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WO2025219528A1 - Administration de plasmide non destructif à des cellules immunitaires - Google Patents

Administration de plasmide non destructif à des cellules immunitaires

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Publication number
WO2025219528A1
WO2025219528A1 PCT/EP2025/060662 EP2025060662W WO2025219528A1 WO 2025219528 A1 WO2025219528 A1 WO 2025219528A1 EP 2025060662 W EP2025060662 W EP 2025060662W WO 2025219528 A1 WO2025219528 A1 WO 2025219528A1
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WO
WIPO (PCT)
Prior art keywords
lipid
cell
pni
cells
mol
Prior art date
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Pending
Application number
PCT/EP2025/060662
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English (en)
Inventor
Mehar Gayatri Devi NAMALA
Anitha THOMAS
Nikita Jain
Zhengyu Chen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Global Life Sciences Solutions Canada ULC
Global Life Sciences Solutions Uk Ltd
Original Assignee
Global Life Sciences Solutions Canada ULC
Global Life Sciences Solutions Uk Ltd
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Publication of WO2025219528A1 publication Critical patent/WO2025219528A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Definitions

  • T cells being “irreversibly electroporated” or killed. There is also some risk to the genetic materials being damaged before or during transfer. In addition, electroporated cells can take a long time to proliferate and a recent study showed that the viability of T cells after electroporation was only 37%.
  • Viral based T cell transfection is labor intensive, expensive and poses manufacturing and regulatory challenges. 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.
  • Recently FDA has given out a guidance that cell therapy treated patients are to be monitored for several years following chimeric antigen receptor (CAR)T or TCR therapy due to possible risk of gene integration caused by the viral means of manipulation of cells.
  • CAR chimeric antigen receptor
  • Examples of cell products available commercially for immune-oncology applications are Kymriah TM for B cell precursor acute lymphoblastic leukemia and Yescarta TM for use in B cell lymphoma.
  • LNPs Lipid nanoparticles
  • Leukemia is the leading cause of mortality in pediatric patients.
  • CAR-T therapy was transformative to the patient’s cancer free recovery.
  • LNPs Lipid nanoparticles
  • These LNPs can have a lipidic or aqueous core and may contain bilayer structures depending on the abundance or structure of each type of lipids used.
  • Recognized challenges to creating successful LNP cell therapy are safety, manufacturability, stability, and efficacy.
  • the invention provides methods of, and compositions for, transfecting cells of hematopoietic lineage, such as an immune cell, with DNA encapsulated in an LNP. 2 P2023-3389-WO [0012]
  • the methods and compositions provide for successful transfer and expression of a protein of interest encoded by a nucleic acid of interest in plasmid DNA (pDNA) into an immune cell.
  • the methods and compositions provide an improved safety profile by eliminating antibiotic resistant genes that are a part of traditional pDNA.
  • the DNA is linear DNA such as single stranded DNA or double stranded DNA, or pDNA such as minicircles, plasmids, self-replicating human episomal plasmids using plasmid as backbone (synthetic), and inducible plasmids to control the gene expression conditions.
  • the LNP is formulated in a lipid composition for cell therapy.
  • the LNP encapsulates a nucleic acid of interest, e.g., a gene of interest, encoding a protein of interest.
  • the LNP encapsulates a gene editing element, such as guide and CRISPR elements.
  • the invention comprises compositions comprising a first population of LNPs encapsulating DNA encoding a protein of interest and a second population of LNPs encapsulating a gene editing element, or combinations thereof.
  • the compositions are administered to biological samples that have been removed from the organism, then those samples treated, washed and restored to the organism.
  • the organism may be a mammal, and in particular may be human. This process is used for cell reprogramming, genetic restoration, or immunotherapy, for example.
  • the biological samples may include immune cells.
  • the drug product is the modified cell.
  • the present invention provides a method of 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.
  • Non-viral delivery can be a safer technology for modulating the T cell than a virus for programming the cells.
  • the present invention provides a method of modulating the T cell receptors to recognize and destroy neoantigens or tumor antigens present on the surface of the tumor cells of the patient, or to modulate T cell populations to treat cancer.
  • T cells may also be modified in other embodiments to ameliorate autoimmune disorders such as celiac disease, Lupus, and diabetes.
  • the present invention provides a lipid nanoparticle (LNP) for transfecting a cell of hematopoietic lineage
  • the LNP includes a lipid mix composition encapsulating a DNA, the lipid mix composition comprising an ionizable lipid, a phospholipid, a stabilizer, and cholesterol.
  • the ionizable lipid includes a cyclopentyl or a tetrahydrofuranyl head group.
  • the ionizable lipid comprises PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or a combination thereof.
  • the DNA is in the form of a linear DNA, a closed DNA, or a circular DNA.
  • the DNA comprises an UTR is downstream or upstream of a gene of interest.
  • the UTR is one or more of a s/MAR, a MAR and a NF sequence element.
  • the DNA is under 5,000 BP in length.
  • the DNA is between 2,500 to 4,500 BP in length.
  • the DNA further comprises a gene editing element.
  • the gene editing element is a PiggyBac transposon element.
  • the PiggyBac based transposon element comprises an oligonucleotide sequence about 80%, about 85%, about 90%, about 95%, about 98%, or about 100% identical to the oligonucleotide sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.
  • the lipid nanoparticle further includes an RNA.
  • the RNA is mRNA.
  • the DNA does not include an antibiotic resistance gene.
  • the cell is a T cell or HSC.
  • the phospholipid comprises distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- 4 P2023-3389-WO phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
  • the stabilizer comprises polyoxyethylene (10) stearyl ether (BrijS10), tocopherol polyethelyne glycol succinate (TPGS), PEG-DMG, dodecyl maltoside, sucrose monolaurate, or any combinations thereof.
  • the lipid mix composition comprises about 35 - 50 Mol% ionizable lipid, about 10 - 30 Mol% structural lipid, about 15 - 50 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%.
  • the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 37.5 Mol% cholesterol, and about 2.5 Mol% polyoxyethylene (10) stearyl ether (BrijS10).
  • the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 39 Mol% cholesterol, and about 0.75 Mol% Tocopherol polyethylene glycol 1000 succinate (TPGS).
  • the present invention provides a lipid mix composition for forming lipid particles in association with a DNA, for use in transfecting a cell of hematopoietic lineage, the lipid mix composition comprising an ionizable lipid, a phospholipid, a stabilizer, and cholesterol.
  • the ionizable lipid includes a cyclopentyl or a tetrahydrofuranyl head group.
  • the ionizable lipid comprises PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or a combination thereof.
  • the stabilizer comprises polyoxyethylene (10) stearyl ether (BrijS10), tocopherol polyethelyne glycol succinate (TPGS), PEG-DMG, dodecyl maltoside, sucrose monolaurate, or any combinations thereof.
  • the lipid mix composition comprises about 35 - 50 Mol% ionizable lipid, about 10 - 30 Mol% structural lipid, about 15 - 50 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%. 5 P2023-3389-WO [0039]
  • the DNA further comprises a gene editing element.
  • the gene editing element comprises a PiggyBac transposon element.
  • the PiggyBac based transposon element comprises an oligonucleotide sequence about 80%, about 85%, about 90%, about 95%, about 98%, or about 100% identical to the oligonucleotide sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.
  • the lipid nanoparticle further including an RNA.
  • the RNA is mRNA.
  • the present invention provides a method of modifying a cell of hematopoietic lineage, the method comprising contacting the cell of hematopoietic lineage with the lipid nanoparticle of any of preceding embodiments.
  • a coding region is expressed in the cell and progeny of the cell at least 10 days after contacting the cell with the LNP.
  • the coding region expresses an RNA.
  • the RNA is tRNA, rRNA, IncRNA, mRNA, saRNA, sgRNA, guide RNA, trcRNA, PiWiRNA, snRNA, snoRNA, crRNA, or combinations thereof.
  • the cell has an unmodified genome.
  • the cell before contacting, has a modified genome.
  • the cell contains extrachromosomal DNA.
  • the present invention provides a modified cell of hematopoietic lineage modified by the method of preceding embodiments.
  • the present invention provides a method of treatment for immune deficiency, cancer, autoimmune disease or genetic insufficiency, the method comprising administering to a subject in need thereof an effective amount of a composition comprising the modified cell of preceding embodiments.
  • FIGS.1A-1B are bar graphs showing the size (FIG. 1A) and encapsulation efficiency (FIG. 1B) of NP-1, NP-2, NP-3 and NP-4.
  • FIG.2A is a line graph showing the viability of T cells treated with 250 ng of NP-1, NP-2, NP-3, and NP-4.
  • FIG. 2B is a line graph showing the viability of T cells treated with 500 ng of NP-1, NP-2, NP-3, and NP-4.
  • FIG.3A is a line graph showing the percent expression of GFP in T cells treated with 250 ng of NP-1, NP-2, NP-3, and NP-4, normalized to untreated cells.
  • FIG.3B is a line graph showing the percent expression of GFP in T cells treated with 500 ng of NP-1, NP-2, NP-3, and NP-4, normalized to untreated cells.
  • FIG.4A is a line graph showing the mean fluorescence intensity (MFI) in T cells treated with 250 ng of NP-1, NP-2, NP-3, and NP-4, normalized to untreated cells.
  • MFI mean fluorescence intensity
  • FIG.4B is a line graph showing the MFI in T cells treated with 500 ng of NP-1, NP-2, NP-3, and NP-4, normalized to untreated cells.
  • FIG.5A shows size and PDI of various NP constructs.
  • FIG.5B shows encapsulation efficiency of the same set of NP constructs. Key parameters measured in this evaluation further include, FIG.5C % viability, FIG.5D % GFP positive cells, FIG. 5E MFI,
  • FIG.5F shows %GFP in PNI 762 -LNP_1 composition screen using donor T cells purchased from Stem Cell Tec Technology.
  • FIG.5H shows transfection efficiency (% TE) at 24, 48 and 72 hours post transfection in LNP_2, LNP_3, LNP_4, LNP_5.
  • FIG. 5I shows MFI for the same compositions of FIG.5H at 24, 48 and 72 hours post transfection.
  • FIGS. 5J and 5K show transfection efficiency (% eGFP) and MFI of the protein expression respectively for PNI 659- LNP_1 encapsulating NP-1 or eGFP mRNA for T-cell DNA delivery.
  • FIGS.6A and 6B show %viability for primary T cells post treatment for two different primary T cell donors.
  • FIGS.7A and 7B show percent GFP integration for Day 1 and Day 2 primary T cells post treatment for the same donor primary T cells used in FIGS.6A (from Donor 1) and 6B (from Donor 2), respectively.
  • FIGS.8A and 8B show the MFI for the same cells used in FIGS.6A-6B with Donor - 1 primary T cells (FIG.8A) and Donor -2 primary T cells (FIG.8B). 7 P2023-3389-WO
  • FIGS.9A – 9H show MFI and % GFP expression assessed on days 2, 3, 5, 7, and 10 post LNP treatment with PNI 550 as ionizable lipid.
  • FIGS.9A, 9C, 9E, and 9G show % GFP expression with PNI-V_PNI22, PNI-IV_PNI22, PNI-V_PNI23, or PNI-IV_PNI23 as payloads, respectively.
  • FIGS.9B, 9D, 9F, and 9H show corresponding MFI.
  • FIGS.10A –10H show MFI and % GFP expression assessed on days 2, 3, 5, 7, and 10 post LNP treatment with PNI 565 as ionizable lipid.
  • FIGS.10A, 10C, 10E, and 10G show % GFP expression with PNI-V_PNI22, PNI-IV_PNI22, PNI-V_PNI23, or PNI-IV_PNI23 as payloads, respectively.
  • FIGS. 10B, 10D, 10F, and 10H show corresponding MFI.
  • FIGS.11A – 11H show MFI and % GFP expression assessed on days 2, 3, 5, 7, and 10 post LNP treatment with PNI 728 as ionizable lipid.
  • FIGS.11A, 11C, 11E, and 11G show % GFP expression with PNI-V_PNI22, PNI-IV_PNI22, PNI-V_PNI23, or PNI-IV_PNI23 as payloads, respectively.
  • FIGS. 11B, 11D, 11F, and 11H show corresponding MFI.
  • FIGS.12A – 12H show MFI and % GFP expression assessed on days 2, 3, 5, 7, and 10 post LNP treatment with PNI 761 as ionizable lipid.
  • FIGS.12A, 12C, 12E, and 12G show % GFP expression with PNI-V_PNI22, PNI-IV_PNI22, PNI-V_PNI23, or PNI-IV_PNI23 as payloads, respectively.
  • FIGS. 12B, 12D, 12F, and 12H show corresponding MFI.
  • FIGS.13A – 13J show MFI and % GFP expression assessed on days 2, 3, 5, 7, and 10 post LNP treatment with PNI 762 as ionizable lipid.
  • FIGS.13A,13C, 13E, 13G and 13I show % GFP expression with GFP-encoding mRNA, PNI-V_PNI22, PNI-IV_PNI22, PNI-V_PNI23, or PNI-IV_PNI23 as payloads, respectively.
  • FIGS.13B, 13D, 13F, 13H and 13J show corresponding MFI. These figures demonstrate that the LNPs of the instant disclosure enable sustained gene expression compared to mRNA (FIG.13A) by delivering the DNA transposons as payloads.
  • FIGS.14A – 14H show MFI and % GFP expression assessed on days 2, 3, 5, 7, and 10 post LNP treatment with PNI 769 as ionizable lipid.
  • FIGS.14A, 14C, 14E, and 14G show % GFP expression with PNI-V_PNI22, PNI-IV_PNI22, PNI-V_PNI23, or PNI-IV_PNI23 as payloads, respectively.
  • FIGS. 14B, 14D, 14F, and 14H show corresponding MFI.
  • FIGS.15A and 15B show effects of s/MAR, MAR and NF Sequence positioning on protein expression levels (% TE, FIG.15A and MFI, FIG. 15B).
  • FIGS.16A-16E show the effects of PNI 768 and PNI 762 LNP_1 and LNP_6 combinations of ionizable lipids and lipid mix compositions on the delivery to human primary T 8 P2023-3389-WO cells of CAR pDNA delivery along with CAR mRNA. Data was measured at 24 hrs post T cell transfection.
  • FIGS.17A-17E show the effects of PNI 768 and PNI 762 LNP_1 and LNP_6 combinations of ionizable lipids and lipid mix compositions on the delivery to human primary T cells of CAR pDNA delivery along with CAR mRNA. Data was measured at 48 hrs post T cell transfection.
  • FIG.16A-16E show the effects of PNI 768 and PNI 762 LNP_1 and LNP_6 combinations of ionizable lipids and lipid mix compositions on the delivery to human primary T cells of CAR pDNA delivery along with CAR mRNA. Data was measured at 48 hrs post T cell transfection
  • FIG. 18A shows specific lysis of leukemic B cell line by CD19 CAR T cells generated using mRNA or transposon LNP.
  • Target cells SUP-B15, CD19+; K562, CD19-
  • FIG.18B shows specific lysis of leukemic B cell line by transposon CD19 CAR T cells 2 or 7 days following LNP addition.
  • Target cells SUP-B15, CD19+; K562, CD19-
  • the invention provides methods for transforming cells of hematopoietic lineage with DNA without disruptive physical methods such as electroporation.
  • the invention provides lipid mix formulations including ionizable lipid, one or more phospholipid(s), and stabilizing agent.
  • the lipid mix formulations according to the invention are provided for modifying a cell ex vivo as cell therapy products, where the modified cell is the drug product.
  • the invention provides lipid mix formulations for formulating DNA-containing LNP.
  • the invention provides a composition for transfecting a cell of hematopoietic lineage comprising a lipid nanoparticle (LNP) encapsulating DNA.
  • the cell is a T cell.
  • the LNP comprises an ionizable lipid, a phospholipid and a stabilizing agent.
  • the ionizable lipid includes a cyclopentyl headgroup or a 9 P2023-3389-WO tetrahydrofuranyl headgroup.
  • the LNP comprises an ionizable lipid including PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 768, PNI 762, PNI 769, PNI 771, or a combination thereof.
  • the DNA is in the form of a plasmid, a linear DNA or a circular DNA.
  • the DNA is under 5,000 base pairs (BP).
  • the DNA is over 5,000 BP in length.
  • the DNA is between 2,500 to 4,500 BP in length.
  • the DNA is from about 30 to 529, 530 to 1029, 1030 to 1529, 1530 to 2029, 2030 to 2529, 2530 to 3029, 3030 to 3529, 3530 to 4029, 4030 to 4529, or 4530 to 5000 base pairs (BP) in length.
  • the DNA is from about 5500 to 6000, 6000 to 6500, 6500 to 7000, 7000 to 7500, 7500 to 8000, 8000 to 8500, or 8500 to 9000 BP in length.
  • the DNA is from about 9000 to 10,000, 10,000 to 11,000, 11,000 to 12,000, 12,000 to 13,000, 13,000 to 14,000, 14,000 to 15,000, 15,000 to 16,000, 16,000 to 17,000, 17,000 to 18,000, 18,000 to 19,000, or 19,000 to 20,000 BP in length.
  • the DNA comprises an integrating protein encoding nucleic acid of interest.
  • the DNA comprises a gene editing element encoding sequence.
  • the gene editing element is a PiggyBac or CRISPR Cas system element.
  • the DNA does not include an antibiotic resistance gene.
  • the invention provides a composition comprising a combination of the LNP compositions as described herein.
  • the invention provides a method of modifying a cell of hematopoietic lineage comprising contacting a cell of hematopoietic lineage with the compositions as described herein.
  • the integrating protein encoding nucleic acid of interest is expressed in the cell and progeny of the cell up to 10 days after contacting the cell with the LNP.
  • the cell before contacting, the cell is unmodified.
  • the cell before contacting, has a modified genome, for example in an ex vivo cell therapy multiple step procedure in which a cell population undergoes multiple modifications.
  • the cell contains exogenous chromosomal material.
  • the cell after contacting, contains integrated exogenous chromosomal material.
  • the invention provides a modified cell of hematopoietic lineage comprising a cell of hematopoietic lineage modified by the methods described herein.
  • the invention provides a method of treatment comprising administering to a subject in need thereof an effective amount of a composition of the modified cells described herein.
  • x and/or y means “one or both of x and y”.
  • x, y, and/or z means any element of the seven-element set ⁇ (x), (y), (z), (x, y), (x, z), (y, z), (x, y, z) ⁇ .
  • x, y and/or z means “one or more of x, y and z”.
  • the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used.
  • the term “about” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is +/-12.5%.
  • administering refers to the physical introduction of an agent to a subject, such as a lipid nanoparticle disclosed herein, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • cancer refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • a “cancer” or “cancer tissue” can include a tumor.
  • in vitro refers to events occurring in an artificial environment, e.g., in a test tube, reaction vessel, cell culture, etc., rather than within a multi-cellular organism.
  • in vitro cell refers to any cell which is cultured ex vivo.
  • an in vitro cell can include a T cell.
  • in vivo refers to events that occur within a multi-cellular organism, such as a human or a non-human animal.
  • pharmaceutically acceptable refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof.
  • a pharmaceutically acceptable carrier means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another (e.g., from one organ to another).
  • Each carrier present in a pharmaceutical composition must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient.
  • materials which may serve as pharmaceutically acceptable carriers comprise: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl ole
  • Treatment refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.
  • treatment or “treating” includes a partial remission. In another embodiment, “treatment” or “treating” includes a complete remission.
  • treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are 16 P2023-3389-WO statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
  • a “disease”, as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject’s health continues to deteriorate.
  • a “disorder” is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
  • a disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.
  • the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals.
  • mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos.
  • the term “animal” as used herein includes humans.
  • the term “subject” may also include a patient, i.e., an animal, having a disease.
  • a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).
  • the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable.
  • the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable.
  • it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder.
  • it can mean decreasing the severity of one or more subsequently developed symptoms.
  • 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 17 P2023-3389-WO 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. [00105] In this disclosure, “transfection” means 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 LIPOFECTAMINETM are established commercial transfecting reagents sold by ThermoFisher Scientific. These research reagents contain permanently cationic lipid(s) and are not suitable for use in vivo or ex vivo.
  • modified or “genetically modified” or “transfected” are used interchangeably, wherein a cell has been manipulated by means of molecular reprogramming of a genomic sequence (e.g. by insertion, deletion, or substitution). Examples include disabling or "knocking out" a specific gene by introducing genetic modifications that prevent its function.
  • CRISPR-Cas9 gene editing techniques enable precise modifications in the DNA sequence to introduce targeted changes, such as inserting, deleting, or modifying specific genes or genetic elements.
  • transgene insertion wherein cells can be genetically modified by introducing new genes or genetic elements from other organisms to confer specific traits or functions onto the cells, such as the production of therapeutic proteins or fluorescent markers.
  • gene silencing sometimes via RNA interference (RNAi), wherein specific genes are selectively silenced or "turned off”.
  • Said modified cells include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein. 18 P2023-3389-WO [00109] “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 C12-200) and are characterized by being insoluble in water but soluble in many organic solvents.
  • lipid mix compositions refers to the components that can be used to prepare the lipid nanoparticles (LNPs) encapsulating a payload.
  • lipid mix compositions for the manufacture of lipid nanoparticles for nucleic acid delivery comprise cationic or ionizable lipid and one or more of phospholipid, cholesterol, or a stabilizer.
  • the stabilizer can include polyethylene glycol conjugated lipids.
  • the lipid mix composition as used in the instant disclosure, are free of the payload.
  • “Lipid mix formulations” refers to the types of components, ratios of components, and the ratio of the total components to the nucleic acid payloads (e.g., LNPs encapsulating a payload).
  • the lipid mix compositions which can be used to mix with the DNA, comprise ionizable lipid as described, a neutral lipid or phospholipid or “structural” lipid which helps with the outer bilayer or monolayer of the LNP, optionally cholesterol and optionally a stabilizer as described above.
  • ionizable lipid as described
  • a neutral lipid or phospholipid or “structural” lipid which helps with the outer bilayer or monolayer of the LNP
  • cholesterol and optionally a stabilizer optionally lipid mix compositions
  • certain ratios of these four components may be optimized.
  • a mole percent ratio of 50 mol% for ionizable lipid has been used successfully in clinical products.
  • targeting gene delivery to cells while maintaining viability of the cells requires a different approach than that used for intramuscular injection and immediate release.
  • lipid mix compositions comprise ionizable lipid (iL), cholesterol, structural lipid, and a stabilizer.
  • the stabilizer includes PEG DMG, TPGS, polyoxyethylene (40) stearate, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, Tridecyl-D-maltoside, Polysorbate 20, Polysorbate 80, or combinations thereof.
  • Lipid mix compositions may optionally include tryglycerides in some embodiments.
  • Non-limiting examples of lipid mix compositions include those disclosed in PCT Publications 2020210901 and 2024006863, which are incorporated by reference herein in their entireties.
  • “Lipid Particles” or “Lipid Nanoparticles” or “LNP” refers to lipid particles manufactured from the lipid mix compositions described above and illustrated below.
  • a 19 P2023-3389-WO therapeutic agent such as a nucleic acid may be encapsulated in the lipid mix composition to provide a nucleic acid-containing lipid nanoparticle or nucleic acid lipid nanoparticle (“NALNP” or “LNP” as used interchangeably in the instant disclosure).
  • the lipid particle represents the physical organization of the lipid mix composition with the therapeutic agent and among the components.
  • a lipid nanoparticle is a lipid particle under 300 nanometers (nm) in diameter.
  • Lipid particles are generally spherical assemblies of lipids, nucleic acid, cholesterol, and stabilizing agents.
  • lipid particles 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.
  • lipid particles are between 1 and 1000 nm in diameter.
  • compositions of the invention comprise ionizable lipids as a component.
  • ionizable lipid refers to a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid but is more neutral at higher pH values. At pH values below the pKa, the lipid is then able to associate with negatively charged 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.
  • suitable ionizable lipids are found in PCT Pub. Nos. WO20252589 and WO21000041, which are incorporated by reference herein in their entireties.
  • the ionizable lipid may be present in the lipid nanoparticle composition in any suitable amount or concentration.
  • the ionizable lipid is present at a concentration of about 10 to about 90 mol% or about 20 to about 70 mol%, e.g., about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%, about 75 mol%, 20 P2023-3389-WO about 80 mol%, about 85 mol%, about 90 mol%, or a concentration within a range defined by any two of the foregoing values.
  • the ionizable lipid is present in lipid compositions preferably in a ratio of about 10 to about 60 Mol%, (“Mol%” means the percentage of the moles that is of a particular component while the total moles of all the components in the lipid compositions is 100 mol%).
  • Mol% means the percentage of the moles that is of a particular component while the total moles of all the components in the lipid compositions is 100 mol%).
  • 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 %, 40 Mol %, 47.5 Mol%, 50 Mol % ionizable lipid would all be in the claimed range of embodiments.
  • the ionizable lipid is present at about 35 to 50 Mol% of the lipid mix composition.
  • DODMA 1,2- dioleyloxy-3-dimethylaminopropane
  • MC3 O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (“MC3”).
  • LNP may be generated from the lipid mix compositions including the ionizable lipids of the invention.
  • Ionizable lipids of the invention include PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, or any combinations thereof, as described herein.
  • Ionizable lipids of the invention include those containing a cyclopentyl or a tetrahydrofuranyl headgroup.
  • Phospholipids, as used herein, also known as “helper lipids,” “structural lipids,” or “neutral lipids” are incorporated into lipid mix compositions and lipid particles of the invention in embodiments.
  • the structural lipid may be present in the lipid nanoparticle composition in any suitable amount.
  • the structural lipid is present in the lipid nanoparticle composition at a concentration of about 1 to about 75 mol% or about 5 to about 60 mol%, e.g., about 1 mol%, about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%, about 75 mol%, or a value within a range defined by any two of the aforementioned values.
  • the lipid mix compositions and lipid particles of the invention include one or more phospholipids at about 10 to 60 Mol% of the lipid mix composition. In some embodiments, the one or more phospholipids is present at about 25 to 60 Mol% of the lipid mix formulation. In some embodiments, the one or more phospholipids is present at about 10 to 40 Mol% of the lipid mix formulation. Suitable phospholipids support the formation of particles during manufacture. Phospholipids refer to any one of several lipid species that exist in either in an anionic, uncharged, or neutral zwitterionic form at physiological pH.
  • Representative phospholipids include diacylphosphatidylcholines, 21 P2023-3389-WO 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 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-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoylphosphatid
  • the phospholipid is distearoylphosphatidylcholine (DSPC). In preferred embodiments, the phospholipid is DOPE. In preferred embodiments, the phospholipid is DSPC. [00118] In another embodiment, 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 lipids.
  • suitable phospholipids include glycolipids (e.g.
  • “Stabilizer” or “stabilizing agent” is a term used to identify the agent that is added to the ionizable lipid, the phospholipid, and the sterol that form the lipid formulation according to the invention.
  • the stabilizing agent may include a non-ionic stabilizing agent.
  • Non-limiting examples of non-ionic stabilizing agents include: Polyethyleneglycol (PEG), DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-200), Polysorbates (Tweens), 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), MyrjTM52 (polyoxyethylene (40) stearate), or any combinations thereof.
  • PEG Polyethyleneglycol
  • DMG-PEG2000 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-200
  • Polysorbates Tweens
  • TPGS Volitamin E polyethylene glycol succ
  • stabilizing agents include those disclosed in PCT applications PCT/EP2024/075129, PCT/EP2024/075124, PCT/EP2024/075128, which are incorporated by reference herein in their entireties.
  • the stabilizing agent includes PEGylated lipids including PEG- DMG 2000 (“PEG-DMG”). Other polyethylene glycol conjugated lipids may also be used. The stabilizing agent may be used alone or in combinations with each other.
  • the stabilizing agent comprises about 0.1 to 5 Mol% of the overall lipid mixture.
  • the stabilizing agent includes about 0.5 to 2.5 Mol% of the overall lipid mixture. In preferred embodiments, the stabilizing agent is present at greater than 1.0 Mol%. In some embodiments the stabilizing agent is present at 5 Mol%. In some embodiments the stabilizing agent is present at 10 to 15 Mol%. In some embodiments, the stabilizing agent is present at 2.5 to 10 Mol%. In other embodiments, the stabilizing agent is present at greater than 10 Mol% of the lipid mixture.
  • the stabilizing agent has a mol% of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or a mol% value within a range defined by any two of the aforementioned values.
  • Sterols are included in some embodiments lipid mix formulations for certain applications, and lipid particles made therefrom include cholesterol, beta-sitosterol, 20-alpha- hydroxysterol, and/or phytosterol.
  • sterol is present at about 15 to 50 Mol% of the lipid mix formulation in some embodiments. In some embodiments, sterol is present at about 15 to 25 Mol% of the lipid mix formulation. In some embodiments, a modified sterol or synthetically derived sterol is present.
  • delivery In the case of cell therapy, delivery is to a particular cell type or population, commonly Ex Vivo. In the case of vaccines, delivery is localized to the skin or muscle.
  • nucleic acid refers to any polymeric chain of nucleotides.
  • a nucleic acid may be DNA, RNA, or a combination thereof.
  • a nucleic acid comprises one or more natural nucleic acid residues.
  • a nucleic acid comprises of one or more nucleic acid analogs.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, or 23 P2023-3389-WO chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues).
  • a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.
  • the term “nucleic acid” is meant to include any oligonucleotide or polynucleotide whose delivery into a cell causes a desirable effect. The definition includes diagnostic agents and research reagents which follow the same physical principles afforded by the invention.
  • oligonucleotides Fragments containing up to 50 nucleotides are generally termed oligonucleotides, and longer nucleotides are called polynucleotides.
  • 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.
  • polynucleotides of the invention include up to 14,000 nucleotides.
  • nucleic acid refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, or combinations thereof, and can be single stranded, double stranded, or contain portions of both double stranded and single stranded sequence, as appropriate.
  • Messenger RNA mRNA
  • mRNA can be modified or unmodified, base modified, and may include different type of capping structures, such as Cap1.
  • nucleic acid refers to self-amplifying RNA (“saRNA”).
  • nucleic acid refers to a plasmid including self-amplifying RNA.
  • polynucleotide and oligonucleotide are used interchangeably and mean 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
  • Polynucleotides have associated counter ions, such as H + , NH 4 + , trialkylammonium, Mg 2+ , Na + , and the like.
  • a polynucleotide 24 P2023-3389-WO may be composed entirely of deoxyribonucleotides (DNA), entirely of ribonucleotides (RNA), or chimeric mixtures thereof.
  • Transposon clinical trials including SuperPiggybac are disclosed in a 2020 paper Magnani CF, et al. Transposon-Based CAR T Cells in Acute Leukemias: Where are We Going? Cells.2020 May 27;9(6):1337. Other examples appear in Table 1.
  • PiggyBac is a type of transposon, a mobile genetic element that can insert and remove DNA sequences in the genome. PiggyBac works by excising itself from one location in the genome and integrating into another, which allows it to be used for gene transfer and integration. PiggyBac insertions are typically stable and can be passed on to the next generation in organisms, making it suitable for long-term genetic modifications. [00128] Examples of suitable DNA vectors include those listed in Table 1. Transposons are mobile genetic elements that can move within a genome. researchers choose the transposon system that best suits their experimental needs, taking into consideration factors such as ease of use, efficiency, and target organism. Table 1.
  • Plasmids are a form of stable nucleic acid that can coexist with an organism’s genome.
  • Cell therapeutics encompass a diverse array of medical treatments utilizing living cells for therapeutic purposes.
  • Immunotherapies such as Chimeric Antigen Receptor T-cell (CAR-T) therapy, T Cell Receptor (TCR) therapy, and Tumor-Infiltrating Lymphocytes (TIL) therapy, which modify and reinfuse immune cells to target neoplastic diseases
  • CAR-T Chimeric Antigen Receptor T-cell
  • TCR T Cell Receptor
  • TIL Tumor-Infiltrating Lymphocytes
  • Hematopoietic cell therapies involving the transplantation and manipulation of hematopoietic stem cells (HSCs) for treating hematological disorders, including leukemia and lymphoma, and especially genetic disorders, using either autologous or allogeneic stem cells
  • HSCs hematopoietic stem cells
  • NK Natural Killer
  • NK Mesenchymal Stem Cell
  • MSC Mesenchymal Stem Cell
  • nucleic acid therapeutic is defined as a substance intended to have a direct effect in the mitigation or prevention of disease, or to act as a research reagent.
  • the nucleic acid cargo is an mRNA or saRNA.
  • the therapeutic agent is a nucleic acid therapeutic, such as a double stranded circular DNA (plasmid), a linearized plasmid DNA, minicircles or msDNA (multicopy single stranded DNA).
  • nucleic acid cargoes include deoxyribonucleic acid, complementary deoxyribonucleic acid, complete genes for gene therapies targeting a variety of diseases, such as cancer, infectious diseases, genetic disorders and neurodegenerative diseases.
  • the nucleic acid therapeutic (NAT), or nucleotide of interest is incorporated into the 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.
  • nucleic acid payload examples include but are not limited to an antisense oligonucleotide (ASO), a ribozyme, a microRNA (miRNA), a messenger RNA (mRNA), a transfer RNA (tRNA), a trans-activating CRISPR RNA (tracrRNA), a guide RNA, a sgRNA, a self-amplifying 27 P2023-3389-WO RNA (SAM or saRNA), a small nuclear RNA (snRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a circular RNA (circRNA), a non-coding RNA (ncRNA), a self- replicating DNA, an LNA, a DNA, a replicon, a pre-condensed DNA, a transposon, a single gene, a vector, a plasmid or a
  • ASO antisense oligonucleotide
  • miRNA messenger
  • nucleic acid payloads or reagents are used to silence genes (with for example siRNA), express genes (with for example mRNA), edit genomes (with for example CRISPR/Cas9), or 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).
  • the nucleic acid is an antigen encoded mRNA for prophylactic or therapeutic vaccine, a nucleic acid for gene therapy, or a nucleic acid for immunogenic cell incorporation, wherein the immunogenic cell is a T cell, natural killer cell, dendritic cell, macrophage, or tumor-infiltrating leukocyte.
  • the nucleic acid that is present in a lipid particle according to this invention may include any form of nucleic acid that is currently known or later developed.
  • the nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA- RNA hybrids. Circular and closed DNA are nucleic acid payloads in some embodiments. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA 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.
  • 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 28 P2023-3389-WO 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.
  • Gene of Interest (“GOI”) is the nucleic acid molecule intended to be integrated and expressed, or exist on an exosome and be expressed, in the cell or in a bioreactor.
  • Genes of Interest include those encoding insulin, human growth hormone, CFTR, ⁇ globin, ⁇ globin, ⁇ globin, BCL11A, KLF1, CCR5, CXCR4, PPP1R12C (AAVS1), HPRT, albumin, Factor VIII, Factor IX, LRRK2, Htt, SOD1, C9orf72, TARDBP, FUS, RHO, CFTR, SFTPB, TRAC, TRBC, PD1, CTLA-4, HLA A, HLA B, HLA C, HLA-DP, HLA-DQ, HLA-DR, LMP7, TAP 1, TAP2, TAPBP, CIITA, DMD, GR, IL2RG, Rag-1, RFX5, FAD2, FAD3, ZP15, KASII, MDH, EPSPS, or a fragment thereof.
  • Gene of Interest may encode a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN- ⁇ , IL-7, IL-21, IL-10, IL-12, IL-15, and TNF- ⁇ ), a chemokine (e.g., MIP-1 ⁇ , MIP-1 ⁇ , MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, and an IL-21 receptor), or an engineered antigen receptor.
  • a cytokine e.g., IL-2, insulin, IFN- ⁇ , IL-7, IL-21, IL-10, IL-12, IL-15, and TNF- ⁇
  • a chemokine e.g., M
  • Nucleic acid payloads may include both coding and non-coding genes of interest. Coding regions are the instructions for building proteins, which are essential molecules for the structure, function, and regulation of the body's cells and tissues. Coding regions, also known as exons, are the segments of DNA that directly encode the amino acid sequence of a protein. These regions are transcribed into messenger RNA (mRNA), which serves as a template for protein synthesis during translation. Mutations in coding regions can lead to changes in the amino acid sequence of the resulting protein, which may affect its structure or function. Coding regions are highly conserved across species and are crucial for understanding the genetic basis of inherited diseases and the development of therapeutic interventions.
  • mRNA messenger RNA
  • Noncoding regions also known as introns and regulatory sequences, are segments of DNA that do not code for proteins. Introns are spliced out during mRNA processing, and only the exons are retained in the mature mRNA. Regulatory sequences, such as promoters, enhancers, and silencers, play critical roles in controlling gene expression by influencing the transcriptional activity of genes. Noncoding regions are involved in various cellular processes, including gene regulation, chromatin structure, and RNA processing. Mutations in noncoding regions can impact gene expression levels or patterns, leading to phenotypic changes or disease 29 P2023-3389-WO susceptibility. Noncoding regions also contain regions of repetitive DNA, such as transposable elements, which can contribute to genome instability and genetic diversity.
  • Noncoding regions of the genome do not directly encode proteins but can code for noncoding RNAs (ncRNAs).
  • Noncoding RNAs are RNA molecules that are transcribed from DNA but are not translated into proteins. Instead, they perform various regulatory and structural functions within the cell. Examples of noncoding RNAs include: tRNA molecules are involved in translating the genetic code from mRNA into amino acid sequences during protein synthesis, rRNA molecules are components of ribosomes, the cellular machinery responsible for protein synthesis.
  • miRNAs are small RNA molecules that regulate gene expression by binding to specific mRNA molecules and either inhibiting their translation or promoting their degradation
  • Long non-coding RNA lncRNAs are RNA molecules longer than 200 nucleotides that do not encode proteins.
  • Nucleic acid payload of the instant disclosure may encode an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof, or a chimeric cytokine receptor.
  • DNA delivery can be used for various CAR T-cell therapy applications (chimeric antigen receptor T-cell therapy, is a type of immunotherapy) that involves genetically modifying a patient’s T cells to recognize and attack cancer cells.
  • CAR T-cell therapy disorders and available treatments include B-cell Acute Lymphoblastic Leukemia (B-ALL): CAR T-cell therapy targeting CD19 has shown significant efficacy in treating relapsed or refractory B-ALL.
  • Approved CAR T-cell therapies for B-ALL include tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta).
  • DLBCL Diffuse Large B-cell Lymphoma
  • DLBCL is a type of non-Hodgkin lymphoma that has also been targeted with CAR T-cell therapy.
  • Axicabtagene ciloleucel 30 P2023-3389-WO (Yescarta) and tisagenlecleucel (Kymriah) have been approved for treating certain patients with DLBCL who have failed other treatments.
  • Multiple Myeloma CAR T-cell therapy is being investigated as a potential treatment for multiple myeloma, a cancer of plasma cells.
  • CAR T-cell therapies could effectively target solid tumors, such as ovarian cancer, pancreatic cancer, and glioblastoma.
  • Autoimmune Diseases CAR T-cell therapy can be engineered to target autoreactive T cells that are responsible for attacking the body’s own tissues in autoimmune diseases such as rheumatoid arthritis, lupus, celiac disease and multiple sclerosis. Research is ongoing to develop CAR T-cell therapies that selectively target and eliminate autoreactive T cells while sparing healthy immune cells.
  • Organ Transplantation By targeting and suppressing the immune response against the transplanted organ, these therapies could potentially reduce the need for lifelong immunosuppressive drugs, which carry risks of infections and other complications.
  • CAR T-cell therapy has been investigated as a potential treatment for allergic diseases, such as asthma and allergic rhinitis. By targeting specific immune cells involved in allergic reactions, CAR T-cell therapy could potentially modulate the immune response and alleviate symptoms associated with allergic diseases.
  • Immunodeficiency Disorders [00150] CAR T-cell therapy and gene editing technologies, such as CRISPR-Cas9, are being explored as potential treatments for primary immunodeficiency disorders, where the immune system is deficient or dysfunctional. [00151] These approaches aim to correct genetic mutations responsible for immunodeficiency and restore normal immune function.
  • IBD Inflammatory Bowel Disease
  • CAR T-cell therapy and other immunomodulatory strategies are being investigated as potential treatments for inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis.
  • these therapies could help reduce inflammation and improve symptoms in patients with IBD.
  • Type 1 Diabetes 31 P2023-3389-WO
  • CAR T-cell therapy and other immunotherapies are being explored as potential treatments for type 1 diabetes, an autoimmune disease characterized by the destruction of insulin-producing beta cells in the pancreas.
  • These approaches aim to target and eliminate autoreactive immune cells responsible for attacking beta cells, thereby preserving or restoring insulin production.
  • “Therapeutic agents” as used herein include nucleic acids as herein described, or nucleic acid therapeutics (“NAT”), proteins, peptides, polypeptides, and small molecules.
  • the term “polypeptides” herein encompasses “oligopeptides” and “proteins” and tertiary and quaternary structures thereof, that are therapeutic agents in some embodiments.
  • An oligopeptide generally consists of from two to twenty amino acids.
  • a polypeptide is a single linear chain of many amino acids of any length held together by amide bonds.
  • a protein consists of one or more and may include structural proteins, energy catalysts, albumin, hemoglobin, immunoglobulins, and enzymes.
  • the lipid particles of the invention can be assessed for size using devices that size particles in solution, such as the MalvernTM ZetasizerTM.
  • the particles generally have a mean particle diameter of from 15 nm to 1000 nm.
  • 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. In one embodiment, the lipid particle has a diameter of from about 50 to about 150 nm.
  • the lipid particle has a diameter from about 15 to about 50 nm.
  • the lipid particles according to embodiments of the invention 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 self-assembly of elements into nanoparticles.
  • a variety of methods have been developed to formulate lipid nanoparticles (LNP) containing genetic drugs. Suitable methods are disclosed in U.S. Pat. No.5,753,613, U.S. Pat. No.6,734,171, and U.S.
  • NanoAssemblrTM instruments automated micro-mixing instruments such as the NanoAssemblrTM instruments (Cytiva, USA) enable the rapid and controlled manufacture of nanomedicines (liposomes, lipid nanoparticles, and polymeric nanoparticles).
  • NanoAssemblrTM 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.
  • Preferred methods incorporate instruments such as the microfluidic mixing devices like the NanoAssemblrTM series including SparkTM, IgniteTM, BlazeTM, GMP system or commercial formulation system, in order to achieve nearly 100% of the nucleic acid used in the formation process is encapsulated in the particles in one step.
  • 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.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 micromixing geometries and their application to microfluidic mixing.
  • PCT Pub. No. WO2018006166 discloses a programmable automated micromixer and mixing chips, therefore.
  • D771834, D771833, D772427, D803416, D800335, D800336 and D812242 disclose mixing cartridges having microchannels and mixing geometries for mixer instruments sold by Cytiva, USA.
  • devices for biological microfluidic mixing are used to prepare the lipid particles according to embodiments of the invention.
  • 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.
  • Non-limiting examples of 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 invention are soluble, and that are miscible with the first solvent.
  • suitable second solvents include 1,4-dioxane, 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 20 to about 300 ⁇ m.
  • the microchannel has a diameter from about 300 to about 1000 ⁇ m.
  • 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. No.9,943,846, or a bifurcating toroidal flow as described in U.S. Pat.
  • lipid mixes of the present invention may be used to deliver a therapeutic agent to a cell, in vitro, ex vivo, or in vivo.
  • the therapeutic agent is a nucleic acid, which is delivered to a cell using lipid particles of the present invention with the nucleic acid encapsulated therein.
  • the nucleic acid may include, but not limited to, an antisense oligonucleotide (ASO), a ribozyme, a microRNA (miRNA), a messenger RNA (mRNA), a transfer RNA (tRNA), a trans-activating CRISPR RNA (tracrRNA), a guide RNA, a single guide RNA, a self-amplifying RNA (SAM or saRNA), a small nuclear RNA (snRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a circular RNA (circRNA), a non- coding RNA (ncRNA), a self-replicating DNA, a locked nucleic acid (LNA), a DNA, a replicon, a pre-condensed DNA, a
  • the nucleic acid cargo of the LNP includes an oligopeptide, polypeptide, or protein which is delivered to a cell using lipid particles of the present invention with the peptide encapsulated therein.
  • the payload or therapeutic agent is a mixture of nucleic acid and protein components, such as Cas9. The methods and lipid mix compositions may be readily adapted for the delivery of any suitable therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment.
  • the present invention provides methods for introducing a nucleic acid payload or cargo into a cell (i.e., transfection).
  • 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 or nucleic acid therapeutic (NAT) 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 35 P2023-3389-WO 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 invention with the cells for a period of time sufficient for intracellular delivery to occur.
  • nucleic acid or nucleic acid therapeutic into the nucleus and chromosomes can be assayed by studying the survival of the gene expression through cell divisions.
  • 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.
  • Methods of the present invention may be practiced in vitro, ex vivo, or in vivo.
  • the lipid mix formulations of the present invention can also be used for delivery of nucleic acids to cells in vivo, using methods which are known to those of skill in the art.
  • the lipid mix formulations of the invention can be used for delivery of nucleic acids to a sample of patient cells that are ex vivo, then are returned to the patient.
  • the delivery of nucleic acid cargo by a lipid particle of the invention is described below.
  • 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.
  • Other routes of administration include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.
  • the pharmaceutical compositions are preferably administered to biological samples that have been removed from the organism, then the cells are washed and restored to the organism.
  • the organism may be a mammal, and in particular may be human. This process is used for cell reprogramming, genetic restoration, or immunotherapy, for example.
  • the present invention provides 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 invention that is associated with a nucleic acid capable of modulating the expression of a target polynucleotide or polypeptide.
  • 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.
  • Cells of the hematopoeitic lineage includes hematopoietic stem cells, precursor immune cells such as T cells and B cells, macrophages, and natural killer cells. The term is intended to encompass cells of both the innate and adaptive immune system.
  • B cells can be isolated from whole blood by cells sorting or magnetic activated bead cell sorting. Moore DK, Motaung B, du Plessis N, Shabangu AN, Loxton AG; SU-IRG Consortium. Isolation of B-cells using Miltenyi MACS bead isolation kits. PLoS One.2019 Mar 20;14(3). White cells in general can be separated from whole blood using density.
  • Immune cell isolation includes methods that enable the enrichment of immune cell subsets using antibody- mediated recognition of specific cell surface antigens, followed by sorting or separation with techniques such as flow cytometry, density centrifugation or magnetic isolation.
  • a T cell, or T lymphocyte is a lymphocyte subtype that has the lead role in cell- mediated immunity. T cells can be distinguished from other white blood cells, (for example, B cells or natural killer cells), by the existence of 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, for example, 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.
  • the triple activation method using anti- CD3/CD28/CD2 antibodies is exemplified below, but dual activation was also effective in our studies. Dual activation is performed using anti CD3/CD28 antibodies. Current clinically used protocols employ the dual activation protocol.
  • T cells may in some cases be derived from differentiated from induced pluripotent stem cells (iPSC) or Embryonic Stem Cells (ESC). 37 P2023-3389-WO [00185] Preparation of T cells for transformation by methods of the invention includes 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.
  • biological tissue such as peripheral blood or arterial blood
  • 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 subject from which the cell is isolated is a healthy donor or volunteer.
  • the cells in some embodiments are primary cells, such as primary human cells.
  • the tissue sources include 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.
  • Exemplary tissue sources include 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.
  • reagents for example, to remove or enrich for certain components.
  • cells from the circulating blood of a subject are obtained by apheresis or leukapheresis.
  • 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 ⁇ kta Flux, for example).
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ++ /Mg ++ free PBS.
  • Unmodified cells mean cells that have not been treated to any structural or genetic changes after removal from a living body.
  • Modified cells mean cells that have been augmented or changed in some way during or after removal from a living body.
  • Separating the T cells from tissue sources may involve density-based cell separation methods, including the preparation of white blood cells from peripheral blood by lysing the red 38 P2023-3389-WO blood cells and centrifugation, for example, through a PercollTM or FicollTM gradient. Other 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.
  • 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.
  • a selection for CD4 + helper cells may be undertaken.
  • naive CD4 + T lymphocytes are CD45RO-, CD45RA + , CD62L + , CD4 + T cells.
  • central memory CD4 + cells are CD62L + and CD45RO + .
  • effector CD4 + cells are CD62L- and CD45RO.
  • 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. [00194]
  • isolation or separation is carried out using an apparatus that carries out one or more of the isolation, cell preparation, separation, processing, and incubation, as required to transform the T cells.
  • 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 A1. 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. (2012) J Immunother. 35(9):689-701. 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 39 P2023-3389-WO stream.
  • 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 non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture.
  • PBMC peripheral blood mononuclear 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 invention 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 invention, wherein the therapeutic agent is selected from a DNA, linear DNA, circular DNA, plasmid DNA, 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 a DNA, linear DNA, circular DNA, plasmid DNA, siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucle
  • the present invention provides 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 invention, wherein the therapeutic agent is selected from a plasmid or DNA which includes a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof.
  • the therapeutic agent is selected from an mRNA, a self-amplifying RNA (saRNA), or an ssODNA, includes a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof.
  • RNA vaccines examples include RNA vaccines, and more particularly self-amplifying mRNA vaccines.
  • a biologically active agent e.g., DNA encoding an immunogen
  • cells of the immune system e.g., antigen-presenting cells, including professional antigen presenting cells
  • formulation of the invention is delivered intramuscularly, after which immune cells can infiltrate the delivery site and process delivered DNA 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 WO2012/006372).
  • 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., human peripheral blood monocytes
  • monocytes e.g., human peripheral blood monocytes
  • the encapsulation efficiency does not have to be 100%. Presence of external DNA molecules (e.g., on the exterior surface of a liposome or LNP) or “naked” DNA molecules (DNA molecules not associated with a liposome or LNP) is acceptable. Preferably, for a formulation comprising lipids and DNA molecules, at least half of the DNA molecules (e.g., at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% of the DNA molecules) are encapsulated in LNPs or complexed with LNPs.
  • the DNA molecules e.g., at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least about 96%, at least about 97%, at least about 98%, or at least
  • Some lipid nanoparticles may comprise a lipid core (e.g., the formulation may comprise a mixture of LNPs and nanoparticles with a lipid core).
  • the DNA molecules may be 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 DNA and cationic lipid). Encapsulation and complexation with LNPs (whether with a lipid or aqueous core) can protect DNA from DNase digestion. The encapsulation/complexation efficiency does not have to be 100%. Presence of “naked” DNA molecules (DNA molecules not associated with the LNP) is acceptable.
  • lipid nanoparticles Preferably, for a formulation comprising a population of LNPs and a population of DNA molecules, at least half of the population of DNA molecules (e.g., at least e.g., at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the DNA molecules) are either encapsulated in LNPs or complexed with LNPs.
  • Some lipid nanoparticles have multilamellar components such as phospholipid bilayers and aqueous pockets.
  • the preferred range of LNP diameters is in the range of 60-180 nm, and in more particular embodiments, in the range of 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.
  • compositions comprising a population of LNPs with different diameters
  • 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; and/or the diameters within the plurality have a polydispersity index ⁇ 0.2.
  • DNA molecules can conveniently be prepared by in vitro transcription (IVT).
  • IVT in vitro transcription
  • 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 invention includes embodiments in which multiple species of RNAs are formulated with a lipid formulation provided by the invention, such as two, three, four or more species of RNA, including different classes of RNA (such as mRNA, siRNA, self- replicating RNAs, and combinations thereof).
  • RNA such as mRNA, siRNA, self- replicating RNAs, and combinations thereof.
  • the DNA codes specific neoantigens in cancer cells or solid tumours.
  • 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), CDK4 (associated with, e.g., melanoma), MUMI (associated with, e.g., melanom
  • tumor immunogens include, but are not limited to, p15, 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, p185erbB2, p180erbB-3, c-met, mn-23HI, TAG-72- 4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, 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
  • compositions in accordance with the present disclosure comprise an effective amount of the lipid formulations described herein (e.g., LNP), as well as any other components, as needed.
  • the pharmaceutical compositions described herein may be prepared by 43 P2023-3389-WO any method known or hereafter developed in the art of pharmacology. In general, 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).
  • the particle size of the lipid particles may be increased and/or decreased.
  • 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 44 P2023-3389-WO mammals by changing biodistribution. Size may also be used to determine target tissue, with larger particles being cleared quickly and smaller one reaching different organ systems.
  • EPO erythropoietin [00213]
  • GFP green fluorescent protein [00214]
  • ug microgram [00215]
  • pg picogram [00216]
  • PBT-1 (or PB-1) and PBT-2 (or PB-2) are piggyBac based transposons designed in- house [00217]
  • ng nanogram [00218]
  • g gram [00219]
  • h hour(s) [00220]
  • HPLC High performance liquid chromatography [00221]
  • mL milliliter(s) [00224]
  • mmol millimole(s) [00225]
  • PBS phosphate buffered solution [00227
  • GOI Genetic element of interest
  • Chimeric antigens, cancer- 45 P2023-3389-WO associated antigens, autoimmune associated antigens, epidermal growth factor (EPO), eGFP are examples of a GOI, but GOI is not limited to these examples.
  • iL ionizable lipid, a lipid that is cationic at lower pH, and converts to uncharged at higher pH. iLs are commonly used in formulations of nucleic acid cargo.
  • MAR stands for “Matrix Attachment Region.” MARs are specific DNA sequences found within the genome that have the ability to bind to the nuclear matrix or scaffold proteins. They affect gene expression by promoting the accessibility of genes to transcription factors and other regulatory elements. S/MAR, or Scaffold/Matrix Attachment Region, refers to a specific type of MARS that serves as a binding site for nuclear scaffold or matrix proteins. S/MARs help tie the chromatin to the nuclear scaffold or matrix, contributing to the formation of chromatin loops and the establishment of functional domains within the nucleus.
  • the woodchuck hepatitis virus post-transcriptional regulatory element increases the transgene (GOI) expression. It is a sequence derived from the Woodchuck Hepatitis Virus (WHV).
  • WPRE element is often used as a tool to enhance gene expression in various systems, including mammalian cells.
  • the WPRE element functions by increasing the stability and efficiency of mRNA, which ultimately leads to higher levels of gene expression. It does this by facilitating nuclear export of the mRNA, preventing its degradation, and increasing its translation efficiency. These properties make the WPRE element a valuable tool for researchers who want to optimize gene expression in their experiments.
  • MAR seq (Ref: MAR characteristic motifs mediate episomal vector in CHO cells, Yan Lin, Zhaoxi Li, TianyunWanga, XiaoyinWang, Li Wang, Weihua Dong, Changqin Jing, Xianjun Yang (dx.doi.org/10.1016/j.gene.2015.01.032).)
  • This example uses a 400 BP sequence of s/MARs, which was previously reported to have the ability to maintain the plasmid episomally.
  • the methods adds a T2A, amino acid sequence downstream to the reporter gene to avoid translation into the s/MARs, as the s/MARs sequence was reported to not promote nuclear integration if it was part of gene transcription.
  • Beta globulin (BGA) poly-A signal was added downstream to the s/MARs.
  • a human Ef1alpha promoter is used as it is one of the strong promoters and the 3NF ( ⁇ B motifs) sequences located both upstream of the promoter and downstream of the reporter gene to enhance the localization 46 P2023-3389-WO of DNA into the nucleus.
  • the examples use a unique RNA out system to avoid using bacterial antibiotic resistance genes.
  • This RNAout mini vector backbone is from NTC.
  • Episomally means a segment of DNA that can exist either autonomously in the cell or as part of a chromosome.
  • NF seq An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression. (DOI: 10.1002/jgm.1312)) Description: This non-viral vector is based on two key features apart from using RNA out bacterial minicircle vectors. First, adding DNA localization signal sequence (DLS) to plasmid DNA (pDNA) will allow intracellular nuclear targeting of pDNA and increase the nuclear import of the pDNA. Using the 3NF DLS sequence will be recognized by the nuclear factor kappa B (NF ⁇ B) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions of T -cell activation.
  • DLS DNA localization signal sequence
  • NF ⁇ B nuclear factor kappa B
  • stabilizing agents include polyethylene glycol derivatives, including PEG-DMG 2000, TPGS (tocopherol polyethylene glycol succinate, e.g., TPGS 1000, TPGS 2000), maltoside, polysorbate 20, polysorbate 80, BRIJ S10 (polyoxyethylene alkyl ether), polyoxyethylene stearyl ether, Myrj52, or other suitable polymers, which have the purpose of extending circulation life, among other things.
  • Components of the lipid mixes may include the ionizable lipid, phospholipid, cholesterol, and stabilizing agent.
  • Low pH formulation buffers (3-6) may be used in formulating the lipid nanoparticle.
  • the pH of the formulation buffer is typically below the pKa of the lipid. Table 2.
  • Non-limiting examples of PNI ionizable lipids 47 P2023-3389-WO [00239]
  • the ionizable lipids of the present disclosure have asymmetric centers.
  • ionizable lipids may occur as racemates, racemic mixtures, individual enantiomers, enantiomeric mixtures, individual diastereomers, or as diastereomeric mixtures, with all possible isomers like tautomers and mixtures thereof.
  • T Cell Cryopreservation and Recovery Isolated T cells were cryopreserved in a cryoprotectant solution (for example, 10% DMSO or a proprietary commercial formulation such as the CryoStor® CS10 medium by STEMCELL Technologies) and stored in liquid or vapor-phase nitrogen. On the day of use, cryovials were thawed at about 37 °C.
  • a cryoprotectant solution for example, 10% DMSO or a proprietary commercial formulation such as the CryoStor® CS10 medium by STEMCELL Technologies
  • T cells must be activated to proliferate and differentiate into effector cells. In the lab, activation can be mimicked by a specific set of cytokines (signalling proteins) such as IL-2 and other proteins like CD2, CD3, CD28. Upon expansion, T cells will grow rapidly. Unless otherwise noted, all reagents were purchased from STEMCELL Technologies, Vancouver, Canada. Also, unless otherwise noted, all biologicals are human derived or human specific.
  • Pan T cells (CD3+), primary, human, Stemcell Technologies, Cat.70024, ImmunoCult-XFTM T Cell Expansion Medium (Stemcell Technologies, Cat. No.10981), recombinant human IL-2 (Stemcell Technologies, Cat. No Cat. No 78036), ImmunoCultTM Human CD3/CD28/CD2 T Cell Activator (Stemcell Technologies, Cat. No.10970), recombinant human ApoE4 (Peprotech Cat. No.350-04), lyophilized human IL-2 (“IL-2”) (Peprotech Inc., Montreal, Canada).
  • Transposon and Transposase [00244] PB-1 and PB-2 were designed based on piggyBac DNA Vector.
  • Transposases were required for efficient transposition of GOI between PB-vectors and chromosomes through a “cut- 49 P2023-3389-WO and-paste” mechanism. Two versions of transposase mRNA were designed, PNI-IV (2128nt) and PNI-V (2128nt). [00245] The transposases were both Clean Capped and include N1 methyl pseudouridine or unmodified uridine. Ten mg batches were analyzed using the TapeStation instrument (Agilent).
  • PiggyBac and Super PiggyBac transposase genes were cloned in PUC19 vector containing poly-A, then the plasmid was subjected to Nde-1 restriction to prepare liner DNA template for IVT.
  • the IVT reaction was carried out using Tris, MgCl 2 , spermidine, and NaCl containing buffer, T7 polymerase from NEB (Cat: M0251L), NTPs from ThermoFisher (Cat: R0481), CleanCap AG from Trilink (Cat: N-7113-100), N1-Methylpseudouridine-5'- Triphosphate from Trilink (Cat: N-1081) and iPPase from Sigma (Cat: I1643-500UN) for 3 hours. After that, the batches were subjected to one hour DNase digestion followed by RNA purification.
  • Cells were the resuspended in 200 uL of 1:1000 FVS660v in PBS, except for unstained and GFP only wells. The cells were incubated at RT in the dark for 10 minutes. Centrifugation at 300xg for 5 minutes was then performed, supernatant removed, and the stain buffer was added. The cells were then centrifuged again, then washed with stain buffer again, and supernatant was removed, and the cells were resuspended by placing the pellet in the stain buffer and performing flow cytometry. CAR staining [00249] Centrifuge the cells at 500 x g, 5 min remove supernatant or media add 200 uL viability stain to needed wells. Add 200 uL PBS to all others.
  • Lipid mix composition solutions were prepared in an organic phase including an organic solvent (e.g., ethanol) by combining prescribed amounts of lipids, including PNI ionizable lipids (e.g., non-limiting examples in Table 2), from individual lipid stocks in the organic solvent (e.g., ethanol).
  • an organic solvent e.g., ethanol
  • PNI ionizable lipids e.g., non-limiting examples in Table 2
  • the pH of the formulation buffer in formulating lipid nanoparticles is typically below the pKa of the lipid.
  • the nanoparticles can be suspended in any physiologically relevant buffer such as PBS, Dextrose, etc.
  • a formulation buffer such as a low pH aqueous buffer (e.g., sodium acetate buffer) to the required concentration.
  • DNA was prepared as described below.
  • All the DNA molecules were cloned and synthesized at Aldevron and dissolved in nuclease free water (1 mg/mL), while making LNPs the desired conc.
  • Lipid nucleic acid particle (LNAP, or interchangeably referred to as “nucleic acid- containing lipid nanoparticles” (NA-LNP) or “lipid nanoparticles” (LNP)) samples were then prepared by running both fluids using mixing instruments such as the NanoAssemblrTM series including SparkTM, IgniteTM, or Ignite PlusTM mixing instrument.
  • the lipid nucleic acid particles (LNAP) made in the instrument were immediately diluted down with Ca++ and Mg++ free 1X PBS at pH 7.4 in the aqueous output well. These LNAP were immediately collected into microcentrifuge tubes containing the same buffer at pH 7.4.
  • Encapsulation efficiency was 51 P2023-3389-WO measured by a modified RibogreenTM assay (Quanti-iT RiboGreenTM RNA assay kit, Thermo Fisher Scientific). This information was used to establish the desired dosage.
  • an aqueous phase of nucleic acids in a low pH buffer e.g., 100 mM sodium acetate buffer
  • an organic phase of lipid mx composition in an organic solvent e.g., lipid mx composition in ethanol
  • a predefined N/P ratio e.g., N/P of 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20.
  • the lipid nucleic acid particles (LNAP) formed were kept at room temperature for a predefined time (e.g., 3 min) and diluted down with Ca++ and Mg++ free 1X PBS at pH 7.4 . These LNAP were kept at 4 deg. C for a predefined time (e.g., 30-60 min) and subjected to a downstream processing such as one using AmiconTM centrifugal filters (Millipore, USA) or using TFF systems. Later, the encapsulation efficiency (EE) was measured by a modified PicoGreen/RibogreenTM assay (Quanti-iT Picogram/ RiboGreenTM RNA assay kit, Thermo Fisher Scientific).
  • Lipid nanoparticles encapsulating the nucleic acid payload were generated by rapidly mixing the organic phase including the lipid mix composition-ethanol solution with the aqueous buffer including the payload inside a microfluidic mixer designed to induce chaotic advection and provide a controlled mixing environment at intermediate Reynolds number (24 ⁇ Re ⁇ 1000).
  • a microfluidic mixer designed to induce chaotic advection and provide a controlled mixing environment at intermediate Reynolds number (24 ⁇ Re ⁇ 1000).
  • the microfluidic channels include, but are not limited to, the channels describes in PCT Pub. No. WO2017117647, U.S. Patent Nos.10,835,878, .
  • mRNA or pDNA was diluted using 100 mM sodium acetate buffer (pH 4) to the required concentration of 0.05 to 0.3 mg/mL depending on N/P ratio of 12, 10, 8, 6 or 4.
  • Lipid nanoparticle samples were then prepared by running both fluids, namely, nucleic acids in aqueous buffer and lipid mix composition in ethanol at a predefined flow ratio and total flow rate (e.g., flow ratio of 3:1 and at a total flow rate of 12 mL/minute).
  • the post cartridge lipid nucleic acid particle sample was diluted 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 desired concentration was achieved, the lipid nucleic acid particles were filter sterilized using 200 ⁇ m filters in aseptic conditions. Final encapsulation efficiency was measured by a modified RibogreenTM assay by using Quant-iTTM RiboGreen® RNA Reagent and Kit (Invitrogen) following manufacturer directions. mRNA and pDNA preparation is described below.
  • P2023-3389-WO particle attributes were generally sized from 50 – 200 nm for mRNA, depending on lipid composition.
  • particle size hydrodynamic diameter of the particles
  • DLS dynamic light scattering
  • He/Ne laser of 633 nm wavelength was used as the light source.
  • Z-Average size was reported as the particle size and defined as the harmonic intensity averaged particle diameter.
  • PDI polydispersity index
  • lipid mix compositions as defined by ionizable lipid/structural lipid/sterol/stabilizing agent mol% ratio, with the total mol% of the components in the lipid mix composition being 100 mol%. 53 P2023-3389-WO 54 P2023-3389-WO 55 P2023-3389-WO [00259]
  • the nucleic acid reagents or payloads used in the following experiments were: Trilink Cleancap eGFP mRNA: Cat.
  • PB mRNA Trilink Cleancap PB mRNA
  • SPB mRNA Trilink Cleancap SPB mRNA
  • Trilink 56 P2023-3389-WO Cleancap CD19 CAR mRNA were made in-house using IVT plasmids.
  • CD19 CAR Plasmid and in-house custom plasmids were synthesized in house, and off-the shelf plasmids were purchased from Aldevron, Fargo, ND (NTC9385R (3xCpG)-CMV-EGFP CpG free). The total size of these custom and off-the shelf plasmids range from 1600-4600 base pair (BP).
  • T cell reagents were from StemCell Technologies unless otherwise stated. T cells were isolated from whole human peripheral blood using a negative selection isolation procedure (EasySep TM Human T Cell Isolation Kit). T cell activation and expansion was carried out using ImmunocultTM Human CD3/CD28/CD2 Activator in ImmunoCultTM Human T Cell Expansion Media supplemented with recombinant human IL-2 (Stemcell). Ap0E4 was purchased from Peprotech Inc., Rocky Hill USA.
  • T cells typically enter a logarithmic phase of growth 48-96 hours after activation, which phase is characterized by a period of rapid proliferation and metabolic activity for 24-72 hours followed by a plateau in the growth curve as the cells start to return to a quiescent state. T cells may be exposed to lipid nucleic acid particle before or during the log phase of growth (e.g., day 0, day 1, day2, day 3, day 4 or day 5).
  • lipid nucleic acid particle e.g., day 0, day 1, day2, day 3, day 4 or day 5.
  • EXAMPLE 2 DNA preparation [00262] Plasmid DNA was purchased from Aldevron/ Nature Technology, while the “PNI” plasmids were designed in house and cloned at Aldevron using NP-1 as the parental molecule or with plasmid as the parental backbone. All plasmid sequences were confirmed using Sanger sequencing and the plasmids stored in Nuclease (DNase/RNase) free water. Moreover, all the plasmids passed the endotoxin test and the amount of endotoxin present in the sample was between of 2 – 200 EU/mg. All of the plasmids were sized from 1600 - 4400 BP including the 500 BP plasmid backbone as mentioned in the following table.
  • Plasmid delivery in T cells using LNPs [00263] Plasmid DNA NP-1, NP-2, NP-3, or NP-4, was prepared via the NanoAssemblrTM SparkTM instrument formulation process and Composition LNP 1. The measurements conducted using Malvern DLS were as follows: The size of all formulated samples was within the range of 85-100 nm, as depicted in FIG. 1.
  • FIG.2A and FIG.2B Two different conditions were examined, namely transfection with 200 ng or 500 ng of plasmid DNA (FIG.2A and FIG.2B, respectively).
  • the results indicate that the use of LNPs for plasmid DNA transfection is not detrimental to the primary T cells’ viability, as shown in FGIS.2A and 2B. Even at the higher concentration of 500 ng, the T cells exhibited good tolerance to the plasmid DNA. This is in contrast to prior studies involving electroporation, where different data trends were observed.
  • the expression of green fluorescent protein was used to visualize the ability of the plasmids in LNP to accomplish the goal of genetic expression.
  • the plasmid transfection efficiency in primary T cells was assessed after introducing plasmid DNA via lipid nanoparticles 59 P2023-3389-WO (LNPs) at different time points (24, 48, 72, and 96 hours). Two distinct conditions were investigated, transfection with 250 ng of plasmid DNA, and transfection with 500 ng of plasmid DNA. Percent GFP expression are shown in FIG. 3A for 250 ng and in FIG. 3B for 500 ng, respectively. MFI is shown in FIG.4A and 4B respectively.
  • the findings demonstrate that the utilization of LNPs for plasmid DNA transfection can yield approximately 80% transfection efficiency for NP-1, while NP-2 to NP-4 can exhibit 55-60% transfection efficacy.
  • FIGS.5F and 5G illustrates MFI of PNI 762 -LNP_1 composition screen, respectively, using donor T cells purchased from Stem Cell Technologies.
  • FIG.5H shows Transfection Efficiency (% TE) at 24, 48 and 72 hours post transfection in LNP_2, LNP_3, LNP_4, LNP_5 with NP1 plasmid DNA or eGFP mRNA encapsulated.
  • FIG.5I shows MFI for the same compositions of FIG.5H at 24, 48 and 72 hours post transfection.
  • FIGS. 5J and 5K show transfection efficiency (% eGFP) and MFI of the protein expression respectively for PNI 659- LNP_1 encapsulating NP-1 or eGFP mRNA in transfecting T-cells.
  • FIG.5J LNP-1 composition with PNI 659 lipid showed around 80% TE for both pDNA (“NP-1”) and mRNA-eGFP in T cells using LNPs.
  • FIG.5K 60 P2023-3389-WO shows that pDNA (“NP-1”) showed higher expression of eGFP compared with eGFP-mRNA expression.
  • Key parameters measured in this evaluation include A) % cell viability post treatment, B) % GFP positive cells or % transfection, and C) MFI or the expression level of GFP.
  • the PiggyBac Transposon includes an ITR, gene of interest or GOI or cargo, another ITR, and is accompanied by PiggyBac Transposase which enables cut and paste into the genomic DNA.
  • Two versions of PiggyBac cassette pDNA capable of delivering up to 200 kb were designed, “PB-1” and “PB-2”.
  • Two transposases were also designed, PNI-IV (2128 nt) and PNI- V (2128 nt) for efficient transposition of GOI between PB-vectors and chromosomes through a “cut-and-paste” mechanism.
  • RNAs were clean capped and modified with N1 methyl pseudouridine in quantity of 10 mg each, and quality tested using the TapeStationTM automated electrophoresis instrument (Agilent, Santa Clara, CA).
  • T cells were thawed and activated, and mRNA and pDNA LNPs were added together to transfect the T cells. Detection was performed at 48 h, 72 h, and days 5, 7, 10 and 15 post transfection. Fresh media was also added to the main culture at 48 h, 72 h, days 5, 7 and 9.
  • Co-dosing of 250 ng of pDNA and 250 ng mRNA was used.
  • the lipid nanoparticle formulation was PNI-550 in LNP 1.
  • FIGS.6A and 6B Viability for primary T cells post LNP treatment/transfection are shown in FIGS.6A and 6B for two different primary T cell donors (FIG.6A, donor-1; FIG.6B, donor-2). Percent GFP integration is shown for Day 1 and Day 2 post LNP treatment for the same cells in FIGS. 7A and 7B. 61 P2023-3389-WO [00275] In this experiment we tested two donors (Donor-1 and 2), and the SPB/PB mRNAs were modified with N1 methyl pseudouridine. PiggyBac insertion of eGFP cassette in primary T cells using LNPs as delivery mode, PNI 550-LNP 1.
  • the MFI is shown for Donor -1 primary T cells in FIG.8A, and for Donor -2 primary T cells in FIG.8B.
  • UT is untreated T cells used as a control.
  • payload encoding mRNA was either added alone (e.g., PNI-IV) or in combination with pDNA (e.g., PNI-IV_PNI22).
  • EXAMPLE 6 Effects of ionizable lipid on gene insertion [00276] Four different combinations of genetic elements were tested for each lipid. LNP 1 composition was used for LNP preparation in all cases while the ionizable lipid was varied.
  • Results are based on the ranges of % eGFP integration during days 2, 3, 5, and 7 post LNP treatment, as illustrated in corresponding figures in FIGS.9A – 14H. 62 P2023-3389-WO [00278]
  • T cell viability was high, at 100% by day 10 for all lipids (results not shown), and the non-limiting examples of the lipid compositions including various ionizable lipids were effective as non-viral delivery of PiggyBac transposons with around 80% gene integration.
  • Both PB and SPB mRNAs showed around 60-80% DNA insertions using PB1 and PB2 pDNA.
  • PNI 762 showed around 70-80% eGFP over period of 10 days with highest MFI at given combinations.
  • FIGS.15A-15B The influence of s/MAR, MAR, and NF sequence positioning in protein expression is illustrated in FIGS.15A-15B, with certain combinations of positions significantly affecting protein expression levels. As illustrated in FIG.15A, certain plasmids with combinations where s/MAR precedes the poly-A signal were less effective than others (e.g., as observed in NP-12 and NP-20 plasmids).
  • Example 8 LNPs for the delivery of CD19 chimeric antigen receptor (CAR) expression pDNA PNI-24 (a plasmid made to order by Aldevron) or transposon insertions of CD19-CAR cassette in human primary cells using LNPs.
  • CAR chimeric antigen receptor
  • pDNA PNI-24 a plasmid made to order by Aldevron
  • transposon insertions of CD19-CAR cassette in human primary cells using LNPs In primary T cells we transfected CD19-CAR DNA encapsulated in lipid nanoparticles and measured % viability, quantified % transfection efficacy (% CAR TE) using flow cytometry, and analyzed the CAR expression profile (e.g., MFI) towards CAR – T cell 63 P2023-3389-WO therapy applications.
  • FIGS.17A-17E illustrated the effects of PNI 768 and PNI 762 LNP_1 and LNP_6 combinations of ionizable lipids and lipid mix formulations on the delivery to human primary T cells of CAR pDNA delivery along with CAR mRNA, at 24 hrs (FIGS.16A-16E) and 48 hrs (FIGS.17A-17E) post T cell transfection, respectively. Above 90% cell viability and transfection efficiency in both 250 ng and 500 ng doses were achieved, as shown in FIGS. 16A-17E. Due to gentle nature of lipid nanoparticle mediated delivery, we achieved transfection efficiency (94% TE) higher than what can be achieved with electroporation.
  • LNPs offer an enhanced platform for the delivery of large DNA like CD19 CAR into primary T cells.
  • the increased transfection efficiency, % viability and cell yield highlight the potential advantages of LNPs for cell-based therapeutics development for various diseases including cancer, HIV, autoimmune disorders and so on.
  • Example 9 Cell killing assay Effector Cell Preparation [00284] Primary human T cells expressing CD19 CAR were prepared and transfection efficiency was determined using flow cytometry. CAR-T cells and untreated (UT) cells were washed and resuspended in RPMI 1640 media (ThermoFisher Scientific, catalog # 11875093) supplemented with 2% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • SUP-B15 cells were obtained from ATCC (Manassas, Virginia, USA ).
  • K562 was obtained from ATCC (Manassas, Virginia, USA) .
  • CD19+ target SUP- B15 cells and CD19- control K562 cells were stained with VPD450 dye (BD Biosciences, catalog # 562158). Cells were washed with PBS, incubated with a 1:1000 dilution of VPD450 dye in PBS for 10 minutes at 37°C, protected from light.
  • VPD450 dye BD Biosciences, catalog # 562158
  • Detection of CD19-specific killing of B Cells Following co-culture, the plate was centrifuged at 300 x g for 5 minutes at room temperature (RT), and the supernatant was removed.
  • FIG. 18A shows specific lysis of leukemic B cell line by CD19 CAR T cells generated using mRNA or transposon LNP.
  • Effector cells were created by expressing CD19 CAR in primary human T cells via mRNA LNP transfection or LNP-mediated co-delivery of CD19 CAR encoded PB2 transposon vector and SPB transposase.
  • Target cells SUP-B15, CD19+; K562, CD19-
  • FVS660 viability dye were stained by flow cytometry. Specific lysis was determined by quantifying VPD450+FVS660+ cells.
  • 18B shows specific lysis of leukemic B cell line by transposon CD19 CAR T cells 2 or 7 days following LNP addition/transfection of cells.
  • Effector cells were generated by 65 P2023-3389-WO expressing CD19 CAR in primary human T cells via LNP-mediated co-delivery of CD19 CAR encoded PB2 transposon vector and SPB transposase.
  • Target cells SUP-B15, CD19+; K562, CD19-
  • Specific lysis of transposon CD19 CAR-T cells was tested 2 days and 7 days after LNP addition.
  • WPRE element is Italic – Woodchuck Hepatitis Virus Postranslational Regulatory Element woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) known to increase the transgene (GOI) ex
  • WPRE element is Italic – Woodchuck Hepatitis Virus Postranslational Regulatory Element woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) known to increase the transgene (GOI) ex
  • MAR seq is non bold Underline text (Ref: MAR characteristic motifs mediate episomal vector in CHO cells, Yan Lin a,c, Zhaoxi Li b, TianyunWanga, ⁇ , XiaoyinWang a, Li Wanga, Weihua Dong a, Changqin Jing b, Xianjun Yang (dx.doi.org/10.1016/j.gene.2015.01.032).)
  • NF seq is bold dashed underline (Ref:
  • the consensus ⁇ B site has a partial palindromic sequence, where R is any purine (A or G), N is any nucleotide, W is A or T, and Y is any pyrimidine (C or T).

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Abstract

L'invention concerne une composition de mélange lipidique permettant de former des nanoparticules lipidiques (LNP) en association avec un ADN, destinée à être utilisée dans la transfection d'une cellule de lignée hématopoïétique, la composition de mélange lipidique comprenant un lipide ionisable, un phospholipide, un stabilisant et un cholestérol. L'invention concerne également les LNP, un procédé de modification de cellules de lignée hématopoïétique à l'aide des LNP, une cellule de lignée hématopoïétique modifiée par le procédé, et un procédé de traitement d'une déficience immunitaire, d'un cancer, d'une maladie auto-immune ou d'une insuffisance génétique.
PCT/EP2025/060662 2024-04-17 2025-04-17 Administration de plasmide non destructif à des cellules immunitaires Pending WO2025219528A1 (fr)

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Citations (23)

* 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
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
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
USD771834S1 (en) 2015-04-28 2016-11-15 University Of British Columbia Microfluidic cartridge
USD771833S1 (en) 2015-04-28 2016-11-15 University Of British Columbia Microfluidic cartridge
USD772427S1 (en) 2015-04-28 2016-11-22 University Of British Columbia Microfluidic cartridge
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
USD800336S1 (en) 2016-07-13 2017-10-17 Precision Nanosystems Inc Microfluidic cartridge
USD800335S1 (en) 2016-07-13 2017-10-17 Precision Nanosystems Inc. Microfluidic chip
WO2018006166A1 (fr) 2016-07-06 2018-01-11 Precision Nanosystems Inc Instrument de mélange microfluidique intelligent et cartouches
USD812242S1 (en) 2016-07-13 2018-03-06 Precision Nanosystems Inc Microfluidic cartridge
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
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
WO2024006960A1 (fr) * 2022-06-29 2024-01-04 Juno Therapeutics, Inc. Nanoparticules lipidiques pour l'administration d'acides nucléiques
WO2024006863A1 (fr) * 2022-06-30 2024-01-04 Precision NanoSystems ULC Formulations de nanoparticules lipidiques pour vaccins

Patent Citations (26)

* 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
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
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
USD772427S1 (en) 2015-04-28 2016-11-22 University Of British Columbia Microfluidic cartridge
USD803416S1 (en) 2015-04-28 2017-11-21 University Of British Columbia Microfluidic cartridge
USD771834S1 (en) 2015-04-28 2016-11-15 University Of British Columbia Microfluidic cartridge
USD771833S1 (en) 2015-04-28 2016-11-15 University Of British Columbia Microfluidic cartridge
US10076730B2 (en) 2016-01-06 2018-09-18 The University Of British Columbia Bifurcating mixers and methods of their use and manufacture
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
WO2018006166A1 (fr) 2016-07-06 2018-01-11 Precision Nanosystems Inc Instrument de mélange microfluidique intelligent et cartouches
USD800335S1 (en) 2016-07-13 2017-10-17 Precision Nanosystems Inc. Microfluidic chip
USD812242S1 (en) 2016-07-13 2018-03-06 Precision Nanosystems Inc Microfluidic cartridge
USD800336S1 (en) 2016-07-13 2017-10-17 Precision Nanosystems Inc Microfluidic cartridge
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
WO2024006960A1 (fr) * 2022-06-29 2024-01-04 Juno Therapeutics, Inc. Nanoparticules lipidiques pour l'administration d'acides nucléiques
WO2024006863A1 (fr) * 2022-06-30 2024-01-04 Precision NanoSystems ULC Formulations de nanoparticules lipidiques pour vaccins

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
"Cell and Tissue Culture: Laboratory Procedures", 1993, J.B. LIPPINCOTT COMPANY
"Current Protocols in Immunology", 1991
"GenBank", Database accession no. M83137.1
"Gene Transfer Vectors for Mammalian Cells", 1987
"Handbook of Experimental Immunology", 1994, ACADEMIC PRESS, INC.
"NCBI", Database accession no. NG 042877.1
"Oligonucleotide Synthesis", 1984
"Remington: The Science and Practice of Pharmacy", 2006, LIPPINCOTT, WILLIAMS AND WILKINS
"Short Protocols in Molecular Biology", 1999, WILEY AND SONS
CA. JANEWAYP. TRAVERS, IMMUNOBIOLOGY, 1997
J.P. MATHERP.E. ROBERTS: "Introduction to Cell and Tissue Culture", 1998, ACADEMIC PRESS
KLEBANOFF, J IMMUNOTHER, vol. 35, no. 9, 2012, pages 689 - 701
MAGNANI CF ET AL.: "Transposon-Based CAR T Cells in Acute Leukemias: Where are We Going?", CELLS, vol. 9, no. 6, 27 May 2020 (2020-05-27), pages 1337
MOORE DKMOTAUNG BDU PLESSIS NSHABANGU ANLOXTON AG: "SU-IRG Consortium. Isolation of B-cells using Miltenyi MACS bead isolation kits", PLOS ONE, vol. 14, no. 3, 20 March 2019 (2019-03-20)
PRAZERES PEDRO HENRIQUE ET AL: "Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells", INTERNATIONAL JOURNAL OF NANOMEDICINE, vol. Volume 18, 18 October 2023 (2023-10-18), New Zealand, pages 5891 - 5904, XP093243617, ISSN: 1178-2013, Retrieved from the Internet <URL:https://www.dovepress.com/getfile.php?fileID=93575> DOI: 10.2147/IJN.S424723 *
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS
TERAKURA ET AL., BLOOD, vol. 1, 2012, pages 72 - 82
YAN LIN A,CZHAOXI LI BTIANYUNWANGAXIAOYINWANG ALI WANGAWEIHUA DONG ACHANGQIN JING BXIANJUN YANG, MAR CHARACTERISTIC MOTIFS MEDIATE EPISOMAL VECTOR IN CHO CELLS

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