running both fluids using the NanoAssemblr® Spark instrument. Briefly, 10-20pg of nucleic acids in 100 mM sodium acetate buffer in a total volume of 32pL was mixed with 16pL of 37.5 mM lipid mix solution as required by the N/P ratios (4, 6, 8, 10 or 12 as described in illustrated examples).
  • LNAP microfluidically mixed lipid nucleic acid particles
  • Lipid mix formulation of lipid particles were generated by rapidly mixing lipidethanol solution with an aqueous buffer inside a microfluidic mixer designed to induce chaotic advection and provide a controlled mixing environment.
  • the microfluidic channels have herringbone features or were configured in a manner as shown in PCT Pub. No. WO2017117647 or U.S. Patent No. 10,835,878.
  • the post cartridge lipid nucleic acid particle sample was diluted by into RNAse free tubes containing three to 40 volumes of phosphate buffered saline (PBS) buffer, pH 7.4. Ethanol was finally removed using AmiconTM centrifugal filters (Millipore, USA) at 3000 RPM, or using TFF systems. Once the required concentration was achieved, the lipid nucleic acid particles were filter sterilized using 200pm filters in aseptic conditions. Final encapsulation efficiency was measured by a modified RibogreenTM assay.
  • PBS phosphate buffered saline
  • DLS Dynamic Light Scattering
  • PDI indicates the width of the particle distribution. This is a parameter calculated from a cumulative analysis of the (DLS)-measured intensity autocorrelation function assuming a single particle size mode and a single exponential fit to the autocorrelation function. From a biophysical point of view, a PDI below 0.1 indicates that the sample is monodisperse. A lower PDI indicates a more homogenous population of lipid particles. 0284] Following mixing in the microfluidic device, the post cartridge lipid nucleic acid particle sample was diluted into RNAse free tubes containing three volumes of PBS, pH 7.4.
  • Ethanol was finally removed through either dialysis in PBS, pH 7, or using AmiconTM centrifugal filters (Millipore, USA) at 3000 RPM, or using TFF systems. Once the required concentration was achieved, the lipid nucleic acid particles were filter sterilized using 0.2pm filters in aseptic conditions. Final encapsulation efficiency was measured by the Ribogreen® assay. Quant-iTTM RiboGreen® RNA Reagent and Kit (Invitrogen) following manufacturer directions. Self-amplifying mRNA plasmid NAT preparation is described below. Observed particle attributes were generally sized from 50 - 200nm for mRNA, depending on lipid composition.
  • This example demonstrates exemplary benefits of the criticality of formulation in various cell types according to principles of embodiments of the disclosure. Specifically, this example demonstrates the suitability of Jurkat cells as a model for assessing T cell transfection of lipid nanoparticles with different lipid formulations.
  • T cells are described above.
  • Jurkat cells are an immortalized T cell model governed by ATCC rules. The inventors discovered that, surprisingly and unexpectedly, transfection in Jurkat cells correlates well with transfection in T cells and with Jurkat cells being much easier to culture, the Jurkat cells have been useful in prescreening.
  • HSC Hematopoietic stem cells
  • BHK cells Baby hamster kidney fibroblasts
  • LNP with payload as well as control were added to HSC four days after stimulation or, for the T cells or Jurkat typically three days after expansion.
  • Staining was performed as follows. On day of detection, the 96-well U bottom plate containing cells were centrifuged at 300xg for 5 minutes at room temperature. Cells were the resuspended in 200ul of 1 : 1000 FVS660v in PBS, except Unstained and GFP only wells. The cells were incubated at RT in the dark for lOminutes.
  • V46, V47, V57 or V22 LNP comprising PNI 516, PNI 659 or PNI 550 ionizable lipids, with mRNA (GFP) concentrations of 84 pg/mL and N/P ratio of 8, the size, poly dispersity index (PDI) and encapsulation efficiency (%EE) were studied.
  • the half maximal effective concentration (ECso) is shown in Table 2.
  • V57 PNI 659 performed the best of all of these.
  • Table 2 depicts the ECso of LNP with different ionizable lipids.
  • V57 PNI 659 showed the lowest ECso suggesting it would be the most effective at transfecting T cells.
  • This example demonstrates the effect of ionizable lipid selection on transfection of baby hamster kidney cell line, Jurkat cell line, and primary T cells according to principles of embodiments of the disclosure. Specifically, this example demonstrates V57 LNPs with at least PNI 659, 721, and 722 can successfully transfect Jurkat cells.
  • T cells On Day 4 after re-activation of human primary CD3+ T cells (treatment media: Serum Free Media containing 100 ng/mL cytokine IL-2 and 1 pg/mL ApoE4), T cells were contacted with serial dilution of LNP starting at 0.5 pg per one million T cells. Batches were made on a NanoAssemblr® Ignite microfluidic mixer. All formulations were made with GFP mRNA at 84 pg/mL (N/P 8) in triplicate, and comprised the V57 ratio and varying ionizable lipids. RibogreenTM data confirmed that the final concentration of nucleic acid was from 22 to 29.9 p/ml. Detection was performed 24h post LNP treatment by flow cytometer. Potency was assessed by titration starting at 0.5 micrograms per million cells and ending at 2.5 micrograms per million cells (5 points).
  • treatment media Serum Free Media containing 100 ng/mL cytokine
  • Table 4 A depicts the effects of various ionizable lipids on survival of primary T cells, nucleic acid cargo expression in baby hamster kidney cells, Jurkat cells, and primary T cells, as well as the size, PDI, and % EE in of the LNPs. Not potent results are labeled as “NP,” while “N/A” is used for any value where a technical issue was experienced.
  • Table 4A V02 and V57 in BHK, Jurkat and Primary T Cells, Varying Ionizable Lipid
  • V57 LNPs surprisingly, do not reduce the viability of primary T cells, and demonstrate high transfection rates, particularly V57 PNI 516 and V57 PNI 659. This suggests V57 LNPs could be useful for cell therapy without impacting T cell survival.
  • Table 4B depicts the effect of different ionizable lipids on V57 LNPs size, PDI, %EE, and percentage of Jurkat cells expressing the nucleic acid cargo.
  • V57 mixes with different ionizable lipids, percent positive in Jurkat cells
  • V57 LNPs had encapsulated over 80% of the nucleic acid cargo and resulted in nucleic acid cargo expression in at least some of the Jurkat cells.
  • the LNPs with PNI 659, 721, and 722 resulted in over 90% of Jurkat cells expressing the nucleic acid cargo. This suggests V57 LNPs with at least PNI 659, 721, or 722 can successfully target and transfect T cells.
  • Table 4C depicts the effect of different lipid mix formulation ratios of PNI-659, DSPC, and DMG-PEG on the percentage of T cells expressing the nucleic acid cargo at different concentrations.
  • Table 4C LNP Efficacy with different formulations and concentrations in T cells [03031 As seen in Table 4C, all the LNPs induced nucleic acid cargo expression in at least some of the T cells. In particular, the V57 and V99 LNPs induced expression of the nucleic acid cargo in at least 75% of the T cells at 2.0 pg/ml. This further supports the data shown in Table 4B and demonstrates that these LNPs, in particular V57 LNPs, can successfully transfect T cells.
  • This example demonstrates the transfection of hematopoietic stem cells and T cells according to principles of embodiments of the disclosure. Specifically, this example demonstrates LNPs described herein can successfully transfect HSC and T cells.
  • HSC are difficult to transfect while preserving their viability. They can be isolated using reagents and starting materials purchased from, for example, Stemcell Technologies in Vancouver, Canada, or enriched for from whole blood.
  • FIG. 1 A shows that all the LNPs were able to transfect HSC cells, with the highest transfection efficiency at a concentration of 0.5 pg / million cells.
  • FIG. IB demonstrates that expression level of the nucleic acid cargo, GFP, increased with increasing concentration, with the concentration of 0.5 pg / million cells showing the highest levels of expression.
  • Table 5 depicts the transduction efficiency (%TE) of different LNPs in HSC and T cells using mRNA GFP as a nucleic acid cargo.
  • This example demonstrates the transfection in natural killer cells 24 h post transfection according to principles of embodiments of the disclosure. Specifically, this example demonstrates LNPs described herein can successfully transfect natural killer cells.
  • Natural Killer cells were thawed, and activator was added prior to incubation at 37 degrees C for 72 hours. On day 4, cells were resuspended an ApoE was added. Cells were seeded in a plate to 6.25 X 10' 5 cells per mL. LNPs were then added at 0.5ug/RNA per 1 million cells and incubated for 24h. On day 5, reading plates were shaken to resuspend the cells. An aliquot of each sample was removed for Calcein staining. After 10 minutes of incubation, the cells were analyzed for GFP expression. V57 showed 60% expression, thus V57 successfully transfected natural killer cells. Data is shown in Table 6 and 7 for eGFP expression, and in Table 8 for cell viability.
  • the LNPs surprisingly maintained cell viability for over 50% of the natural killer cells up to 7 days after transfection. These results demonstrate that these LNPs could be effective for cell therapies targeting natural killer cells due to their transfection ability and effects on viability.
  • This example demonstrates gene editing enabled by compositions according to principles of embodiments of the disclosure. Specifically, this example demonstrates that certain LNPs described herein successfully deliver gene editing nucleic acid cargos to T cells.
  • Cryopreserved primary human pan T cells were thawed and activated. LNPs were produced with RNA encapsulation efficiency assessed to determine dosing. On day 4, activated T cells were incubated with the gene editing RNA-LNPs Cas9 and sgRNA. From days 4-11, cells were expanded to increase cell numbers for subsequent experiments. On day 13, gene-edited cells were treated with CAR mRNA-optimally while editing with V57- PNI-516 shows modest activity.
  • Table 9 depicts ECso of gene editing nucleic acid cargo in different LNP formulations in primary T cells. Payload was mRNA-GFP and Cas-9/guide RNA were included.
  • FIG. 2 shows the effect of the concentration of the nucleic acid cargo on percentage of cells with TCR knockout (TCR KO) in the different LNPs.
  • TCR KO TCR knockout
  • Table 9 ECso in pg/mL for the TCR Gene editing data in primary T cells.
  • V57 PNI 659 was the best performer for achieving successful gene editing via LNP delivery, followed by V22 PNI 550. This demonstrates that these LNPs can successfully transfect T cells with gene editing cargos, suggesting they could be useful for editing specific genes in cell therapies.
  • This example demonstrates the results of LNP versus electroporation with mRNA eGFP payloads according to principles of embodiments of the disclosure. Specifically, this example demonstrates that certain LNPs described herein had improved transfection efficiency, nucleic acid cargo expression, and cell viability compared to electroporation.
  • mRNA was diluted in PBS and stored on ice. For each condition, 10 5 cells were electroporated at a dose of 0.5 or 2 pg mRNA/ million cells and Mock (no mRNA) was included in each experiment as a control. Cell pellets were diluted in the nucleofection buffer as per the instruction in the nucleofection kit, Cat.#. V4XP-3012 (Lonza).
  • the cells were electroporated using the Lonza 4D-NucleofectorTM X unit instrument, cell type program “T cell, human, stim” pulse code EH 140, immediately diluted in lOOuL culture media, and aliquoted into a 96 well plate and incubated at 37°C/5% CO2 for 24 hours. Next day cells were stained for live/dead discrimination, measured on the CytoFLEXTM flow cytometer (Beckman Coulter), then analyzed using FlowJoTM V10.7 software (BD Research).
  • T cell complete media was added and the samples were transferred to a 6 well plate.
  • Activator was removed at day 4, TCR KO Detection with Flow Cytometry was performed on day 7 or 8.
  • V57 PNI 659 showed better transfection efficiency than electroporation (payload was GFP, FIG. 3 A).
  • payload was GFP, FIG. 3 A.
  • mRNA delivery by electroporation appears to be less effective when measured by expression (GFP MFI).
  • GFP MFI mRNA delivery by electroporation appears to be less effective when measured by expression
  • FIG. 3C all three LNP formulations were less detrimental to cell viability compared to electroporation.
  • LNP comprising GFP mRNA payload in V57-PNI-516, V57-PNI-659 and V22-PNI-550 LNP were studied at 0.5 and 2 ug/million cells for effect on cell count and cell viability at 24 hours post treatment with untreated cells as a control. Electroporation was run for no payload, 0.5 and 2.0 ug/million cells. Cell count is shown in FIG. 4A, percent viability in FIG. 4B.
  • This example demonstrates transfection efficiency of antigen presenting cells according to principles of embodiments of the disclosure. Specifically, this example demonstrates how culture time prior to transfection, cell maturation, LNP lipid formulation, LNP concentration, and the duration of culture post transfection effected transfection in antigen presenting cells.
  • 03281 First Variable Culture time prior to transfection over four different conditions.
  • monocytes previously isolated from a LeukopakTM and cryopreserved were thawed and resuspended in ImmunoCult- ACFTM Dendritic Cell Medium (STEMCELL), supplemented with 1% ImmunoCultTM- ACF Dendritic Cell Differentiation Supplement (STEMCELL), or “APC Media.”
  • the cells were evaluated with an NC200 NucleocounterTM instrument and seeded onto T-25 flasks at a concentration of 1.0E+06 cells/mL with 5 mL per flask.
  • D3 three-day culture
  • Second Variable Cell Maturation.
  • LNP formulation used for transfection V46 PNI 516, V47 PNI 516, V47 PNI 659, V57 PNI 659, and V22 PNI 550 and the effect of concentration of the different formulations on transfection efficiency.
  • the various cell suspensions with ApoE4 were transferred into 96-well U-bottom TC-treated plates (Falcon) in aliquots of 125 pL/well.
  • the diluted LNPs were transferred to the plates containing the various cell culture conditions for a total well volume of 250 pL/well. Following LNP addition, the plates were incubated at 37°C at 5% CO2.
  • Viability was reported as % cells positive for Calcein Red-Orange. Percent GFP was reported from % cells positive for FITC, and MFI was calculated as Median FITC-A+ (FITC- A)/ Average Unstained Median Single Cells (FITC-A).
  • Table 10A depicts the different set up conditions for the dendritic cell LNP transfection evaluation described in this example.
  • Table 10A Variable Set-Up for Dendritic cell LNP Transfection Evaluation [0337] Two hundred conditions were evaluated. Table 10B depicts culture workflow for different conditions.
  • Table 11 depicts culture conditions and cell density. The different culture conditions were resuspended at various concentrations, and the variable representing the culture day varied both in the cell concentration seeded at the time of transfection as well as the LNP concentration.
  • Table 12 depicts the intended LNP concentration and the calculated LNP concentration based on the number of seeded cells for different culture conditions. Table 12. Recalculated LNP Concentrations Based on Seeded Cell Numbers.
  • EXAMPLE 8 0340 This example demonstrates the transfection efficiency of natural killer cells (NK) using various lipid nanoparticle (LNP) compositions according to principles of embodiments of the disclosure.
  • the assay protocol was set up to evaluate five variables with different conditions evaluated across each variable (See Table 13).
  • the first variable defined the media used for culturing the NK cells. Three different media were tested during the experiment and their effect on LNP transfection of the cells was evaluated. The first media condition, consisted of XVIVO 15 Serum Free Hematopoietic Cell Medium (Lonza) with 10% Heat Inactivated Human AB Serum (GeminiBio), 1% GlutamaxTM (Gibco), IL-2, IL- 12, IL-21 (STEMCELL), IL- 15 (Cytiva), and IL- 18 (R&D Systems).
  • the second media condition was composed of RPMI 1640 (ATCC), 10% Heat Inactivated Human AB Serum (GeminiBio) 1% GlutamaxTM (Gibco), IL-2, IL- 12, IL-21 (STEMCELL), IL- 15 (Cytiva), and IL- 18 (R&D Systems).
  • the final media condition (“SC”) used the ImmunoCultTM NK Cell Expansion Kit (STEMCELL), which included a coating material applied to all culture plates prior to cell seeding.
  • the second variable was defined as culture time prior to transfection, with five different conditions evaluated. On the day of transfection, variable three was introduced in the form of the LNP formulation used for transfection: V46 PNI 516, V47 PNI 516, V47 PNI 659, V57 PNI 659, and V22 PNI 550. The fourth variable was concentration of the different formulations: 0.25, 0.5, 1.0, and 2.0 pg/L0E+06 cells. [0344] The lipid nanoparticles were thawed on ice and diluted to the desired concentrations with the three corresponding media in 1.5 mL Eppendorf tubes.
  • the cells were and resuspended in their respective culture conditions to a final concentration of 5.0E+05 cells/mL or 2.0E+04 cells/mL (because of cell culture density).
  • ApoE4 (Peprotech) in PBS was suspended to 100 pg/mL and added to the resuspended cell suspension for a final concentration of 2 pg/mL, then transferred into 96-well U-bottom TC-treated plates (Falcon) at 125 pL/well.
  • the diluted LNPs were then transferred to the plates containing the various cell culture conditions for a total well volume of 250 pL/well. Following LNP addition, the plates were incubated at 37°C and 5% CO2.
  • the fifth variable was duration of culture post transfection: 1 day and 2 days.
  • a solution of CellTraceTM Calcein Red-Orange, AM was used as an indicator of cell viability.
  • the indicator powder was resuspended in Dimethyl sulfoxide (DMSO) to stock solution.
  • DMSO Dimethyl sulfoxide
  • the stock was then further diluted in Flow Cytometry Staining Buffer (FACS, eBioscience) to 7.5 pM.
  • FACS Flow Cytometry Staining Buffer
  • the culture plates were removed from the incubator, the contents gently resuspended by pipetting, and an aliquot of 100 pL was removed from each well and transferred to a new 96-well U-bottom plate.
  • Table 13 depicts the different set up conditions for the dendritic cell LNP transfection evaluation described in this example.
  • Tables 15-19 depict the cell viability and GFP expression levels at 24 and 48 hours.
  • V57 PNI 659 and V22 PNI-550 were better at transfecting APC in this study. This demonstrates a high level of transfection efficiency in these LNPs, suggesting they could be useful for cell therapies targeting APCs.
  • V57 PNI 659 performs the best at 48 H. APC culture was difficult as a whole, but V57 still outperformed the other compositions. V46 also showed promise in this application. This demonstrates that APCs transfected by these LNPS still express detectable levels of the nucleic acid cargo up to 7 days after transfection, suggesting these LNPs could be useful for cell therapies targeting APCs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pulmonology (AREA)
  • Biomedical Technology (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Dermatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne une formulation lipidique qui est sensiblement exempte de cholestérol et qui est mélangée avec de l'acide nucléique pour former des nanoparticules d'acide nucléique lipidique qui sont capables de transfecter des cellules du système immunitaire d'une manière moins destructive que l'électroporation.
PCT/US2023/085851 2023-06-28 2023-12-22 Formulations de nanoparticules lipidiques pour thérapie cellulaire et procédés associés WO2025005990A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
USPCT/US2023/069303 2023-06-28
PCT/US2023/069303 WO2024006863A1 (fr) 2022-06-30 2023-06-28 Formulations de nanoparticules lipidiques pour vaccins

Publications (1)

Publication Number Publication Date
WO2025005990A1 true WO2025005990A1 (fr) 2025-01-02

Family

ID=89772120

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2023/085851 WO2025005990A1 (fr) 2023-06-28 2023-12-22 Formulations de nanoparticules lipidiques pour thérapie cellulaire et procédés associés
PCT/US2023/085852 WO2025005991A1 (fr) 2023-06-28 2023-12-22 Formulations de nanoparticules lipidiques pour thérapie cellulaire et procédés associés

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US2023/085852 WO2025005991A1 (fr) 2023-06-28 2023-12-22 Formulations de nanoparticules lipidiques pour thérapie cellulaire et procédés associés

Country Status (1)

Country Link
WO (2) WO2025005990A1 (fr)

Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5753613A (en) 1994-09-30 1998-05-19 Inex Pharmaceuticals Corporation Compositions for the introduction of polyanionic materials into cells
US6040177A (en) 1994-08-31 2000-03-21 Fred Hutchinson Cancer Research Center High efficiency transduction of T lymphocytes using rapid expansion methods ("REM")
US6734171B1 (en) 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
US20040262223A1 (en) 2001-07-27 2004-12-30 President And Fellows Of Harvard College Laminar mixing apparatus and methods
WO2009016515A2 (fr) 2007-08-01 2009-02-05 Novartis Ag Compositions comprenant des antigènes pneumococciques
WO2010033140A2 (fr) 2008-05-06 2010-03-25 Innovative Micro Technology Appareil amovible/jetable pour dispositif de tri de particules de mems
US20110003380A1 (en) 2007-12-07 2011-01-06 Stefan Miltenyi Sample Processing System and Methods
WO2011005799A2 (fr) 2009-07-06 2011-01-13 Novartis Ag Molécules d'arn autorépliquantes et leurs utilisations
US7901708B2 (en) 2002-06-28 2011-03-08 Protiva Biotherapeutics, Inc. Liposomal apparatus and manufacturing methods
WO2012006372A1 (fr) 2010-07-06 2012-01-12 Novartis Ag Administration d'arn à différents types cellulaires
WO2017117647A1 (fr) 2016-01-06 2017-07-13 The University Of British Columbia Mélangeurs à bifurcation et leurs procédés d'utilisation et de fabrication
US9758795B2 (en) 2009-11-04 2017-09-12 The University Of British Columbia Nucleic acid-containing lipid particles and related methods
WO2018006166A1 (fr) 2016-07-06 2018-01-11 Precision Nanosystems Inc Instrument de mélange microfluidique intelligent et cartouches
US9943846B2 (en) 2011-10-25 2018-04-17 The University Of British Columbia Limit size lipid nanoparticles and related methods
US10159652B2 (en) 2013-10-16 2018-12-25 The University Of British Columbia Device for formulating particles at small volumes
WO2020252375A1 (fr) * 2019-06-14 2020-12-17 Dnalite Therapeutics, Inc. Compositions et procédés pour véhicules d'administration biologiques
WO2020252589A1 (fr) 2019-06-20 2020-12-24 Precision Nanosystems Inc. Lipides ionisables pour administration d'acides nucléiques
WO2021000041A1 (fr) 2019-06-29 2021-01-07 Precision Nanosystems Inc. Lipides ionisables pour l'administration d'acides nucléiques
WO2021216577A1 (fr) * 2020-04-20 2021-10-28 Board Of Regents, The University Of Texas System Systèmes d'administration de nanoparticules lipidiques (lnp) et leurs utilisations
WO2022101486A1 (fr) * 2020-11-16 2022-05-19 BioNTech SE Compositions pharmaceutiques comprenant des particules et de l'arnm et leurs procédés de préparation et de stockage
WO2023001952A1 (fr) * 2021-07-21 2023-01-26 Universidad De Granada Nanoparticules de phosphate de calcium chargées de jasmonate pour induire des réponses efficaces de défense des plantes
WO2023057979A1 (fr) 2021-10-07 2023-04-13 Precision NanoSystems ULC Nanoparticules lipidiques de vaccin à arn

Patent Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6040177A (en) 1994-08-31 2000-03-21 Fred Hutchinson Cancer Research Center High efficiency transduction of T lymphocytes using rapid expansion methods ("REM")
US5753613A (en) 1994-09-30 1998-05-19 Inex Pharmaceuticals Corporation Compositions for the introduction of polyanionic materials into cells
US6734171B1 (en) 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
US20040262223A1 (en) 2001-07-27 2004-12-30 President And Fellows Of Harvard College Laminar mixing apparatus and methods
US7901708B2 (en) 2002-06-28 2011-03-08 Protiva Biotherapeutics, Inc. Liposomal apparatus and manufacturing methods
WO2009016515A2 (fr) 2007-08-01 2009-02-05 Novartis Ag Compositions comprenant des antigènes pneumococciques
US20110003380A1 (en) 2007-12-07 2011-01-06 Stefan Miltenyi Sample Processing System and Methods
WO2010033140A2 (fr) 2008-05-06 2010-03-25 Innovative Micro Technology Appareil amovible/jetable pour dispositif de tri de particules de mems
WO2011005799A2 (fr) 2009-07-06 2011-01-13 Novartis Ag Molécules d'arn autorépliquantes et leurs utilisations
US9758795B2 (en) 2009-11-04 2017-09-12 The University Of British Columbia Nucleic acid-containing lipid particles and related methods
WO2012006372A1 (fr) 2010-07-06 2012-01-12 Novartis Ag Administration d'arn à différents types cellulaires
US9943846B2 (en) 2011-10-25 2018-04-17 The University Of British Columbia Limit size lipid nanoparticles and related methods
US10159652B2 (en) 2013-10-16 2018-12-25 The University Of British Columbia Device for formulating particles at small volumes
US10835878B2 (en) 2016-01-06 2020-11-17 The University Of British Columbia Bifurcating mixers and methods of their use and manufacture
WO2017117647A1 (fr) 2016-01-06 2017-07-13 The University Of British Columbia Mélangeurs à bifurcation et leurs procédés d'utilisation et de fabrication
US10076730B2 (en) 2016-01-06 2018-09-18 The University Of British Columbia Bifurcating mixers and methods of their use and manufacture
WO2018006166A1 (fr) 2016-07-06 2018-01-11 Precision Nanosystems Inc Instrument de mélange microfluidique intelligent et cartouches
WO2020252375A1 (fr) * 2019-06-14 2020-12-17 Dnalite Therapeutics, Inc. Compositions et procédés pour véhicules d'administration biologiques
WO2020252589A1 (fr) 2019-06-20 2020-12-24 Precision Nanosystems Inc. Lipides ionisables pour administration d'acides nucléiques
WO2021000041A1 (fr) 2019-06-29 2021-01-07 Precision Nanosystems Inc. Lipides ionisables pour l'administration d'acides nucléiques
WO2021216577A1 (fr) * 2020-04-20 2021-10-28 Board Of Regents, The University Of Texas System Systèmes d'administration de nanoparticules lipidiques (lnp) et leurs utilisations
WO2022101486A1 (fr) * 2020-11-16 2022-05-19 BioNTech SE Compositions pharmaceutiques comprenant des particules et de l'arnm et leurs procédés de préparation et de stockage
WO2023001952A1 (fr) * 2021-07-21 2023-01-26 Universidad De Granada Nanoparticules de phosphate de calcium chargées de jasmonate pour induire des réponses efficaces de défense des plantes
WO2023057979A1 (fr) 2021-10-07 2023-04-13 Precision NanoSystems ULC Nanoparticules lipidiques de vaccin à arn

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 2006, LIPPINCOTT, WILLIAMS AND WILKINS
GIULIANI ET AL., PROC NATL ACAD SCI U S A., vol. 103, no. 29, 6 July 2006 (2006-07-06), pages 10834 - 9
KLEBANOFF, J IMMUNOTHER, vol. 35, no. 9, 2012, pages 651 - 660
TERAKURA ET AL., BLOOD, vol. 1, 2012, pages 72 - 82
WANG, JIMMUNOTHER, vol. 35, no. 9, 2012, pages 689 - 701

Also Published As

Publication number Publication date
WO2025005991A1 (fr) 2025-01-02

Similar Documents

Publication Publication Date Title
EP3986866B1 (fr) Lipides ionisables pour administration d'acides nucléiques
CN115279743B (zh) 用于核酸递送的可离子化脂质
US20230285538A1 (en) Efficacious mrna vaccines
KR102732019B1 (ko) T 세포 유전자 발현의 비바이러스 변형
EP4413144A1 (fr) Nanoparticules lipidiques de vaccin à arn
WO2025005990A1 (fr) Formulations de nanoparticules lipidiques pour thérapie cellulaire et procédés associés
WO2024006863A1 (fr) Formulations de nanoparticules lipidiques pour vaccins
AU2023300349A1 (en) Lipid nanoparticle formulations for vaccines
WO2020072699A1 (fr) Agents thérapeutiques à cargaison d'exosome destinés au traitement de la drépanocytose
Bevers Systemic cancer vaccination: tuning mRNA-LNP towards splenic uptake
KR20240161965A (ko) 이종 프라임 부스트 백신 조성물 및 사용 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23848236

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载