WO2025019869A2 - Cell culture medium and compositions thereof - Google Patents

Abstract

Provided are, inter alia, cell culture media for cell-transfection or intracellular delivery, and methods and kits using the same.

Description

CELL CULTURE MEDIUM AND COMPOSITIONS THEREOF

The present application claims the benefit of U.S. provisional application no. 63/527,910 filed July 20, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND

[0001] Gene therapy and cell therapy has become an increasingly valuable modality for treating or preventing various diseases. Many of these therapies require the production of vectorized viruses, such as lentivirus (LVVs) and adeno-associated virus (AAVs), or genetically engineering cells ex vivo, such as CAR-T cell or CAR-macrophage therapy. 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.

[0002] It would be desirable to have improved systems for gene and cell therapies.

SUMMARY

[0003] We now provide compositions and methods for transfecting a desired molecule into a cell at greater efficiency.

[0004] In particular, we have now found that increasing cell medium viscosity through addition of one or more thickening agents in the transfection process can notably increase the efficiency of transfer of nucleic acid or other molecules into a target cell. In aspects, other than increasing the transfection efficiency, the thickening agent is substantially biologically inert and does not alter the transferred biological material or cell.

[0005] In one aspect, 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. Preferably, the media and methods as described herein can be used for intracellular delivery of other therapeutic substances into cells. [0006] In one aspect, the disclosure provides cell culture media. Preferably, a cell culture medium includes a liquid medium that comprises: a thickening agent that can provide increased viscosity of the fluid medium composition.

[0007] Suitably, the cell culture medium may comprise a cell-growth component.

[0008] In particular, in one aspect, 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. In another aspect 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. In at least certain systems, 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.

[0009] 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. In at least certain system, 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.

[00010] 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.

[00011] 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. [00012] 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('). hydroxypropyl methylcellulose (HPMC), or hydroxyethyl cellulose; xanthan gum, guar gum, dextran, dextran sulfate, hyaluronic acid, alginate, chondroitin sulfate, or a derivative, or combination thereof.

[00013] 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.

[00014] In one aspect, polymeric thickening agents may comprise one or more naturally occurring polymers. In a further aspect, polymeric thickening agents may comprise one or more synthetic occurring polymers. In a still further aspect, polymeric thickening agents may comprise one or more synthetic occurring polymers and one or more naturally occurring polymers.

[00015] 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. In certain aspects, 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.

[00016] In particular aspects, 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.

[00017] In particular aspects, 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). [00018] 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. For example, 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.

[00019] In certain aspects, the material to be transfected (sometimes referred to herein as biological substance) may suitably include a polymer, a lipid nanoparticle or liposome. For example, the nucleic acid biological substance may be suitably combined, or encapsulated in the polymer, lipid nanoparticle or liposome. In certain embodiments, the polymer may form a vesicle together with lipids for delivery of cargo material (e.g., biological substance contained inside the vesicle).

[00020] Suitably and preferred cell culture medium compositions may comprise additional components. In particular, 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). In another aspect, 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.

[00021] In another aspect, the disclosure provides a composition for producing a transfected cell. The composition preferably includes a cell; and the cell culture medium as described herein.

[00022] In another aspect, 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.

[00023] 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.

[00024] In particular, 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.

[00025] In certain aspects, 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.

[00026] 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.

[00027] In preferred aspects, the cell involved in the transduction method is a mammalian cell. In other aspects, the present methods and compositions may be employed to transduce molecules into other animal cells, plant cells, yeast cells, insect cells, or bacterial cells. Thus, in certain aspects, 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.

[00028] In further aspects, pharmaceutical compositions are provided that suitably comprise a viscosity modifier as disclosed herein a molecule of interest for transduction. In aspects, a pharmaceutical composition comprises a viscosity modifier as disclosed herein. In aspects, the molecule of interest and viscosity modifier components are administered simultaneously or sequentially.

[00029] Also provided are a 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.

[00030] As referred to 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, 10⁴ or more, 10⁵ or more, 10⁶ or more, 10⁷ or more, 10⁸ or more cells.

[00031] Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[00032] 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. The transfection efficiency was assessed at 24 h after commencing transfection and reported as the amount of luciferase protein expressed per mg of luciferase per mg of total protein in culture (n = 3 for B16F10 cells, HEK293T cells, and Jurkat cells; n = 4 for all other cell types).

[00033] 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. The transfection efficiency was analyzed by FACScan at 24 h after transfection and reported as the percentage of cells positive for mCherry expression (bars) and mean fluorescence intensity per cell (line) (n = 3).

[00034] 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. Cellular uptake was analyzed at 24 h after transfection by FACScan and reported as percentage of cells with Cy5-labeled mRNA LNPs (n = 3). [00035] FIG. 4A and 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. Transfection was conducted in a culture medium with defined viscosity ranging from 0.77 cP to 15 cP at dose equivalent to 1 pg of plasmid DNA or mRNA; and transfection efficiency was analyzed by flow cytometry at 24 h after transfection (n = 3).

[00036] 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 IxlO¹⁰ AAV9 per well. The “0.77 cP” group refers to the standard cell culture medium. The transfection efficiency was analyzed by flow cytometry at 48 h after transfection (n = 3).

[00037] 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.

[00038] FIG. 7A - FIG. 7D. Extracellular fluid viscosity-dependent transfection efficiency mediated by mRNA LNPs. FIG. 7A, Normalized luciferase expression level mediated by mLuc LNPs in B16-F10, HEK293T, Jurkat, Ramos cells in media with different viscosities (0.77-15 cP), measured at 24 h after transfection at 2 pg mLuc/mL in 0.5 mL media per well for 8-15 x 10⁴ cells in a 24-well plate (n = 3). FIG. 7B and 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.

[00039] 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 io⁴ cells in a 24-well plate). FIG. 8A and 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.

[00040] 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 10⁴ cells in a 24-well plate). FIG. 9B, Fold change in luciferase expression level at 24 h after transfection.

[00041] 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.

[00042] 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. 11A, B16-F10 cells, FIG. 11B, Jurkat cells, FIG. 11C, HEK293T cells, and FIG. 11D, Ramos B cells.

[00043] FIG. 12A and FIG. 12B. Viability of B16-F10 cells and Jurkat cells cultured in media with different viscosities. Alamar Blue assay was performed to test the viability of FIG. 12A, B16-F10 and FIG. 12B, Jurkat cells cultured in media at different viscosity levels (0.77, 2, 8, and 15 cP) for 24 h. (n = 3). [00044] FIG. 13A and FIG. 13B Enhanced transfection efficiency across various cell types using mRNA LNPs. Transfection efficiency of LNPs on a wider range of cell types with media at different viscosity levels (0.77 - 15 cP) (n = 3). FIG. 13A, Cells showing a bell-shaped curve in dose-response study. FIG. 13B, Cells showing a declining trend in dose-response study.

[00045] FIG. 14A - FIG. 14C Media viscosity-dependent transfection efficiency mediated by mLuc LNPs across various cell types. FIG. 14A, Normalized luciferase expression level mediated by mRNA LNPs in 21 cell types in media with different viscosity levels (0.77-15 cP) (n = 3). 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. 14B, n = 3) and in 4T1, BMDM, RAW 264.7 and PC-12 (FIG. 14C, n = 3). P values were determined in a two-tailed t-test. Data are presented as mean ± SEM. ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[00046] 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. 14B, Normalized luminescence of LNPs on RAW264.7 (n = 3, 2 pg/mL mRNA LNPs, 0.5 mL each well in a 24-well plate), c.f. Supplementary FIG. 13B.

[00047] 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. 16A, B16-F10 cells, FIG. 16B, Jurkat cells, FIG. 16C, HEK293T cells, and FIG. 16D, Ramos B cells.

[00048] 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. 17A and FIG. 17B, Cellular uptake of Cy5-labeled pDNA LNPs (0.5 pg/mL of pDNA in LNPs in 0.5 mL media per well for 8 x 10⁴ cells in a 24-well plate) in B16-F10 (FIG. 17A) and HEK293T (FIG. 17B) cells at different viscosity levels ranging from 0.77 to 15 cP (n = 3). FIG. 17C and FIG. 17D, Representative histograms of B16-F10 (FIG. 17C) and HEK293T cells (FIG. 17D) at different viscosities. At this pDNA dose level, 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.

[00049] FIG. 18A - FG. 18E Effect of media viscosity on cellular uptake and endosomal escape efficiency of mRNA LNPs. FIG. 18A, Cellular uptake of LNPs (2 pg/mL of mRNA using LNPs prepared with 75% mCherry and 25% Cy5-labeled mRNA) in B16-F10, HEK293T, Jurkat, and Ramos cells at different viscosity levels ranging from 0.77 to 15 cP (n = 3). FIG. 18B, Schematic illustration of the Gal8-GFP spots formation in Gal8-GFP-engineered cell lines. FIG. 18C and FIG. 18D, Quantitative Cellomics high-content analysis (HCA) of endosomal escape by LNPs conducted at 4 h after transfection in media with defined (0.77-8 cP) (n = 3). 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.

[00050] 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.

[00051] 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. 20B, Relative cellular uptake level of cells treated with specific pathway inhibitors and Cy5-labeled LNPs in media with 0.77 cP or 2 cP (n = 3). FIG. 20C, Schematic of the proposed viscosity-sensing pathway. FIG. 20D, Actin remodeling/dynamics and NHEl-mediated swelling and pH homeostasis involved in LNP uptake. Data are presented as mean ± SEM. P values were determined via oneway ANOVA with Dunnett’s multiple comparisons test, ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < o 001, ****P < 0.0001.

[00052] 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.

[00053] 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.

[00054] FIG. 23A - FIG. 23D Transfection/transduction efficiency of different nucleic acids and vehicles on HEK 293T cells under different media viscosity conditions. FIG. 23A and FIG. 23B, Transfection efficiency of PEIpro/nucleic acid nanoparticles containing mCherry mRNA (FIG. 23 A) or pDNA (FIG. 23B) in a media with a viscosity of 0.77, 2, 8, or 15 cP) (n = 3).

FIG. 23C, Transfection of HEK293T cells using LNPs containing mCherry pDNA under the same media conditions (n = 3). FIG. 23D, Transduction efficiency of AAV encoding GFP DNA in HEK293T cells under the same media conditions (n = 3). 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.

[00055] 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.

[00056] FIG. 25A and FIG. 25B. Cell uptake and viscosity-sensing pathways on endocytosis of GFP AAV at different viscosity levels in HEK293T cells. FIG. 25A, Relative transduction level of cells treated with endocytic pathways inhibitors and GFP AAV in media with 0.77 cP or 8 cP (n = 3). FIG. 25B, Relative transduction level of cells treated with viscosity-sensing pathway inhibitors and GFP AAV in different media with 0.77 cP or 8 cP (n = 3). Data are presented as mean ± SEM.

[00057] FIG. 26A - FIG. 26D. Alexa fluor® 647 chromPure™ mouse transferrin on Bl 6- F10 cells at different viscosities. FIG. 26A and FIG. 26B, Uptake efficiency at the cellular level was evaluated by flow cytometry following the treatment of transferrin (25 pg/mL). The percentage of AF647+ cells (FIG. 26A) and MFI among the AF647+ cells (FIG. 26B) were shown (n = 3). FIG. 26C, Representative histograms ofB16-F10 cells at different viscosities.

FIG. 26D, Representative flow cytometry panels showing the comparison of cells under different viscosities.

[00058] 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. 27B, Normalized luciferase expression level mediated by pLuc LNPs in HEK293F cells conducted in media with different viscosities in a range of 0.77-15 cP (n = 3). FIG. 27C, Transduction efficiency of LVV-CMV-mCherry-Puro on CD3+ T cells of PBMCs in media with different viscosity levels (MON 10, n = 3). 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.

[00059] FIG. 28A - FIG. 28F. Human primary B cells treated with LNPs loading mCherry mRNA for flow cytometry analysis. FIG. 28A and FIG. 28B, Percentage of cells transfected and MFI of mCherry+ human primary B cells treated with mCherry LNPs in media with different viscosity levels (0.77, 2, 8, and 15 cP) (n = 3). 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.

[00060] FIG. 29. Multiplicity of infection (MOI) selection of LVV transduction of Jurkat cells. Jurkat cells were treated with LVVs with different MOI doses. The transduction efficiency was assessed at 72 h and 96 h after treatment (n = 3).

[00061] FIG. 30A - FIG. 30D. Effect of mRNA LNP dose on viscosity-dependent transfection efficiency in B16-F10, HEK293T, Jurkat, and MOLT-4 cells. Cells were treated with different doses of LNPs with reference to mRNA concentration (1, 2, and 4 pg/mL) under different media viscosity levels (0.77, 2, and 8 cP), a, B16-F10 cells, b, HEK293T cells, c, Jurkat cells, and d, MOLT-4 cells, (n = 3). DETAILED DESCRIPTION

[00062] Provided herein, inter alia, are cell culture media that can significantly alter (e.g., increase or improve) cell uptake and transfection efficiency. Among other things, 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.

[00063] In particular, disclosed herein are 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. Cells subjected to optimized media viscosity exhibit a different profile of uptake pathways, and higher levels of cell cortical tension, contractility, and swelling dynamics, compared with that cultured in media with the standard viscosity of 0.77 cP. These results demonstrate the role of media viscosity in mediating transfection process and offers a practical approach to further optimization of various gene delivery and cell programming processes, thereby reducing production costs, and advancing the accessibility of gene and cell therapies.

[00064] Previously, in vitro cell transfection methods have been conducted using a cell culture media with a fluid viscosity similar to that of water, which is 0.77 centipoise (cP) at 37°C, substantially lower than the extracellular fluids (4-8 cP) of the human body, such as interstitial fluid and blood.

[00065] As set forth in the examples which follow, through screening in different cell types, the effect of extracellular fluid viscosity ranging from 0.77 to 15 cP (at 37°C) was examined on transfection efficiency using plasmid DNA (pDNA) or mRNA-based lipid nanoparticles, polyplex nanoparticles, and viral vectors. Through this screening assay in different cell types, the optimal media viscosity for the highest transfection efficiency and mechanisms responsible for the media viscosity-dependent transfection enhancement effect were examined by correlating with cellular uptake and endosomal escape efficiency.

Definitions [00066] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[00067] In this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein.

[00068] Wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided.

[00069] Unless otherwise indicated, all numbers, values, and/or expressions referring to quantities of ingredients, reaction conditions, polymer compositions, and formulations used herein are to be understood as modified in all instances by the term "about" as such numbers are inherently approximations that are reflective of, among other things, the various uncertainties of measurement encountered in obtaining such values. Further, unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[00070] In the disclosure when a range is described for a variable, it will be understood that the variable includes all values including the end points described within the stated range. For example, the 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. Also, for example, 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. [00071] As used herein, 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). In certain aspects of the disclosure, the culture media is in a liquid form that does not contain any solidified nor gelated portion.

[00072J As used herein, the term “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).

[00073] As used herein, the term “transfecting”, “transfection” or similar terms 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. In “transfecting” or “transfection” as referred to herein, 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. In certain aspects, nucleic acid is delivered into cells. In certain aspects, nanoparticles are delivered into cells.

[00074] As used herein, the term “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. In certain aspects, 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. In certain embodiments of the disclosure, one or more thickening agents may control the viscosity of the cell culture medium (e.g., liquid medium).

[00075] As used herein, the term “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. Tn certain aspect of the disclosure, 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. In some embodiments, 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. In certain aspects of the disclosure, the culture media is in a liquid form that can be defined by measuring a viscosity thereof.

[00076] As used herein, 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). In certain aspect, 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.

[00077] As used herein, the term “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).

[00078] As used herein, the term “zzz vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe, cell, or tissue thereof).

[00079] As used herein, 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. In certain embodiments, the nanoparticles include lipid components (e.g., phospholipid) so as to form lipid nanoparticles (LNPs). In some embodiments, 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. In some embodiments of the disclosure, the term “lipid nanoparticle” may be interchangeably used with the term for “liposome.”

[00080] In some aspects, 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. In some embodiments, 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.

[00081] As used herein, 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, 10⁴ or more, or 10⁵ or more cells.

[00082] The phrase “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.

[00083] The term “pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” 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. One of skill in the art will recognize that other pharmaceutical excipients are useful in the disclosure.

[00084] As used herein “stable” refers to a compound or composition (e g., culture medium) that is sufficiently robust in a storage condition.

[00085] The term "derivative" as used herein 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. For example, 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.

Cell Culture Media Composition

[00086] The disclosure provides a culture medium composition that suitably include a thickening agent to adjust or modulate the viscosity.

[00087] In an aspect, provided is a cell culture medium or composition thereof including: a liquid medium including a thickening agent. Suitably, the cell culture medium also may comprise a cell-growth component. In particular aspects, 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.

[00088] In some embodiments, 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. In certain embodiments, thickening agent includes methylcellulose for adjusting or controlling viscosity. [00089] In certain embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[00090] 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 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. In some embodiments, 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.

[00091] In certain embodiments, 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). In certain embodiments, the liquid medium (media) can be formulated to regulate, control, or adjust surrounding effects which may affect cell growth or proliferation.

[00092] In certain embodiments, the cell culture medium further includes a cell transfection agent. In some embodiments, 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).

[00093] In certain embodiments, the cell culture medium further includes a biological substance to be transfected into a cell. In some embodiments, the biological substance may be a virus. In some embodiments, the biological substance may be a vector. In some embodiments, the biological substance may be a nucleic acid (e.g., microRNA, siRNA, mRNA, viral RNA, RNA oligo, DNA, ribozyme, or aptamer). In some embodiments, the biological substance may an oligonucleotide. In some embodiments, the biological substance may include one or more biological substances selected from virus, a vector, a nucleic acid, an oligonucleotide, or debris thereof.

[00094] In certain embodiments, the biological substance may suitably include a viral vector. Exemplary 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.

[00095] In certain embodiments, 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.

[00096] The term "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. In some embodiments, a therapeutic and/or prophylactic is an RNA. 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. In certain embodiments, the RNA is an mRNA.

[00097] 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. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

[00098] In other aspects, 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. For example, 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. In some embodiments, the siRNA may be an immunomodulatory siRNA. [00099] In some agents, 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.

[000100] 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. In some embodiments, a nucleic acid or polynucleotide further includes a poly-A region or a Kozak sequence (e.g. , in the 5 -UTR). In some cases, polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, a polynucleotide or nucleic acid (e.g. , an mRNA) 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). For example, 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).

[000101] Generally, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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.

[000103] 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). In one embodiment, 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).

[000104] 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. For example, 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.

[000105] 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. Similarly, 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.

[000106] Nanoparticle compositions may be designed for one or more specific applications or targets. For example, 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. For example, 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). In certain embodiments, 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. In some embodiments, a composition may be designed to be specifically delivered to a mammalian liver.

[000107] 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. For example, 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. In some embodiments, 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. For example, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic may be from about 10: 1 to about 40: 1. In certain embodiments, 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).

[000108] In certain aspects, 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.

[000109] In some embodiments, 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.

[000110] In an aspect, provided is a composition for producing a transfected cell. In particular, the composition includes the cell culture medium as described herein.

[000111] In certain aspect, the composition for producing the transfected cell includes a cell and a suitably selected cell culture medium described herein. [000112] Biological material (e g. nucleic acid) and the thickening agent are contacted with the cell in combination, either simultaneously, sequentially, or separately in any order. In a preferred embodiment, they are administered simultaneously (e.g. from a container containing the combination). Thus, in some embodiments, the thickening agent comprises the biological material in admixture.

[000113] 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. Suitably the cell may be contacted with a culture medium during transfection.

[000114] A cell culture medium may comprise additional materials for use with live cells or live cell culture or application in vivo. For example, 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.

[000115] In some aspects, the cell culture medium or composition is used for producing a transfected mammalian cell. In some embodiments, the cell may include a T-cell. In some aspects, the cell may include an embryonic cell. In some aspects, the cell may include a stem cell. In some aspects, the cell may include a mammalian tissue. In certain aspects, the mammalian cell may be a cancer cell line.

[000116] 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. MDA-MB-231, MDA-MB-436, MDA-MB-157, and MDA-MB-453), 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, SW1417, LS411N, NCLH508, SK-CO-1, SW1116, SW948, T84, LS123, SW837 and HT-29), bone cancer cell line (HOS, A-673, SK-PN-DW, U-2 OS, and Saos- 2), bladder cancer cell line (e.g., 5637, HT-1197, HT-1376, RT4, SW780, T-24, TCCSUP, and UM-UC-3), brain cancer cell line (e.g., KASUMI-1, HL-60, THP-1, K-562, RS4;11, MOLT-4, and CCRF-CEM), brain cancer cell line (e.g., A172, SW1088, H4, U118-MG, U87-MG, A172, SW1088, H4, U118-MG, and U87-MG), pancreatic cancer cell line (e.g., Capan-2, Pane 10.05, CFPAC-1, HPAF-II, SW 1990, BxPC-3, and AsPC-1), leukemia cell line (e g., KASUMI-1, HL- 60, THP-1, K-562, RS4;11, MOLT-4, and CCRF-CEM), lung cancer cell line (e.g., NCI-H2126, NCI-H1299, NCI-H1437, NCLH1563, NCI-H1573, NCLH1975, NCI-H661, BEAS-2B, A549, NCI-H1299, NCI-H596, NCI-H1770, NCLH1975, BEAS-2B, NCLH1882, NCLH1417, NCL H719, NCI-H1105, and NCI-H1048), and renal cancer cell line (e.g, Caki-1, HCC89, CAL-54, KMRC-3, KMRC-20, SLR21, TUHR14TKB, UO-31, VMRC-RCZ, SNU-1272, RCC10RGB, SNU-349, Caki-2, ACHN, VMRC-RCW, SLR24, TUHR10TKB, TUHR4TKB, HCC1011, 769- P, HCC1319, SK-RC-20, OS-RC-2, Caki-1, SLR26, A-704, A-498, SLR23, SLR25, KMRC-2, UOK101, BFTC-909, 786-0, KMRC-1, or G-402). 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.

[000117] 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.

[000118] In certain aspects, the cell involved in the transfection method is a mammalian cell. In other aspects, 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. In preferred aspects, 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. In some aspects, the cell is a non-human cell. [000119] In aspects, 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.

[000120] In other aspects, the cell is in vitro or ex vivo. For example, the cell may be in a culture medium, wherein the culture medium optionally supports the maintenance, differentiation and/or expansion of the cell.

[000121] In some embodiments, the cell is derived from an established cell line, such as an established human cell line. In some embodiments, the established cell line is an immortalised cell line. In other embodiments the cell line is a primary cell line.

[000122] 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.

[000123] In some aspects, 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.

[000124] In some aspects, the cell is a stem cell or a cell derived by differentiation of a stem cell. In some embodiments the stem cell is a pluripotent stem cell, such as an embryonic stem cell, optionally a human embryonic stem cell. In some aspects, the cell is not a human embryonic stem cell. In some aspects, the stem cell is not obtained by methods that involve the use of human embryos for commercial or industrial purposes. In some aspects, the stem cell is not obtained by methods that necessarily involve the destruction of a human embryo. In some aspects the stem cell is a murine embryonic stem cell. In other embodiments, the stem cell is an adult stem cell, such as a neural, adipose or hematopoietic stem cell. In some aspects the cell is a murine or human neural stem cell, neuron cell or glia cell. In some aspects the stem cell is an induced pluripotent stem cell. In some embodiments the cell is a somatic cell or a germ cell. [000125] In some aspects, 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.

[000126] In some aspects 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.

Methods and Kits

[000127] In an aspect, provided is a method of producing a transfected cell. The method includes steps of incubating a cell with the cell culture medium as described herein. In some embodiments, the cell is incubated at a temperature range of about 10 to 37 °C for about 1 to 24 hours. In certain embodiments, 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.

[000128] In some embodiments, the method may further include conducting electroporation, sonoporation, or laser irradiation to the cell culture to increase transfection efficiency.

[000129] In transfection, 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. In certain aspects, the incubation time is 1 to 24 hours, or 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[000130] In an aspect, also provided is kit for producing a transfected cell. The kit includes the cell culture medium as described above.

[000131] In certain aspects, 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.

[000132] Cell Viability

[000133] As discussed, in preferred aspects, 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. In a preferred embodiment, 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.

[000134] Efficiency/Time for Transduction

[000135] In order to transduce a molecule of interest into a cell, the molecule of interest and cell are in contact for a sufficient length of time for the molecule to transduce into the cell. [000136] Generally, 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. [000137] In a preferred aspect, 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. In some aspects 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

[000138] 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).

[000139] In aspects, 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).

[000140] In some aspects, a present method may comprise one round of transduction.

However, in other embodiments, multiple rounds of transduction may be desirable. For example, in some embodiments 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.

[000141] In between 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. In some embodiments, the recovery period is at least 10, at least 20, at least 30, at least 40 or at least 50 minutes.

[0001421 Iⁿ some aspects there may be no recovery period, or there is a recovery period of less than 24 hours, less than 12 hours, less than 6 hours, less than 3 hours, less than 1 hour, less than 30 minutes or less than 10 minutes.

[000143] During the recovery periods, the transduction buffer is removed from the cells and the cells are typically cultured in cell culture medium suitable for the particular cell type.

[000144] Exemplary Transduction Buffers and Methods

[000145] Non-limiting examples of 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.

[000146] In certain asepcts, there is provided a method for transducing a molecule of interest into a cell, wherein the method comprises 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. In certain aspects, the transduction compound is a small molecule compound and is not a detergent. In certain aspects, 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.

[000147] In certain aspects, 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. [000148] In some aspects, a present method comprises contacting the cell with the transduction buffer for a period of at least 30 minutes, preferably for about 12 hours. In some aspects, 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.

[000149] In certain aspects, the method for modifying a nucleic acid, such as a genetic sequence, in a cell, further comprises isolating or using the modified cell.

[000150] As discussed, a modified cell obtainable or obtained by the present cells is also provided. In certain aspects, the modified cell comprises a transduced gene editing system. In some embodiments, the modified cell does not comprise a viral vector. In certain aspects, the modified cell does not comprise a nanoparticle carrier.

[000151] Pharmaceutical Composition

[000152] In further aspects, a pharmaceutical composition comprising a viscosity modifier as disclosed herein and a molecule of interest for transduction. In some aspects, a pharmaceutical composition comprises viscosity modifier as disclosed herein. In some aspects, the molecule of interest and viscosity modifier as disclosed herein are administered simultaneously or sequentially.

[000153] The pharmaceutical composition can include further components in addition to the viscosity modifier and a molecule of interest. For example, 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.

[000154] The pharmaceutical composition may be sterile and/or pyrogen-free.

[000155] The invention also provides a container (e.g. vial) or delivery device (e.g. syringe) pre-filled with a pharmaceutical composition as disclosed herein.

[000156] 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.

[000157] Compositions of the invention may be prepared in various liquid forms. For example, 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.

[000158] Further provided are 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.

[000159] In certain aspects, 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.

[000160] In some embodiments, the invention provides a transduction buffer or pharmaceutical composition, for use in therapy, prophylaxis or diagnosis.

[000161] Further provided are methods for therapy or diagnosis comprising transducing a molecule of interest into a cell. The cell may be an in vivo cell, in which case the treatment is a direct treatment. Alternatively, the cell may be transduced in vitro, e.g. for in vitro diagnosis. Alternatively, 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.

EXAMPLES

Example 1 : Media Composition for Viscosity Adjustment [000162] To adjust the viscosity of the cell culture media, 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.

[000163] In addition to 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.

Example 2: Cells and transfection methods

Experimental Methods

[000164] For monolayer culture studies, cells including 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) and B16F10 cells (American Type Culture Collection, USA; maintained in DMEM + 10% FBS, at 37 °C, 5% CO2, and saturated humidity) were seeded into 24-well plates at a cell density of 25,000 cells/well at 1 day prior to transfection. For transfection, the original medium in the wells was first drained and replaced by the methylcellulose (thickening agent)-containing medium, and then the particles were pipetted into the well. For suspension culture studies, 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. When characterizing luciferase as the reporter, 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). When characterizing GFP or mCherry as the reporter, 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). Results

[000165] To comprehensively assessed the effect of media viscosity on transfection efficiency mediated by nanoparticles in different cell types, 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 cells), HeLa cells (immortalized human cervical cancer cells), CT26 cells (mouse colorectal carcinoma cells), DC2.4 cells (mouse dendritic cells), and X9 cells (mouse fat cells) . By adjusting concentration of methylcellulose, the culture media viscosity is adjusted from 0.77 cP (0% methylcellulose) to 15 cP. As shown in FIG. 1A - FIG. 1C, 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.

[000166] To access the effect of media viscosity on cellular uptake level, which is an essential step for the transfection process, the Cy5-labeled mRNA was loaded into the LNPs. As the FIG. 3A - FIG. 3C showed, a clear and consistent trend for cellular uptake was observed, which strongly correlated with the enhancement of transfection efficiency. These results demonstrated that the viscosity-dependent transfection efficiency was strongly contributed by improved cellular uptake.

[000167] Furthermore, 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. As shown in FIG. 4A and FIG. 4B, 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. [000168] In another example, as shown in 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.

[000169] These results demonstrate that the viscosity of the cell culture media significantly influences endocytic activity of cells, which in turn modulates transfection efficiency of several different viral and non-viral nanoparticle carriers, including AAV, lipid nanoparticles, and polymer-based gene carriers. A cell medium viscosity of between 1 and 8 cP could be optimal for at least certain systems to increase cell transfection efficiency substantially.

[000170] Example 3

[000171] I) Extracellular fluid viscosity influences mRNA LNP-mediated transfection efficiency in both adherent and suspension cells

[000172] We first examined the effect of media viscosity (0.77-15 cP) on the transfection efficiency mediated by an d mRNA LNP formulation that has been used in Spikevax²⁹ (FIG. 6 the below Table 1). Its transfection activity has also been demonstrated in both in vivo and in vitro cell transfection applications^8,30. This LNP formulation contains SM-102, DSPC, cholesterol, and DMG-PEG2000 at an SM-102/DSPC/cholesterol/DMG-PEG2000 molar ratio of 50: 10:38.5: 1.5. To adjust the viscosity of media during in vitro cell transfection, we introduced varying concentrations of 65-kDa methylcellulose into the cell culture medium²². This allowed us to achieve media with viscosities spanning from 0.77 cP (0% w/v) to 15 cP (0.65% w/v) at 37 °C, without significantly affecting the osmolarity of the media (less than 0.03% increase)²². When transfecting B 16-F 10 cells (a murine melanoma cell line) and HEK 293T cells (a human embryonic kidney cell line) using the LNPs containing 2 pg luciferase mRNA (mLuc)/mL in 5 x 10⁴ cells/well, the luciferase expression level increased substantially as a function of the media viscosity when measured at 24 h after transfection, peaking at 2-3 cP before subsequently decreasing. Compared to the standard culture media at 0.77 cP, a media viscosity of 2 cP resulted in a 11 -fold increase in mRNA expression in Bl 6-F 10 cells and a 4-fold increase in HEK 293T cells (FIG. 7A).

Table 1. Concentrations of methylcellulose added to cell culture media to achieve different viscosities.¹ Media viscosity (cP) Methylcellulose (w/v%)

0.77 0

1.0 0.110

1.5 0.210

2.0 0.281

3.0 0.382

4.0 0.453

6.0 0.554

8.0 0.625

10 0.680

12 0.725

15 0.781

[000173] In addition to adherent cells, we examined the effect of media viscosity in two suspension cell lines, including Jurkat cells (an immortal human T lymphocyte cell line) and Ramos cells (a human B lymphocyte cell line). The normalized luciferase expression level measured at 24 h after transfection peaked at 1.5 cP and 3 cP in Jurkat and Ramos cells, showing a 1.8-fold (P < 0.05) and 2.6-fold increases (P < 0.0001), respectively, compared to that in the 0.77-cP media (FIG. 1C). In addition, we monitored the normalized luciferase expression levels mediated by mRNA or pDNA LNPs during the first 48 h following transfection in HEK293T cells; and similar enhancement effects were observed (FIG. 8A and FIG. 8B). The normalized luciferase expression level consistently peaked at 2 cP at all points, particularly for mRNA LNPs. Interestingly, luciferase expression level mediated by pDNA LNPs at 2 cP continued to increase with time until at least 48 h, whereas the luciferase expressed peaked at 36 h at 8 cP (FIG. 8C). For mRNA LNPs, 2 cP was the optimal viscosity at all time points tested. At 2 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). Besides, we further tested the effect of dynamic culture, i.e., introducing shaking to a traditionally adherent cell culture, on luciferase expression level in HEK293T cells. We found that introducing shaking condition (120 rpm) to the HEK293T cell culture following transfection did not alter the viscosity-dependent relationship of luciferase expression; the peak luciferase expression also occurred at 2 cP with an average of 3.2-fold higher luciferase expression over the 0.77 -cP viscosity (FIG. 9A and FIG. 9B), even though the luciferase expression level was reduced under the shaking condition, as compared with the static culture. [000174] We further conducted a detailed assessment of LNP-mediated transfection efficiency in these cell types using mRNA encoding mCherry under varying media viscosity conditions utilizing flow cytometry analysis (FIG. 2A - FIG. 2F). As depicted in FIG. 7C - FIG. 7D and FIG. 10 and FIG. 11A - FIG. 11D, even though the percentage of cells transfected at this mRNA dose remained the same due to the already high level of transfection (93.8% for B16-F10 cells, 85.7% for HEK293 cells, and 72.8% for Jurkat cells), substantial levels of increase in median fluorescence intensity (MFI) were observed, reaching a peak level of 2.47-fold (P < 0.0001) for B16-F10 cells, 6.8-fold (P < 0.0001) for HEK293T cells, and 2.94-fold (P < 0.0001) for Jurkat cells at 2-cP media viscosity compared to the 0.77-cP condition. In Ramos cells, both the percentage of transfected cells (88.5% vs. 62.7%) and MFI (2.31-fold) increased with the media viscosity, peaking at 2-3 cP. It is important to note that when the media viscosity increased beyond 6-8 cP, the transfection efficiency measured in either normalized luciferase expression (FIG. 7A), percentage cells transfected (FIG. 7B), and/or MFI of the expressed mCherry (FIG. 7C and FIG. 7D) were reduced to the level below the 0.77-cP condition. Under this tested media viscosity range from 0.77 to 15 cP, which is a much narrower range compared to that tested in previous studies³¹, no significant change in cell viability was observed (FIG.

12A and FIG. 12B).

[000175] ii) Increased mRNA LNP-mediated transfection efficiency is commonly observed in a wide range of cell types under optimal media viscosity

[000176] To compare the effect of media viscosity on mRNA LNP-mediated transfection efficiency across various cell types, we conducted additional experiments using mLuc LNPs under different viscosity conditions (FIG. 14A and FIG. 13A - FIG. 13B). 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, Neuro-2a: mouse neuroblasts). 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. Notably, 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. Furthermore, we observed insignificant change in normalized luciferase expression level in macrophages (BMDMs and RAW 264.7 cells), PC 12 cells, and 4T1 cells (FIG. 14C). This phenomenon is likely attributed to the inherently high endocytic activities in these cell types³² (FIG. 9A and FIG. 9B).

[000177] iii) Media viscosity influences cellular uptake and endosomal escape efficiencies of mRNA LNPs

[000178] We have previously shown that increasing extracellular fluid viscosity substantially enhances actin polymerization kinetics and promotes activation of NHE, thereby altering the cellular membrane activity²². We postulated that culturing cells in media with specified viscosity levels during LNP -mediated transfection can enhance endocytic efficiency and likely change the intracellular trafficking kinetics of mRNA LNPs. To characterize the effect of media viscosity on the uptake of mRNA LNPs under varying viscosity conditions, we labeled the mRNA cargo with Cy5 dye. We assessed cellular uptake and endosomal escape kinetics through flow cytometry and high-content analysis (HCA), employing the quantitative Cellomics technique³³.

[000179] Four representative cell lines, including Bl 6-F 10, HEK293T, Jurkat, and Ramos cells, were treated with mRNA LNPs containing Cy5-labeled mRNA. The cell uptake level of mRNA LNPs as a function of media viscosity was evaluated at 24 h after transfection (FIG. 18A). 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). In addition, the enhanced uptake efficiency was observed for pDNA LNPs in B16-F10 and HEK293T cells at around 2 cP viscosity at the LNP dose tested, whereas the average levels of uptake among the Cy5-positive cells were 45.6% and 56.0% higher than that at the 0.77-cP condition, respectively (FIG. 17A - FIG. 17D). In general, the bell-shaped curve depicting cell uptake level under varying viscosity conditions closely resembled the trends for transfection efficiency. This result suggests that the moderately elevated viscosity levels in transfection media, i.e., 2-3 cP, substantially enhanced the cellular uptake of mRNA LNPs, which is a crucial factor contributing to the subsequent improvement in transfection efficiency.

[000180] Endosomal escape is also a critical step in a successful transfection process mediated by vectors entering the cells through endocytic pathways. To probe whether the 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.

[000181] After transfecting these cells with mCherry LNPs in media with defined viscosities for 4 h, we imaged the cells after fixation and quantify the number of GFP-Gal8 spots by Cellomics high-content analysis (HCA)³³. The number of Gal8-GFP spots increased with media viscosity and peaked or plateaued at the media viscosity of 2 cP (FIG. 18C- FIG. 18E and FIG. 19). The GFP spot counts for C2C12-Gal8-GFP cells and B16-Gal8-GFP cells were 1.9-fold (P < 0.05) and 2.3-fold (P < 0.001) higher at 2 cP compared to 0.77 cP, respectively. These results confirmed the enhanced endosomal escape activities in C2C12 cells and B16-F10 cells under the elevated viscosity conditions, although quantitatively separating the increase in levels of Gal8- GFP into contributions from cellular uptake and endosomal escape is challenging. Taken together, by altering the media viscosity, both cellular uptake and endosomal escape levels were substantially enhanced. These critical effects are consistent with and may be responsible, at least partially, for observed media viscosity-dependent transfection enhancement outcome.

[000182] We next investigated whether the uptake pathways for mRNA LNPs were altered when transfection was conducted in media with different viscosities (FIG. 20A). Using B16-F10 cells as a model, we selected several inhibitors targeting various cellular uptake pathways (see Table 2 below) to probe the mechanism responsible for the enhancement effect on transfection efficiency observed at 2 cP as compared with the standard viscosity (0.77 cP) (FIG. 20B and FIG. 21). For both standard viscosity and 2 cP viscosity, the 4°C group showed no uptake, confirming that the uptake of LNPs is ATP-dependent^35,36. When transfection was conducted at 37°C and the standard viscosity, all pathways were actively involved in uptake of the LNPs, as adding inhibitors brought the transfection efficiency to the background level, except for phagocytosis pathway. When the inhibitor for phagocytosis was added, the amount of uptake was reduced by about 50% (P < 0.0001). In contrast, the inhibition pattern observed in the 2-cP media was substantially different. In the presence of the respective inhibitor, uptake via phagocytosis (ns, P > 0.05) was not affected, whereas uptake via clathrin/caveolin-independent endocytosis and caveolin-mediated endocytosis were reduced by 10% (P < 0.0001) and 25% (P < 0.0001), respectively. In addition, the enhancement effect at the 2-cP condition was completely abolished by the inhibitor for macropinocytosis; and the uptake was nearly completed blocked by the inhibitor for clathrin-mediated endocytosis. These data reveal that under an elevated viscosity condition (2 cP), different from the 0.77 cP condition where uptake of LNPs was critically dependent upon nearly all the pathways, LNP uptake was primarily through the clathrin- mediated endocytosis and macropinocytosis pathways. The elevated media viscosity drastically altered the profde of uptake pathways through which mRNA LNPs entered the cells.

[000183] Our previous study has revealed that the elevated media viscosity increases mechanical loading on cells and induces an ARP2/3 -complex-dependent dense actin network remodeling, which then enhances NHE1 polarization through its actin-binding partner ezrin. NHE1 promotes membrane swelling and increases membrane tension, which in turn activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RhoA-dependent cell contractility^21,22. We postulated that the coordinated action of actin remodeling/dynamics, NHE1 -mediated swelling, and RhoA-based contractility can increase endocytosis of LNPs at elevated viscosities. To test this hypothesis, we used specific inhibitors that target various viscosity-sensing pathways (see Table 2 below) to explore the connection between the effect of media viscosity and cellular uptake outcomes in B16-F10 cells (FIG. 20D). The cellular uptake of LNPs in the presence of inhibitors for various receptors and key components for viscosity-sensing pathways (FIG. 20D and FIG. 22), all but myosin Il-inhibitor reduced the uptake activity observed at 2-cP viscosity to the baseline level at the standard viscosity (P < 0.0001); and ARP2/3 -inhibitor had the strongest effect and reduced the uptake to nearly 30% of the baseline level (P < 0.01). These data revealed the interplay between these viscosity-sensing pathways and enhanced cellular uptake at 2-cP viscosity condition; and confirmed that the increased cell cortical tension, contractility, and swelling, triggered by the increased levels of actin-network remodeling and NHE1 polarization in response to the elevated media viscosity (1.5—3 cP), are directly correlated with the improved cellular uptake level of the LNPs.

Table 2. Inhibitors used for the uptake pathway and cell membrane receptor studies.

Reagent Pathway target Concentration Reference #

Dynasore Clathrin-mediated uptake 80 pM 2

MpCD Caveolae-mediated uptake 10 mM 3

EIPA Macropinocytosis 75 pM 4

Cytochalasin D Phagocytosis 2 pM 5

„ Clathrin- and Caveolae- ,

Rottenn . , , 6 pM 6 independent uptake ( PKCo)

CK666 ARP2/3 100 pM 1

Latrunculin A Actin 2 pM 1

NSC 668394 Ezrin 10 pM 1

Y-27632 Rho-associated protein kinase 10 pM 1

Blebbistatin Myosin 50 pM 1

BAPTA RhoA GTPase 50 pM 1

BAPTA AM Ca²⁺ 25 pM 1

EIPA NHE1 40 pM 1

HC-067047 TRPV4 1 pM 1

Reference # for Table 2:

1. Bera, K. et al. Extracellular fluid viscosity enhances cell migration and cancer dissemination. Nature 611, 365-373 (2022).

2. Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839-850 (2006).

3. Zidovetzki, R. & Levitan, I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim. Biophys. Acta 1768, 1311-1324 (2007).

4. Koivusalo, M. et al. Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rael and Cdc42 signaling. J. Cell Biol. 188, 547-563 (2010).

5. Goddette, D. W. & Frieden, C. Actin polymerization. The mechanism of action of cytochalasin D. J. Biol. Chem. 261, 15974-15980 (1986). 6. Davies, S. P., Reddy, H., Caivano, M. & Cohen, P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95-105 (2000).

[000184] iv) Enhanced transfection or transduction efficiencies of polyplex nanoparticles and viral vectors were observed under optimized media viscosity

[000185] We next explored whether the media viscosity-dependent enhancement effect is applicable to other transfection vectors or carriers such as polyplex nanoparticles and viral vectors, and additional payloads such as pDNA instead of mRNA. In viral vector production process, poly(ethylenimine) (PEI)-pro is widely used as a polycation to form PEIpro/pDNA nanoparticles containing a pDNA cocktail, which are then used to transfect a production cell line for manufacturing AAV or LVV³⁷. In CAR-T therapy, AAV^38,39, LVV⁴⁰’⁴¹, and LNPs^42,43 have all been used for T cell engineering. In this study, we investigated the transfection efficiency of mRNA/PEIpro particles on HEK293T cells using mCherry-encoding mRNA (FIG. 23A and FIG. 23B and FIG. 24A - FIG. 24B). The transfection efficiency of mRNA/PEIpro and pDNA/PEIpro nanoparticles in HEK293T cells showed media viscosity dependence that is similar to that with mRNA LNPs: both peaking at a media viscosity of 2 cP, showing 1.3-fold (P < 0.01) and 2.5-fold (P < 0.01) increases in percent cell transfected, compared to 0.77 cP media. MFI values increased by 59% (P < 0.01) and 73% (P < 0.0001), respectively. In addition, LNPs containing mCheriy pDNA, when tested in HEK293T cells, showed a 10.9-fold increase (P < 0.0001) in transfected cell percentage under the 2-cP viscosity condition, compared with that in 0.77-cP viscosity media; and the MFI increased by 48% (P < 0.0001, FIG. 23C and FIG. 24C). Finally, we examined the effect of media viscosity on transduction efficiency of GFP-encoding AAV in HEK293T cells. Following a 48-h incubation with AAV⁴⁴, it is interesting that cell transduction efficiency increased with media viscosity gradually; at 8 cP media viscosity, the percent cell transfected increased from 35% to 62% and MFI increased by 50% (P < 0.0001), compared with media at standard viscosity (FIG. 23D and FIG. 24D). We confirmed that under both 0.77 and 8-cP conditions, the major uptake pathways for AAV were all involved, including clathrin/caveolin-independent endocytosis, macropinocytosis, and phagocytosis (FIG. 25A). Different from results of LNPs (FIG. 20B), 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 transferrin⁴⁵ (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.

[000186J v) Media viscosity-enhanced transfection was observed in HEK293F suspension cells and LVV-mediated immune cell programming

[000187] In gene and cell therapy, transfection plays a pivotal role in the entire therapeutic production process (FIG. 27A). For one, the production of transduction vectors like AAV and LVV heavily depends on the transfection efficiency of viral production cell lines such as HEK293F cells. We already showed that 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). Here we also tested the transfection of pLuc LNPs in HEK293F cells suspended in media with different viscosities. The 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). In addition, we showed that 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). Additionally, 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).

Table 3. Characterization of various nanoparticles used in Example 3

Particles Z-Average (nm) PD1 Zeta Potential Encapsulation

(n = 3) fmV) Efficiency

References:

1. Zu, H. & Gao, D. Non-viral vectors in gene therapy: recent development, challenges, and prospects. AAPS J. 23, 78 (2021).

2. Butt, M. H. et al. Appraisal for the potential of viral and nonviral vectors in gene therapy: a review. Genes (Basel) 13, 1370 (2022).

3. Fan, G. et al. Bio-inspired polymer envelopes around adenoviral vectors to reduce immunogenicity and improve in vivo kinetics. Acta Biomater. 30, 94-105 (2016).

4. Zhu, Y. el al. Optimization of lipid nanoparticles for gene editing of the liver via intraduodenal delivery. Biomaterials 308, 122559 (2024).

5. Goncalves, G. A. R. & Paiva, R. de M. A. Gene therapy: advances, challenges and perspectives. Einstein (Sao Paulo) 15, 369-375 (2017).

6. Tian, Y., Hu, D., Li, Y. & Yang, L. Development of therapeutic vaccines for the treatment of diseases. Mol. Biomed. 3, 40 (2022).

7. Kacherovsky, N. et al. Traceless aptamer-mediated isolation of CD8+ T cells for chimeric antigen receptor T-cell therapy. Nat. Biomed. Eng. 3, 783-795 (2019).

8. Zhu, Y. et al. Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity. Nat. Biomed. Eng. 8, 544-560 (2024).

9. Zhang, Y. et al. Close the cancer-immunity cycle by integrating lipid nanoparticle-mRNA formulations and dendritic cell therapy. Nat. Nanotechnol. 18, 1364-1374 (2023). 10. Billingsley, M. M. etal. In vivo mRNA CAR T cell engineering via targeted ionizable lipid nanoparticles with extrahepatic tropism. Small 20, e2304378 (2024).

11. Zhao, Y. etal. Polymetformin combines carrier and anticancer activities for in vivo siRNA delivery. Nat. Commun. 7, 11822 (2016).

12. Xiong, M. P. et al. Poly(aspartate-g-PEI800), a polyethylenimine analogue of low toxicity and high transfection efficiency for gene delivery. Biomaterials 28, 4889-4900 (2007).

13. Wang, G. P. etal. Analysis of lentiviral vector integration in HIV+ study subjects receiving autologous infusions of gene modified CD4+ T Cells. Mol. Ther. 17, 844-850 (2009).

14. Ghassemi, S. etal. Rapid manufacturing of non-activated potent CAR T cells. Nat. Biomed. Eng. 6, 118-128 (2022).

15. Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 18, 358-378 (2019).

16. Pekrun, K. et al. Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors. JCI Insight 4, el 31610 (2019).

17. Metzloff, A. E. et al. Antigen presenting cell mimetic lipid nanoparticles for rapid mRNA CAR T cell cancer immunotherapy. Adv. Mater. 36, e2313226 (2024).

18. Nemir, S. & West, J. L. Synthetic materials in the study of cell response to substrate rigidity. Ann. Biomed. Eng. 38, 2-20 (2010).

19. Yankaskas, C. L. et al. The fluid shear stress sensor TRPM7 regulates tumor cell intravasation. Sci. Adv. 7, eabh3457 (2021).

20. Zhao, R. et al. Cell sensing and decision-making in confinement: The role of TRPM7 in a tug of war between hydraulic pressure and cross-sectional area. Sci. Adv. 5, eaaw7243 (2019).

21. Maity, D. et al. Extracellular hydraulic resistance enhances cell migration. Adv. Sci. 9, e2200927 (2022). 22. Bera, K. et al. Extracellular fluid viscosity enhances cell migration and cancer dissemination. Nature 611, 365-373 (2022).

23. Zhang, Y. el al. Polarized NHE1 and SWELL 1 regulate migration direction, efficiency and metastasis. Nat. Comm. 13, 6128 (2022).

24. Goult, B. T., von Essen, M. & Hytonen, V. P. The mechanical cell - the role of force dependencies in synchronising protein interaction networks. J. Cell Sci. 135, jcs259769 (2022).

25. Sun, L., Suo, C., Li, S.-T., Zhang, H. & Gao, P. Metabolic reprogramming for cancer cells and their microenvironment: Beyond the Warburg Effect. Biochim. Biophys. Acta Rev. Cancer 1870, 51-66 (2018).

26. Mayor, S. & Pagano, R. E. Pathways of clathrin-independent endocytosis. Nat. Rev. Mol. Cell Biol. 8, 603-612 (2007).

27. Parton, R. G. et al. Caveolae: The FAQs. Traffic 21, 181-185 (2020).

28. Donahue, N. D., Acar, H. & Wilhelm, S. Concepts of nanoparticle cellular uptake, intracellular trafficking, and kinetics in nanomedicine. Adv. Drug Deliv. Rev. 143, 68-96 (2019).

29. Pegu, A. et al. Durability of mRNA-1273 vaccine-induced antibodies against SARS-CoV- 2 variants. Science 373, 1372-1377 (2021).

30. Zhang, L. et al. Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability. NPJ Vaccines 8, 156 (2023).

31. Pittman, M. et al. Membrane ruffling is a mechanosensor of extracellular fluid viscosity. Nat. Phys. 18, 1112-1121 (2022).

32. Ryter, A. Relationship between ultrastructure and specific functions of macrophages. Comp. Immunol. Microbiol. Infect. Dis. 8, 119-133 (1985).

33. Hu, Y. et al. Size-controlled and shelf-stable DNA particles for production of lentiviral vectors. Nano Lett. 21, 5697-5705 (2021). 34. Rui, Y. et al. High-throughput and high-content bioassay enables tuning of polyester nanoparticles for cellular uptake, endosomal escape, and systemic in vivo delivery of mRNA. Sci. Adv. 8, eabk2855 (2022).

35. Rennick, J. J., Johnston, A. P. R. & Parton, R. G. Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics. Nat. NanotechnoL 16, 266-276 (2021).

36. Zhu, Y. et al. Albumin-biomineralized nanoparticles to synergize phototherapy and immunotherapy against melanoma. J. Control. Rel. 322, 300-311 (2020).

37. Nyamay’Antu, A., Kedinger, V. & Erbacher, P. Simplifying the efficient clinical -grade production of viruses. Genet. Eng. Biotechnol. News 37, 24-25 (2017).

38. Meng, M. & Wu, Y.-C. Combination of AAV-CCL19 and GPC3 CAR-T cells in the Treatment of hepatocellular carcinoma. J. Immunol. Res. 2021, 1782728 (2021).

39. M090, P. D., Farnos, O., Sharon, D. & Kamen, A. A. Targeted delivery of chimeric antigen receptor into T cells via CRISPR-mediated homology-directed repair with a dual-AAV6 transduction system. Curr. Issues Mol. Biol. 45, 7705-7720 (2023).

40. Sawaisom, P. et al. Comparison of the efficacy of second and third generation lentiviral vector transduced CAR CD 19 T cells for use in the treatment of acute lymphoblastic leukemia both in vitro and in vivo models. PLoS One 18, e0281735 (2023).

41. Yang, P. et al. CD24 is a novel target of chimeric antigen receptor T cells for the treatment of triple negative breast cancer. Cancer Immunol. Immunother. 'll, 3191-3202 (2023).

42. Zhou, J.-E. et al. Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo. J. Control. Rel. 350, 298-307 (2022).

43. Billingsley, M. M. et al. Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering. Nano Lett. 20, 1578-1589 (2020).

44. Gray, S. J. et al. Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration. Curr. Protoc. Neurosci. Chapter 4, Unit 4.17 (2011). 45. Edwards, D. A., Gooch, K. J., Zhang, I., McKinley, G. H. & Langer, R. The nucleation of receptor-mediated endocytosis. Proc. Natl. Acad. Sci. USA 93, 1786-1791 (1996).

46. Muto, S., Ohtaka, A., Nemoto, J., Kawakami, K. & Asano, Y. Effects of hyperosmolality on Na, K-ATPase gene expression in vascular smooth muscle cells. J. Membr. Biol. 162, 233-245 (1998).

47. Yue, Y. & Wu, C. Progress and perspectives in developing polymeric vectors for in vitro gene delivery. Biomater. Sci. 1, 152-170 (2013).

48. Kanasty, R., Dorkin, J. R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967-977 (2013).

49. Dahlman, J. E. et al. Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics. Proc. Natl. Acad. Sci. 114, 2060-2065 (2017).

50. Da Silva Sanchez, A. J. el al. Universal barcoding predicts in vivo ApoE-independent lipid nanoparticle delivery. Nano Lett. 22, 4822-4830 (2022).

[000188] It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the invention. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A fluid cell culture medium comprising: a water-soluble thickening agent, wherein the cell culture medium has a viscosity in a range of at least about 0.8 cP.

2. The cell culture medium of claim 1 further comprising a cell-growth component.

3. The cell culture medium of claim 1 or 2 wherein the thickening agent is substantially biologically inert in a transfection process.

4. The cell culture medium of any one of claims 1 through 3 wherein the thickening agent comprises one or more compounds having a molecular weight of 800 Daltons or greater.

5. The cell culture medium of any one of claims 1 through 4 wherein the thickening agent comprises one or more polymers.

6. The cell culture medium of any one of claims 1 through 5, wherein the thickening agent comprises one or more water-soluble polymers selected from a group including a glycol ether polymer such as polyethylene glycol (PEG), a glycerol polymer such as polypropylene glycol), polyvinyl alcohol (PVA), poly(vinyl pyrrolidone) (PVP), carbomers, polyacrylamide, or a derivative thereof.

7. The cell culture medium of any one of claims 1 through 6 wherein the thickening agent comprises one or more water-soluble polymers, or their derivatives, or combinations thereof.

8. The cell culture medium of claim 7 wherein the one or more water-soluble polymers is polysaccharide or its derivatives.

9. The cell culture medium of claim 8 wherein the one or more water-soluble polysaccharides are selected from a group including a methyl cellulose polymer, carboxymethylcellulose (CM? •, hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC), xanthan gum, guar gum, a dextran, dextran sulfate, alginate, hyaluronic acid, chondroitin, chondroitin sulfate, or a derivative thereof.

10. The cell culture medium of any one of claims 1 through 9, wherein the cell culture medium comprises an amount of about 0.1 to 60 wt% of the thickening agent based on the total weight of the cell culture medium.

11. The cell culture medium of any one of claims 1 to 10, wherein the cell culture medium has a viscosity in a range of about 0.8 to 15 cP.

12. The cell culture medium of any one of claims 1 to 10, wherein the cell culture medium has a viscosity in a range of about 0.8 to 10 cP.

13. The cell culture medium of any one of claims 1 to 10, wherein the cell culture medium has a viscosity in a range of about 2 to 10 cP.

14. The cell culture medium of any one of claims 1 to 10, wherein the cell culture medium has a viscosity in a range of about 2 to 6 cP.

15. The cell culture medium of any one of claims 1 to 10, wherein the cell culture medium has a viscosity in a range of about 2 to 5 cP.

16. The cell culture medium of any one of claims 1 to 15, further comprising a cell transfection agent.

17. The cell culture medium of 16, wherein the cell transfection agent comprises polyethyleneimine (PEI) and its derivatives, poly(beta-amino ester) (PBAE) and its derivatives, lipid nanoparticles (LNPs), and adeno associate virus (AAV).

18. The cell culture medium of any one of claims 1 to 17, further comprising a material to be transfected to a cell.

19. The cell culture medium of claim 18, wherein the material to be transfected comprises a virus, a vector, a nucleic acid, an oligonucleotide, or a combination thereof.

20. The cell culture medium of claim 18 or 19, wherein the material to be transfected comprises microRNA, siRNA, mRNA, viral RNA, RNA oligo, DNA, ribozyme, or aptamer.

21. The cell culture medium of any one of claims 18 through 20, wherein the material to be transfected comprises a polymer, a lipid nanoparticle or liposome.

22. The cell culture medium of any one of claims 18 through 21 wherein the material to be transfected comprises a therapeutic agent or can provide a therapeutic agent.

23. A composition for producing a transfected cell comprising: a cell; and a cell culture medium of any one of claims 1 to 22.

24. The composition of claim 23, wherein the cell is a mammalian cell.

25. The composition of claim 23, wherein the cell is a T-cell, an embryonic cell, or a stem cell.

26. A method of producing a transfected cell, comprising: incubating a cell with a cell culture medium of any one of claims 1 to 22.

27. The method of claim 26 wherein the method is performed in vitro.

28. The method of claim 26 wherein the method is performed in vivo.

29. A pharmaceutical composition comprising a water-soluble thickening agent that provides a viscosity in a range of at least about 0.8 cP.

30. The pharmaceutical composition of claim 29 further comprises a material to be transfected to a cell.

31. The pharmaceutical composition of claim 30 wherein the material to be transfected comprises a virus, a vector, a nucleic acid, an oligonucleotide, or a combination thereof.

32. The pharmaceutical composition of claim 30 or 31 wherein the material to be transfected comprises microRNA, siRNA, mRNA, viral RNA, RNA oligo, DNA, ribozyme, or aptamer.

33. The pharmaceutical composition of any one of claims 30 through 32, wherein the material to be transfected comprises a polymer, a lipid nanoparticle or liposome.

34. The pharmaceutical composition of any one of claims 30 through 33 wherein the material to be transfected comprises a therapeutic agent or can provide a therapeutic agent.