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WO2025240411A1 - Méthodologie de production de vecteur viral économique et conviviale - Google Patents

Méthodologie de production de vecteur viral économique et conviviale

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Publication number
WO2025240411A1
WO2025240411A1 PCT/US2025/029052 US2025029052W WO2025240411A1 WO 2025240411 A1 WO2025240411 A1 WO 2025240411A1 US 2025029052 W US2025029052 W US 2025029052W WO 2025240411 A1 WO2025240411 A1 WO 2025240411A1
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WIPO (PCT)
Prior art keywords
virus
collection medium
pva
mammalian cells
particles
Prior art date
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Pending
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PCT/US2025/029052
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English (en)
Inventor
Hiromitsu Nakauchi
Joydeep BHADURY
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Leland Stanford Junior University
Original Assignee
Leland Stanford Junior University
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Publication of WO2025240411A1 publication Critical patent/WO2025240411A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16023—Virus like particles [VLP]
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material

Definitions

  • Recombinant retrovirus vectors including recombinant lentivirus vectors are commonly used in biotechnology research and for development of gene therapies, including for gene transfer into pluripotent stem cells.
  • a gene of interest including coding and non-coding RNAs, may be cloned into a recombinant lentivirus DNA vector.
  • the recombinant lentivirus vector is then co-transfected with packaging helper plasmids into mammalian cells in order to produce particles in which the recombinant lentivirus is packaged into a recombinant virion.
  • packaging helper plasmids into mammalian cells in order to produce particles in which the recombinant lentivirus is packaged into a recombinant virion.
  • Various methods are known for packaging recombinant lentivirus particles, including those described in U.S.
  • Patent 11,414,675 U.S. Patent Publications 2022/0235371 and US 2018/0135077.
  • Another challenge among many is to obtain a composition comprising virus-like particles that do not trigger unwanted differentiation of sensitive cells types like hematopoietic stem cells (HSCs), pluripotent stem cells (PSCs), etc. and deliver recombinant viral RNA capable upon reverse transcription of integrating into a host genome.
  • HSCs hematopoietic stem cells
  • PSCs pluripotent stem cells
  • this disclosure aims to address these and other needs by providing virus-like particle (VLP) preparations, tissue-culture medium compositions, tissue-culture supplements, kits and VLP production methods.
  • VLP virus-like particle
  • this disclosure provides an in vitro method for producing a virus- like particle, the method comprising the following steps: a) contacting mammalian cells with a collection medium supplemented with at least a propionate, a butyrate and caffeine, wherein the collection medium contains about 5% v/v or less of heat-inactivated serum and a pyruvate, or the collection medium is serum-free and contains polyvinyl alcohol (PVA), wherein the mammalian cells being seeded in a tissue culture vessel and wherein the mammalian cells express one or more of recombinant viral proteins for an assembly into virus-like particles; b) culturing the mammalian cells in the collection medium; and c) collecting the collection medium from step b), said collection medium comprising virus-like
  • the steps may be repeated at least once and/or step b) may performed for about 1 to about 24 hours.
  • the method may further comprise prior to step a), co- transfecting mammalian cells with DNA premixed with a transfection reagent, wherein the DNA comprises a mixture of a recombinant retroviral vector, a first recombinant retrovirus packaging plasmid and a second recombinant retrovirus packaging plasmid.
  • Present methods may further comprise purifying the virus-like particles by centrifuging and/or filtering the collection medium of step c).
  • the methods may include one or more of the following features: the mammalian cells are attached to a solid support; the virus-like particles comprise one or more lentivirus proteins and a recombinant RNA sequence in which a payload is flanked with truncated long terminal repeats (LTRs); and/or the collection medium is serum-free and comprises polyvinyl alcohol (PVA).
  • the solid support may comprise a tissue culture plastic coated with a poly-lysine solution.
  • Preferred mammalian cells may include human embryonic kidney (HEK) 293T cells.
  • the collection medium may further comprise one or more of the following: L-Glutamine or its stabilized dipeptide form L-alanyl-L-glutamine, Insulin, Transferrin, Selenium, Ethanolamine, essential amino acids, or any combination thereof.
  • L-Glutamine or its stabilized dipeptide form L-alanyl-L-glutamine, Insulin, Transferrin, Selenium, Ethanolamine, essential amino acids, or any combination thereof.
  • the collection medium comprises Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s Patent Application 4227.155879PCT F-12 Nutrient medium (F12), Roswell Park Memorial Institute (RPMI) 1640 medium, or any mixture thereof, supplemented with at least a combination of sodium propionate, sodium butyrate, caffeine and polyvinyl alcohol (PVA).
  • the collection medium may comprise the following supplement: about 3.5 mM to about 5.5 mM of the propionate; about 1 mM to about 3 mM of the butyrate; about 0.5 mM to about 2.5 mM of caffeine; and about 0.05 % wt/vol to about 0.2 % wt/vol of PVA.
  • the collection medium does not comprise beta- mercaptoethanol and/or the collection medium may comprise from about 5mM to about 15 mM of sodium pyruvate.
  • PVA is at least partially hydrolyzed and having a degree of hydrolyzation of at least 85% or higher, or PVA is highly hydrolyzed and having a degree of hydrolyzation of 98% or higher.
  • co-transfections can be performed with the transfection reagent which comprises a polyethyleneimine and preferably, a ratio of the DNA to the transfection reagent may be in the range from about 1:1 to about 1:6.
  • the DNA may be used in a total concentration ranging from about 0.01 to about 0.02 pM per one square centimeter (cm 2 ) of the tissue culture vessel surface onto which the mammalian cells are plated.
  • the cells may be co- transferred with less than 0.01 pM of each: the recombinant retrovirus vector, the first retrovirus packaging plasmid and the second retrovirus packaging plasmid, per one square centimeter (cm 2 ) of the tissue culture vessel surface onto which the mammalian cells are plated.
  • the first retrovirus packaging Patent Application 4227.155879PCT plasmid may encode and express in co-transfected mammalian cells a lentivirus reverse transcriptase and a lentivirus capsid structural protein GAG; and/or the second retrovirus packaging plasmid may encode and express in mammalian cells vesicular stomatitis virus glycoprotein (VSV-G protein) and/or lentivirus REV protein.
  • VSV-G protein vesicular stomatitis virus glycoprotein
  • the methods according to this disclosure include those, wherein the virus-like particle carries a payload for transduction of mammalian cells.
  • this disclosure relates to a method for transducing pluripotent stem cells, the method comprising: i) producing virus-like particles by the methods according to this disclosure; and ii) contacting the pluripotent stem cells with the virus-like particles.
  • this disclosure relates to a serum-free collection medium for producing a virus-like particle
  • the serum-free collection medium comprises, consists essentially of, or consists of a basic tissue culture medium supplemented with at least: - L-Glutamine and/or its stabilized dipeptide form L-alanyl-L- glutamine; - Insulin, Transferrin, Selenium, Ethanolamine, or any mixture thereof; - essential amino acids; - a propionate; - a butyrate; - Caffeine; and Patent Application 4227.155879PCT - PVA, wherein PVA is at least partially hydrolyzed and having a degree of hydrolyzation of at least 85% or higher, or PVA is highly hydrolyzed and having a degree of hydrolyzation of at least 98% or higher.
  • Embodiments of the serum-free collection medium may include those, wherein the basic tissue culture medium is a 50:50 v/v liquid mixture of DMEM/F12 and RPMI 1640 supplemented with: about 0.1-5 mM L-Glutamine or its stabilized dipeptide form L-alanyl-L- glutamine; about 1-5 ⁇ M Insulin; about 0.5-1 ⁇ M Transferrin; about 0.02-0.5 ⁇ M Selenium; about 25-35 ⁇ M Ethanolamine; about 3.5 mM-5.5 mM Sodium Propionate; about 1 mM-3 mM Sodium Butyrate; about 0.5 mM - 2.5 mM Caffeine; and about 0.05 % - 0.2 % wt/vol PVA at 0.1% (wt/vol).
  • the basic tissue culture medium is a 50:50 v/v liquid mixture of DMEM/F12 and RPMI 1640 supplemented with: about 0.1-5 mM L-Glutamine or its stabilized di
  • this disclosure relates to a preparation comprising virus-like particles mixed with the serum-free collection medium according to this disclosure.
  • Patent Application 4227.155879PCT [00021]
  • this disclosure relates to a use of a preparation comprising virus-like particles mixed with the serum-free collection medium according to this disclosure for genome manipulation of a cell, including but not limited to, stem cells, pluripotent stem cells, hematopoietic stem cells and any other cells.
  • this disclosure relates to a supplement for producing virus-like particles, the supplement comprising: a propionate; a butyrate; caffeine; and partially or highly hydrolyzed PVA; wherein the supplement components are supplied in concentrations dilutable to the following final concentrations: about 3.5 mM to about 5.5 mM of the propionate; about 1 mM to about 3 mM of the butyrate; about 0.5 mM to about 2.5 mM of caffeine; and about 0.05 % wt/vol to about 0.2 % wt/vol of PVA.
  • the components may be provided in separate containers.
  • Fig.1 is a block diagram of one embodiment of a method for producing a preparation of virus-like particles according to this disclosure.
  • Fig.2 is a comparative analysis for co-transfecting mammalian cells at low or high DNA concentrations.
  • Fig.3 reports that infectivity of virus-like particles depends on DNA concentration and on a composition of collection medium.
  • Fig.4 reports a comparative analysis of infectivity of virus-like particles depends on a composition of collection medium.
  • % wt means percentage by weight and % vol means percentage by volume.
  • % wt/vol means weight in volume and % v/v means volume in volume.
  • mM or “pM” refers to a concentration and stands for millimoles or picomoles of a compound in one liter of solution, respectively.
  • a 1 mM caffeine solution refers to a composition with 1 millimoles of caffeine per one liter of solution.
  • DMEM stands for Dulbecco’s Modified Eagle’s Medium, the composition of which was reported in Dulbecco, R. and Freeman, G. (1959) Virology Patent Application 4227.155879PCT 8:396.
  • DMEM comprises amino acids, vitamins, inorganic salts, glucose (dextrose) and phenol red and is commercially available from ThermoFisher Scientific, USA.
  • DMEM high glucose may comprise 25 mM of D-glucose (dextrose) as shown in Table A below: Table A.
  • RPMI 1640 medium refers to Roswell Park Memorial Institute 1640 medium, a composition of which was initially reported in Moore, G.E., Gerner, R.E. and Franklin, H.A. (1967) J.A.M.A., 199:519 and Mossinger, J. (1991) Parasitol., 103:85 (Nematodes, Litomosoides carinii).
  • RPMI 1640 is commercially available from ThermoFisher Scientific, USA.
  • RPMI 1640 comprises amino acids, vitamins, inorganic salts, D-glucose (dextrose), Glutathione (reduced) and Phenol Red, as shown in Table B below.
  • Table B. RPMI 1640 (ThermoFisher Scientific Cat NO 11875119) Patent Application 4227.155879PCT [00036]
  • DMEM/F12 medium refers to 1:1 mixture of DMEM with Ham’s F-12 Nutrient medium, reported in Ham, R.G., (1965) Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Natl. Acad. Sci., 53:288.
  • This formulation combines DMEM’s with high concentration of glucose, amino acids, and vitamins with F-12’s wide variety of components.
  • This composition comprises amino acids, vitamins, inorganic salts, D-glucose (dextrose), Phenol Red, Hypoxanthine Na, Linoleic Acid, Lipoic Acid, Putrescine 2HCl, Sodium Pyruvate and Thymidine, as shown in Table C below.
  • DMEM/F12 ThermoFisher Scientific Cat NO 11320082
  • Patent Application 4227.155879PCT [00037]
  • FBS refers to Fetal Bovine Serum.
  • HEPES stands for 4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid.
  • mammalian cells are cultured in vitro in tissue culture in a sterile tissue culture vessel placed in an incubator maintained at the optimal temperature of about 37 ° C with about 5% v/v CO2, unless specified otherwise.
  • the term “plasmid” is used in its common meaning and refers to a circular double-stranded DNA construct which carries an origin of replication sequence and is capable of replication independently from chromosomal DNA.
  • virus-like particle refers to a nanoscale structure of assembled recombinant viral proteins, including as described in Charlesworth et al. BioRxiv, January 12, 2024 and Nooraei et al. J Nanobiotechnol, 2021, 19: 59. VLPs may be used for genome editing methods, including as described in Charlesworth et al. BioRxiv, January 12, 2024 and in Banskota et al. Cell, 2022 Jan 20; 185 (2):250-265.
  • the VLPs may be composed of recombinant viral proteins Patent Application 4227.155879PCT of one virus type, for example, recombinant lentivirus proteins, including but not limited to one or more of GAG, REV and/or Retrovirus Polymerase or any portion thereof; or the VLPs may be composed of recombinant viral proteins of several different virus types, e.g. recombinant lentivirus proteins and additional recombinant proteins from a virus which is not a lentivirus. Examples of plasmids for expression of suitable recombinant viral proteins and in particular of recombinant retrovirus proteins may be found in Mangoet et al. Nat Commun.2019, Jan 3; 10(1): 45.
  • the VLPs may comprise a payload (cargo) for delivery to a target mammalian cell.
  • the VLPs may or may not contain a recombinant nucleic acid.
  • payloads include, but are not limited to, nucleic sequences, RNA sequences, DNA sequences, genes or any portions thereof, proteins, peptides, antigens, epitopes, small molecules, biologics, anti-cancer drugs, vaccine, an imaging agent and/or any other biologics or small molecule to be delivered into a cell.
  • the VLPs lack at least a portion of a viral genetic material necessary for replication and multiplication of a virus.
  • VLP preparations may be transfection-competent to transfect cells once, VLPs cannot replicate and multiply into new viral particles in transfected cells for future rounds of infection.
  • VLPs do not encode viral structural proteins and these structural proteins may be instead supplied by a helper plasmid or from some other source during production of VLPs.
  • the VLP may be a retrovirus- like particle, including a lentivirus-like particle, in which a cell-derived lipid bilayer membrane envelops an inner core in which recombinant retroviral, preferably lentiviral, proteins are assembled into a shell (capsid) around a recombinant nucleic acid which may include a recombinant sequence to be integrated into a mammalian Patent Application 4227.155879PCT cell.
  • the lipid envelope may display one or more recombinant viral proteins that may facilitate anchoring of the VLP to a target cell membrane and initiating a VLP fusion and entry (transfection, infection) into the target mammalian cell.
  • VLPs may also include those VLPs which are non-enveloped.
  • VLPs are lentivirus VLPs carrying a recombinant nucleic acid sequence in the form of an RNA for reverse transcription and integration into a mammalian genome, the recombinant nucleic acid sequence being flanked with truncated lentivirus long terminal repeats (LTRs).
  • LTRs lentivirus VLPs carrying a recombinant nucleic acid sequence in the form of an RNA for reverse transcription and integration into a mammalian genome, the recombinant nucleic acid sequence being flanked with truncated lentivirus long terminal repeats (LTRs).
  • LTRs truncated LTRs are necessary in order to facilitate integration of the recombinant nucleic acid sequence into a target cell genome.
  • the LTRs are truncated and are preferably missing the LTR U3 portion such that upon integration into the target cell genome, the L
  • this disclosure provides methods for production of virus- like particles.
  • this disclosure relates to a collection medium suitable for production and harvesting of virus-like particles. It has been unexpectedly found that culturing mammalian cells co-transfected with a recombinant retrovirus vector and packaging helper plasmids in a collection medium according to this disclosure and then also harvesting (collecting) virus-like particles in the collection medium provides significant technical advantages, including one or more of the following: 1) virus-like particles may be produced in a high titer and these VLPs are capable of transfecting (infecting) cells; 2) the virus-like particles are suitable for integration into mammalian genome; Patent Application 4227.155879PCT 3) this culturing protocols minimize contamination of the virus-like particle preparations with cell debris and growth factors; 4) the culturing medium may be also used as a freezing medium for VLP preparations; and 5) the collection medium in which the virus-like particles are harvested does not interfere with re
  • the above technical advantages and at least some others are attributed at least in part to the following compositions of the collection medium.
  • the collection medium according to this disclosure may be referred in this disclosure as the Lenti Virus Collection Medium (LVCTM). It will be appreciated that the LVCTM collection medium may be used for production of any virus-like particles (VLPs), including, but not limited to, recombinant lentivirus-like particles.
  • VLPs virus-like particles
  • the collection medium according to this disclosure is a composition which comprises a water-based basic tissue culture medium supplemented with at least: - propionic acid or its conjugate base propionate (C2H5COO-), preferably in the form of sodium propionate Na(C 3 H 7 COO) and/or any other salt, ester and/or amide of propionic acid, - butyric acid or its conjugate base butyrate (C 3 H 7 COO-), preferably in the form of sodium butyrate Na(C 3 H 7 COO) and/or any other salt, ester and/or amide of butyric acid; and Patent Application 4227.155879PCT - caffeine.
  • C2H5COO- propionic acid or its conjugate base propionate
  • C2H5COO- preferably in the form of sodium propionate Na(C 3 H 7 COO) and/or any other salt
  • ester and/or amide of propionic acid - butyric acid or its conjugate base butyrate (C 3
  • the collection basic tissue culture medium may be one or more of the following tissue culture media: DMEM, DMEM high glucose, F-12, RPMI 1640, or any combination thereof.
  • the basic tissue culture medium may comprise a combination of DMEM, F-12 and/or RPMI 1640.
  • Other basic tissue culture media, especially those basic tissue culture media which are suitable for growth of epithelial cells in tissue culture may be also used instead of, or in addition to DMEM, F-12 and/or RPMI 1640.
  • Table 1 One embodiment of the basic tissue culture medium according to this disclosure is shown in Table 1 below: Table 1.
  • the collection medium comprises the basic medium, one preferred embodiment of which is shown in Table 1, and is further supplemented with at least a propionate, preferably sodium propionate, a butyrate, preferably sodium butyrate, and caffeine.
  • the collection medium is in liquid form when in use, with water being used as solvent. Powdered formulations of the collection medium may be also prepared for easy storage and to be mixed with water prior to use.
  • the serum can be exposed to an elevated temperature for a period of time sufficient to Patent Application 4227.155879PCT inactivate complement, preferably at a temperature of about 55-56 °C for a period of time, preferably for about 30 minutes.
  • a pyruvate preferably sodium pyruvate, may be used in any concentrations and preferably, in an amount ranging from about 5 mM to about 15 mM, and more preferably from about 7.5 mM to about 12.5 mM or about 8 mM to about 13 mM.
  • the collection medium may comprise polyvinyl alcohol (PVA).
  • PVA polyvinyl alcohol
  • the collection medium may comprise polyvinyl alcohol (PVA).
  • PVA is partially hydrolyzed and having a degree of hydrolyzation of at least 85% or higher, preferably about 87% to about 90%.
  • PVA may be highly hydrolyzed and having a degree of hydrolyzation of 98% or higher.
  • PVA variants of different molecular weights may be used.
  • Molecular Weight Range Commonly between 30,000 to 70,000 g/mol. Highly Hydrolyzed PVA: c. Degree of Hydrolyzation: Above 98%. d. Molecular Weight Range: Typically, from 31,000 to 50,000 g/mol. Low Molecular Weight PVA: e. Molecular Weight Range: Around 9,000 to 10,000 g/mol. f. Degree of Hydrolyzation: Often highly hydrolyzed (around 80% to 90%).
  • the collection medium may comprise one or more of the following additional supplements: 1) L-Glutamine or its stabilized dipeptide form L-alanyl-L-glutamine, commercially available under trade name GLUTAMAX TM from ThermoFisher Scientific, USA; 2) Insulin-Transferrin-Selenium-Ethanolamine, commercially available under tradename ITS-X from ThermoFisher Scientific, USA; and/or 3) Patent Application 4227.155879PCT essential amino acids, a composition of which may be commercially available under trade name MEM- amino acids from ThermoFisher Scientific, USA.
  • L-Glutamine preferably its stabilized dipeptide form L-alanyl-L- glutamine may be supplied in any suitable concentration, and preferably from about 1.5 mM to about 2 mM.
  • Insulin-Transferrin-Selenium-Ethanolamine refers to a composition comprising insulin, transferrin, selenium and ethanolamine, commercially available under tradename ITS-X as a 100Xstock.
  • composition can be used in any suitable amounts, but preferably the final concentrations when in use may be as follows: [00058] about 1.7 ⁇ M Insulin; [00059] about 0.068 ⁇ M Transferrin; [00060] about 0.038 ⁇ M Selenium; and [00061] about 32.7 ⁇ M Ethanolamine.
  • a mixture of essential amino acids may include amino acids shown in Table 2 and can be supplied as a 50X solution which can be referred in this disclosure as MEM essential amino acids, commercially available from ThermoFisher Scientific, USA. Table 2.
  • Table 3 lists an embodiment of a collection medium composition according to this disclosure, which comprises a reduced amount of heat-inactivated serum: Patent Application 4227.155879PCT Table 3.
  • LVCTM #2.1 Collection Medium Composition in Liquid Form [00064] The LVCTM #2.1 collection medium composition of Table 3 can be prepared by adding components listed in Table 3 into DMEM-High Glucose (Table A) to their final concentrations listed in Table 3.
  • Table 4 lists an embodiment of a serum-free collection medium composition according to this disclosure: Patent Application 4227.155879PCT Table 4.
  • LVCTM #3 Collection Medium Composition in Liquid Form [00066]
  • the LVCTM #3 collection medium composition of Table 4 can be prepared by adding components listed in Table 4 into the Basic Tissue Culture Medium of Table 1 to achieve final concentrations as listed in Table 4.
  • Patent Application 4227.155879PCT [00067] It has been unexpectedly found, as demonstrated by a comparative analysis in Figs.2-4, that the collection medium according to this disclosure and in particular the serum-free collection medium according to this disclosure is technically advantageous for producing a virus-like particle preparation which is infectious and genome integration competent.
  • this disclosure relates to a supplement for producing virus-like particles, the supplement comprising: a propionate, preferably sodium propionate; a butyrate, preferably sodium butyrate; caffeine; and partially or highly hydrolyzed PVA.
  • a propionate preferably sodium propionate
  • a butyrate preferably sodium butyrate
  • caffeine partially or highly hydrolyzed PVA.
  • at least some components of the supplement are provided in separate containers.
  • the supplement components are dilutable to the following final concentrations: about 3.5 mM to about 5.5 mM of the propionate; about 1 mM to about 3 mM of the butyrate; about 0.5 mM to about 2.5 mM of caffeine; and about 0.05 % wt/vol to about 0.2 % wt/vol of PVA.
  • step (12) mammalian cells are contacted with the collection medium according to this disclosure, supplemented with at least a propionate, a butyrate and caffeine, wherein the collection medium contains about 5% v/v or less of heat- inactivated serum and a pyruvate, or the collection medium is serum-free and contains polyvinyl alcohol (PVA).
  • the mammalian cells are seeded in a tissue culture vessel.
  • the mammalian cells express one or more of recombinant viral proteins for an assembly into virus-like particles.
  • the DNA may comprise a mixture of a recombinant retroviral vector, a first recombinant retrovirus packaging plasmid and a second recombinant retrovirus packaging plasmid; and wherein the mammalian cells are seeded in a tissue culture vessel having a first tissue culture medium.
  • the first tissue culture medium may be any tissue culture medium known to be used for DNA transfection of mammalian, and in particular, epithelial cells.
  • the first tissue culture medium may comprise 50:50 mix of DMEM/F12 and RPMI, supplemented with 7.5 mM 4-(2-Hydroxyethyl)piperazine- Patent Application 4227.155879PCT 1-ethane-sulfonic acid (HEPES), but any other tissue culture medium suitable for transfection of mammalian cells with plasmids can be also used.
  • Transfected cells may be cultured in the first tissue culture for a period of time of about 5 to about 20 hours.
  • Any mammalian cells which are suitable for packaging of retrovirus vectors can be used.
  • the cells may be an immortalized cell line and even more preferably an immortalized cell line derived from epithelial cells.
  • the cells may be HEK 273T cells (for example, HEK 293T cell with ATCC access number CRL-3216 or HEK 293T/17 with ATCC access number CRL- 11268), but any other immortalized cell lines, preferably epithelial immortalized cell lines can be also used, including Chinese Hamster Ovary (CHO) cells.
  • CHO Chinese Hamster Ovary
  • a poly-lysine (D or L) solution can be used in any suitable concentration, for example in the range of about 0.001 % wt/vol to about 0.01 % wt/vol in phosphate buffer saline (PBS) or any other buffer suitable for tissue culture cells. This lower poly-lysine concentration may facilitate cell adhesion and eliminates the need to rinse the tissue culture plastic before cells can be plated.
  • Patent Application 4227.155879PCT [00077] The cells can be seeded at different densities, depending upon how soon a co-transfection is to take place.
  • cells can be seeded at 20,000 cells/cm2 for transfection in 3 days; or at 40,000 cells/cm2 for transfection in 2 days; or at 85,000 to 90,000 cells/cm2 for transfection in 1 day.
  • the cells may be seeded at 85,000 to 90,000 cells per one square centimeter (cm 2 ) of the tissue culture vessel surface onto which the mammalian cells are plated for transfection to be performed in one day post-seeding.
  • tissue culture vessels may be used without limitations, including, but not limited to, a tissue culture plate, a tissue culture flask and/or microplate, such as a microplate with 6, 8, 12, or more (e.g.96) wells.
  • the cells are co-transfected with a mixture of DNA and a transfection reagent which complexes with DNA and facilitates its delivery into a mammalian cell.
  • the transfection reagent may comprise a cationic lipid and/or a branched or linear polyamine polymer polyethyleneimine (PEI).
  • Any commercially available reagents for transfection of mammalian cells can be used, including a 3:1 mixture of cationic lipid DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1- propaniminium trifluoroacetate) with neutral phospholipid DOPE (dioleoylphosphatidylethanolamine), available under tradename LIPOFECTAMINE from Thermo Fisher Scientific, Inc. or polyethyleneimine, commercially available from many different sources.
  • DOSPA dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1- propaniminium trifluoroacetate
  • DOPE dioleoylphosphatidylethanolamine
  • LIPOFECTAMINE dioleoylphosphatidylethanolamine
  • retroviral vector plasmid which encodes a payload to be delivered and integrated into a cell genome by transducing the cell with a virus-like particle that will be produced from the retroviral vector by the present methods.
  • the payload include, but are not limited to, a gene of interest, a marker (a fluorescent protein) and/or any other nucleotide sequence such as for example as a sequence encoding guide RNA (gRNA), single guide RNA (sgRNA) or any other nucleic sequence that will be delivered and integrated into a cell genome by transducing the cell with a virus-like particle produced accordingly to the methods of disclosure.
  • gRNA sequence encoding guide RNA
  • sgRNA single guide RNA
  • the payload is flanked by truncated retrovirus long terminal repeats (LTRs).
  • LTRs truncated retrovirus long terminal repeats
  • the LTR-flanked payload is operably linked to a promoter, e.g. a CMV promoter, suitable for driving expression (production of RNA) of the flanked payload in mammalian cells.
  • the payload may include another promoter downstream of an LTR, but upstream of the payload sequence to be transferred and expressed in target cells upon infection.
  • the payload may be expressed in a targeted cell from this second promoter after infection and genome integration is completed.
  • retrovirus vectors in this disclosure include GOI (pJB010) in which the payload is mNeon protein, a bright green fluorescent protein. (Shaner, N., Lambert, G., Chammas, A. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10, 407–409 (2013).
  • the pCS-CAG-mNEON lentiviral Patent Application 4227.155879PCT plasmid was generated by cloning mNeon into the pCS-CAG-eGFP plasmid (Masaki et al. Cell Stem Cell.2016 Nov 3; 19(5):587-592).
  • the DNA may include at least one helper plasmid, and preferably two helper plasmids, a first recombinant retrovirus packaging plasmid which may be referred in some embodiments of this disclosure as psPAX2 (psPAX2 was a gift from Didier Trono, Addgene plasmid # 12260; http://n2t.net/addgene:12260; RRID:Addgene_12260), and a second recombinant retrovirus packaging plasmid which may be referred in some embodiments of this disclosure as pMD2.g (pMD2.G was a gift from Didier Trono, Addgene plasmid # 12259; http://n2t.net/addgene:12259; RRID:Addgene_12259).
  • the first retrovirus packaging plasmid encodes and expresses in mammalian cells a retrovirus polymerase, e.g. HIV-pol, which includes reverse transcriptase necessary for producing DNA from recombinant RNA after a VLP is transduced into a cell, and a retrovirus capsid structural protein (GAG) necessary for a structural assembly (capsid) of a virus-like particle.
  • retrovirus polymerase e.g. HIV-pol
  • GAG retrovirus capsid structural protein
  • capsid structural protein
  • the second retrovirus packaging plasmid may encode and expresses in mammalian cells vesicular stomatitis virus glycoprotein (VSV-G protein) which is displayed on a cell-derived envelope that will envelope the recombinant capsid upon its lytic release from a transfected cell.
  • VSV-G protein vesicular stomatitis virus glycoprotein
  • the second retrovirus packaging plasmid may also encode and express retrovirus REV protein which Patent Application 4227.155879PCT includes a nuclear localization signal for delivery of recombinant RNA into a nucleus of a target cell.
  • the helper plasmids may encode additional regulatory elements and/or proteins useful for improving transduction of the virus-like particle and integration of the payload.
  • helper plasmids examples include those described at a research repository Addgene, accessible online.
  • the DNA mixture of the three plasmids may be co-mixed with a transfection reagent in any concentration. However, it has been discovered by a comparative analysis and as is shown in Fig.2 that keeping DNA low concentration helps with increasing production of virus-like particles in higher titers.
  • the DNA (a mixture of the 3 plasmids) may be used in a total concentration ranging from about 0.01 to about 0.02 pM per one square centimeter (cm 2 ) of the tissue culture vessel surface onto which the mammalian cells are plated.
  • the cells may be co-transferred with less than 0.01 pM of each: the recombinant retrovirus vector which is a lentivirus vector, the first retrovirus packaging plasmid and the second retrovirus packaging plasmid, per one square centimeter (cm 2 ) of the tissue culture vessel surface onto which the mammalian cells are plated.
  • low DNA concentrations may include those listed in Table 5 below. Table 5.
  • the co-transfected mammalian cells may be cultured for about 5 to about 20 hours, but other time periods may be also used, until step (12) is performed by which the co-transfected mammalian cells are contacted with the collection medium according to this disclosure.
  • step (12) is performed by which the co-transfected mammalian cells are contacted with the collection medium according to this disclosure.
  • the co-transfected mammalian cells are then cultured in step (14) in the collection medium. During this incubation period, virus-like particles are released from the mammalian cells into the collection medium.
  • the collection medium is then collected in step (16), said collection medium comprising virus-like particles.
  • the steps (12, 14 and 16) may be repeated with a fresh volume of the collection medium at least once.
  • Each of the culturing steps may be performed for any suitable period of time.
  • step (14) can be performed for about 1 to about 24 hours, preferably for about 8 to about 24 hours, and more preferably for about 10 to about 20 hours.
  • the collection medium comprising the virus-like particles may be further processed by centrifugation and/or filtration in order to Patent Application 4227.155879PCT separate virus-like particles from cell debris and/or reduce the solution volume and increase concentration of the virus-like particles.
  • Suitable centrifugation procedures may include centrifuging at 500-1000 g for a period of time, for example, for about 1 to about 10 minutes.
  • Suitable filtration procedures may include, but are not limited to, using a low protein binding filter made of a membrane material which has a low affinity for proteins.
  • Suitable filters include, but are not limited to, low protein affinity polyethersulfone (PES) and/or low protein affinity polyvinylidene fluoride (PVDF) membranes.
  • the collection medium is compatible with transfection/transduction/infection protocols for mammalian cells and is determined not to be interfering with manipulations of hematopoietic stem cells or pluripotent stem cells.
  • present methods allow for production of high titer transfection- competent (infectious) virus-like particles which can be used in many different applications, including for research and gene therapy treatments.
  • Fig.3 it illustrates production of virus-like particles according to the methods of this disclosure. In this experiment, three different batches of virus-like particles, previously stored at -80°C in a freezing media (which also served as collection media), were thawed on ice.
  • Fig.4 it illustrates production of virus-like particles according to the methods of this disclosure and as in connection with Fig.3, but all transfections were completed with low DNA concentrations and a PEI as a transfection reagent.
  • this disclosure replates to virus-like particle preparations in which virus-like particles are mixed with the collection medium accordingly to this disclosure, preferably the serum-free collection medium.
  • the VLPs may be preferably produced by the methods according to this disclosure, but other methods may be also used. It has been unexpectedly found that the collection medium of this disclosure can be used as a freezing medium. Furthermore, the VLP preparations in the collection medium can be used for transduction of mammalian cells, including pluripotent stem cells. The preparations are substantially free of cell debris and allow for using the collected virus-like particles without the need to undergo extensive purification and separation from medium components.
  • the LVCTM#3 collection medium maintains effective concentrations of sodium propionate, sodium butyrate, and caffeine, identified as key to enhancing virus-like particle production, while also eliminating the need for fetal bovine serum (FBS), thus sidestepping the issues related to cost, variability, and specialized equipment.
  • FBS fetal bovine serum
  • the LVCTM#2.1 follows a reduced Patent Application 4227.155879PCT serum approach with a higher DNA input, employing cost-effective transfection agents like Linear PEI.
  • LVCTM#3 distinguishes itself with a unique basal media composition of table 1, optimized for serum-free recombinant virus production, one embodiment of which is shown in Table 4.
  • a standout feature of LVCTM#3 is using it in conjunction with PEI transfection reagent, which significantly reduces plasmid DNA required to generate high-titer infectious VLPs, enhancing both accessibility and efficiency. This development is particularly beneficial for producing lentivirus VLPs for sensitive applications, such as those involving hematopoietic stem cells (HSCs), which risk differentiation when exposed to FBS or other contaminants like BSA.
  • HSCs hematopoietic stem cells
  • LVCTM3 By eliminating FBS, LVCTM3 overcomes issues of batch variability and the carryover of serum components, enabling cleaner and more consistent LV preparations.
  • Reduced Plasmid DNA Requirement The utilization of PEI Max as a transfection agent facilitates a 66% reduction in the plasmid DNA needed to produce high-titer infectious lentivirus-like particles, thus enhancing efficiency, and reducing production costs. This reduction is particularly crucial for genome- wide LV-based screens, such as CRISPR/Cas9 libraries, which require a substantial amount of plasmids and tissue culture (TC) reagents to generate the plasmid library necessary for the screen.
  • TC tissue culture
  • LVCTM#3 By decreasing the plasmid DNA requirement while increasing the LV titters, LVCTM#3 not only streamlines a Patent Application 4227.155879PCT production process but also substantially lowers the costs associated with these large-scale genomic screens. • Ease of Use: Designed for simplicity and accessibility, LVCTM#3 can be easily integrated into existing laboratory workflows without the need for specialized equipment or proprietary components, making advanced LV production techniques more accessible to a wider range of researchers. [000100] The following non-limiting examples are provided in order to further aid a person of skill in practicing methods according to this disclosure. Example 1.
  • Coating Tissue Culture Plates Prepare a coating solution by diluting 10 ⁇ l of the 10% poly-lysine (D or L) stock solution into 50 ml of Dulbecco’s Phosphate-Buffered Saline (dPBS) to achieve a final concentration of 0.002%. This lower concentration facilitates direct use without the need for rinsing the plates with PBS, which is often required at higher concentrations (0.1% or 0.01%) Incubate the TC plates with this solution for 10 minutes at room temperature (RT), then aspirate and return the solution to the tube. This reused solution can be stored at 4°C for up to 3 months.
  • D or L 10% poly-lysine
  • dPBS Phosphate-Buffered Saline
  • Cell Seeding Seed cells at specific densities depending on the time before transfection: Patent Application 4227.155879PCT For transfection in 3 days: Seed at 20,000 cells/cm2. For transfection in 2 days: Seed at 40,000 cells/cm2. For transfection in 1 day: Seed at 85,000 to 90,000 cells/cm2. [000103] These seeding densities are based on the growth rates of HEK-293T and HEK-293T/17 cells. [000104] Preparation for Transfection (for a single 15cm dish): Tube A: Add 400 ⁇ l of Transfection media and the appropriate volume of PEI Max (not exceeding 10% of the total culture volume, e.g., 15 ml for a 15 cm plate).
  • Tube B Add 400 ⁇ l of Transfection media, psPAX2 (packing plasmid) at 0.0045 pM/cm2, pMD2.g (VSVG envelope) at 0.0045 pM/cm2, and the transfer plasmid (gene of interest) at 0.0090 pM/cm2.
  • Transfection Process Combine the contents of Tubes A and B, mix by pipetting or brief vortexing, and incubate for 5-20 minutes at RT (typically 15 minutes).
  • Virus-like particles prepared according to methods of this disclosure were used to infect mammalian cells in comparison to other preparations. Results of these comparative analysis are reported in Figs.3 and 4. Table 6 below describes concentrations for each label point in Fig.3 and Table 7 below describes concentrations for each label point in Fig.4.
  • Patent Application 4227.155879PCT Table 6. Compositions And Concentrations For Fig.3 Patent Application 4227.155879PCT Patent Application 4227.155879PCT
  • Ethanolamine a precursor in phospholipid biosynthesis, particularly phosphatidylethanolamine (PE), plays a critical role in membrane fusion, cell signaling, and membrane curvature regulation. Nonetheless, combining 2-ME and Ethanolamine showed no added advantage over LVCTM3.
  • PE phosphatidylethanolamine

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Abstract

L'invention concerne des compositions de milieux de collecte complétées par au moins un propionate, un butyrate et de la caféine, le milieu de collecte contenant environ 5 % v/v ou moins de sérum inactivé par la chaleur et un pyruvate, ou le milieu de collecte étant exempt de sérum et contenant de l'alcool polyvinylique (PVA) et des procédés de culture tissulaire pour produire une préparation de particules de type virus compétentes pour la transfection et à intégration génomique, ainsi que des préparations de particules de type viral dans le milieu de collecte, les préparations appropriées pour des modifications génétiques de cellules.
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