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WO2025019869A2 - Milieu de culture cellulaire et compositions associées - Google Patents

Milieu de culture cellulaire et compositions associées Download PDF

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Publication number
WO2025019869A2
WO2025019869A2 PCT/US2024/039036 US2024039036W WO2025019869A2 WO 2025019869 A2 WO2025019869 A2 WO 2025019869A2 US 2024039036 W US2024039036 W US 2024039036W WO 2025019869 A2 WO2025019869 A2 WO 2025019869A2
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Prior art keywords
cell
culture medium
cell culture
cells
viscosity
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WO2025019869A3 (fr
Inventor
Hai-Quan Mao
Yining ZHU
Jingyao MA
Sean X. Sun
Zhuoxu GE
Qin NI
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Johns Hopkins University
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Johns Hopkins University
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—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
    • C12N2521/00—Culture process characterised by the use of hydrostatic pressure, flow or shear forces

Definitions

  • HIVs lentivirus
  • AAVs adeno-associated virus
  • CAR-T cell or CAR-macrophage therapy adeno-associated virus
  • All these approaches required efficient transfection of the cell lines (e.g. HEK 293 cells for virus production) or primary cells (immune cells for cell therapy) via nanoparticles or virus.
  • the production yield and cost continue to be adversely affected by inadequate transfection, despite the utilization of diverse methods.
  • the thickening agent is substantially biologically inert and does not alter the transferred biological material or cell.
  • a cell culture medium composition is provided that can suitably improve transfection efficiency and a method of producing a transfected cell using the cell culture medium composition.
  • the media and methods as described herein can be used for intracellular delivery of other therapeutic substances into cells.
  • the disclosure provides cell culture media.
  • a cell culture medium includes a liquid medium that comprises: a thickening agent that can provide increased viscosity of the fluid medium composition.
  • the cell culture medium may comprise a cell-growth component.
  • the cell culture medium suitably has a viscosity in a range of 0.8 cP to 10 cP, or up to or at least 1, 1.2, 1.4, 1.6, 1.8, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cP.
  • the cell culture medium suitably has a viscosity in a range of 0.8 cP to 15 cP, or up to or at least 1, 1.2, 1.4, 1.6, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14 or 15 cP.
  • a cell culture medium with a viscosity greater than 15, 14, 13 or 12 cP it can be less preferred use a cell culture medium with a viscosity greater than 15, 14, 13 or 12 cP, and thus preferred cell culture mediums may have an enhanced viscosity but less than 15, 14, 13, 12, 11, 10 or 9 cP. It was found that a preferred viscosity for transfection of non-viral vectors can be less than 12, 11 or 10 cP. It also was found that for viral including AAV transfection, efficiency may plateau at about 8 cP. Thus, a preferred viscosity for transfection of viral vectors can be less than 8.5 or 8.0 or 7.5 cP. Unless otherwise, indicated, references herein to cP values of a fluid sample are as determined at 37°C.
  • the thickening agent suitably provides increased viscosity to the cell culture medium.
  • suitable thickening agents include high molecular materials such as those that have a molecular weight of at least about 800, 1000, 1500 or 2000 Daltons, or molecular weight (Mn or Mw) of at least about 5,000, 10,000, 50,000, 100,000, 200,000, 3000,000, 400,000, 500,000 or 1,000,000.
  • Mn or Mw molecular weight
  • use of a higher molecular weight thickening agent may enable use of a relatively lower amount (e.g. weight amount) of a thickening agent to achieve a desired transfection efficiency.
  • Polymer materials may be preferred thickening agents, including water soluble or water miscible polymers.
  • Suitable polymers may be non-aromatic or have one or more portions or repeat units that comprise aromatic groups such as optionally substituted phenyl, naphthyl and the like. In at least certain aspects, non-aromatic polymers are preferred.
  • Preferred polymer thickening agents may comprise moi eties or repeat units that contain or more N, O or S atoms, for example one or more ether, ester, hydroxy, carboxy, keto, amino, cyano, nitro, amide, thioether, sulfone or sulfoxide moieties.
  • Particularly thickening agents include one or more of water soluble polysaccharides, such as an optionally substituted alkyl cellulose such as methyl cellulose, carboxymethylcellulose (C Xl(').
  • HPMC hydroxypropyl methylcellulose
  • HPMC hydroxypropyl methylcellulose
  • xanthan gum guar gum
  • dextran dextran sulfate
  • hyaluronic acid alginate
  • chondroitin sulfate or a derivative, or combination thereof.
  • a thickening agent also may be a water soluble and/or crosslinked synthesized polymers of distinct components, for example, one, two, or more of selected glycol polymers such as polyethylene glycol (PEG), propylene glycol polymers, polyvinyl alcohol (PVA), poly(vinyl pyrrolidone) (PVP), carbomers, polyacrylamide, or a derivative, or combination thereof.
  • selected glycol polymers such as polyethylene glycol (PEG), propylene glycol polymers, polyvinyl alcohol (PVA), poly(vinyl pyrrolidone) (PVP), carbomers, polyacrylamide, or a derivative, or combination thereof.
  • polymeric thickening agents may comprise one or more naturally occurring polymers.
  • polymeric thickening agents may comprise one or more synthetic occurring polymers.
  • polymeric thickening agents may comprise one or more synthetic occurring polymers and one or more naturally occurring polymers.
  • the components of a cell culture medium composition suitably may be present in varying amounts. Optimal amounts for a particular system can be readily determined empirically, for example the transfection efficiency cells in the cell medium with varying one amounts of one or more cell culture medium can be assessed.
  • the cell culture medium suitably comprises an amount of about 0.1 to 60 wt% of the thickening agent based on the total weight of the cell culture medium. In particular aspects, the cell culture medium comprises an amount of about 0.1 to 20 wt% of the thickening agent based on the total weight of the cell culture medium.
  • a cell culture medium comprises at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wt% of the thickening agent based on the total weight of the cell culture medium.
  • a cell culture medium suitably will contain at least an amount of a thickening agent to provide a 5, 10 or 20 percent increase in transfection efficiency relative to a control composition (same cell culture medium but without the thickening agent).
  • a cell culture medium also suitably includes one or more substances (may be referred to as biological substances or materials) to be transfected or transduced to a cell, for example a vector, a nucleic acid, an oligonucleotide, or a combination thereof, which may include microRNA, siRNA, mRNA, viral RNA, RNA oligo, DNA, ribozyme, or aptamer, and/or a polymer, a lipid nanoparticle or liposome.
  • the material to be transfected may provide for a therapeutic agent.
  • the material to be transfected may comprise a therapeutic agent or can provide a therapeutic agent such as upon expression of a nucleic acid molecule in the transfected cell.
  • the material to be transfected may suitably include a polymer, a lipid nanoparticle or liposome.
  • the nucleic acid biological substance may be suitably combined, or encapsulated in the polymer, lipid nanoparticle or liposome.
  • the polymer may form a vesicle together with lipids for delivery of cargo material (e.g., biological substance contained inside the vesicle).
  • a culture medium may comprise one or more cell transfection agents, including, but not be limited to, polyethyleneimine (PEI), adeno associated virus (AAV), polybeta-amino esters (PBAE), or lipid nanoparticles (LNPs).
  • the cell culture medium may comprise non-packaged genetic or biologic agents, including but not limited to plasmid DNA, mRNA, circular RNA, self-replicating RNA, siRNA, microRNA, or proteins, and the transfection is achieved through physical methods such as electroporation, mechanical shear, or transient cell membrane disruption.
  • the disclosure provides a composition for producing a transfected cell.
  • the composition preferably includes a cell; and the cell culture medium as described herein.
  • the disclosure provides method of producing a transfected cell.
  • the method includes a step of incubating a cell with the cell culture medium as described herein.
  • Preferred cell culture medium compositions can enhance the delivery of cargo and macromolecules into cells by improving transfection efficiency cells, including those cells that are considered difficult to transfect, e.g. those cells that are refractory to transfection or that exhibit substantially lower transfection efficiency than standard transformed cell lines routinely used.
  • preferred cell medium compositions can increase the transfection efficiency of up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%>, up to 90%, up to 95%, up to 100% or in excess of 100% relative to a comparable composition that does not contain a thickening agent as disclosed herein.
  • a cell medium composition does not a lipid material, or does not contain a lipid material in greater than 0.25, or 0.5 or 1 weight percent based on total weight of the cell medium composition. In certain aspects, a cell medium composition does not a peptide material, or does not contain a peptide material in greater than 0.25, or 0.5 or 1 weight percent based on total weight of the cell medium composition. In certain aspects, a cell medium composition does not either a lipid material or a peptide material, or does not contain either a peptide material or a lipid material each or either in greater than 0.25, or 0.5 or 1 weight percent based on total weight of the cell medium composition.
  • the present transduction compositions and methods can be used to transduce a molecule of interest into any cell, including a primary cell or a stem cell (including their derivatives, such as progenitor cells), a normal healthy cell or a diseased cell.
  • the cell involved in the transduction method is a mammalian cell.
  • the present methods and compositions may be employed to transduce molecules into other animal cells, plant cells, yeast cells, insect cells, or bacterial cells.
  • the cell is an animal cell, a plant cell, a yeast cell, an insect cell or a bacterial cell. In some embodiments, the cell is not a bacterial cell.
  • compositions are provided that suitably comprise a viscosity modifier as disclosed herein a molecule of interest for transduction.
  • a pharmaceutical composition comprises a viscosity modifier as disclosed herein.
  • the molecule of interest and viscosity modifier components are administered simultaneously or sequentially.
  • transduced cell or population of cells obtained or obtainable using the transduction buffer and/or the methods described herein.
  • a cell or population of cells comprising a molecule of interest may be provided wherein the molecule of interest has been transduced into the cell using the transduction buffer and/or methods described herein.
  • references to a “cell” are inclusive or also apply to a “cell population”, for example of 2 or more, 10 or more, 100 or more, 1000 or more, 10 4 or more, 10 5 or more, 10 6 or more, 10 7 or more, 10 8 or more cells.
  • FIG. 1A, FIG. IB and FIG. 1C show effects of enhanced viscosity on transfection efficiencies of Spikevax® LNPs carrying mRNA encoding luciferase in Bl 6F 10, HEK293T, Jurkat, PC12, C2C12, 4T1, NIH-3T3, Caco2, RAW264.7, Neuro2A, MOLT4, HepG2, PC3, MDA-MB-231, Hela, CT26, DC2.4, and X9 cells at an mRNA dose of 1 pg per well in a culture medium with a defined ranging from 0.77 cP to 15 cP.
  • the “Untreated” group refers to nontransfected cells.
  • the “0.77 cP” group refers to the standard cell culture medium.
  • FIG. 2A- FIG. 2F show effects of medium viscosity on transfection efficiency of Spikevax® LNPs carrying mRNA encoding mCherry in Bl 6F 10 cells (FIG. 2A and FIG. 2B), HEK293T cells (FIG. 2C and FIG. 2D), and Jurkat cells (FIG. 2E and FIG. 2F) at an mRNA dose of 1 pg per well in a culture medium with a defined ranging from 0.77 cP to 15 cP.
  • the “0.77 cP” group refers to the standard cell culture medium.
  • FIG. 3A, FIG. 3B and FIG. 3C show effects of medium viscosity on cellular uptake of Cy5-labeled mRNA LNPs (Spikevax® formulation) in B16F10, HEK293T, and Jurkat cells at an mRNA dose of 1 pg per well in a culture medium with a defined ranging from 0.77 cP to 15 cP.
  • the “0.77 cP” group refers to the standard cell culture medium.
  • LNPs pDNA lipid nanoparticles
  • mRNA LNPs mRNA LNPs
  • PEI polyethyleneimine
  • FIG. 4B show effects of medium viscosity on transfection efficiencies of pDNA lipid nanoparticles (LNPs) or mRNA LNPs (FIG. 4A) or polyethyleneimine (PEI)ZpDNA or PEI/mRNA nanoparticles (FIG. 4B) in HEK293 T cells.
  • the mCherry construct was used as the reporter gene for both pDNA and mRNA payloads.
  • PEIpro® was used as the PEI carrier and Spikevax® was used as the LNP carrier.
  • the “0.77 cP” group refers to the standard cell culture medium.
  • FIG. 5A, FIG. 5B and FIG. 5C show effects of medium viscosity on AAV9- mediated transfection in HEK293T cells.
  • Transfection was conducted in a culture medium with defined viscosity of 0.77, 2, 8, and 15 cP using IxlO 10 AAV9 per well.
  • the “0.77 cP” group refers to the standard cell culture medium.
  • FIG. 6 Schematic of luciferase assay and FACS analysis.
  • Adherent cells were preseeded and suspension cells were ready for use in the flask. Cells were seeded in 24-well plates at a specific density tailored for each cell type. Cell culture media will be refreshed with one supplemented with viscosity modifying agent then treated with mRNA LNPs. Cells were then incubated overnight at 37°C and then analyzed for luciferase activity or flow cytometry analysis according to the protocols described in the Method section.
  • FIG. 7A - FIG. 7D Extracellular fluid viscosity-dependent transfection efficiency mediated by mRNA LNPs.
  • FIG. 7C Transfection efficiency at the cellular level was evaluated by flow cytometry at 24 h following the treatment of LNPs 1.5 pg/mL mCherry mRNA.
  • the percentage of mCherry+ cells (FIG. 7B) and MFI among the mCherry+ cells (FIG. 7C) were shown (n 3).
  • FIG. 7D Representative histograms of the transfected culture of the four cell types. For each cell type, non-treated cells were used as a control for the initial SSC-FSC gating to measure background fluorescence. Data are presented as mean ⁇ SEM. P values were determined via one-way ANOVA with Dunnett’s multiple comparisons test, ns: P > 0.05, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG. 8A - FIG. 8D Kinetics of the overall luciferase expression level mediated by mRNA/pDNA LNPs under different viscosities.
  • HEK293T cells were treated with LNPs loaded with mRNA or pDNA encoding luciferase (2 pg/mL mRNA or pDNA LNPs, 2 pg/mL of mRNA or pDNA, 0.5 mL per well for 8 x io 4 cells in a 24-well plate).
  • FIG. 8C Luciferase expression levels at different time points (6, 12, 24, 36, and 48 h) in various viscosity levels (0.77, 2, 8, and 15 cP) in cells transfected with pDNA LNPs.
  • FIG. 8B and FIG. 8D Luciferase expression levels at different time points in various viscosity levels in cells transfected with mRNA LNPs.
  • FIG. 9A and FIG. 9B Comparison of viscosity-dependent luciferase expression levels mediated by mRNA LNPs in HEK293T cells under static and dynamic culture conditions.
  • HEK293T cells were treated with mRNA LNPs and chemiluminescence signals were measured at 24 h after transfection conducted under different viscosities in two different culture conditions.
  • FIG. 9A Normalized luciferase expression levels at 24 h after transfection (2 pg/mL mRNA LNPs in 0.5 mL media per well for 8 x 10 4 cells in a 24-well plate).
  • FIG. 9B Fold change in luciferase expression level at 24 h after transfection.
  • FIG. 10 Representative gating strategy examples of cells treated with LNPs loaded with mCherry mRNA for flow cytometry analysis. Gating was first based on FSC/SSC together with FSC-A/FSC-H (singlet populations). The cells within the gate were further analyzed based on mCherry signal.
  • FIG. 11A - FIG. 11D Representative flow cytometry panels showing comparison of cells transfected under baseline viscosity (0.77 cP), optimized viscosity and untreated group.
  • FIG. 13A Cells showing a bell-shaped curve in dose-response study.
  • FIG. 13B Cells showing a declining trend in dose-response study.
  • FIG. 14A - FIG. 14C Media viscosity-dependent transfection efficiency mediated by mLuc LNPs across various cell types.
  • FIG. 14B and FIG. 14C Comparison of luciferase expression level under optimal media viscosity with that in the standard culture media (0.77 cP) in CT26, Neuro-2a, DC2.4, HeLa, Hep G2, NIH/3T3, BMDC, X9, MOLT-4, Caco-2, C2C12, MDA-MB-231, and PC-3 (FIG.
  • FIG. 15A and FIG. 15B The normalized luciferase expression level mediated by mRNA LNPs and uptake of Cy5-labeled mRNA LNPs in Raw264.7 cells.
  • FIG. 14A Cy5- labeled mRNA LNP uptake efficiency in Raw 264.7 was evaluated by flow cytometry.
  • FIG. 16A - FIG. 16D Representative flow cytometry panels showing comparison of cellular uptake of LNPs loaded with Cy5-labeled mRNA in cells transfected under baseline viscosity (0.77 cP), optimized viscosity, and untreated group.
  • FIG. 17A - FIG. 17D Effect of media viscosity on cellular uptake of pDNA LNPs. Effect of media viscosity on cellular uptake of pDNA LNPs.
  • FIG. 17D Representative histograms of B16-F10 (FIG. 17C) and HEK293T cells (FIG. 17D) at different viscosities.
  • 100% of cells were positive in terms of cell uptake under all viscosity levels (0.77, 2, 8, and 15 cP); nonetheless, MFI levels of uptake varied as a function of media viscosity. When media viscosity was increased to 8 cP or higher, MFI was reduced to a similar or lower level than the standard 0.77-cP condition.
  • FIG. 18A - FG. 18E Effect of media viscosity on cellular uptake and endosomal escape efficiency of mRNA LNPs.
  • FIG. 18B Schematic illustration of the Gal8-GFP spots formation in Gal8-GFP-engineered cell lines.
  • FIG. 18E Representative images collected by the confocal laser scanning microscopy of C2C12-Gal8-GFP cells at 4 h after transfection with LNPs carrying Cy5-mRNA in media with defined viscosities. P values were determined via one-way ANOVA with Dunnett’s multiple comparisons test, ns: P > 0.05, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG. 19 Representative confocal laser scanning microscopy images of C2C12-Gal8- GFP and B16-F10-Gal8-GFP cells treated with mRNA LNPs in media with different viscosities.
  • Cells were treated with mRNA LNPs carrying Cy5-mRNA in media with different viscosity levels (0.77, 2, and 8 cP), a, C2C12-Gal8-GFP cells, and b, B16-F10-Gal8-GFP cells.
  • FIG. 20A - FIG. 20D Cell uptake pathways and actin remodeling/dynamics, NHE1- mediated swelling, and RhoA-based contractility on endocytosis of mRNA LNPs at different viscosity levels in B16-F10 cells.
  • FIG. 20A Four major endocytic pathways, including clathrin- mediated, caveolae-mediated, macropinocytosis, and phagocytosis.
  • FIG. 20C Schematic of the proposed viscosity-sensing pathway.
  • FIG. 20A Four major endocytic pathways, including clathrin- mediated, caveolae-mediated, macropinocytosis, and phagocytosis.
  • FIG. 20B Relative cellular uptake level of cells treated with specific pathway inhibitors
  • FIG. 21 Schematic of pathway inhibition experiment. B16-F10 cells were seeded in 24-well plates one day before the transfection experiment. After incubation overnight, media was refreshed with that supplemented with viscosity-modifying agent. The cells were then treated with different inhibitors for 1 h and transfected with mRNA LNPs loaded with Cy5-labeled mRNA. After incubation for 2 h, cells were analyzed by FACScan.
  • FIG. 22 Gating strategy example of pathway inhibition experiments for flow cytometry data analysis. Gating was first based on FSC/SSC together with FSC-A/FSC-H and SSC-A/SSC-H (singlet populations). The live cell population within the gate was further analyzed based on Cy5 signal.
  • FIG. 23A - FIG. 23D Transfection/transduction efficiency of different nucleic acids and vehicles on HEK 293T cells under different media viscosity conditions.
  • FIG. 24A - FIG. 24D Representative flow cytometry panels showing comparison of transfection or transduction efficiency using different agents under baseline viscosity (0.77 cP, left) and 2-cP viscosity (right) in HEK293T cells.
  • Cells were transfected with FIG. 24A, mRNA PEI nanoparticles, FIG. 24B, pDNA PEI nanoparticles, FIG. 24C, pDNA LNPs, or FIG. 24D, GFP AAV, respectively.
  • FIG. 25A and FIG. 25B Cell uptake and viscosity-sensing pathways on endocytosis of GFP AAV at different viscosity levels in HEK293T cells.
  • FIG. 26A - FIG. 26D Alexa fluor® 647 chromPureTM mouse transferrin on Bl 6- F10 cells at different viscosities.
  • FIG. 26C Representative histograms ofB16-F10 cells at different viscosities.
  • FIG. 26D Representative flow cytometry panels showing the comparison of cells under different viscosities.
  • FIG. 27A - FIG. 27C Viscosity-enhanced transfection/transduction of viral production in HEK293F suspension cells and human PBMCs.
  • FIG. 27A Schematic representation of the production process for virus vectors and their subsequent application in cell programming, highlighting the steps where media viscosity-enhanced transfection may be applied.
  • FIG. 28A - FIG. 28F Human primary B cells treated with LNPs loading mCherry mRNA for flow cytometry analysis.
  • FIG. 28C - FIG. 28E The comparison of transfected B cells at 0.77 cP, 2 cP and untreated group.
  • FIG. 28F Representative histograms of the transfected B cells.
  • FIG. 29 Multiplicity of infection (MOI) selection of LVV transduction of Jurkat cells.
  • FIG. 30A - FIG. 30D Effect of mRNA LNP dose on viscosity-dependent transfection efficiency in B16-F10, HEK293T, Jurkat, and MOLT-4 cells.
  • cell culture media that can significantly alter (e.g., increase or improve) cell uptake and transfection efficiency.
  • modulating the viscosity of the culture media may be important for optimizing cellular uptake and transfection efficiency, which can be demonstrated in multiple cell types such as hard-to- transfect cell types, like T cells and other cell types with strong therapeutic values.
  • results demonstrating an effect of culture media viscosity on transfection efficiency of gene delivery vehicles including lipid nanoparticles, polyplex nanoparticles, adeno-associated vectors, and lentiviral vectors in a wide range of cell types.
  • Substantially enhanced levels of transfection efficiency are observed by optimizing the media viscosity around the range found in biological fluids, 2-4 centipoise (cP), for lipid nanoparticles and polyplex nanoparticles, and correlated with increased levels of cellular uptake and endosomal escape.
  • cP centipoise
  • variable includes all values including the end points described within the stated range.
  • range of “5 to 10” will be understood to include any subranges, such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, and the like, as well as individual values of 5, 6, 7, 8, 9 and 10, and will also be understood to include any value between valid integers within the stated range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, 6.5 to 9, and the like.
  • the range of “10% to 30%” will be understood to include subranges, such as 10% to 15%, 12% to 18%, 20% to 30%, etc., as well as all integers including values of 10%, 11%, 12%, 13% and the like up to 30%, and will also be understood to include any value between valid integers within the stated range, such as 10.5%, 15.5%, 25.5%, and the like.
  • the term “culture media” refers to a material (e.g., matrix or medium) that suitably may contain and provide cell-growth components or essential nutrients (e.g., carbon sources, nitrogen sources, phosphorus sources, and growth factors) and minerals (e.g., salts, or metal ions) for sustaining biological activities and supporting growth of organisms (e.g., microorganism, cells, tissues, bacteria, virus, and the like).
  • the culture media is in a liquid form that does not contain any solidified nor gelated portion.
  • cell-growth component refers to essential nutrients (e.g., carbon sources, nitrogen sources, phosphorus sources, and growth factors) and minerals (e.g., salts, or metal ions) for sustaining biological activities and supporting growth of organisms (e.g., microorganism, cells, tissues, bacteria, virus, and the like).
  • essential nutrients e.g., carbon sources, nitrogen sources, phosphorus sources, and growth factors
  • minerals e.g., salts, or metal ions
  • transfecting means or includes intracellular delivery of a material into a cell, i.e. uptake of a cell of material of interest (e.g. biological material) including nucleic acid. “Transfecting”, “transfection” or other similar term thus includes for example uptake of foreign DNA via a viral vector (which also may be referred to transduction). As used herein, “transfecting”, “transfection” or similar terms include transduction.
  • materials that may be delivered into cells include nucleic acids (DNA and RNA), proteins, peptides, small molecules, nanomaterials including synthetic nanomaterials and nanoparticles and others.
  • nucleic acid is delivered into cells.
  • nanoparticles are delivered into cells.
  • thickening agent refers to a substance or components in a cell culture medium (e.g., liquid medium) that does not affect the cell viability, growth or other biological activity but changes physical property or characteristics of the cell culture medium.
  • a cell culture medium e.g., liquid medium
  • one or more thickening agents may be used, for example, each and respective thickening agents may function to provide same or different characteristics, such as viscosity, density, solid content, flow rate, or light scattering property.
  • one or more thickening agents may control the viscosity of the cell culture medium (e.g., liquid medium).
  • viscosity refers to a physical property of a liquid or fluid as a measurement of resistance against fluidity or deformation.
  • the viscosity of a material may vary based on the liquid or fluid’s condition such temperature, pressure, and rate (velocity) of flow.
  • the viscosity is presented using measuring unit centipoise (cP) that is based on the viscosity of water, for example, the viscosity of water at 20 °C is defined as 1 cP.
  • the viscosity may be controlled, modulated or adjusted using ingredients dissolved in the liquid or fluid, for example, by adding or reducing the weight of certain ingredients or inducing a chemical reaction or physical adherence.
  • the culture media is in a liquid form that can be defined by measuring a viscosity thereof.
  • the term “cell transfection agent” refers to a substance or a chemical/biological material that can facilitate or promote entry of transfecting material (e.g., a virus, a vector, a nucleic acid, an oligonucleotide, and proteins, or particularly RNA and DNA) into a cell (e.g., prokaryotic and eukaryotic cells).
  • transfecting material e.g., a virus, a vector, a nucleic acid, an oligonucleotide, and proteins, or particularly RNA and DNA
  • the cell transfection agent may induce changes in cellular membranes, e.g., by changing ionic or electric characteristics of the cell membrane, or surface physiology or inducing a membrane fusion.
  • Exemplary cell transfection agent may include, but not be limited to, calcium phosphate, DOTMA (N-[l-(2,3,- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) liposomes, polyethyleneimine (PEI) and its derivatives, lipid nanoparticles (LNPs), and poly(beta-amino ester) (PBAE) and its derivatives.
  • DOTMA N-[l-(2,3,- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • PEI polyethyleneimine
  • LNPs lipid nanoparticles
  • PBAE poly(beta-amino ester)
  • zzz vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
  • zzz vivo refers to events that occur within an organism (e.g., animal, plant, or microbe, cell, or tissue thereof).
  • the term “nanoparticle” refers to a particular or spherical substance having a size range from about 1 to about 900 nm, from about 10 to about 800 nm, from about 10 to about 700 nm, from about 10 to about 600 nm, from about 10 to about 500 nm, from about 10 to about 400 nm, from about 10 to about 300 nm, or from about 10 to about 200 nm.
  • the nanoparticles include lipid components (e.g., phospholipid) so as to form lipid nanoparticles (LNPs).
  • the lipid nanoparticles carry cargo molecules such as biological substances (e.g., a virus, a vector, a nucleic acid, an oligonucleotide, and the like) in internal spaces.
  • biological substances e.g., a virus, a vector, a nucleic acid, an oligonucleotide, and the like
  • the term “lipid nanoparticle” may be interchangeably used with the term for “liposome.”
  • the lipid nanoparticles have a zeta potential (mV) ranging from about -50 to 50 mV. In some embodiments, the lipid nanoparticles prior to incorporating other components (nucleic acid components) have the zeta potential (mV) ranging from about -50 to about 0 mV.
  • the nucleic acids (e.g., viral vector, RNA and DNA) on the surface of the lipid nanoparticles alter the surface charge of the nanoparticle, e.g., by exposing additional anionic charges from the backbone of the nucleic acids.
  • the zeta potential of the nanoparticle may be controlled by adjusting the amounts of the nucleic acids (e.g., viral vector, RNA and DNA) incorporated.
  • the term “cell” refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA.
  • a cell can be identified by well- known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring.
  • Cells may include prokaryotic and eukaroytic cells.
  • Prokaryotic cells include but are not limited to bacteria.
  • Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect and human cells.
  • the term “cell” as referred to herein indicates a single cell and also indicates to a cell population e.g. 2 or more, 10 or more, 100 or more, 1000 or more, 10 4 or more, or 10 5 or more cells.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • compositions of the disclosure refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the disclosure without causing a significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the vaccines of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the vaccines of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the vaccines of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers,
  • stable refers to a compound or composition (e g., culture medium) that is sufficiently robust in a storage condition.
  • derivative means a compound that may be produced from another compound of similar structure in one or more steps.
  • a “derivative” or “derivatives” of a compound retains at least a degree of the desired function of the compound. Accordingly, an alternate term for “derivative” may be "functional derivative.
  • a derivative of a polymer used as a thickening agent as disclose herein may be able to provide increased transfection efficiency such as at least a 3, 5, 10 or 20 percent increase in transfection efficiency relative to a control composition that does not contain the thickening agent.
  • the disclosure provides a culture medium composition that suitably include a thickening agent to adjust or modulate the viscosity.
  • a cell culture medium or composition thereof including: a liquid medium including a thickening agent.
  • the cell culture medium also may comprise a cell-growth component.
  • the cell culture medium has a viscosity in a range of about 0.8 to 15 or 0.8 to 10 cP, more typically 1.0 or 2.0 cP to 8.0, 9.0 or 10 cP.
  • the thickening agent includes one or more selected from methylcellulose, hyaluronic acid, polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxyethyl cellulose, poly(vinyl pyrrolidone), and a polysaccharide.
  • thickening agent includes methylcellulose for adjusting or controlling viscosity.
  • the cell culture medium may include an amount of about 0.1 to 10 wt% of the thickening agent based on the total weight of the cell culture medium.
  • the content of the thickening agent may range from about 0.1 to 5 wt% based on the total weight of the cell culture medium.
  • the content of the thickening agent may range from about 0.1 to 3 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 0.1 to 1 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 1 to 10 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 1 to 9 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 1 to 8 wt% based on the total weight of the cell culture medium.
  • the content of the thickening agent may range from about 1 to 7 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 1 to 6 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 1 to 5 wt% based on the total weight of the cell culture medium.
  • the content of the thickening agent may range from about 0.1 to 1 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 1 to 2 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 2 to 3 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 3 to 4 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 4 to 5 wt% based on the total weight of the cell culture medium.
  • the content of the thickening agent may range from about 5 to 6 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 6 to 7 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 7 to 8 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 8 to 9 wt% based on the total weight of the cell culture medium. In some embodiments, the content of the thickening agent may range from about 9 to 10 wt% based on the total weight of the cell culture medium.
  • the liquid medium (media) may be selected from Dulbecco medium or modified product thereof (e.g., DMEM (Dulbecco's Modified Eagle Medium)), Minimum Essential Medium (MEM) (e.g., Eagle's minimum essential medium), Roswell Park Memorial Institute (RPMI) 1640 Medium, serum-free media (e.g., CHO cell culture media, hybridoma media, and protein expression media), human plasma-like medium (HPLM), and combinations thereof.
  • the liquid medium (media) can be selected based on nature of the cells to be incubated or cultivated, or the purpose or process of cell utilization (e.g., transfection).
  • the liquid medium (media) can be formulated to regulate, control, or adjust surrounding effects which may affect cell growth or proliferation.
  • the cell culture medium further includes a cell transfection agent.
  • the cell transfection agent may suitably include polyethyleneimine (PEI) and its derivatives, adeno associated virus (AAV), poly(beta-amino esters) (PBAE) and its derivatives, and lipid nanoparticles (LNPs).
  • the cell culture medium further includes a biological substance to be transfected into a cell.
  • the biological substance may be a virus.
  • the biological substance may be a vector.
  • the biological substance may be a nucleic acid (e.g., microRNA, siRNA, mRNA, viral RNA, RNA oligo, DNA, ribozyme, or aptamer).
  • the biological substance may an oligonucleotide.
  • the biological substance may include one or more biological substances selected from virus, a vector, a nucleic acid, an oligonucleotide, or debris thereof.
  • the biological substance may suitably include a viral vector.
  • viral vectors may include, but not be limited to retroviruses, lentivirus (LVVs), adenovirus, adeno-associated virus (AAVs), plant virus (e.g., Tobacco mosaic virus (TMV)), or genetically engineered hybrid viral vector.
  • the biological substance may suitably include microRNA. In certain embodiments, the biological substance may suitably include siRNA. In certain embodiments, the biological substance may suitably include mRNA. In certain embodiments, the biological substance may suitably include viral RNA or DNA. In certain embodiments, the biological substance may suitably include RNA oligo. In certain embodiments, the biological substance may suitably include DNA (e.g., genomic DNA, cDNA, or isolated DNA). In certain embodiments, the biological substance may suitably include ribozyme. In certain embodiments, the biological substance may suitably include aptamer.
  • polynucleotide in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc.
  • RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicersubstrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof.
  • the RNA is an mRNA.
  • An mRNA may encode any polypeptide of interest, including any naturally or non- naturally occurring or otherwise modified polypeptide.
  • a polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity.
  • a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.
  • a therapeutic agent (which may be referred to as biological material herein) is an siRNA.
  • An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest.
  • an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA.
  • An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest.
  • the siRNA may be an immunomodulatory siRNA.
  • a therapeutic agent is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus.
  • Nucleic acids and polynucleotides useful in the disclosure typically include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5 -terminus of the first region (e.g. , a 5’-UTR), a second flanking region located at the 3 ’-terminus of the first region (e.g., a 3’-UTR), at least one 5 -cap region, and a 3 ’-stabilizing region.
  • a nucleic acid or polynucleotide further includes a poly-A region or a Kozak sequence (e.g. , in the 5 -UTR).
  • polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide.
  • a polynucleotide or nucleic acid e.g. , an mRNA
  • a polynucleotide or nucleic acid may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a poly A sequence, and/or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e g., an alternative nucleoside).
  • the 3 - stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2 -0-methyl nucleoside and/or the coding region, 5 -UTR, 3 -UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5- methoxy uridine), a 1 -substituted pseudouridine (e.g. , 1 -methyl-pseudouridine or 1 -ethyl- pseudouridine), and/or a 5-substituted cytidine (e.g., 5-methyl-cytidine).
  • a 5-substituted uridine e.g., 5- methoxy uridine
  • a 1 -substituted pseudouridine e.g. , 1 -methyl-pseudouridine or 1 -ethyl- pseudouridine
  • the shortest length of a polynucleotide can be the length of the polynucleotide sequence that is sufficient to encode for a dipeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a tripeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a tetrapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a pentapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a hexapeptide.
  • the length of the polynucleotide sequence is sufficient to encode for a heptapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for an octapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a nonapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a decapeptide [000102] In certain aspects, a polynucleotide is greater than 30 nucleotides in length. In another embodiment, the polynucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides.
  • the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides.
  • the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides.
  • the length is at least 1 100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.
  • Nucleic acids and polynucleotides may include one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).
  • all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the poly A tail, and any combination of (a, b, c, or d above) comprise naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).
  • Nucleic acids and polynucleotides may include one or more alternative components, as described herein, which impart useful properties including increased stability and/or the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.
  • an alternative polynucleotide or nucleic acid exhibits reduced degradation in a cell into which the polynucleotide or nucleic acid is introduced, relative to a corresponding unaltered polynucleotide or nucleic acid.
  • These alternative species may enhance the efficiency of protein production, intracellular retention of the polynucleotides, and/or viability of contacted cells, as well as possess reduced immunogenicity.
  • Nanoparticle compositions may include a lipid component and one or more additional components, such as a therapeutic and/or prophylactic.
  • a nanoparticle composition may be designed for one or more specific applications or targets.
  • the elements of a nanoparticle composition may be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements.
  • the particular formulation of a nanoparticle composition may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combinations of elements.
  • Nanoparticle compositions may be designed for one or more specific applications or targets.
  • a nanoparticle composition may be designed to deliver a therapeutic and/or prophylactic such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal's body.
  • Physiochemical properties of nanoparticle compositions may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs.
  • the therapeutic and/or prophylactic included in a nanoparticle composition may also be selected based on the desired delivery target or targets.
  • a therapeutic and/or prophylactic may be selected for a particular indication, condition, disease, or disorder and/or for delivery to a particular cell, tissue, organ, or system or group thereof (e g., localized or specific delivery).
  • a nanoparticle composition may include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest.
  • Such a composition may be designed to be specifically delivered to a particular organ.
  • a composition may be designed to be specifically delivered to a mammalian liver.
  • the amount of a therapeutic agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic and/or prophylactic.
  • the amount of an RNA useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA.
  • the relative amounts of a therapeutic and/or prophylactic and other elements (e.g. , lipids) in a nanoparticle composition may also vary.
  • the wt/wt ratio of the lipid component to a therapeutic agent in a nanoparticle composition may be from about 5: 1 to about 60: 1 , such as 5: 1 , 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 1 1 : 1, 12: 1 , 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, and 60: 1.
  • the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic may be from about 10: 1 to about 40: 1.
  • the wt/wt ratio is about 20: 1.
  • the amount of a therapeutic agent in a nanoparticle composition may, for example, be measured using spectroscopy (e.g., ultraviolet-visible spectroscopy).
  • the cell culture medium has a viscosity in a range of about 0.8 to 10 cP, preferably, in a range of about 1 or 2 cP to 8, 9 or 10 cP. In some embodiments, the cell culture medium has a viscosity in a range of about 1 to 8 cP. In some embodiments, the cell culture medium has a viscosity in a range of about 2 to 4, 5, 6, 7 or 8 cP. In some embodiments, the cell culture medium has a viscosity in a range of about 3 to 8 cP. In some embodiments, the cell culture medium has a viscosity in a range of about 4 to 8 cP.
  • the cell culture medium has a viscosity in a range of about 0.8 to 5 cP. In some embodiments, the cell culture medium has a viscosity in a range of about 2 to 4 cP. In some embodiments, the cell culture medium has a viscosity in a range of about 3 to 4 cP.
  • compositions for producing a transfected cell include the cell culture medium as described herein.
  • the composition for producing the transfected cell includes a cell and a suitably selected cell culture medium described herein.
  • Biological material e g. nucleic acid
  • the thickening agent is administered simultaneously (e.g. from a container containing the combination).
  • the thickening agent comprises the biological material in admixture.
  • a cell suitably may be is plated in a culture medium as disclosed herein including a culture medium with a thickening agent component as disclosed herein, suitable for the particular cell, prior to transfection.
  • a culture medium including a culture medium with a thickening agent component as disclosed herein, suitable for the particular cell, prior to transfection.
  • the cell may be contacted with a culture medium during transfection.
  • a cell culture medium may comprise additional materials for use with live cells or live cell culture or application in vivo.
  • a cell culture medium suitably may contain one or more of a biological pH buffer, one or more growth factors, amino acids, vitamins and/or nutrients.
  • the cell culture medium or composition is used for producing a transfected mammalian cell.
  • the cell may include a T-cell.
  • the cell may include an embryonic cell.
  • the cell may include a stem cell.
  • the cell may include a mammalian tissue.
  • the mammalian cell may be a cancer cell line.
  • Exemplary T-cell lines include for example a Jurkat cell, HEL cell line, TK-1, BW5 147.3, T ALL-104, MJ, J45.01, HH, Loucy, EL4, MOLT-3, and MOLT-4 cell line.
  • Exemplary cancer cell lines may include, but not be limited to, liver cancer cell line (e.g., BRAF, CDKN2A, CTNNB1, NRAS, STK11, SNU-475, C3A, SNU-449, PLC/PRF/5, SNU-387, SK- HEP-1, SNU-423, and TP53), breast cancer cell line (e.g., HCC1599, HCC1937, HCC1143, MDA-MB-468, HCC38, HCC70, HCC1806, HCC1187, DU4475, HCC1599, HCC1937, HCC1143, MDA-MB-468, HCC38, HCC70, HCC1806, HCC1187, DU4475, T-549, Hs 578T, MDA-MB-231, MDA-MB-436, MDA-MB-157,MDA-MB-453, BT-20, HCC1395, BT-549, Hs 578T.
  • liver cancer cell line e.g.,
  • stomach cancer cell e.g., KATOIII, NCI-N87, SNU-16, SNU-5, AGS, and SNU-1
  • cervical and gynecological cancer cell e.g., Ca-Ski, DoTc2,-4510, SiHa, C-33-A, SK-LMS-1, HT-3, ME-180, Caov-3, SW626, MES-SA, SK-UT-1, KLE, AN3-CA
  • head and neck cancer cell line e.g., A-253, SCC- 15, SCC-25, SCC-9, Detroit 562, and FaDu
  • skin cancer cell or melanoma line e.g., SK-MEL- 3, SH-4, SK-MEL-24, and RPMI-795, and B16-F10)
  • colon cancer cell line e.g., SNU-C1, SW48, RKO, COLO 205, SW
  • Exemplary embryonic or stem cell lines may include, but not be limited to, JI, Rl/E, MITC-STO (ATCC 56-X), MEF, SNL76/7, MEF (CF- 1), C57BL/6, L2-RYC, B104-1-1, SNLP 76/7-4, CE-1, ESF 158, G-01ig2, AB2.2, B6/BLU, RW.4, Rl, ES-E14TG2a, and ES-D3.
  • Exemplary virus production cell lines may include, but not be limited to, HEK293 (human embryonic kidney) cells and their derivatized cells including HEK293T cells, HEK293F, HEK293.2sus cells.
  • the cell involved in the transfection method is a mammalian cell.
  • the cell is an animal cell, a plant cell, a yeast cell, an insect cell or a bacterial cell. In some embodiments, the cell is not a bacterial cell.
  • a mammalian cell is a human, primate, rodent (e.g. mouse or rat), rabbit, dog, cat, horse, cow or pig cell. These mammals are useful for research purposes.
  • the cell is a non-human cell.
  • the cell is in vivo, or optionally in situ. For example, when treating or diagnosing a medical condition, the biological material could be administered in combination with the thickening agent to an organism or tissue in need thereof.
  • the cell is in vitro or ex vivo.
  • the cell may be in a culture medium, wherein the culture medium optionally supports the maintenance, differentiation and/or expansion of the cell.
  • the cell is derived from an established cell line, such as an established human cell line.
  • the established cell line is an immortalised cell line.
  • the cell line is a primary cell line.
  • Examples of established human cell lines suitable for use in the present compositons, medium and methods include for example HeLa, ESTD AB database, DU145 (prostate cancer), Lncap (prostate cancer), MCF-7 (breast cancer), MDA-MB-438 (breast cancer), PC3 (prostate cancer), T47D (breast cancer), THP-1 (acute myeloid leukemia), COS7 (immortalised CV-1 cells from kidney tissue), U87 (glioblastoma), SHSY5Y human neuroblastoma cells, cloned from a myeloma, Saos-2 cells (bone cancer), HEK293 (human embryonic kidney) cells and their derivatized cells including HEK293T cells, HEK293F, HEK293.2sus cells.
  • the cell is a primary cell.
  • a primary cell or cell line is derived from a cell taken directly from a living organism, and has not been immortalized. In other words, a primary cell or cell line is genetically and phenotypically stable.
  • the cell is a stem cell or a cell derived by differentiation of a stem cell.
  • the stem cell is a pluripotent stem cell, such as an embryonic stem cell, optionally a human embryonic stem cell.
  • the cell is not a human embryonic stem cell.
  • the stem cell is not obtained by methods that involve the use of human embryos for commercial or industrial purposes.
  • the stem cell is not obtained by methods that necessarily involve the destruction of a human embryo.
  • the stem cell is a murine embryonic stem cell.
  • the stem cell is an adult stem cell, such as a neural, adipose or hematopoietic stem cell.
  • the cell is a murine or human neural stem cell, neuron cell or glia cell.
  • the stem cell is an induced pluripotent stem cell.
  • the cell is a somatic cell or a germ cell.
  • the cell is a cell relating to the immune system, such as a T cell, B cell or leukocyte, including but not limited to a phagocyte (macrophage, neutrophil, or dendritic cell), mast cell, eosinophil, basophil, and natural killer cell.
  • the cells for transfection are cultured in an atmosphere comprising between about 4% and about 10% CO2. In some aspects, the cells for transduction are cultured in an atmosphere comprising between about 5% and about 9% CO2, or about 6% and about 8% CO2, suitably about 5% CO2.
  • a method of producing a transfected cell includes steps of incubating a cell with the cell culture medium as described herein.
  • the cell is incubated at a temperature range of about 10 to 37 °C for about 1 to 24 hours.
  • the cell is incubated at a temperature range of about 25 to 37 °C, or preferably at 37 °C, for about 1 to 24 hours.
  • the method may further include conducting electroporation, sonoporation, or laser irradiation to the cell culture to increase transfection efficiency.
  • the biological material of interest and cell are in contact for a sufficient length of time (incubation time or transfection time) for the molecule to transfection into the cell.
  • the incubation time is 1 to 24 hours, or 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
  • kit for producing a transfected cell includes the cell culture medium as described above.
  • a cell culture medium as disclosed does not contain an effective amount or any amount of a transduction compound and/or an associated salt as disclosed in U.S. Patent 10,883,116.
  • the present viscosity modifiers and methods preferably have minimal impact on the viability of the cells.
  • An example of an assay that measures proliferation is the BrdU incorporation assay, which measures BrdU incorporation into cellular DNA during cell proliferation.
  • BrdU incorporation assay measures BrdU incorporation into cellular DNA during cell proliferation.
  • the BrdU incorporation assay when the cells being subjected to the transduction methods of the invention are subjected to the BrdU incorporation assay, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99% or all cells demonstrate incorporation of BrdU into cellular DNA of the cells.
  • the molecule of interest and cell are in contact for a sufficient length of time for the molecule to transduce into the cell.
  • the amount of uptake into the cell correlates with the amount of time (incubation time) the cell is in contact with the viscosity modifier and molecule of interest.
  • the incubation time is between about 1 and about 24 hours, for example between about 2 and about 12 hours or between about 2 and about 5 hours.
  • the incubation time is at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours or more than 13 hours. In some aspects the incubation time is less than 48 hours, less than 24 hours, less than 20 hours, less than 15 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour
  • the rate of transduction may depend upon the cell type and the molecule of interest to be transduced (e.g. the molecule’s size, charge, hydrophobicity).
  • transduction can be detected using reporter constructs including reporter constructs that are commercially available such as a luciferase or a GFP reporter construct, wherein levels of fluorescence correspond to levels of expression (see the Examples section for more details).
  • reporter constructs including reporter constructs that are commercially available such as a luciferase or a GFP reporter construct, wherein levels of fluorescence correspond to levels of expression (see the Examples section for more details).
  • a present method may comprise one round of transduction.
  • multiple rounds of transduction may be desirable.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of transduction are carried out on the same cells.
  • Each round of transduction may involve transduction of the same molecule or of different molecules of interest.
  • each round of transduction there may be a “recovery period” of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 hours.
  • the recovery period is at least 10, at least 20, at least 30, at least 40 or at least 50 minutes.
  • the transduction buffer is removed from the cells and the cells are typically cultured in cell culture medium suitable for the particular cell type.
  • transduction buffers and methods are provided below. It is to be understood that any combination of compatible embodiments described herein can be used for a transduction buffer or method for transduction comprising a transduction buffer. Some examples of combinable embodiments are provided below.
  • a method for transducing a molecule of interest into a cell comprising contacting the cell with a molecule of interest and contacting the cell with a transduction buffer comprising a salt which binds to and/or activates a sodium/hydrogen transporter protein, a transduction compound and optionally glycine and/or glycerol as osmoprotectants, wherein the transduction compound is a small molecule compound.
  • the transduction buffer (or cell culture media) also may include a viscosity modifier as disclosed herein.
  • the transduction compound is a small molecule compound and is not a detergent.
  • the transduction compound is a small molecule compound and is not a detergent and is a zwitterion or a non-zwitterionic compound with a group that is bioisoteric to a negatively charged functional group. In certain aspects, the transduction compound is a small molecule compound and is not a detergent and is a zwitterion. In certain aspects, the transduction compound is a small molecule compound and is a zwitterion or a non- zwitterionic compound with a group that is bioisoteric to a negatively charged functional group. In certain aspects, the transduction buffer comprises a cell-permeable antibiotic, such as for example doxycycline or tetracycline.
  • the transduction buffer additionally comprises one or more (e.g. 1, 2, 3, 4 or 5) of a viscosity enhancer, growth factor, cytokine, neurotransmitter, or agonists thereof, such as a GABA agonist.
  • a present method comprises contacting the cell with the transduction buffer for a period of at least 30 minutes, preferably for about 12 hours.
  • a present method involves at least two rounds of transduction i.e. the cell is contacted by the transduction buffer and the molecule of interest for at least two continuous periods of at least 30 minutes with a recovery period in between.
  • the method for modifying a nucleic acid, such as a genetic sequence, in a cell further comprises isolating or using the modified cell.
  • the modified cell comprises a transduced gene editing system.
  • the modified cell does not comprise a viral vector.
  • the modified cell does not comprise a nanoparticle carrier.
  • a pharmaceutical composition comprising a viscosity modifier as disclosed herein and a molecule of interest for transduction.
  • a pharmaceutical composition comprises viscosity modifier as disclosed herein.
  • the molecule of interest and viscosity modifier as disclosed herein are administered simultaneously or sequentially.
  • the pharmaceutical composition can include further components in addition to the viscosity modifier and a molecule of interest.
  • a pharmaceutical composition suitably may comprise a pharmaceutically acceptable carrier, which can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity.
  • Suitable pharmaceutically acceptable carriers are well known in the art.
  • Pharmaceutically acceptable carriers can, for example, include liquids such as water, saline, glycerol and ethanol.
  • Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like.
  • the pharmaceutical composition may be sterile and/or pyrogen-free.
  • the invention also provides a container (e.g. vial) or delivery device (e.g. syringe) pre-filled with a pharmaceutical composition as disclosed herein.
  • a container e.g. vial
  • delivery device e.g. syringe
  • the appropriate dose may vary depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. human, non-human primate, primate, etc.), the degree of transduction desired, the formulation of the pharmaceutical composition, the treating doctor's assessment of the medical situation, and other relevant factors.
  • the dose may fall in a relatively broad range that can be determined through routine trials.
  • Effective dosage volumes can be routinely established, depending on the purpose of the composition.
  • Typical human dose of the composition might be, for example about 0.5 ml e.g. for intramuscular injection (e.g. local injection into the muscle or tissue of interest). Similar doses may be used for other delivery routes.
  • compositions of the invention may be prepared in various liquid forms.
  • the compositions may be prepared as injectables, either as solutions or suspensions.
  • injectables for local sub-cutaneous or intramuscular administration are typical. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used.
  • kits comprising a pharmaceutical composition as disclosed herein.
  • the kit may additionally comprise cells and/or molecules of interest for transduction.
  • the kit may also comprise instructions for use.
  • the kit may include the various components of the transduction buffer in one or more separate containers, e.g. 1, 2, 3, 4, 5, 6 or more separate containers.
  • the invention provides a cell obtainable or obtained by the transduction methods of the present invention, for example, wherein the cell does not comprise a viral vector (for example, does not comprise the viral vectors encoding proteins that can modify genes), or for example, wherein the cell does not comprise carrier nanoparticles, micelles or liposomes.
  • a viral vector for example, does not comprise the viral vectors encoding proteins that can modify genes
  • the cell does not comprise carrier nanoparticles, micelles or liposomes.
  • the invention provides a transduction buffer or pharmaceutical composition, for use in therapy, prophylaxis or diagnosis.
  • the cell may be an in vivo cell, in which case the treatment is a direct treatment.
  • the cell may be transduced in vitro, e.g. for in vitro diagnosis.
  • the cell may be transduced in vitro prior to transplantation of the cell into a patient.
  • the transplantation may be autologous or allogenic, i.e. the transduced cell may be transplanted back into the same patient that it was taken from (autologous) or into a different person (allogenic). In a preferred aspects the transplantation is autologous.
  • Example 1 Media Composition for Viscosity Adjustment
  • a thickening agent of is added into the commonly used cell culture media, including but not limited to 1640 RPMI (Roswell Park Memorial Institute), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's minimum essential medium), or freestyle 293 expression medium at different concentration to achieve the appropriate viscosity from 0.77 cP to 15 cP.
  • methylcellulose there are many other cell compatible polymers that can be used to adjust the fluid viscosity (i.e. function as a thickening agent) such as hyaluronic acid, polyethylene glycol (PEG), polyvinyl alcohol (PVA), and hydroxyethyl cellulose, poly(vinyl pyrrolidone), naturally occurring polysaccharides (e.g., guar gum, xanthan gum, and carrageenan), and their derivatives or combination thereof.
  • hyaluronic acid polyethylene glycol (PEG), polyvinyl alcohol (PVA), and hydroxyethyl cellulose, poly(vinyl pyrrolidone), naturally occurring polysaccharides (e.g., guar gum, xanthan gum, and carrageenan), and their derivatives or combination thereof.
  • PEG polyethylene glycol
  • PVA polyvinyl alcohol
  • hydroxyethyl cellulose poly(vinyl pyrrolidone)
  • HEK293T cells American Type Culture Collection, USA; maintained in DMEM + 10% FBS and 2 mM L-glutamine, at 37 °C, 5% CO2, and saturated humidity
  • B16F10 cells American Type Culture Collection, USA; maintained in DMEM + 10% FBS, at 37 °C, 5% CO2, and saturated humidity
  • HEK293T cells American Type Culture Collection, USA; maintained in DMEM + 10% FBS, at 37 °C, 5% CO2, and saturated humidity
  • B16F10 cells American Type Culture Collection, USA; maintained in DMEM + 10% FBS, at 37 °C, 5% CO2, and saturated humidity
  • Jurkat cells (American Type Culture Collection, USA; maintained in 1640 RPMI + 10% FBS, at 37 °C, 5% CO2, and saturated humidity) were collected and seeded into 24-well plates at a cell density of 25,000 cells/well with the methylcellulose- containing medium right before the transfection, and then the particles were pipetted into the well. At 24 h post-dosing, the transfection was examined.
  • the cells were lysed by reporter lysis buffer (Promega, USA) using two freeze-thaw cycles, with the lysate characterized by a luminometer upon addition of luciferin assay solution (Promega, USA) against a ladder generated by the standardized luciferase samples (Promega, USA).
  • reporter lysis buffer Promega, USA
  • GFP or mCherry the cells were suspended by trypsin-EDTA in PBS supplemented with 1% FBS and 0.5 mM EDTA and analyzed by a Attune flow cytometer (ThermoFisher, USA).
  • HEK293T cells immortalized human embryonic kidney epithelial cells, B16F10 cells (a mouse melanoma cell line), Jurkat cells (immortalized human T lymphoblasts), PC12 cells (rat adrenal pheochromocytoma cells), C2C12 cells (mouse myoblast cells), 4T1 cells (mouse mammary carcinoma cells), NIH-3T3 cells (mouse embryonic fibroblast cells), Caco2 cells (human colorectal adenocarcinoma cells), RAW264.7 cells (mouse macrophage cells), Neuro2A cells (mouse neuroblastoma cells), MOLT4 cells (human acute lymphoblastic leukemia cells), HepG2 cells (human hepatocellular carcinoma cells), PC3 cells (human prostate cancer cells), MDA-MB-231 cells (human breast adenocarcinoma
  • the culture media viscosity is adjusted from 0.77 cP (0% methylcellulose) to 15 cP.
  • concentration of methylcellulose By adjusting concentration of methylcellulose, the culture media viscosity is adjusted from 0.77 cP (0% methylcellulose) to 15 cP.
  • the transgene level increased by several fold at the same dosage of mRNA LNPs (1 pg per well) with the highest level observed at around 2-4 cP for all cell lines.
  • FIG. 2A - FIG. 2F further confirms the 3 representative cell lines results using a different mRNA construct using flow cytometry.
  • the effect of medium viscosity on transfection efficiency is applicable to other transfection agents and types of payloads, such as PEI/nucleic acid nanoparticles and pDNA as a payload.
  • mRNA as a payload showed higher transfection efficiency than pDNA payload when LNPs and PEI/nucleic acid nanoparticles were tested. Nonetheless, the peak transfection efficiency appeared to be maintained at 2 cP for HEK293T cells for these carriers and payloads.
  • FIG. 5A - FIG. 5C when AAV9 was tested as the transfection vector in HEK293T cells, the viscosity of culture media also influenced the transfection efficiency, which increased from about 35% positive at 0.77 cP to 65% at 8 cP.
  • the luciferase expression level was further increased until 36 h, compared to all other viscosity conditions where the peak expression was observed at 24 h (FIG. 8D).
  • the tested cell types included cancer cell lines (B16-F10: mouse melanoma cell, 4T1: mouse breast cancer cell, MDA-MB-231: human breast cancer cell, PC-3: human prostatic adenocarcinoma, Hep G2: human hepatocellular carcinoma, Caco-2: human colon epithelial cancer cell, CT26: mouse colorectal carcinoma cell, HeLa: human cervical carcinoma, PC-12: rat pheochromocytoma,), immune cells (Jurkat: human T-cell Leukemia, MOLT-4: human T lymphoblast cell, Ramos: human B lymphocyte cell, DC2.4: mouse dendritic cell, RAW 264.7: mouse macrophage cell, BMDMs: mouse bone marrow-derived macrophage, BMDCs: mouse bone marrow-derived dendritic cells), and other frequently used cell lines (293 T: human kidney cell, NIH/3T3: fibroblast cell, C2C12: mouse myoblast cell, X9: mouse fat cell
  • the normalized luciferase expression level increased by 2 to 60-fold among these cell types (FIG. 14B and FIG. 13A), excluding RAW264.7, PC12, BMDM and 4T1 cells (FIG. 14C and FIG. 13B), when subjected to a higher media viscosity.
  • the optimal media viscosity varied among different cell types, though within the range of 1 to 4 cP (FIG. 14A). This variability might be originated from distinct cell profdes, given their diverse tissue origins and species-specific factors.
  • the highest cellular uptake was achieved at around 2-3 cP, where the cellular uptake levels were about 1.5-fold (P ⁇ 0.0001), 5.8-fold (P ⁇ 0.0001), 2.5-fold (P ⁇ 0.0001), and 1.5- fold (P ⁇ 0.0001) higher in B16-F10, HEK293T, Jurkat, and Ramos cells, respectively, at optimized viscosity compared to the 0.77-cP condition (FIG. 18A and FIG. 16A - FIG. 16D).
  • Endosomal escape is also a critical step in a successful transfection process mediated by vectors entering the cells through endocytic pathways.
  • endosomal escape efficiency of the mRNA LNPs was also altered by media viscosity, we used two engineered cell lines (C2C12-Gal8-GFP cells and B16-Gal8-GFP cells) both expressing galectin- 8 (Gal8) fused with GFP for this experiment.
  • Gal8 protein present in the cytosol, binds to glycans exposed on the cell membrane after endosomal vesicles are damaged, leading to the aggregation of Gal8-GFP protein and formation of GFP spots (FIG. 18B) 34 .
  • the uptake pathway distributions were similar between the two viscosity conditions, although the clathrin-mediated pathway was utilized slightly more under the 8-cP condition as compared with the 0.77-cP condition; and similar to non-viral particles at the 2-cP condition, all viscosity-sensing pathways played a critical role in the enhanced transduction process for AAV at the 8-cP viscosity (FIG. 25B). Additionally, the viscosity-enhanced particle internalization process can be extended to some other traditional endocytic cargos, such as transferrin 45 (FIG. 26A - FIG. 26D). These results collectively confirmed that the viscosity-mediated enhancement in cell transfection applies not only to lipid- based nanoparticles, but also to polyplex nanoparticles and viral vectors. Moreover, this enhancement effect appears to be independent of the type of cargo used.
  • transfection plays a pivotal role in the entire therapeutic production process (FIG. 27A).
  • the production of transduction vectors like AAV and LVV heavily depends on the transfection efficiency of viral production cell lines such as HEK293F cells.
  • the transfection efficiency of pDNA/PEIpro nanoparticles in HEK293T cells was enhanced by 2.5-fold (P ⁇ 0.01) in percentage of cells transfection with 73% increase in MFI (P ⁇ 0.0001), when the media viscosity was increased from 0.77 cP to 2 cP (FIG. 23B).
  • P ⁇ 0.01 in percentage of cells transfection with 73% increase in MFI (P ⁇ 0.0001)
  • luciferase expression level in HEK293F cells mediated by pDNA LNPs increased by over 30-fold under elevated viscosities, compared with 0.77 cP (FIG. 23C).
  • the transfection of human primary B cells mediated by mCherry mRNA LNPs was also increased by about 2-fold in terms of percent of cells transfected and the MFI increased by about 80% at 2 cP compared with 0.77 cP condition (FIG. 28B).
  • LVV-mediated transduction of peripheral blood mononuclear cells (PBMCs) also showed a similar media viscosity-dependence as AAVs on HEK293T cells (FIG. 23D), which was plateaued at the 8cP condition (FIG. 27C and FIG. 29).
  • Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo. J. Control. Rel. 350, 298-307 (2022).

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Abstract

L'invention concerne, entre autres, des milieux de culture cellulaire pour la transfection cellulaire ou l'administration intracellulaire, ainsi que des procédés et des kits les utilisant.<i />
PCT/US2024/039036 2023-07-20 2024-07-22 Milieu de culture cellulaire et compositions associées Pending WO2025019869A2 (fr)

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