Compositions for maintaining lentiviral vectors and uses thereof

Abstract

The present disclosure provides improved aqueous formulations for storing viral vectors and methods of making and using the same.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 346,001, filed on May 26, 2022. The entire contents of the foregoing application are expressly incorporated herein by reference. Technical Field

[0003] The present disclosure relates to viral vector compositions having improved stability at room temperature, refrigerated and frozen temperatures. More specifically, the present disclosure relates to improved aqueous and frozen liquid viral vector compositions that exhibit high titer recovery, particle integrity and potency after storage under various conditions. Background Art

[0004] Viral vectors have emerged as a significant way to deliver therapeutic cargo to target cells. Such vectors include systems derived from adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV) and lentivirus. These vectors can introduce and / or integrate a gene of interest (e.g., a therapeutic gene) into the genome of a target cell. Therefore, target cells can be edited in theory to express any gene of interest, which can also have therapeutic benefits. Such therapies have entered the clinic (see, for example, Contemporary Gene Therapy (Curr Gene Ther.) 2015; 15 (1): 64-81 and Blood Review (Blood Rev.) January 21, 2022; 100929), and represent a revolutionary approach to treating certain diseases.

[0005] One factor limiting the use of viral vectors in clinical settings (including lentiviruses) is low stability, aggregation, and loss of viral titer after storage at temperatures above and below freezing (see, e.g., Kumru et al., J Pharm Sci. 2018 November; 107(11): 2764-2774). In fact, viral vectors can be subjected to a wide variety of temperatures during preparation, storage, and final research or commercial use. Viral vectors can be subjected to physiological temperatures (e.g., 25°C and / or 37°C) during preparation and transduction, cooler temperatures (e.g., 2-8°C) during purification and short-term storage, and freezing temperatures (e.g., 0°C or lower) during long-term storage. In addition, viral vectors can undergo several freeze-thaw cycles during their service life and storage.

[0006] Therefore, there remains a need for improved compositions for long-term storage of viral vectors. Summary of the invention

[0007]

[0013] The present disclosure generally relates, in part, to improved compositions for storing viral vectors, including but not limited to retroviral vectors or lentiviral vectors.

[0008] In one aspect, an aqueous viral composition is provided, comprising a viral vector, a HEPES or L-histidine buffer, a carbohydrate, and an amino acid.

[0009] In another aspect, an aqueous viral composition is provided, wherein the composition comprises a viral vector; a HEPES buffer; a carbohydrate; a salt; and a poloxamer.

[0010] In various embodiments, the buffer is present at a concentration of about 25 mM to about 30 mM, about 26 mM to about 29 mM, about 27 mM to about 28 mM, or about 27.5 mM. In specific embodiments, the buffer is a HEPES buffer. In specific embodiments, the buffer is an L-histidine buffer.

[0011] In various embodiments, the carbohydrate is present at a concentration of about 66mM to about 80mM, about 67mM to about 79mM, about 68mM to about 78mM, about 69mM to about 77mM, about 70mM to about 76mM, about 71mM to about 75mM, about 72mM to about 74mM, or about 73mM. In various embodiments, the carbohydrate is present at a concentration of about 2.0% to about 3.0% by weight per volume of the composition, about 2.1% to about 2.9% by weight per volume of the composition, about 2.2% to about 2.8% by weight per volume of the composition, about 2.3% to about 2.7% by weight per volume of the composition, about 2.4% to about 2.6% by weight per volume of the composition, or about 2.5% by weight per volume of the composition. In various embodiments, the carbohydrate is a disaccharide. In various embodiments, the carbohydrate is lactose, glucose, mannose, mannitol, sorbitol, sucrose, trehalose and / or glycerol. In some embodiments, the carbohydrate is sucrose and / or trehalose. In specific embodiments, the carbohydrate is sucrose. In specific embodiments, the carbohydrate is trehalose.

[0012] In various embodiments, the amino acid is present at a concentration of about 40mM to about 60mM, about 41mM to about 59mM, about 42mM to about 58mM, about 43mM to about 57mM, about 44mM to about 56mM, about 45mM to about 55mM, about 46mM to about 54mM, about 47mM to about 53mM, about 48mM to about 52mM, about 49mM to about 51mM, or about 50mM. In some embodiments, the amino acid is a non-polar amino acid. In some embodiments, the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, methionine, isoleucine phenylalanine, tyrosine, and tryptophan. In some embodiments, the amino acid is selected from the group consisting of phenylalanine, tyrosine, tryptophan, and proline. In a specific embodiment, the amino acid is L-proline.

[0013] In various embodiments, the composition further comprises a salt. In certain embodiments, the salt is present at a concentration of about 65mM to about 85mM, about 66mM to about 84mM, about 67mM to about 83mM, about 68mM to about 82mM, about 69mM to about 81mM, about 70mM to about 80mM, about 71mM to about 79mM, about 72mM to about 78mM, about 73mM to about 77mM, about 74mM to about 76mM, about 75mM. In certain embodiments, the salt is a chloride salt, KCl or NaCl. In a specific embodiment, the salt is NaCl.

[0014] In various embodiments, the composition further comprises a poloxamer. In some embodiments, the poloxamer is present at a concentration of about 0.01 mg / ml to about 1 mg / ml, about 0.02 mg / ml to about 0.9 mg / ml, about 0.03 mg / ml to about 0.8 mg / ml, about 0.04 mg / ml to about 0.7 mg / ml, about 0.05 mg / ml to about 0.6 mg / ml, about 0.06 mg / ml to about 0.5 mg / ml, about 0.07 mg / ml to about 0.4 mg / ml, about 0.08 mg / ml to about 0.3 mg / ml, about 0.09 mg / ml to about 0.2 mg / ml, about 0.1 mg / ml to about 0.8 mg / ml, about 0.1 mg / ml to about 0.5 mg / ml, or about 0.2 mg / ml to about 0.4 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.1 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.3 mg / ml. In some embodiments, the poloxamer is poloxamer 188, poloxamer 288, poloxamer 335, poloxamer 338, or poloxamer 407. In specific embodiments, the poloxamer is poloxamer 188 (P188).

[0015] In various embodiments, the composition comprises a pH of about 6.5 to about 8. In some embodiments, the composition comprises a pH of about 6.5. In some embodiments, the composition comprises a pH of about 7. In some embodiments, the composition comprises a pH of about 7.5. In some embodiments, the composition comprises a pH of about 8.

[0016] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; and about 50 mM L-proline; wherein the composition comprises a pH of about 7.

[0017] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM L-histidine; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; and about 50 mM L-proline; wherein the composition comprises a pH of about 7.

[0018] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; about 50 mM L-proline; and about 0.1 to about 0.8 mg / mL poloxamer 188; wherein the composition comprises a pH of about 7.

[0019] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; about 50 mM L-proline; and about 0.3 mg / mL poloxamer 188; wherein the composition comprises a pH of about 7.

[0020] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; about 50 mM L-proline; and about 75 mM NaCl; wherein the composition comprises a pH of about 7.

[0021] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; about 50 mM L-proline; about 75 mM NaCl; and about 0.1 to about 0.8 mg / mL poloxamer 188; wherein the composition comprises a pH of about 7.

[0022] In various embodiments, the composition further comprises a salt. In some embodiments, the salt is present at a concentration of about 65mM to about 85mM, about 66mM to about 84mM, about 67mM to about 83mM, about 68mM to about 82mM, about 69mM to about 81mM, about 70mM to about 80mM, about 71mM to about 79mM, about 72mM to about 78mM, about 73mM to about 77mM, about 74mM to about 76mM, about 75mM. In a specific embodiment, the salt is NaCl.

[0023] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 2.5 wt % sucrose per volume of the composition; about 75 mM NaCl; and about 0.1 to about 0.8 mg / ml poloxamer 188 or about 0.01 wt % to about 0.08 wt % poloxamer 188 per volume of the composition; wherein the composition comprises a pH of about 7.

[0024] In any of the aspects and embodiments contemplated herein, the viral vector is present in an amount of about 1×10 8 About 2x 10 9 In various embodiments, the viral vector is present at a titer of about 1 x 10 8 TU / ml, about 2x10 8 TU / ml, about 3x 10 8 TU / ml, about 4x 10 8 TU / ml, about 5x 10 8 TU / ml, about 6x 10 8 TU / ml, about 7x 10 8 TU / ml, about 8x 10 8 TU / ml, about 9x 10 8 TU / ml, about 1x 10 9 TU / ml or about 2x 10 9 Titers of TU / ml are present.

[0025] In various embodiments, the vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, the lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1) or human immunodeficiency virus 2 (HIV-2). In specific embodiments, the lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1).

[0026] In any aspect and embodiment of the aspects and embodiments contemplated herein, the viral vector is pseudotyped. In various embodiments, the viral vector is pseudotyped with an envelope protein from a strain of vesicular stomatitis virus. In some embodiments, the strain of vesicular stomatitis virus is selected from the group consisting of Indiana, Alagoas, New Jersey, Isfahan, CoCal, Maraba, or Piry. In a specific embodiment, the viral vector is pseudotyped with a vesicular stomatitis virus G (VSV-G) protein. In some embodiments, the viral vector is pseudotyped with an envelope derived from: Measles envelope protein, Sindbis envelope protein, Morbillivirus F and H proteins, Sendai F and HN proteins, or Paramyxoviridae F and H proteins.

[0027] In any of the aspects and embodiments contemplated herein, the viral vector comprises a polynucleotide comprising a transgene. In some embodiments, the transgene encodes a therapeutic protein. In some embodiments, the transgene or therapeutic protein is a chimeric antigen receptor (CAR), a chimeric co-stimulatory receptor (CCR), an αβT cell receptor (αβ-TCR), a γδT cell receptor (γδ-TCR), a dimerizing agent-regulated immune receptor complex (DARIC) or a switch receptor. In some embodiments, the transgene or therapeutic protein is used to treat a monogenic disease, disorder or condition. In some embodiments, the transgene or therapeutic protein is a therapeutic globin for treating hemoglobinopathies or an ABCD1 gene for treating CALD.

[0028] In specific embodiments, the compositions contemplated herein do not comprise serum, human serum albumin (HSA), PIPES, sodium citrate, sodium phosphate, and / or Tris. In some embodiments, the compositions contemplated herein do not comprise salt. In some embodiments, the compositions contemplated herein do not comprise NaCl. In some embodiments, the compositions contemplated herein do not comprise KCl. In some embodiments, the compositions contemplated herein do not comprise trehalose.

[0029] In any of the aspects and embodiments contemplated herein, the viral vector maintains an infectious titer recovery rate of greater than about 75%, about 80%, about 85%, about 90% or about 95% in HOS cells after storage at 25°C for 24 hours, storage at 2-8°C for 48 hours, storage at 2-8°C for 168 hours and / or at least 1 freeze-thaw cycle relative to the infectious titer of the viral vector in the composition before storage or at least one freeze-thaw cycle.

[0030] In any of the aspects and embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 56° to about 62°C as measured by differential scanning fluorimetry (DSF).

[0031] In any of the aspects and embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 58° to about 60°C as measured by differential scanning fluorimetry (DSF).

[0032] In any of the aspects and embodiments contemplated herein, the viral vector maintains a hydrodynamic diameter of about 150 nm to about 170 nm relative to the hydrodynamic diameter of the viral vector in the composition prior to storage or at least one freeze-thaw cycle, as measured by dynamic light scattering (DLS) at 25°C and a viscosity value of 0.967 centipoise (cP).

[0033] In any of the aspects and embodiments contemplated herein, the viral vector maintains at least 78%, at least 80%, at least 85%, at least 90% or at least 95% potency after storage as compared to a reference standard as measured by transgene expression in PBMCs.

[0034] In any of the aspects and embodiments contemplated herein, the composition has no visible fiber particles (bundled fibers) after storage. In any of the aspects and embodiments contemplated herein, the composition has 5 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has 4 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has 3 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has 2 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has 1 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has no visible particles after storage.

[0035] In various embodiments, the storage is at 25° C., 2-8° C., or 37° C. In some embodiments, the storage is for 24 hours, 48 ​​hours, or 72, 96, 120, 144, or 168 hours or more.

[0036] In various embodiments, the storage comprises one or more freeze-thaw cycles. In some embodiments, the one or more freeze-thaw cycles are 1, 2, 3, 4 or 5 freeze-thaw cycles. In some embodiments, the one or more freeze-thaw cycles comprise freezing the composition at about -65° or lower for about 1.5 hours or longer, and thawing at 30°C for 1.5 hours.

[0037] In any of the aspects and embodiments contemplated herein, the composition is frozen.

[0038] In another aspect, a method for storing a viral vector is provided, the method comprising providing a viral vector, contacting the viral vector with any of the compositions contemplated herein, and storing the viral composition at about 25°C or less.

[0039] In another aspect, a method for storing a viral vector is provided, the method comprising providing a viral vector, contacting the viral vector with any of the compositions contemplated herein, and storing the viral composition at about 2-8°C or lower for at least about 24 hours.

[0040] In another aspect, a method for cryopreserving a viral vector is provided, the method comprising providing a viral vector, contacting the viral vector with any of the compositions contemplated herein, freezing the viral composition, and storing the viral composition at about 0°C or below.

[0041] In various embodiments, the methods contemplated herein comprise storing the viral composition for at least about 24 hours, 48 ​​hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 148 hours, or at least about 168 hours.

[0042] In another aspect, a method of expressing a transgene in a cell is provided, the method comprising contacting the cell with any of the compositions contemplated herein. In various embodiments, the cell is a mammalian cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a hematopoietic stem cell or progenitor cell. In some embodiments, the cell is a human CD34+ hematopoietic cell or progenitor cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is an αβ T cell. In some embodiments, the cell is a γδ T cell. In some embodiments, the cell is a CD3 + 、CD4 + and / or CD8 + Cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 A diagram showing the screening process / steps and formulation components tested during the screening process.

[0044] Figure 2 A diagram of the process flow chart, test conditions, and quality assessment techniques / assays are shown.

[0045] Figure 3 Shown are the infectious titers and percent recovery (%) of various lentiviral vector compositions after storage at 25°C for 24 hours or at 2-8°C for 48 hours.

[0046] Figure 4 Shown are the hydrodynamic sizes of lentiviral vector particles suspended in various compositions after storage at 37°C for 48 hours, at 25°C for 24 hours, or at 2-8°C for 48 hours.

[0047] Figure 5 Thermal development of lentiviral vector compositions in the range of 20-90°C is shown.

[0048] Figure 6 Shown are the infectious titers and percent recovery (%) of various lentiviral vector compositions after storage at 25°C for 24 hours or at 2-8°C for 48 hours.

[0049] Figure 7 Shown are the hydrodynamic sizes of lentiviral vector particles suspended in various compositions after storage at 37°C for 48 hours, at 25°C for 24 hours, or at 2-8°C for 48 hours.

[0050] Figure 8 Thermal development of lentiviral vector compositions in the range of 20-90°C is shown.

[0051] Fig. 9 Shown are the infectious titers and percent recovery (%) of various lentiviral vector compositions after storage at 25°C for 24 hours or at 2-8°C for 48 hours.

[0052] Fig.10 Shown are the infectious titers and percent recovery (%) of various lentiviral vector compositions after storage at 25°C for 24 hours or at 2-8°C for 48 hours.

[0053] Fig.11 Shown are the infectious titers and percent recovery (%) of various lentiviral vector compositions after 5 freeze-thaw cycles.

[0054] Fig.12 Shown are the hydrodynamic sizes of lentiviral vector particles suspended in various compositions after storage at 37°C for 48 hours, at 25°C for 24 hours, or at 2-8°C for 48 hours.

[0055] Fig.13 Shown are the hydrodynamic sizes of lentiviral vector particles suspended in various compositions after 5 freeze-thaw cycles.

[0056] Fig.14 Shown are the infectious titers and percent recovery (%) of various HEPES lentiviral vector compositions after storage at 25°C for 24 hours or at 2-8°C for 168 hours.

[0057] Fig.15Shown are the hydrodynamic sizes of lentiviral vector particles suspended in various HEPES compositions after storage at 2-8°C for 168 hours.

[0058] Fig.16 Shown are visible particle assessments of various HEPES lentiviral compositions after storage at 2-8°C for 48, 96 or 168 hours.

[0059] Fig.17 Shown are the infectious titers and percent recovery (%) of various HEPES lentiviral vector compositions after 5 freeze-thaw cycles.

[0060] Fig.18 Shown are the hydrodynamic sizes of lentiviral vector particles suspended in various HEPES compositions after 5 freeze-thaw cycles.

[0061] Fig.19 Shown are visible particle assessments of various HEPES lentiviral compositions after 5 freeze-thaw cycles.

[0062] Fig. 20 Shown are the infection titers and percent recovery rates (%) of various L-histidine lentiviral vector compositions after storage at 25°C for 24 hours or at 2-8°C for 168 hours.

[0063] Fig.21 Shown are the hydrodynamic sizes of lentiviral vector particles suspended in various L-histidine compositions after storage at 2-8°C for 168 hours.

[0064] Fig. 22 Shown are visible particle evaluations of various L-histidine lentiviral compositions after storage at 2-8°C for 48, 96 or 168 hours.

[0065] Fig.23 Shown are the infectious titers and recovery percentages (%) of various L-histidine lentiviral vector compositions after 5 freeze-thaw cycles.

[0066] Fig.24 Shown are the hydrodynamic sizes of lentiviral vector particles suspended in various L-histidine compositions after 5 freeze-thaw cycles.

[0067] Fig.25 Shown are visible particle evaluations of various L-histidine lentiviral compositions after 5 freeze-thaw cycles.

[0068] Fig.26 Shown are the intermediate bulk titer recovery rates of different lentiviral vectors after storage at 2-8°C for 24, 120 or 168 hours.

[0069] Fig. 27Shown are visible particle assessments of various lentiviral compositions with and without P188 after preparation.

[0070] Fig.28 Visible particle formation of lentiviral compositions with and without P188 is shown.

[0071] Fig.29 Shown are the recovery rates and infectious titers of different batches of lentiviral vectors formulated with and without P188 after storage at 2-8°C for 24, 120 or 168 hours.

[0072] Fig.30 Shown are the infectious titers of lentiviral compositions with and without P188 after storage for 6 months at ≤ -65°C.

[0073] Fig.31 Relative potency evaluation of lentiviral compositions is shown. DETAILED DESCRIPTION

[0074] A. Overview

[0075] The present disclosure relates generally, in part, to viral vector (e.g., lentiviral vector) compositions that exhibit high titer recovery, thermal stability, particle integrity, no to low visible particle formation, and potency after storage under various conditions. Without wishing to be bound by any particular theory, many biological formulations (including viruses) lose protein structure and activity during the preparation process and storage if an optimal solution or formulation has not yet been determined. When subjected to similar preparation and storage stresses in various solutions, viral vectors also lose their ability to effectively transduce cells and structural changes.

[0076] Therefore, formulations are contemplated herein that surprisingly minimize viral vector loss and maintain viral vector integrity and activity under conditions known to affect viral integrity. Specifically, formulations contemplated herein exhibit high titer recovery, particle integrity, thermal stability, no visible particle formation to low visible particle formation (e.g., only 1, 2 or 3 visible particles) and efficacy after storage under various conditions including storage at 37 °, 25 ° C, 2-8 ° C and / or after one or more freeze-thaw cycles. Storage, cryopreservation and transduction methods are also contemplated.

[0077] Techniques for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification, and related techniques and procedures can be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology as cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, Volumes I and II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober (John Wiley & Sons, New York, NY, 2001); Real-Time PCR: Current Technology and Applications, edited by Julie Logan, Kirstin Edwards, and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes (Academic Press, NY, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, NY, 1991); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (N. Gait, ed., 1984). The Hybridization (B.Hames and S.Higgins, 1985); Transcription and Translation (B.Hames and S.Higgins, 1984); Animal Cell Culture (R.Freshney, ed., 1986; Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, ed., 3rd ed., 2010 Humana Press); Immobilized Cells and Enzymes (IRL Press, 1986); Methods in Enzymology (Academic Press, Inc., New York, NY); Gene Transfer Vectors for Mammalian Cells (Gene Transfer Vectors for Mammalian Cells). =Cells (JH Miller and MP Calos, eds., 1987, Cold Spring Harbor Laboratory Press); Harlow and Lane, Antibodies (Cold Spring Harbor, NY, 1998); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th ed. (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Current Protocols in Immunology, 1996). Immunology" (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0078] B. Definition

[0079] Before describing the present disclosure in greater detail, it may be helpful to understand the present disclosure by providing definitions of certain terms to be used herein.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any method and material similar to or equivalent to the method and material described herein may be used to practice or test specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For purposes of this disclosure, the following terms are defined hereinafter.

[0081] The articles "a," "an," and "the" are used herein to refer to one or more than one (ie, at least one, or one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0082] The use of alternatives (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0083] The term "and / or" should be understood to mean one or both of the alternatives.

[0084] As used herein, the term "about" or "approximately" refers to an amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of an amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length relative to a reference amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length. For example, a composition having a pH of "about 7" means that the composition has a pH of 7±1%-15% (e.g., ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%).

[0085] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5 refers to every value encompassed within the range. 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0086] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of the stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or facilitate the activities or actions specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that no other elements are present that materially affect the activities or actions of the listed elements.

[0087] References throughout this specification to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "another embodiment," or "another embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the above phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for excluding the feature in a specific embodiment.

[0088] As used herein, the term "buffer" refers to a mixture of a weak acid and its conjugated base or a weak base and its conjugated acid.

[0089] As used herein, the term "freeze-thaw" or "freeze-thaw cycle" refers to exposing a liquid mixture, such as an aqueous solution or suspension, to a temperature at or below its freezing point until the mixture is frozen, and then thawing the mixture at a temperature greater than its freezing point. The freezing step can be performed, for example, by placing the mixture in an environment where the temperature is about 0°C to about -80°C. In some embodiments, the freezing temperature is about -20°C to about -80°C. In some embodiments, the freezing temperature is about -65°C. The frozen aqueous solution or suspension can be stored for a period of one or more hours, one or more days, one or more weeks, one or more months, or one or more years before thawing. The thawing of a solution or suspension can be performed by exposing the solution or suspension to a condition where the temperature is about 2°C to about 8°C or by storing the mixture at room temperature (e.g., about 25°C). In some embodiments, the solution or suspension is thawed at 30°C. In some embodiments, the solution or suspension is thawed at 37°C.

[0090] As used herein, the term "percent by weight per volume" or "% w / v" means the weight percentage (in grams) of a single component relative to the total volume of the mixture containing the component. For example, 500 mg of a component in a total volume of 8 ml is 6.25% w / v, and 500 mg of a component in a total volume of 5 ml is 10% w / v.

[0091] As used herein, the term "titer" or "viral titer" refers to the number of infectious vector particles or "transduction units" that produce transgene products in target cells. Viral titer can be measured by functional assays such as Xiao et al., Exp. Neurobiol. 144: 113-124, 1997; Fisher et al., J. Viral. 70: 520-532, 1996; and GENE Therapy (2002), 9, 1155-1162; each of which is incorporated herein by reference in its entirety. Alternatively, viral titer can be measured by determining the amount of viral DNA that has been integrated into the host cell genome, for example, using polymerase chain reaction (PCR) techniques known in the art.

[0092] Additional definitions are set forth throughout this disclosure.

[0093] C. Preparation

[0094] Disclosed herein are formulations that surprisingly minimize viral vector loss and maintain activity under conditions known to affect viral integrity. The formulations contemplated herein comprise at least a buffer and a carbohydrate.

[0095] In certain embodiments, the buffer is N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer. The chemical formula of HEPES is C8H 18 N2O4S, and its molecular weight is 238.3012 g / mol. The IUPAC ID of HEPES is 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid.

[0096] In various embodiments, the buffer is an L-histidine buffer. L-histidine is the L-enantiomer of the amino acid histidine. The chemical formula of L-histidine is C6H9N3O2, and its molecular weight is 155.157 g / mol.

[0097] In some embodiments, the buffer is present at a concentration of about 25 mM to about 30 mM. In some embodiments, the buffer is present at a concentration of about 26 mM to about 29 mM. In some embodiments, the buffer is present at a concentration of about 27 mM to about 28 mM.

[0098] In some embodiments, the buffer is present at a concentration of about 25mM. In some embodiments, the buffer is present at a concentration of about 25.5mM. In some embodiments, the buffer is present at a concentration of about 26mM. In some embodiments, the buffer is present at a concentration of about 26.5mM. In some embodiments, the buffer is present at a concentration of about 27mM. In some embodiments, the buffer is present at a concentration of about 27.5mM. In certain embodiments, the buffer is present at a concentration of 27.5mM. In some embodiments, the buffer is present at a concentration of about 28mM. In some embodiments, the buffer is present at a concentration of about 28.5mM. In some embodiments, the buffer is present at a concentration of about 29mM. In some embodiments, the buffer is present at a concentration of about 29.5mM. In some embodiments, the buffer is present at a concentration of about 30mM.

[0099] In various embodiments, the carbohydrate is present at a concentration of about 66mM to about 80mM. In some embodiments, the carbohydrate is present at a concentration of about 67mM to about 79mM. In some embodiments, the carbohydrate is present at a concentration of about 68mM to about 78mM. In some embodiments, the carbohydrate is present at a concentration of about 69mM to about 77mM. In some embodiments, the carbohydrate is present at a concentration of about 70mM to about 76mM. In some embodiments, the carbohydrate is present at a concentration of about 71mM to about 75mM. In some embodiments, the carbohydrate is present at a concentration of about 72mM to about 74mM.

[0100] In some embodiments, the carbohydrate is present at a concentration of about 66mM. In some embodiments, the carbohydrate is present at a concentration of about 67mM. In some embodiments, the carbohydrate is present at a concentration of about 68mM. In some embodiments, the carbohydrate is present at a concentration of about 69mM. In some embodiments, the carbohydrate is present at a concentration of about 70mM. In some embodiments, the carbohydrate is present at a concentration of about 71mM. In some embodiments, the carbohydrate is present at a concentration of about 72mM. In some embodiments, the carbohydrate is present at a concentration of about 73mM. In certain embodiments, the carbohydrate is present at a concentration of 73mM. In some embodiments, the carbohydrate is present at a concentration of about 74mM. In some embodiments, the carbohydrate is present at a concentration of about 75mM. In some embodiments, the carbohydrate is present at a concentration of about 76mM. In some embodiments, the carbohydrate is present at a concentration of about 77mM. In some embodiments, the carbohydrate is present at a concentration of about 78mM. In some embodiments, the carbohydrate is present at a concentration of about 79mM. In some embodiments, the carbohydrate is present at a concentration of about 80mM.

[0101] In various embodiments, the carbohydrate is present at a concentration of about 15mM to about 45mM. In some embodiments, the carbohydrate is present at a concentration of about 20mM to about 40mM. In some embodiments, the carbohydrate is present at a concentration of about 25mM to about 35mM. In some embodiments, the carbohydrate is present at a concentration of about 25mM to about 34mM. In some embodiments, the carbohydrate is present at a concentration of about 25mM to about 33mM. In some embodiments, the carbohydrate is present at a concentration of about 26mM to about 32mM. In some embodiments, the carbohydrate is present at a concentration of about 27mM to about 31mM. In some embodiments, the carbohydrate is present at a concentration of about 28mM to about 30mM.

[0102] In some embodiments, the carbohydrate is present at a concentration of about 15mM. In some embodiments, the carbohydrate is present at a concentration of about 16mM. In some embodiments, the carbohydrate is present at a concentration of about 17mM. In some embodiments, the carbohydrate is present at a concentration of about 18mM. In some embodiments, the carbohydrate is present at a concentration of about 19mM. In some embodiments, the carbohydrate is present at a concentration of about 20mM. In some embodiments, the carbohydrate is present at a concentration of about 21mM. In some embodiments, the carbohydrate is present at a concentration of about 22mM. In some embodiments, the carbohydrate is present at a concentration of about 23mM. In some embodiments, the carbohydrate is present at a concentration of about 24mM. In some embodiments, the carbohydrate is present at a concentration of about 25mM. In some embodiments, the carbohydrate is present at a concentration of about 26mM. In some embodiments, the carbohydrate is present at a concentration of about 27mM. In some embodiments, the carbohydrate is present at a concentration of about 28mM. In some embodiments, the carbohydrate is present at a concentration of about 29mM. In some embodiments, the carbohydrate is present at a concentration of about 30mM. In some embodiments, the carbohydrate is present at a concentration of about 31mM. In some embodiments, the carbohydrate is present at a concentration of about 32mM. In some embodiments, the carbohydrate is present at a concentration of about 33mM. In some embodiments, the carbohydrate is present at a concentration of about 34mM. In some embodiments, the carbohydrate is present at a concentration of about 35mM. In some embodiments, the carbohydrate is present at a concentration of about 36mM. In some embodiments, the carbohydrate is present at a concentration of about 37mM. In some embodiments, the carbohydrate is present at a concentration of about 38mM. In some embodiments, the carbohydrate is present at a concentration of about 39mM. In some embodiments, the carbohydrate is present at a concentration of about 40mM. In some embodiments, the carbohydrate is present at a concentration of about 41mM. In some embodiments, the carbohydrate is present at a concentration of about 42mM. In some embodiments, the carbohydrate is present at a concentration of about 43mM. In some embodiments, the carbohydrate is present at a concentration of about 44mM. In some embodiments, the carbohydrate is present at a concentration of about 45mM.

[0103] In various embodiments, the carbohydrate is present in a concentration of about 2.0% to about 3.0% by weight per volume of the composition. In some embodiments, the carbohydrate is present in a concentration of about 2.1% to about 2.9% by weight per volume of the composition. In some embodiments, the carbohydrate is present in a concentration of about 2.2% to about 2.8% by weight per volume of the composition. In some embodiments, the carbohydrate is present in a concentration of about 2.3% to about 2.7% by weight per volume of the composition. In some embodiments, the carbohydrate is present in a concentration of about 2.4% to about 2.6% by weight per volume of the composition. In some embodiments, the carbohydrate is present in a concentration of about 2.5% by weight per volume of the composition. In certain embodiments, the carbohydrate is present in a concentration of 2.5% by weight per volume of the composition.

[0104] In various embodiments, the carbohydrate is present at a concentration of about 0.5% to about 1.5% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.6% to about 1.4% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.7% to about 1.3% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.8% to about 1.2% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.9% to about 1.1% by weight per volume of the composition.

[0105] In some embodiments, the carbohydrate is present at a concentration of about 0.5% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.6% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.7% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.8% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.9% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.0%. In some embodiments, the carbohydrate is present at a concentration of about 1.1% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.2% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.3% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.4% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.5% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.6% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.7% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.8% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.9% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.1% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.2% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.3% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.4% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.5% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.6% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.7% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.8% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.9% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3% by weight per volume of the composition.

[0106] In various embodiments, the carbohydrate is present at a concentration of about 100mM to about 135mM. In some embodiments, the carbohydrate is present at a concentration of about 105mM to about 130mM. In some embodiments, the carbohydrate is present at a concentration of about 110mM to about 125mM. In some embodiments, the carbohydrate is present at a concentration of about 111mM to about 124mM. In some embodiments, the carbohydrate is present at a concentration of about 111mM to about 123mM. In some embodiments, the carbohydrate is present at a concentration of about 112mM to about 122mM. In some embodiments, the carbohydrate is present at a concentration of about 113mM to about 121mM. In some embodiments, the carbohydrate is present at a concentration of about 114mM to about 120mM. In some embodiments, the carbohydrate is present at a concentration of about 115mM to about 119mM. In some embodiments, the carbohydrate is present at a concentration of about 116mM to about 118mM.

[0107] In some embodiments, the carbohydrate is present at a concentration of about 100 mM. In some embodiments, the carbohydrate is present at a concentration of about 101 mM. In some embodiments, the carbohydrate is present at a concentration of about 102 mM. In some embodiments, the carbohydrate is present at a concentration of about 103 mM. In some embodiments, the carbohydrate is present at a concentration of about 104 mM. In some embodiments, the carbohydrate is present at a concentration of about 105 mM. In some embodiments, the carbohydrate is present at a concentration of about 106 mM. In some embodiments, the carbohydrate is present at a concentration of about 107 mM. In some embodiments, the carbohydrate is present at a concentration of about 108 mM. In some embodiments, the carbohydrate is present at a concentration of about 109 mM. In some embodiments, the carbohydrate is present at a concentration of about 110 mM. In some embodiments, the carbohydrate is present at a concentration of about 111 mM. In some embodiments, the carbohydrate is present at a concentration of about 112 mM. In some embodiments, the carbohydrate is present at a concentration of about 113 mM. In some embodiments, the carbohydrate is present at a concentration of about 114 mM. In some embodiments, the carbohydrate is present at a concentration of about 115 mM. In some embodiments, the carbohydrate is present at a concentration of about 116mM. In some embodiments, the carbohydrate is present at a concentration of about 117mM. In some embodiments, the carbohydrate is present at a concentration of about 118mM. In some embodiments, the carbohydrate is present at a concentration of about 119mM. In some embodiments, the carbohydrate is present at a concentration of about 120mM. In some embodiments, the carbohydrate is present at a concentration of about 121mM. In some embodiments, the carbohydrate is present at a concentration of about 122mM. In some embodiments, the carbohydrate is present at a concentration of about 123mM. In some embodiments, the carbohydrate is present at a concentration of about 124mM. In some embodiments, the carbohydrate is present at a concentration of about 125mM. In some embodiments, the carbohydrate is present at a concentration of about 126mM. In some embodiments, the carbohydrate is present at a concentration of about 127mM. In some embodiments, the carbohydrate is present at a concentration of about 128mM. In some embodiments, the carbohydrate is present at a concentration of about 129mM. In some embodiments, the carbohydrate is present at a concentration of about 130mM. In some embodiments, the carbohydrate is present at a concentration of about 131mM. In some embodiments, the carbohydrate is present at a concentration of about 132 mM. In some embodiments, the carbohydrate is present at a concentration of about 133 mM. In some embodiments, the carbohydrate is present at a concentration of about 134 mM.In some embodiments, the carbohydrate is present at a concentration of about 135mM. In some embodiments, the carbohydrate is present at a concentration of about 136mM. In some embodiments, the carbohydrate is present at a concentration of about 137mM. In some embodiments, the carbohydrate is present at a concentration of about 138mM. In some embodiments, the carbohydrate is present at a concentration of about 139mM. In some embodiments, the carbohydrate is present at a concentration of about 140mM. In some embodiments, the carbohydrate is present at a concentration of about 141mM. In some embodiments, the carbohydrate is present at a concentration of about 142mM. In some embodiments, the carbohydrate is present at a concentration of about 143mM. In some embodiments, the carbohydrate is present at a concentration of about 144mM. In some embodiments, the carbohydrate is present at a concentration of about 145mM. In some embodiments, the carbohydrate is present at a concentration of about 146mM. In some embodiments, the carbohydrate is present at a concentration of about 147mM. In some embodiments, the carbohydrate is present at a concentration of about 148mM. In some embodiments, the carbohydrate is present at a concentration of about 149mM. In some embodiments, the carbohydrate is present at a concentration of about 150mM.

[0108] In various embodiments, the carbohydrate is present at a concentration of about 265mM to about 285mM. In some embodiments, the carbohydrate is present at a concentration of about 266mM to about 284mM. In some embodiments, the carbohydrate is present at a concentration of about 267mM to about 283mM. In some embodiments, the carbohydrate is present at a concentration of about 268mM to about 282mM. In some embodiments, the carbohydrate is present at a concentration of about 269mM to about 281mM. In some embodiments, the carbohydrate is present at a concentration of about 270mM to about 280mM. In some embodiments, the carbohydrate is present at a concentration of about 271mM to about 279mM. In some embodiments, the carbohydrate is present at a concentration of about 272mM to about 278mM. In some embodiments, the carbohydrate is present at a concentration of about 273mM to about 277mM. In some embodiments, the carbohydrate is present at a concentration of about 274mM to about 276mM.

[0109] In some embodiments, the carbohydrate is present at a concentration of about 265mM. In some embodiments, the carbohydrate is present at a concentration of about 266mM. In some embodiments, the carbohydrate is present at a concentration of about 267mM. In some embodiments, the carbohydrate is present at a concentration of about 268mM. In some embodiments, the carbohydrate is present at a concentration of about 269mM. In some embodiments, the carbohydrate is present at a concentration of about 270mM. In some embodiments, the carbohydrate is present at a concentration of about 271mM. In some embodiments, the carbohydrate is present at a concentration of about 272mM. In some embodiments, the carbohydrate is present at a concentration of about 273mM. In some embodiments, the carbohydrate is present at a concentration of about 274mM. In some embodiments, the carbohydrate is present at a concentration of about 275mM. In some embodiments, the carbohydrate is present at a concentration of about 276mM. In some embodiments, the carbohydrate is present at a concentration of about 277mM. In some embodiments, the carbohydrate is present at a concentration of about 278mM. In some embodiments, the carbohydrate is present at a concentration of about 279mM. In some embodiments, the carbohydrate is present at a concentration of about 280mM. In some embodiments, the carbohydrate is present at a concentration of about 281 mM. In some embodiments, the carbohydrate is present at a concentration of about 282 mM. In some embodiments, the carbohydrate is present at a concentration of about 283 mM. In some embodiments, the carbohydrate is present at a concentration of about 284 mM. In some embodiments, the carbohydrate is present at a concentration of about 285 mM.

[0110] In various embodiments, the carbohydrate is present at a concentration of about 3.5% to about 4.5% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.6% to about 4.4% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.7% to about 4.3% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.8% to about 4.2% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.9% to about 4.1% by weight per volume of the composition.

[0111] In some embodiments, the carbohydrate is present at a concentration of about 3.5% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.6% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.7% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.8% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.9% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.0%. In some embodiments, the carbohydrate is present at a concentration of about 4.1% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.2% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.3% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.4% by weight per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.5% by weight per volume of the composition.

[0112] In specific embodiments, the carbohydrate is a disaccharide. In various embodiments, the carbohydrate is lactose, glucose, mannose, mannitol, sorbitol, sucrose, trehalose, inulin and / or glycerol. In some embodiments, the carbohydrate is sucrose and / or trehalose. In specific embodiments, the carbohydrate is sucrose. In specific embodiments, the carbohydrate is trehalose.

[0113] In various embodiments, the composition comprises an amino acid. In some embodiments, the amino acid is present at a concentration of about 40mM to about 60mM. In some embodiments, the amino acid is present at a concentration of about 41mM to about 59mM. In some embodiments, the amino acid is present at a concentration of about 42mM to about 58mM. In some embodiments, the amino acid is present at a concentration of about 43mM to about 57mM. In some embodiments, the amino acid is present at a concentration of about 44mM to about 56mM. In some embodiments, the amino acid is present at a concentration of about 45mM to about 55mM. In some embodiments, the amino acid is present at a concentration of about 46mM to about 54mM. In some embodiments, the amino acid is present at a concentration of about 47mM to about 53mM. In some embodiments, the amino acid is present at a concentration of about 48mM to about 52mM. In some embodiments, the amino acid is present at a concentration of about 49mM to about 51mM.

[0114] In some embodiments, the amino acid is present at a concentration of about 40mM. In some embodiments, the amino acid is present at a concentration of about 41mM. In some embodiments, the amino acid is present at a concentration of about 42mM. In some embodiments, the amino acid is present at a concentration of about 43mM. In some embodiments, the amino acid is present at a concentration of about 44mM. In some embodiments, the amino acid is present at a concentration of about 45mM. In some embodiments, the amino acid is present at a concentration of about 46mM. In some embodiments, the amino acid is present at a concentration of about 47mM. In some embodiments, the amino acid is present at a concentration of about 48mM. In some embodiments, the amino acid is present at a concentration of about 49mM. In some embodiments, the amino acid is present at a concentration of about 50mM. In certain embodiments, the amino acid is present at a concentration of 50mM. In some embodiments, the amino acid is present at a concentration of about 51mM. In some embodiments, the amino acid is present at a concentration of about 52mM. In some embodiments, the amino acid is present at a concentration of about 53mM. In some embodiments, the amino acid is present at a concentration of about 54mM. In some embodiments, the amino acid is present at a concentration of about 55mM. In some embodiments, the amino acid is present at a concentration of about 56 mM. In some embodiments, the amino acid is present at a concentration of about 57 mM. In some embodiments, the amino acid is present at a concentration of about 58 mM. In some embodiments, the amino acid is present at a concentration of about 59 mM. In some embodiments, the amino acid is present at a concentration of about 60 mM.

[0115] In various embodiments, the amino acid is present at a concentration of about 15mM to about 35mM. In some embodiments, the amino acid is present at a concentration of about 16mM to about 34mM. In some embodiments, the amino acid is present at a concentration of about 17mM to about 33mM. In some embodiments, the amino acid is present at a concentration of about 18mM to about 32mM. In some embodiments, the amino acid is present at a concentration of about 19mM to about 31mM. In some embodiments, the amino acid is present at a concentration of about 20mM to about 30mM. In some embodiments, the amino acid is present at a concentration of about 21mM to about 29mM. In some embodiments, the amino acid is present at a concentration of about 22mM to about 28mM. In some embodiments, the amino acid is present at a concentration of about 23mM to about 27mM. In some embodiments, the amino acid is present at a concentration of about 24mM to about 26mM.

[0116] In some embodiments, the amino acid is present at a concentration of about 15mM. In some embodiments, the amino acid is present at a concentration of about 16mM. In some embodiments, the amino acid is present at a concentration of about 17mM. In some embodiments, the amino acid is present at a concentration of about 18mM. In some embodiments, the amino acid is present at a concentration of about 19mM. In some embodiments, the amino acid is present at a concentration of about 20mM. In some embodiments, the amino acid is present at a concentration of about 21mM. In some embodiments, the amino acid is present at a concentration of about 22mM. In some embodiments, the amino acid is present at a concentration of about 23mM. In some embodiments, the amino acid is present at a concentration of about 24mM. In some embodiments, the amino acid is present at a concentration of about 25mM. In some embodiments, the amino acid is present at a concentration of about 26mM. In some embodiments, the amino acid is present at a concentration of about 27mM. In some embodiments, the amino acid is present at a concentration of about 28mM. In some embodiments, the amino acid is present at a concentration of about 29mM. In some embodiments, the amino acid is present at a concentration of about 30mM. In some embodiments, the amino acid is present at a concentration of about 31mM. In some embodiments, the amino acid is present at a concentration of about 32 mM. In some embodiments, the amino acid is present at a concentration of about 33 mM. In some embodiments, the amino acid is present at a concentration of about 34 mM. In some embodiments, the amino acid is present at a concentration of about 35 mM.

[0117] In various embodiments, the amino acid is present at a concentration of about 65mM to about 85mM. In some embodiments, the amino acid is present at a concentration of about 66mM to about 84mM. In some embodiments, the amino acid is present at a concentration of about 67mM to about 83mM. In some embodiments, the amino acid is present at a concentration of about 68mM to about 82mM. In some embodiments, the amino acid is present at a concentration of about 69mM to about 81mM. In some embodiments, the amino acid is present at a concentration of about 70mM to about 80mM. In some embodiments, the amino acid is present at a concentration of about 71mM to about 79mM. In some embodiments, the amino acid is present at a concentration of about 72mM to about 78mM. In some embodiments, the amino acid is present at a concentration of about 73mM to about 77mM. In some embodiments, the amino acid is present at a concentration of about 74mM to about 76mM.

[0118] In some embodiments, the amino acid is present at a concentration of about 65mM. In some embodiments, the amino acid is present at a concentration of about 66mM. In some embodiments, the amino acid is present at a concentration of about 67mM. In some embodiments, the amino acid is present at a concentration of about 68mM. In some embodiments, the amino acid is present at a concentration of about 69mM. In some embodiments, the amino acid is present at a concentration of about 70mM. In some embodiments, the amino acid is present at a concentration of about 71mM. In some embodiments, the amino acid is present at a concentration of about 72mM. In some embodiments, the amino acid is present at a concentration of about 73mM. In some embodiments, the amino acid is present at a concentration of about 74mM. In some embodiments, the amino acid is present at a concentration of about 75mM. In some embodiments, the amino acid is present at a concentration of about 76mM. In some embodiments, the amino acid is present at a concentration of about 77mM. In some embodiments, the amino acid is present at a concentration of about 78mM. In some embodiments, the amino acid is present at a concentration of about 79mM. In some embodiments, the amino acid is present at a concentration of about 80mM. In some embodiments, the amino acid is present at a concentration of about 81mM. In some embodiments, the amino acid is present at a concentration of about 82 mM. In some embodiments, the amino acid is present at a concentration of about 83 mM. In some embodiments, the amino acid is present at a concentration of about 84 mM. In some embodiments, the amino acid is present at a concentration of about 85 mM.

[0119] In some embodiments, the amino acid is a non-polar amino acid. In some embodiments, the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, methionine, isoleucine phenylalanine, tyrosine and tryptophan. In some embodiments, the amino acid is selected from the group consisting of phenylalanine, tyrosine, tryptophan and proline. In some embodiments, the amino acid is phenylalanine. In some embodiments, the amino acid is tyrosine. In some embodiments, the amino acid is tryptophan. In some embodiments, the amino acid is proline. In a specific embodiment, the amino acid is L-proline.

[0120] In various embodiments, the composition further comprises salt. In some embodiments, the salt is present at a concentration of about 65mM to about 85mM. In some embodiments, the salt is present at a concentration of about 66mM to about 84mM. In some embodiments, the salt is present at a concentration of about 67mM to about 83mM. In some embodiments, the salt is present at a concentration of about 68mM to about 82mM. In some embodiments, the salt is present at a concentration of about 69mM to about 81mM. In some embodiments, the salt is present at a concentration of about 70mM to about 80mM. In some embodiments, the salt is present at a concentration of about 71mM to about 79mM. In some embodiments, the salt is present at a concentration of about 72mM to about 78mM. In some embodiments, the salt is present at a concentration of about 73mM to about 77mM. In some embodiments, the salt is present at a concentration of about 74mM to about 76mM.

[0121] In some embodiments, the salt is present at a concentration of about 65mM. In some embodiments, the salt is present at a concentration of about 66mM. In some embodiments, the salt is present at a concentration of about 67mM. In some embodiments, the salt is present at a concentration of about 68mM. In some embodiments, the salt is present at a concentration of about 69mM. In some embodiments, the salt is present at a concentration of about 70mM. In some embodiments, the salt is present at a concentration of about 71mM. In some embodiments, the salt is present at a concentration of about 72mM. In some embodiments, the salt is present at a concentration of about 73mM. In some embodiments, the salt is present at a concentration of about 74mM. In some embodiments, the salt is present at a concentration of about 75mM. In certain embodiments, the salt is present at a concentration of 75mM. In some embodiments, the salt is present at a concentration of about 76mM. In some embodiments, the salt is present at a concentration of about 77mM. In some embodiments, the salt is present at a concentration of about 78mM. In some embodiments, the salt is present at a concentration of about 79mM. In some embodiments, the salt is present at a concentration of about 80mM. In some embodiments, the salt is present at a concentration of about 81 mM. In some embodiments, the salt is present at a concentration of about 82 mM. In some embodiments, the salt is present at a concentration of about 83 mM. In some embodiments, the salt is present at a concentration of about 84 mM. In some embodiments, the salt is present at a concentration of about 85 mM. In certain embodiments, the salt is a chloride salt. In a specific embodiment, the salt is KCl. In a specific embodiment, the salt is NaCl.

[0122] In various embodiments, the composition further comprises a poloxamer. "Poloxamer" refers to a nonionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene. Poloxamers are also known by the trade names "Pluronics" or "Synperonics" (BASF). The block copolymer can be represented by the following formula: HO(C2H4O) x (C3H60) y (C2H40) z H.

[0123] The synthesis of block copolymers produces a polymer population with an average molecular weight. Therefore, in a specific embodiment, if not otherwise explicitly stated, the term "poloxamer" as used herein can be used interchangeably with the term "poloxamers" (representing an entity of several poloxamers, also referred to as a mixture of poloxamers). The term "average" about the number of monomer units or molecular weights of one or more poloxamers as used herein is due to the technical inability to produce all poloxamers with the same composition and therefore the same molecular weight. The poloxamers produced according to the most advanced methods will exist as a mixture of poloxamers, each of which shows variability about its molecular weight, but the mixture averages the molecular weight specified herein as a whole. BASF and Sigma Aldrich are suitable sources for the poloxamers in the specific embodiments contemplated herein.

[0124] In some embodiments, the poloxamer is present at a concentration of about 0.01 mg / ml to about 2 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.1 mg / ml to about 2 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.2 mg / ml to about 2 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.3 mg / ml to about 2 mg / ml.

[0125] In some embodiments, the poloxamer is present at a concentration of about 0.01 mg / ml to about 1 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.1 mg / ml to about 1 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.1 mg / ml to about 0.8 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.2 mg / ml to about 1 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.3 mg / ml to about 1 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.02 mg / ml to about 0.9 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.03 mg / ml to about 0.8 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.04 mg / ml to about 0.7 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.05 mg / ml to about 0.6 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.06 mg / ml to about 0.5 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.07 mg / ml to about 0.4 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.08 mg / ml to about 0.3 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.09 mg / ml to about 0.2 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.1 mg / ml to about 0.5 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.2 mg / ml to about 0.4 mg / ml.

[0126] In certain embodiments, the poloxamer is present at a concentration of about 0.1 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.2 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.3 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.4 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.5 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.6 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.7 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.8 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 0.9 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.0 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.1 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.2 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.3 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.4 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.5 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.6 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.7 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.8 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 1.9 mg / ml. In certain embodiments, the poloxamer is present at a concentration of about 2.0 mg / ml. In some embodiments, the poloxamer is present at a concentration of about 0.1 mg / ml. In certain embodiments, the poloxamer is present at a concentration of 0.1 mg / ml. In some embodiments, the poloxamer is poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, In certain embodiments, the poloxamer is poloxamer 407.

[0127] In various embodiments, the composition comprises a pH of about 6.5 to about 8.0. In some embodiments, the composition comprises a pH of about 6.5. In some embodiments, the composition comprises a pH of about 6.6. In some embodiments, the composition comprises a pH of about 6.7. In some embodiments, the composition comprises a pH of about 6.8. In some embodiments, the composition comprises a pH of about 6.9. In some embodiments, the composition comprises a pH of about 7.0. In some embodiments, the composition comprises a pH of about 7.1. In some embodiments, the composition comprises a pH of about 7.2. In some embodiments, the composition comprises a pH of about 7.3. In some embodiments, the composition comprises a pH of about 7.4. In some embodiments, the composition comprises a pH of about 7.5. In some embodiments, the composition comprises a pH of about 7.6. In some embodiments, the composition comprises a pH of about 7.7. In some embodiments, the composition comprises a pH of about 7.8. In some embodiments, the composition comprises a pH of about 7.9. In some embodiments, the composition comprises a pH of about 8.0.

[0128] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; and about 50 mM L-proline; wherein the composition comprises a pH of about 7.

[0129] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM L-histidine; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; and about 50 mM L-proline; wherein the composition comprises a pH of about 7.

[0130] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; about 50 mM L-proline; and about 0.1 to about 0.8 mg / mL poloxamer 188; wherein the composition comprises a pH of about 7.

[0131] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; about 50 mM L-proline; and about 75 mM NaCl; wherein the composition comprises a pH of about 7.

[0132] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 weight % sucrose per volume of the composition; about 50 mM L-proline; about 75 mM NaCl; and about 0.1 to about 0.8 mg / mL poloxamer 188; wherein the composition comprises a pH of about 7.

[0133] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 wt % sucrose per volume of the composition; about 75 mM NaCl; and about 0.1 to about 0.8 mg / ml poloxamer 188 or about 0.01 wt % poloxamer 188 per volume of the composition; wherein the composition comprises a pH of about 7.

[0134] In a specific embodiment, an aqueous viral composition is provided, wherein the composition comprises a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 wt % sucrose per volume of the composition; and about 75 mM NaCl; wherein the composition comprises a pH of about 7.

[0135] In any of the embodiments contemplated herein, the viral vector is present in an amount of about 1 x 10 8 About 2x10 9 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 1 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 2 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 3 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 4 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 5 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 6 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 7 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 8 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 9 x 10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 1 x 109 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 2 x 10 9 The titer or concentration is present in TU / ml.

[0136] In certain embodiments contemplated herein, the formulation does not include a specific component. Thus, in any of the embodiments contemplated herein, the composition does not include PIPES. In any of the embodiments contemplated herein, the composition does not include sodium citrate. In any of the embodiments contemplated herein, the composition does not include sodium phosphate. In any of the embodiments contemplated herein, the composition does not include Tris. In any of the embodiments contemplated herein, the composition does not include salt. In any of the embodiments contemplated herein, the composition does not include NaCl. In any of the embodiments contemplated herein, the composition does not include KCl. In any of the embodiments contemplated herein, the composition does not include serum. In any of the embodiments contemplated herein, the composition does not include human serum albumin (HSA). In any of the embodiments contemplated herein, the composition contemplated herein does not include trehalose.

[0137] In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 75% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 76% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 77% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 78% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 79% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 80% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 81% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 82% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains greater than about 83% recovery of the infectious titer in HOS cells after storage and / or freeze-thaw cycles relative to the infectious titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains greater than about 84% recovery of the infectious titer in HOS cells after storage and / or freeze-thaw cycles relative to the infectious titer of the viral vector in the composition before storage or freeze-thaw cycles.In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 85% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 86% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 87% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 88% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 89% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 90% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 91% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains an infection titer recovery of greater than about 92% in HOS cells after storage and / or freeze-thaw cycles relative to the infection titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains greater than about 93% recovery of the infectious titer in HOS cells after storage and / or freeze-thaw cycles relative to the infectious titer of the viral vector in the composition before storage or freeze-thaw cycles. In any of the embodiments contemplated herein, the viral vector maintains greater than about 94% recovery of the infectious titer in HOS cells after storage and / or freeze-thaw cycles relative to the infectious titer of the viral vector in the composition before storage or freeze-thaw cycles.In any of the embodiments contemplated herein, the viral vector maintains greater than about 95% recovery of the infectious titer in HOS cells after storage and / or freeze-thaw cycles relative to the infectious titer of the viral vector in the composition before storage or freeze-thaw cycles.

[0138] In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 56° to about 62°C, as measured by differential scanning fluorimetry (DSF). In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 58° to about 60°C, as measured by differential scanning fluorimetry (DSF). In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 56°, as measured by differential scanning fluorimetry (DSF). In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 57°, as measured by differential scanning fluorimetry (DSF). In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 58°, as measured by differential scanning fluorimetry (DSF). In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 59°, as measured by differential scanning fluorimetry (DSF). In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 60°, as measured by differential scanning fluorimetry (DSF). In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 61°, as measured by differential scanning fluorimetry (DSF). In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 62°, as measured by differential scanning fluorimetry (DSF).

[0139] In any of the embodiments contemplated herein, the viral vector maintains a hydrodynamic diameter of about 150 nm to about 170 nm after storage and / or at least one freeze-thaw cycle relative to the hydrodynamic diameter of the viral vector in the composition before storage or at least one freeze-thaw cycle, as measured by dynamic light scattering (DLS) at 25°C and a viscosity value of 0.967 centipoise (cP).

[0140] In any of the embodiments contemplated herein, the viral vector maintains at least 78% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 79% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 80% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 81% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 82% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 83% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 84% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 85% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 86% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 87% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 88% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 89% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 90% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 91% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 92% potency as measured by transgene expression in PBMCs compared to a reference standard.In any of the embodiments contemplated herein, the viral vector maintains at least 93% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 94% potency as measured by transgene expression in PBMCs compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 95% potency as measured by transgene expression in PBMCs compared to a reference standard.

[0141] In any of the aspects and embodiments contemplated herein, the composition has no visible particles or visible fiber particles (bundled fibers) after storage. In any of the aspects and embodiments contemplated herein, the composition has 5 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has 4 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has 3 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has 2 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has 1 or fewer visible particles or microparticles after storage. In any of the aspects and embodiments contemplated herein, the composition has no visible particles after storage.

[0142] In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has no visible particles or visible fiber particles (bundled fibers) after storage for every 50 ml of the composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 5 or less visible particles or microparticles after storage for every 50 ml of the composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 4 or less visible particles or microparticles after storage for every 50 ml of the composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 3 or less visible particles or microparticles after storage for every 50 ml of the composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 2 or less visible particles or microparticles after storage for every 50 ml of the composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 1 or less visible particles or microparticles after storage for every 50 ml of the composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has no visible particles after storage for every 50 ml of the composition.

[0143] In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has no visible particles or visible fiber particles (bundled fibers) after storage for every 36 ml of composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 5 or less visible particles or microparticles after storage for every 36 ml of composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 4 or less visible particles or microparticles after storage for every 36 ml of composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 3 or less visible particles or microparticles after storage for every 36 ml of composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 2 or less visible particles or microparticles after storage for every 36 ml of composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has 1 or less visible particles or microparticles after storage for every 36 ml of composition. In any aspect and embodiment of the aspects and embodiments contemplated herein, the composition has no visible particles after storage for every 36 ml of composition.

[0144] In various embodiments, the storage is at 2-8°C. In some embodiments, the storage is at 2°C. In some embodiments, the storage is at 3°C. In some embodiments, the storage is at 4°C. In some embodiments, the storage is at 5°C. In some embodiments, the storage is at 6°C. In some embodiments, the storage is at 7°C. In some embodiments, the storage is at 8°C. In various embodiments, the storage is at 25°C. In various embodiments, the storage is at 37°C.

[0145] In various embodiments, the storage lasts for 24 hours, 48 ​​hours, or 72, 96, 120, 144, or 168 hours or longer. In some embodiments, the storage lasts for at least 24 hours. In some embodiments, the storage lasts for at least 48 hours. In some embodiments, the storage lasts for at least 72 hours. In some embodiments, the storage lasts for at least 96 hours. In some embodiments, the storage lasts for at least 120 hours. In some embodiments, the storage lasts for at least 144 hours. In some embodiments, the storage lasts for at least 168 hours or 1 week.

[0146] In some embodiments, the storage lasts at least 1 week. In some embodiments, the storage lasts at least 2 weeks. In some embodiments, the storage lasts at least 3 weeks. In some embodiments, the storage lasts at least 4 weeks. In some embodiments, the storage lasts at least 1 month. In some embodiments, the storage lasts at least 2 months. In some embodiments, the storage lasts at least 3 months. In some embodiments, the storage lasts at least 4 months. In some embodiments, the storage lasts at least 5 months. In some embodiments, the storage lasts at least 6 months. In some embodiments, the storage lasts at least 7 months. In some embodiments, the storage lasts at least 8 months. In some embodiments, the storage lasts at least 9 months. In some embodiments, the storage lasts at least 10 months. In some embodiments, the storage lasts at least 11 months. In some embodiments, the storage lasts at least 1 year. In some embodiments, the storage lasts at least 2 years. In some embodiments, the storage lasts at least 3 years. In some embodiments, the storage lasts at least 4 years. In some embodiments, the storage lasts at least 5 years or more.

[0147] In various embodiments, the storage comprises one or more freeze-thaw cycles. In some embodiments, the storage comprises at least 1 freeze-thaw cycle. In some embodiments, the storage comprises at least 2 freeze-thaw cycles. In some embodiments, the storage comprises at least 3 freeze-thaw cycles. In some embodiments, the storage comprises at least 4 freeze-thaw cycles. In some embodiments, the storage comprises at least 5 freeze-thaw cycles. In some embodiments, the one or more freeze-thaw cycles comprise freezing the composition at about -65° or lower for about 1.5 hours or more, and thawing at 30°C for 1.5 hours.

[0148] In any of the embodiments contemplated herein, the composition is frozen.

[0149] D. Method

[0150] Methods for storage, cryopreservation, transduction, and expression of transgenes are also considered.

[0151] In one aspect, a method for storing a viral vector is provided, the method comprising providing a viral vector, contacting the viral vector with any of the compositions contemplated herein, and storing the viral composition at about 25° C. or less. In some embodiments, the storage temperature is 2-8° C. or less. In some embodiments, the storage temperature is 0° C. or less.

[0152] In another aspect, a method for cryopreservation of a viral vector is provided, the method comprising providing a viral vector, contacting the viral vector with any of the compositions contemplated herein, freezing the viral composition, and storing the viral composition at about 0°C or below. In some embodiments, the composition is stored at -20°C or below. In some embodiments, the composition is stored at -65°C or below. In some embodiments, the composition is stored at -80°C or below.

[0153] In various embodiments, the storage lasts for at least 24 hours. In some embodiments, the storage lasts for at least 48 hours. In some embodiments, the storage lasts for at least 72 hours. In some embodiments, the storage lasts for at least 96 hours. In some embodiments, the storage lasts for at least 120 hours. In some embodiments, the storage lasts for at least 144 hours. In some embodiments, the storage lasts for at least 168 hours. In some embodiments, the storage lasts for at least 1 week. In some embodiments, the storage lasts for at least 2 weeks. In some embodiments, the storage lasts for at least 3 weeks. In some embodiments, the storage lasts for at least 4 weeks. In some embodiments, the storage lasts for at least 1 month. In some embodiments, the storage lasts for at least 2 months. In some embodiments, the storage lasts for at least 3 months. In some embodiments, the storage lasts for at least 4 months. In some embodiments, the storage lasts for at least 5 months. In some embodiments, the storage lasts for at least 6 months. In some embodiments, the storage lasts for at least 7 months. In some embodiments, the storage lasts for at least 8 months. In some embodiments, the storage lasts for at least 9 months. In some embodiments, the storage lasts for at least 10 months. In some embodiments, the storage lasts for at least 11 months. In some embodiments, the storage lasts for at least 1 year. In some embodiments, the storage lasts for at least 2 years.

[0154] In another aspect, a method of transduction is provided, the method comprising contacting a cell with any of the compositions contemplated herein comprising a viral vector, thereby transducing the cell.

[0155] In another aspect, a method of expressing a transgene in a cell is provided, the method comprising contacting the cell with any of the compositions contemplated herein comprising a viral vector, wherein the viral vector comprises the transgene.

[0156] In various embodiments, the cell is a mammalian cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a hematopoietic stem cell or progenitor cell. In some embodiments, the cell is a CD34+ hematopoietic stem cell or progenitor cell. In some embodiments, the cell is a human CD34+ hematopoietic stem cell or progenitor cell.

[0157] In some embodiments, the cell is a peripheral blood mononuclear cell (PMBC). In some embodiments, the cell is a T cell. In some embodiments, the cell is an αβT cell. In some embodiments, the cell is a γδT cell. In some embodiments, the cell is a CD3 + 、CD4 + and / or CD8 + Cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0158] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC). Illustrative immune effector cells contemplated herein are T lymphocytes, including but not limited to cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTL; CD4 + In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T (NKT) cells.

[0159] Immune effector cells can be autologous / self ("self") or non-autologous ("non-self", e.g., allogeneic, isogenic, or xenogeneic). As used herein, "autologous" refers to cells from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the compared cells. As used herein, "isogenic" refers to cells of different subjects that are genetically identical to the compared cells. As used herein, "xenogeneic" refers to cells of a different species than the compared cells. In preferred embodiments, the cells are autologous.

[0160] Illustrative immune effector cells for use with the methods contemplated in the specific embodiments include T lymphocytes. The terms "T cell" or "T lymphocyte" are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) cell or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4-CD8 - T cells or any other subset of T cells. Other illustrative populations of T cells suitable for use in specific embodiments include naive T cells (T N ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ) and effector T cells (T EFF ).

[0161] As will be appreciated by the technician, other cells can also be used together with the method envisioned herein as immune effector cells. Specifically, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include the progenitor cells of effector cells, wherein such progenitor cells can be induced in vivo or in vitro to differentiate into immune effector cells. Therefore, in a particular embodiment, immune effector cells include the progenitor cells of immune effector cells, such as hematopoietic stem cells (HSC) contained in CD34+ cell colonies derived from cord blood, bone marrow or mobilized peripheral blood, and the progenitor cells are differentiated into mature immune effector cells when applied to a subject, or the progenitor cells can be induced in vitro to differentiate into mature immune effector cells.

[0162] As used herein, the term "CD34+ cells" refers to cells expressing CD34 protein on their cell surface. As used herein, "CD34" refers to cell surface glycoproteins (e.g., sialomucin) that generally act as cell-cell adhesion factors and participate in the entry of T cells into lymph nodes. The CD34+ cell population contains hematopoietic stem cells (HSCs), which differentiate and contribute to all hematopoietic lineages when administered to patients, including cells of T cell, NK cell, NKT cell, neutrophil and monocyte / macrophage lineages.

[0163] In a specific embodiment, a method for preparing an immune effector cell expressing a transgenic or therapeutic protein is provided herein. In one embodiment, the method comprises transducing an immune effector cell separated from an individual so that the immune effector cell expresses a transgenic or therapeutic protein with a viral vector composition (e.g., a lentiviral composition) considered herein. In a specific embodiment, the transduced cell is subsequently cultured to amplify before being administered to a subject.

[0164] In certain embodiments, immune effector cells are separated from individuals and genetically modified without further manipulation in vitro. Then, such cells can be directly applied to individuals again. In other embodiments, immune effector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express transgenic, for example, engineered chimeric receptors (e.g., CAR, CCR, DARIC or switch receptors), TCR or other transgenics (e.g., proteins or cytokines). In this regard, immune effector cells can be cultured before and / or after being genetically modified.

[0165] In specific embodiments, prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, a source of cells is obtained from a subject. In specific embodiments, the modified immune effector cells comprise T cells.

[0166] In specific embodiments, PBMCs can be directly genetically modified to express transgenic or therapeutic proteins. In certain embodiments, after PBMC separation, T lymphocytes are further separated, and in certain embodiments, cytotoxic and helper T lymphocytes can be sorted into naive, memory and effector T cell subpopulations before or after genetic modification and / or amplification.

[0167] Immune effector cells such as T cells can be genetically modified using known methods after separation, or immune effector cells can be activated and amplified in vitro (or differentiated in the case of progenitor cells) before being genetically modified. In a specific embodiment, immune effector cells such as T cells are activated and stimulated to amplify, and then genetically modified with a transgene or therapeutic protein (e.g., transduced with a viral vector composition contemplated herein). In various embodiments, T cells can be activated and expanded before or after genetic modification using methods such as those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; 7,572,631; and U.S. Patent Application Publication No. 20170051252.

[0168] In one embodiment, CD34 + Hematopoietic stem cells or progenitor cells are transduced with the nucleic acid constructs contemplated herein. In certain embodiments, the transduced CD34 + The cells differentiate into mature immune effector cells in vivo after administration to a subject, typically the subject from which the cells were initially isolated. + Cells can be stimulated in vitro prior to exposure to or after genetic modification with one or more of the following cytokines: Flt-3 ligand (FLT3), stem cell factor (SCF), megakaryocyte growth factor and differentiation factor (TPO), IL-3, and IL-6 according to the methods described above (Asheuer et al., 2004; Imren, et al., 2004).

[0169] In certain embodiments, the cells are transduced with a vector as contemplated herein in the presence of a polycationic polymer. In some embodiments, the polycationic polymer is polybrene, protamine sulfate, polyethyleneimine, or polyethylene glycol / poly-L-lysine block copolymer. In some embodiments, the cells are transduced in the presence of polybrene. In some embodiments, the cells are transduced in the presence of about 2 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 3 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 4 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 5 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 6 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 7 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 8 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 2 μg / ml to about 8 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 3 μg / ml to about 8 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 4 μg / ml to about 8 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 5 μg / ml to about 8 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 6 μg / ml to about 8 μg / ml polybrene. In some embodiments, the cells are transduced in the presence of about 7 μg / ml to about 8 μg / ml polybrene.

[0170] E. Viral vector

[0171] Compositions and methods encompassed herein include viral vectors. The term "vector" is used herein to refer to a nucleic acid molecule that is capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically connected to (e.g., inserted into) a carrier nucleic acid molecule. The vector may include a sequence that directs the autonomous replication in the cell, or may include a sequence that is sufficient to allow integration into the host cell DNA. In a specific embodiment, the vector is a viral vector or a non-viral vector.

[0172] As is apparent to those skilled in the art, the term "viral vector" is widely used to refer to nucleic acid molecules (e.g., transfer plasmids) comprising viral-derived nucleic acid elements that generally promote the transfer of nucleic acid molecules or are integrated into the genome of a cell, or to viral particles that mediate nucleic acid transfer. In addition to one or more nucleic acids, viral particles will typically include various viral components and sometimes host cell components. The term "viral vector" or "slow virus vector" can refer to viruses or viral particles that can transfer nucleic acid into a cell or to the nucleic acid itself that is transferred. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from viruses.

[0173] Illustrative examples of viral vector systems suitable for use in the embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus (HSV), adenovirus, and vaccinia virus vectors.

[0174] In various embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral vector.

[0175] AAV is a small (about 26nm) replication-deficient, mainly free-type, non-enveloped virus. AAV can infect dividing and non-dividing cells, and its genome can be incorporated into the genome of the host cell. Recombinant AAV (rAAV) is usually composed of at least one transgene and its regulatory sequence and 5' and 3' AAV inverted terminal repeats (ITR). The length of the ITR sequence is about 145bp. In a specific embodiment, the rAAV comprises ITR and capsid sequences separated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10.

[0176] In some embodiments, chimeric rAAV is used. In some embodiments, rAAV comprises one or more ITR sequences. In some embodiments, the ITR sequence is isolated from one AAV serotype, and the capsid sequence is isolated from another AAV serotype. For example, rAAV having an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6 is referred to as AAV2 / AAV6. In a specific embodiment, the rAAV vector may comprise an ITR from AAV2 and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10. In a preferred embodiment, rAAV comprises an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6. In a preferred embodiment, rAAV comprises an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

[0177] In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.

[0178] The construction, production, and purification of rAAV vectors have been disclosed, for example, in U.S. Pat. Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated herein by reference in its entirety.

[0179] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in specific embodiments include, but are not limited to, Moloneymurine leukemia virus (M-MuLV), Moloneymurine sarcoma virus (MoMSV), Harveymurine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV) and Rous Sarcoma Virus (RSV) and lentivirus.

[0180] In some embodiments, the viral vector is a lentiviral vector. As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus, including HIV type 1 and HIV 2); Vesnamedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0181] In some embodiments, the lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1) or human immunodeficiency virus 2 (HIV-2). In specific embodiments, the lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1).

[0182] In various embodiments, the lentiviral vectors contemplated herein comprise one or more LTRs and one or more or all of the following accessory elements: cPPT / FLAP, Psi(Ψ) packaging signal, export element, poly(A) sequence, and may optionally comprise WPRE or HPRE, insulator elements, selectable markers, and cellular suicide genes, as discussed elsewhere herein.

[0183] In specific embodiments, the lentiviral vectors contemplated herein may be integrative or non-integrative or integration-defective lentiviruses. As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus having an integrase that lacks the ability to integrate the viral genome into the genome of a host cell. Integration-incompetent viral vectors have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety.

[0184] Illustrative mutations of the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116 N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E 157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K1 86T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0185] In one embodiment, the HIV-1 integrase-deficient pol gene comprises D64V, D116I, D116A, E152G or E152A mutations; D64V, D116I and E152G mutations; or D64V, D116A and E152A mutations.

[0186] In one embodiment, the HIV-1 integrase-deficient pol gene comprises a D64V mutation.

[0187] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA that in its native sequence context is a direct repeat and contains the U3, R and U5 regions.

[0188] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus (e.g., HIV-1 or HIV-2). Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou et al., 2000, Cell, 101:173. In another embodiment, the lentiviral vector contains a FLAP element having one or more mutations in the cPPT and / or CTS element. In yet another embodiment, the lentiviral vector comprises a cPPT or CTS element. In yet another embodiment, the lentiviral vector does not comprise a cPPT or CTS element.

[0189] As used herein, the term "packaging signal" or "packaging sequence" refers to a psi[Ψ] sequence located within the retroviral genome that is required for inserting the viral RNA into the viral capsid or particle, see, e.g., Clever et al., 1995. Journal of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0190] The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Culle et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the hepatitis B virus post-transcriptional regulatory element (HPRE).

[0191] In a specific embodiment, the expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally transcription termination signals into the vector. A variety of post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, Journal of Virology, 73:2886); the post-transcriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); etc. (Liu et al., 1995, Genes Dev, 9:1766).

[0192] Because the LTR is modified, the lentiviral vector preferably contains several safety enhancements. A "self-inactivating" (SIN) vector refers to a replication-defective vector, for example, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription outside the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0193] As used herein, the term "pseudotype" or "pseudotyped" refers to a virus having a viral envelope protein that has been replaced with an envelope protein of another virus that has preferred properties. For example, HIV can be pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope protein, which allows HIV to infect a wider range of cells because the HIV envelope protein (encoded by the env gene) normally targets the virus to the CD4 + Presenting cells. Other envelope proteins for pseudotyping include, but are not limited to, envelopes from other vesicular virus strains (e.g., Indiana, Alagoas, New Jersey, Isfahan, Kokar, Malaba, or Piri), measles envelope proteins, Sindbis envelope proteins, measles virus proteins (e.g., F and H proteins), Sendai virus proteins (e.g., F and HN proteins), or Paramyxoviridae proteins (e.g., F and H proteins).

[0194] In certain embodiments, the lentiviral vector is produced according to known methods. See, for example, Kutner et al., BMC Biotechnol. 2009; 9: 10. Doi: 10.1186 / 1472-6750-9-10; Kutner et al., Nat. Protoc. 2009; 4(4): 495–505. Doi: 10.1038 / nprot.2009.22 and WO2023 / 003844.

[0195] According to certain specific embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from lentiviruses, e.g., HIV-1. However, it should be understood that retroviral and / or lentiviral sequences from a variety of different sources can be used, or combinations and a large number of substitutions and changes in certain lentiviral sequences in lentiviral sequences can be accommodated without compromising the ability of the transfer vector to perform the functions described herein. In addition, various lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, many of which may be suitable for producing viral vectors or transfer plasmids contemplated herein.

[0196] In various embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is an AnkT9W vector Lenti-D vector. In some embodiments, the lentiviral vector is an AnkT9W vector, a T9Ank2W vector, a TNS9 vector, a TNS9.3 vector, a TNS9.3.55 vector, a lentiglobin HPV569 vector, a blue globin BB305 vector, a BG-1 vector, a BGM-1 vector, a GLOBE vector, a G-GLOBE vector, a βAS3-FB vector, or a derivative thereof. In some embodiments, the lentiviral vector is an AnkT9W vector or a derivative thereof. In some embodiments, the lentiviral vector is a T9Ank2W vector or a derivative thereof. In some embodiments, the lentiviral vector is a TNS9 vector or a derivative thereof. In some embodiments, the lentiviral vector is a TNS9 vector or a derivative thereof. In some embodiments, the lentiviral vector is a TNS9.3 vector or a derivative thereof. In some embodiments, the lentiviral vector is a TNS9.3 vector or a derivative thereof. In some embodiments, the lentiviral vector is a blue globin HPV569 vector or a derivative thereof. In some embodiments, the lentiviral vector is a blue teglobin BB305 vector or a derivative thereof. In some embodiments, the lentiviral vector is a BG-1 vector or a derivative thereof. In some embodiments, the lentiviral vector is a BGM-1 vector or a derivative thereof. In some embodiments, the lentiviral vector is a GLOBE vector or a derivative thereof. In some embodiments, the lentiviral vector is a G-GLOBE vector or a derivative thereof. In some embodiments, the lentiviral vector is a βAS3-FB vector or a derivative thereof. In specific embodiments, the lentiviral vector is BB305.

[0197] F. Transgenic

[0198] In any of the embodiments contemplated herein, the viral vector comprises a polynucleotide comprising a transgene. In some embodiments, the transgene encodes a therapeutic protein.

[0199] As used herein, the term "transgene" refers to an exogenous nucleic acid sequence encoding a protein or functional nucleotide. A transgene may encode a non-natural or naturally occurring protein or polypeptide. As used herein, the term "therapeutic protein" refers to a protein or polypeptide encoded by a transgene that can be used to treat a patient's disease (e.g., cancer).

[0200] In various embodiments, the transgenic or therapeutic protein is a chimeric antigen receptor (CAR) that specifically binds to a target antigen, a chimeric co-stimulatory receptor (CCR), an αβT cell receptor (αβ-TCR), a γδT cell receptor (γδ-TCR), an immune receptor complex regulated by a dimerizer (DARIC), or a switch receptor. In some embodiments, the transgenic or therapeutic protein is an exogenous co-stimulatory factor, an immunomodulatory factor, an agonist of a co-stimulatory factor, an antagonist of an immunosuppressive factor, an immune cell conjugate, or a fusion protein. In some embodiments, the transgenic or therapeutic protein is a co-stimulatory factor. In some embodiments, the transgenic or therapeutic protein is a cytokine.

[0201] As used herein, the term "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specific targeting" describes the binding affinity of a binding domain to a target antigen above background binding. 5 M -1 Affinity or K a The binding domain "specifically binds" to a target antigen if the binding domain (or fusion protein comprising the binding domain) binds or associates with the target antigen at a specific equilibrium association constant of greater than or equal to about 10 (i.e., the equilibrium association constant for a specific binding interaction in units of 1 / M). In certain embodiments, the binding domain (or fusion protein comprising the binding domain) binds or associates with the target antigen at a specific equilibrium association constant of greater than or equal to about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 1013 M -1 K a Binds to the target. A "high affinity" binding domain (or a single-chain fusion protein thereof) refers to a K a For at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 or larger binding domains.

[0202] The terms "selectively binds" or "selectively bound" or "selectively binding" or "selectively targeting" and describe the preferential binding of a molecule to a target molecule in the presence of multiple off-target molecules (on-target binding).

[0203] In various embodiments, the CAR, CCR, DARIC or switch receptor specifically binds to a target antigen selected from the group consisting of: alpha folate receptor (FRα), alpha vβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGF R), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), IL-10Rα, IL-13Rα2, κ, cancer / testis antigen 2 (LAGE-1A), K-Ras, K-Ras G12C, K-RasG12D, λ, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC Class I chain-associated protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, breakpoint X 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL16 binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1).

[0204] In various embodiments, the CAR, CCR or DARIC specifically binds to a target antigen selected from the group consisting of BCMA, CD33, CD20, CD79a, CD79b, CLL-1, IGF2BP3 / A3, MUC16, NY-ESO, PRAME, PSA, TACI and TP53.

[0205] In various embodiments, the αβ-TCR or γδ-TCR specifically binds to a target antigen selected from the group consisting of α-fetoprotein (AFP), members of the B melanoma antigen (BAGE) family, brothers of regulatory factors at imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, antigen recognized by cytotoxic T cells (CTL) on melanoma (CAMEL), Epstein-Barr virus (Epstein-Barr virus, EBV) antigen, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE–6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), K-Ras, K-Ras G12C, K-Ras G12D, latent membrane protein 2 (LMP2), melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), P53, P antigen (PAGE) family member, placenta-specific 1 (PLAC1), antigen preferentially expressed in melanoma (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X 2 (SSX2), synovial sarcoma X 3 (SSX3), synovial sarcoma X 4 (SSX4), synovial sarcoma X 5 (SSX5), synovial sarcoma X 8 (SSX8), thyroglobin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms' tumor protein (WT-1), X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).

[0206] In various embodiments, the αβ-TCR or γδ-TCR specifically binds to the MAGE-A4 peptide.

[0207] Illustrative transgenes, therapeutic proteins, including but not limited to CARs, CCRs, αβ-TCRs, γδ-TCRs, DARICs and switch receptors, and methods of making and using the same are disclosed in any one or more of WO2021 / 067347, WO2020 / 252110, WO2020 / 227474, WO2020 / 227475, WO2020 / 227481, WO2020 / 193767, WO2020 / 123947, WO2019 / 126724, WO2018 / 094244, WO2017 / 180993, WO2016094304, WO2015017214, WO2013154760, which are incorporated herein by reference in their entirety.

[0208] In various embodiments, the transgene or therapeutic protein is a therapeutic globin for treating a hemoglobinopathy or an ABCD1 gene for treating CALD.

[0209] In certain embodiments, the transgenic or therapeutic protein is a globin. In certain embodiments, the globin is human β-globin, human δ-globin, anti-sickling globin, human γ-globin, human β-globin, A-T87Q -Globin, human beta A-G16D / E22A / T87Q -globin or human β A-T87Q / K95E / K120E -globin. In certain embodiments, the globin is human β-globin. In certain embodiments, the globin is anti-sickling globin. In certain embodiments, the globin is human γ-globin. In certain embodiments, the globin is human β-globin. A-T87Q -globin. In certain aspects, the globin is human β A-G16D / E22A / T87Q -globin. In certain aspects, the globin is human β A-T87Q / K95E / K120E In certain embodiments, the β-globin is human β-globin. In specific embodiments, the β-globin is β A-T87Q Globin.

[0210] In specific embodiments, polynucleotides encoding a transgene or a therapeutic protein as described herein are provided.

[0211] As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded and are recombinant, synthetic or isolated. Polynucleotides include, but are not limited to: precursor messenger RNA (precursor RNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, genomic RNA (gRNA), positive strand RNA (RNA (+)), negative strand RNA (RNA (-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA or recombinant DNA. Polynucleotides refer to polymeric forms of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, as well as all intermediate lengths, whether ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. It will be readily understood that in this context, "intermediate lengths" means any length between the cited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. In specific embodiments, the polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence.

[0212] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from the sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that are normally adjacent to the fragment. In specific embodiments, an "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not occur in nature and have been prepared by the hand of man. In specific embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semisynthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0213] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0214] In specific embodiments, the polynucleotides described herein, including polynucleotides encoding transgenic or therapeutic proteins as described herein, can be codon optimized. As used herein, the term "codon optimized" refers to replacing codons in polynucleotides encoding polypeptides to increase the expression, stability and / or activity of the polypeptides. Factors that affect codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias or synthetically constructed bias tables between two or more organisms or genes; (ii) variation in codon bias within an organism, gene, or genome; (iii) systematic variation in codons, including background; (iv) variation in the tRNAs for which codons decode; (v) variation in codons, either overall or at one position in a triplet, according to GC%; (vi) variation in similarity to a reference sequence, such as a naturally occurring sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge about the function of a DNA sequence on which a codon substitution set is designed; (x) systematic variation in the codon set for each amino acid; and / or (xi) isolated removal of spurious translation start sites.

[0215] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base that is N-glycosidically linked to a phosphorylated sugar. Nucleotides are understood to include natural bases and a variety of modified bases recognized in the art. Such bases are generally located at the 1' position of the nucleotide sugar portion. Nucleotides generally comprise a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose.

[0216] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that differ from a reference polynucleotide by the addition, deletion, substitution or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted or modified or replaced with a different nucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0217] As used herein, the statement "sequence identity" or, for example, "a sequence that is 50% identical to the following" refers to the extent to which the sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within a comparison window. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions where the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein.

[0218] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a gene sequence in a vector that can express RNA and subsequently a polypeptide. In one embodiment, the nucleic acid cassette contains one or more genes of interest, for example, one or more polynucleotides of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, for example, a promoter, an enhancer, a poly (A) sequence and one or more genes of interest, for example, one or more polynucleotides of interest. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes. The nucleic acid cassette is positioned and sequentially oriented within the vector so that the nucleic acid in the cassette can be transcribed into RNA, and translated into a protein or polypeptide when necessary, undergoes appropriate post-transcriptional modifications required for activity in a transformed cell, and translocated to a suitable compartment by targeting to a suitable intracellular compartment or secreting to an extracellular compartment to have biological activity. Preferably, the cassette has its 3' end and 5' end adapted to be ready for insertion into a vector, for example, the cassette has a restriction endonuclease site at each end. The cassette can be removed as a single unit and inserted into a plasmid or viral vector.

[0219] Polynucleotides include one or more polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide or a polynucleotide that serves as a template for a transcriptional inhibitory polynucleotide, as contemplated herein.

[0220] As disclosed elsewhere herein or as known in the art, regardless of the length of the coding sequence itself, the polynucleotides contemplated herein can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP sites, FRT sites, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, such that the overall length of the polynucleotides can vary significantly. Thus, it is contemplated that polynucleotide fragments of virtually any length can be employed, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0221] Any of the various mature techniques known and available in the art can be used to prepare, manipulate, express and / or deliver polynucleotides. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0222] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as Sleeping Beauty, PiggyBac.

[0223] Additional illustrative examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.

[0224] Illustrative examples of viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0225] Illustrative examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; the pLenti4 / V5-DEST vector (Promega) for lentivirus-mediated gene transfer and expression in mammalian cells; TM 、pLenti6 / V5-DEST TMand pLenti6.2 / V5-GW / lacZ (Invitrogen). In specific embodiments, the coding sequence of the polypeptide disclosed herein can be ligated into such expression vectors for expressing the polypeptide in mammalian cells.

[0226] The "expression control sequence", "control element" or "regulatory sequence" present in the expression vector is the non-translated region of the vector, including the replication origin, selection box, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), intron, polyadenylation sequence, 5' and 3' non-translated region, all of which interact with host cell proteins for transcription and translation. The strength and specificity of such elements can vary. Depending on the vector system and host utilized, any number of suitable transcription and translation elements can be used, including ubiquitous promoters and inducible promoters.

[0227] In a specific embodiment, the polynucleotide comprises a vector, and the vector includes but is not limited to expression vectors and viral vectors. The vector may include one or more exogenous, endogenous or heterologous control sequences, such as promoters and / or enhancers. "Endogenous control sequences" are sequences naturally connected to a given gene in a genome. "Exogenous control sequences" are control sequences that are placed in juxtaposition with a gene by genetic manipulation (i.e., molecular biotechnology) so that the transcription of the gene is guided by the connected enhancer / promoter. "Heterologous control sequences" are exogenous sequences from different species from cells manipulated by genes. "Synthetic" control sequences may include a variety of endogenous and / or exogenous sequences and / or elements of sequences that provide the best promoter and / or enhancer activity for a particular therapy as determined in vitro or in a computer.

[0228] As used herein, the term "promoter" refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In a specific embodiment, a promoter operable in a mammalian cell comprises an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site and / or another sequence found 70 to 80 bases upstream of the transcription start site: a CNCAAT region, wherein N can be any nucleotide.

[0229] The term "enhancer" refers to a segment of DNA that contains sequences that can provide enhanced transcription and that can function independently of its orientation relative to another control sequence in some cases. An enhancer can function synergistically or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences that can provide both promoter and enhancer functions.

[0230] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship that permits the components to function in their intended manner. In one embodiment, the term refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0231] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that continuously or continually permits transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a wide variety of cell and tissue types or a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in a restricted variety of cell and tissue types, respectively.

[0232] Exemplary ubiquitous expression control sequences suitable for use in specific embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5 promoter and P11 promoter from vaccinia virus, elongation factor 1-alpha (EF1a) promoter, early growth factor promoter, EGR1, ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (β-KIN), human ROSA26 locus (Irions et al., Nature Biotechnology Biotechnology)》25,1477-1482(2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site replaced (MND) U3 promoter (Haas et al. "Journal of Virology" 2003;77(17):9439-9450).

[0233] In one embodiment, the vector comprises the MNDU3 promoter. In one embodiment, the vector comprises the EF1a promoter including the first intron of the human EF1a gene. In one embodiment, the vector comprises the EF1a promoter lacking the first intron of the human EF1a gene.

[0234] In particular embodiments, it may be desirable to use cell-, cell type-, cell lineage-, or tissue-specific expression control sequences to achieve cell type-specific, lineage-specific, or tissue-specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues, or during a particular developmental stage).

[0235] In specific embodiments, it may be desirable to express a polynucleotide under a T cell-specific promoter.

[0236] As used herein, "conditional expression" may refer to any type of conditional expression, including but not limited to inducible expression; repressible expression; expression in cells or tissues having a specific physiological, biological or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, by controlling expression by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that increases or decreases the expression of a polynucleotide encoded by the polynucleotide of interest.

[0237] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferons), the "GeneSwitch" mifepristone regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switch (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducing agents include, but are not limited to, glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0238] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into a cistron (protein coding region) such as an initiation codon such as ATG, thereby allowing the gene to undergo cap-independent translation. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83 and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly used by those skilled in the art include those described in U.S. Pat. No. 6,692,736. Additional examples of "IRES" known in the art include, but are not limited to, IRES obtainable from picornaviruses (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as immunoglobin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2) and insulin-like growth factor (IGFII), translation initiation factor eIF4G and yeast transcription factors TFIID and HAP4, encephalomyocarditis virus (EMCV) commercially available from Novagen (Duke et al., 1992. J. Virol. 66(3):1602-9), and VEGF IRES (Huez et al., 1998. Mol. Cell. Biol. 18(11):6178-90). IRES have been reported in viral genomes of species of the Picornaviridae, Dicistroviridae, and Flaviviridae families, as well as in HCV, Friedreich's murine leukemia virus (FrMLV), and Moloney's murine leukemia virus (MoMLV).

[0239] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0240] In a specific embodiment, the polynucleotide comprises a consensus Kozak sequence. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 198), where R is a purine (A or G) (Kozak, 1986, Cell 44 (2): 283-92 and Kozak, 1987, Nucleic Acids Res. 15 (20): 8125-48).

[0241] The elements that guide the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase the expression of heterologous genes. The transcription termination signal is usually present downstream of the polyadenylation signal. In a specific embodiment, the vector comprises a polyadenylation sequence at the 3' of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that guides the termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, and therefore helps to improve translation efficiency. Cutting and polyadenylation are guided by the poly (A) sequence in RNA. The core poly (A) sequence of mammalian precursor RNA has two recognition elements flanking the cutting-polyadenylation site. Generally, the almost unchanged AAUAAA hexamer is located 20-50 nucleotides upstream of the more variable element rich in U or GU residues. The cutting of the nascent transcript occurs between these two elements and is coupled to up to 250 adenosines added to the 5' cleavage product. In a specific embodiment, the core poly (A) sequence is an ideal poly A sequence (e.g., AATAAA, ATTAAA, AGTAAA). In a specific embodiment, the poly (A) sequence is an SV40 poly A sequence, a bovine growth hormone poly A sequence (BGHpA), a rabbit β-globin poly A sequence (rβgpA), a variant thereof, or another suitable heterologous or endogenous poly A sequence known in the art. In a specific embodiment, the poly (A) sequence is synthetic.

[0242] The polynucleotides encoding one or more polypeptides or fusion polypeptides can be introduced into immune effector cells, e.g., T cells, by both non-viral and viral methods. In a specific embodiment, the delivery of one or more polynucleotides can be provided by the same method or by different methods and / or by the same vector or by different vectors.

[0243] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0244] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art, based on the teachings contemplated herein, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. One of ordinary skill in the art will readily recognize various non-critical parameters that may be changed or modified to produce substantially similar results.

[0245] Examples

[0246] Example 1

[0247] Lentiviral vector purification and screening preparation

[0248] The basic process for formulating lentiviral vectors (LVV) begins with ultrafiltration (UF) and diafiltration (DF) (collectively referred to as UFDF) by tangential flow filtration (TFF), followed by dilution with 2X / 1X stem cell growth medium (SCGM). A hollow fiber membrane with a cutoff of 500 kDa is used to concentrate the purified LVV 4-5 times. The product is then exchanged into a diafiltration buffer of 50 mM HEPES (pH 7.5) and 100 mM NaCl. The resulting material is a UFDF pool. To obtain a control formulated product, the UFDF pool is diluted with 2X / 1X SCGM at a weight ratio of 1:1. The formulated product is then filtered through a 0.22 micron polyethersulfone (PES) membrane to obtain an intermediate body. In order to achieve different formulated products for screening as described herein, the method remains the same as described, except that the diafiltration buffer is changed using different buffers, and the UFDF pool is formulated at a ratio of 1:1 with a 2-fold concentrated formulation solution. After production, approximately 1 mL of the intermediate bulk was filled into 2 mL plastic vials and closed with rubber serum stoppers.

[0249] A broad overview of the screening process / steps and formulation components tested is shown in Figure 1 To determine the optimal formulation, the stability of the intermediate bulk was evaluated under the expected or desired manufacturing conditions, such as storage at 25°C for up to 24 hours and storage at 2-8°C for up to 48 or 168 hours. These conditions were used for screening. Stress conditions were also evaluated to assess formulation robustness, including (i) up to five freeze-thaw cycles, with freezing at ≤-65°C for >1.5 hours and thawing at 30°C for 1.5 hours, or (ii) storage at 37°C for up to 48 hours ( Figure 2 ).

[0250] Example 2

[0251] Analysis of Lentiviral Cryopreserved Formulations

[0252] Stability was assessed by the infection titer measured by viral transduction of HOS cells, the hydrodynamic size measured by dynamic light scattering (DLS), and the number of visible particles observed with a black or white background under fluorescence. The development temperature of some formulations in the formulation was evaluated by differential scanning fluorimetry (DSF). The formulations stored under different temperatures and / or freeze-thaw conditions were compared with initial values ​​or baselines. The CAR expression of the formulations maintaining LVV integrity in PBMCs was further evaluated.

[0253] Infectious titer is used as the main indicator of stability. The infectious titer or the number of transduced units is measured by viral transduction of HOS cells using standard techniques (see, for example, "Gene Therapy" (2002), 9, 1155-1162). The selection criterion is that the recovery rate is higher than 75% (infectious titer after storage / initial infectious titer x 100%).

[0254] Changes in hydrodynamic size measured by dynamic light scattering (DLS) were noted when the size increased by approximately 10 nm or more after storage.

[0255] The number of visible particles was monitored by visual observation under light with an output greater than 2000 lux. Particles can be attributed to impurities such as host cell proteins (HCP) or lentiviral aggregates. The formulation was evaluated to determine whether a particle-free solution could be maintained for an extended period of time. The appearance was monitored to distinguish between intrinsic, internal, and foreign particles after vial filling.

[0256] DSF is used to characterize the conformational stability of lentiviruses to differentiate formulations. A lower thermal unfolding temperature may indicate that the lentivirus is less stable in a particular formulation.

[0257] -Summary of experimental results of buffer species screening

[0258] For buffer selection, the buffers evaluated were HEPES, sodium citrate, PIPES, L-histidine (L-His), sodium phosphate, and Tris at a concentration of 27.5 mM and a pH of 7. These formulations also contained 73 mM trehalose. Based on >75% recovery of infection titers in transduced HOS cells, the HEPES, PIPES, and L-His formulations had minimal changes after storage at 25°C for 24 hours and at 2-8°C for 48 hours ( Figure 3 By hydrodynamic sizing, no size changes were observed for any formulation after storage at 25°C for 24 hours and 2-8°C for 48 hours. After storage at 37°C for 48 hours, no size changes were observed for the HEPES, L-His, and Tris formulations, while there were size changes for the sodium citrate and sodium phosphate formulations ( Figure 4By thermal development measurement results, all buffers have a comparative development onset ranging between 45-47°C ( Figure 5 ). Sodium citrate, sodium phosphate and PIPES appear to have two unfolding transitions, while L-His, HEPES and Tris buffers appear to have one unfolding transition. Based on the first unfolding transition, L-His, HEPES and Tris have the highest thermal unfolding temperatures of 54-57°C, while sodium citrate, sodium phosphate and PIPES have unfolding temperatures of 48-49°C.

[0259] -Summary of pH screening experimental results

[0260] The target pH was assessed by evaluating LVV in 27.5 mM HEPES and 73 mM trehalose at pH 6.5, 7, or 8. The study also evaluated LVV in 27.5 mM PIPES and 73 mM trehalose at pH 6.5 or 7. Minimal changes were observed in HEPES (pH 7), HEPES (pH 8), and PIPES (pH 7) formulations based on >75% recovery of infectious titers in transduced HOS cells after storage at 25°C for 24 hours and 2-8°C for 48 hours ( Figure 6 By hydrodynamic size, the HEPES (pH 8) formulation had an observed change after storage at 25°C for 24 hours and at 2-8°C for 48 hours. All formulations except the HEPES (pH 7) formulation had a size change after storage at 37°C for 48 hours ( Figure 7 By thermal development measurements, HEPES (pH 6.5) and PIPES (pH 6.5) formulations had an earlier onset of development at approximately 40°C ( 2.2 Å) compared to PIPES (pH 7), HEPES (pH 7), and HEPES (pH 8) formulations which had an onset of development between 45-48°C. Figure 8 ). PIPES (pH 7) and HEPES (pH 8) formulations appear to have two unfolding transitions, while the remaining formulations do not. Based on the first unfolding transition, PIPES (pH 6.5) and HEPES (pH 6.5) formulations have the highest unfolding temperatures of approximately 60°C, followed by the HEPES (pH 7) formulation, which has a unfolding temperature of 54°C. Based on the overall evaluation, LVV HEPES (pH 7) performed best, meeting many of the screening criteria.

[0261] -Summary of experimental results of disaccharide and salt screening

[0262] Comparison of carbohydrates trehalose and sucrose in HEPES and PIPES buffers. Based on LVV stability ( Fig. 9), no differences were observed, so only the sucrose combination was also evaluated further.

[0263] 27.5 mM HEPES (pH 7) formulations were compared with 73 mM sucrose and 73 mM sucrose in combination with 75 mM NaCl or KCl. The HEPES-based formulations and the salt formulations were comparable by infection titer results after storage at 25°C for 24 hours and at 2-8°C for 48 hours ( Fig.10 Only the formulations with 73 mM sucrose or with 75 mM NaCl had minimal changes as measured by infection titer measurements through freeze-thaw stress (5 cycles) ( Fig.11 By hydrodynamic size, no changes were observed in HEPES-based formulations after storage at 25°C for 24 hours, 2-8°C for 48 hours, and 37°C for 48 hours ( Fig.12 After five freeze-thaw cycles, no size changes were observed for the HEPES-based formulations ( Fig.13 ). From this study, 27.5 mM HEPES (pH 7) formulations containing 73 mM sucrose with or without 75 mM NaCl were the best formulation choices.

[0264] PIPES-based formulations were further evaluated because they have been reported in the literature to be effective against lentiviruses. 27.5 mM PIPES (pH 6.5) with 73 mM trehalose was compared to 27.5 mM PIPES (pH 6.5) with 73 mM sucrose and 75 mM NaCl. In addition, 27.5 mM PIPES (pH 7) with 73 mM sucrose or 73 mM sucrose in combination with 75 mM NaCl or 75 mM KCl was evaluated. Based on >80% recovery of infection titers in transduced HOS cells, PIPES (pH 6.5) formulations with 73 mM sucrose and 75 mM NaCl had minimal changes after storage at 25°C for 24 hours and 2-8°C for 48 hours ( Fig.10 After freeze-thaw (5 cycles), the PIPES (pH 6.5) formulation with 73 mM sucrose and 75 mM NaCl had the highest recovery of infectious titers ( Fig.11 By hydrodynamic size, no changes were observed for PIPES-based formulations after storage at 25°C for 24 hours and at 2-8°C for 48 hours. Size changes were observed for all PIPES-based formulations after storage at 37°C for 48 hours ( Fig.12 After five freeze-thaw cycles, only the PIPES (pH 6.5) formulation with 73 mM sucrose and 75 mM NaCl increased in size ( Fig.13 ).

[0265] Based on a comparison of PIPES- and HEPES-based formulations performed at similar pH, LVV was surprisingly more stable in HEPES-based buffers, particularly as assessed by particle size and titer after freeze-thaw under various conditions.

[0266] -Summary of experimental results of additional component screening

[0267] Screening studies were completed with several additional buffer and sucrose compositions to see if the additional compositions improved LVV stability at 5°C at 168 hours and by monitoring the number of visible particles with longer temperature hold durations.

[0268] The formulation contained 27.5 mM HEPES (pH 7) and the following:

[0269] -73mM sucrose and 75mM NaCl

[0270] -73mM sucrose and 75mM NaCl and 0.1mg / mL P188

[0271] -73mM sucrose and 50mM L-Pro

[0272] -146mM sucrose

[0273] -146 mM sucrose and 75 mM NaCl

[0274] -146 mM sucrose and 50 mM L-Arg

[0275] -146mM sucrose and 50mM L-Arg and 50mM L-Glu

[0276] -146 mM sucrose and 0.1 mg / mL P188

[0277] -146mM sucrose and 16mg / mL inulin

[0278] The following formulations were also evaluated with 27.5 mM L-His (pH 7):

[0279] -146mM sucrose

[0280] -146mM sucrose and 50mM L-Pro

[0281] -146mM sucrose and 16mg / mL inulin

[0282] -275mM sorbitol

[0283] -73mM sucrose and 75mM NaCl

[0284] -73mM sucrose and 50mM L-Pro

[0285] -73mM sucrose and 75mM NaCl and 0.1mg / mL P188

[0286] All 27.5 mM HEPES formulations (pH 7) composed of 146 mM sucrose performed poorly after storage at 25°C for 24 hours and at 2-8°C for 168 hours as measured by infectious titers ( Fig.14 ). 27.5 mM HEPES (pH 7) and 73 mM sucrose formulations with 75 mM NaCl, 75 mM NaCl and 0.1 mg / mL P188 or 50 mM L-proline had the highest titer recovery at different storage temperatures ( Fig.14 After storage at 2-8°C for 168 hours, the hydrodynamic size of all formulations remained unchanged ( Fig.15 ). Finally, comparing only the 73 mM sucrose based formulations, the least number of visible particles was observed for the formulations containing 0.1 mg / mL poloxamer 188 and 50 mM L-proline after storage at 2-8°C. Similarly, formulations 27.5 mM HEPES (pH 7) with 73 mM sucrose, 75 mM NaCl and 0.1 mg / mL poloxamer 188 or 73 mM sucrose and 50 mM L-proline had high titer recovery, no change in hydrodynamic size, and very few visible particles after freezing for greater than 1.5 hours at ≤-65°C and thawing for 1.5 hours at 30°C. ( Figure 16-19 In a similar evaluation, a 27.5 mM L-histidine (pH 7) formulation with 73 mM sucrose and 50 mM L-Pro showed the highest recovery of infectious titers after 24 h storage at 25°C, 168 h storage at 2-8°C, and after one freeze-thaw cycle, with no change in hydrodynamic size and the lowest number of visible particles ( Figure 20-25 ).

[0287] 27.5 mM HEPES (pH 7), 73 mM sucrose and 50 mM L-Pro formulations were evaluated in multiple lentiviral vectors (e.g., LVV1, LVV2, LVV3, LVV4 and LVV5) encoding various constructs targeting different genes of interest. Titer recovery after storage at 2-8°C for up to 168 hours ( Fig.26 ). The analysis was grouped by retention time of 24, 120 and 168 hours. A one-way ANOVA was performed followed by a figure-Kramer test analysis. The p-values ​​comparing the mean titer recovery rates were not significant (greater than 0.05) and indicated that the formulations of each LVV performed similarly.

[0288] Additional evaluations were performed with Poloxamer 188 to reduce the number of visible particles sometimes observed after preparation. Formulations with only 27.5 mM HEPES (pH 7), 73 mM sucrose, and 50 mM L-Pro and with the addition of 0.08 g / L, 0.3 g / L, or 0.8 g / L Poloxamer 188 were visually compared after storage at 2-8°C ( Fig. 27 and 28 ). Addition of at least 0.3 g / L reduced the appearance of particles after 24 hours and minimized the type of particles after 120 hours of storage compared to formulations without P188 and with 0.08% P188. The infectious titer of LVV formulated with 27.5 mM HEPES (pH 7), 73 mM sucrose, 50 mM L-Pro and 0.3 g / L P188 was monitored for 168 hours at 2-8°C and compared to formulations without P188 ( Fig.29 ). Both formulations had similar performance, indicating that the addition of P188 did not affect the recovery rate of infectious titers.

[0289] The final concern was long term storage of LVV at ≤ -65°C. Formulations with and without P188 were compared for LVV1, LVV2, and LVV3 at ≤ -65°C for up to 6 months. All LVVs and formulations maintained infectious titer levels for up to 6 months of storage ( Fig.30 ).

[0290] Example 3

[0291] Lentiviral efficacy analysis

[0292] The efficacy of slow virus is measured in peripheral blood mononuclear cells (PBMC).PBMC is transduced with slow viruses of various infection multiplicities (MOI of 2.5, 5, 10 and 20), and the relative frequency (% CAR+ cells) of chimeric antigen receptor expression is measured.CAR+ is detected by fluorescently labeled proteins combined with CAR, and cell colonies are evaluated by flow cytometry.Relative efficacy is reported as relative measurement results relative to reference standards.

[0293] The following formulations had the highest recovery of infectious titers after storage at 25°C, 2-8°C, and multiple freeze-thaw cycles associated with the lentiviral preparation process. As a confirmation of compatibility with the drug product process, relative potency measurements were performed and confirmed that there was no significant impact on potency (expression of CAR+) and stable LVV ( Fig.31 ).

[0294] -27.5 mM HEPES (pH 7), 73 mM sucrose and 75 mM NaCl

[0295] -27.5 mM HEPES (pH 7), 73 mM sucrose, 75 mM NaCl and 0.1 mg / mL P188

[0296] -27.5 mM HEPES (pH 7), 73 mM sucrose and 50 mM L-Pro

[0297] -27.5 mM L-His (pH 7), 73 mM sucrose and 50 mM L-Pro

[0298] The top formulations evaluated here meet the standards required for the manufacturing process. The extended duration at 25°C and 2-8°C allows flexibility for many manufacturing operations, and the combination of HEPES (pH 7) and L-His (pH 7) and sucrose appears to maintain stability. In addition, the freeze-thaw process significantly reduces the recovery rate of lentiviral vectors. Studies have shown that in some cases, the addition of sodium chloride or L-proline improves the recovery rate after a single freeze-thaw and sometimes up to five freeze-thaw cycles. Finally, during the filling completion process, the reduction of visible particles can indicate the aggregation of particles in the solution. Poloxamer 188 and L-proline appear to have minimized these interactions.

[0299] Example

[0300] The present disclosure contemplates the following embodiments:

[0301] 1. An aqueous virus composition comprising:

[0302] a) Viral vectors

[0303] b) HEPES or L-histidine buffer;

[0304] c) carbohydrates; and

[0305] d) Amino acids.

[0306] 2. The composition of embodiment 1, wherein the buffer is present at a concentration of about 25 mM to about 30 mM.

[0307] 3. The composition of any preceding embodiment, wherein the buffer is present at a concentration of about 26 mM to about 29 mM.

[0308] 4. The composition of any preceding embodiment, wherein the buffer is present at a concentration of about 27 mM to about 28 mM.

[0309] 5. The composition of any preceding embodiment, wherein the buffer is present at a concentration of about 27.5 mM.

[0310] 6. The composition of any one of embodiments 1 to 5, wherein the buffer is a HEPES buffer.

[0311] 7. The composition of any one of embodiments 1 to 5, wherein the buffer is an L-histidine buffer.

[0312] 8. The composition of any preceding embodiment, wherein the carbohydrate is present at a concentration of about 66 mM to about 80 mM.

[0313] 9. The composition of any preceding embodiment, wherein the carbohydrate is present at a concentration of about 67 mM to about 79 mM.

[0314] 10. The composition of any preceding embodiment, wherein the carbohydrate is present at a concentration of about 68 mM to about 78 mM.

[0315] 11. The composition of any preceding embodiment, wherein the carbohydrate is present at a concentration of about 69 mM to about 77 mM.

[0316] 12. The composition of any preceding embodiment, wherein the carbohydrate is present at a concentration of about 70 mM to about 76 mM.

[0317] 13. The composition of any preceding embodiment, wherein the carbohydrate is present at a concentration of about 71 mM to about 75 mM.

[0318] 14. The composition of any preceding embodiment, wherein the carbohydrate is present at a concentration of about 72 mM to about 74 mM.

[0319] 15. The composition of any preceding embodiment, wherein the carbohydrate is present at a concentration of about 73 mM.

[0320] 16. The composition of any one of embodiments 1 to 12 wherein the carbohydrate is present at a concentration of about 2.0% to about 3.0% by weight per volume of the composition.

[0321] 17. The composition of any one of embodiments 1 to 12 wherein the carbohydrate is present at a concentration of about 2.1% to about 2.9% by weight per volume of the composition.

[0322] 18. The composition of any one of embodiments 1 to 12 wherein the carbohydrate is present at a concentration of about 2.2% to about 2.8% by weight per volume of the composition.

[0323] 19. The composition of any one of embodiments 1 to 12 wherein the carbohydrate is present at a concentration of about 2.3% to about 2.7% by weight per volume of the composition.

[0324] 20. The composition of any one of embodiments 1 to 12 wherein the carbohydrate is present at a concentration of about 2.4% to about 2.6% by weight per volume of the composition.

[0325] 21. The composition of any one of embodiments 1 to 12 wherein the carbohydrate is present at a concentration of about 2.5% by weight per volume of the composition.

[0326] 22. The composition of any one of embodiments 1 to 21, wherein the carbohydrate is a disaccharide.

[0327] 23. The composition of any one of embodiments 1 to 21, wherein the carbohydrate is lactose, glucose, mannose, mannitol, sorbitol, sucrose, trehalose, and / or glycerol.

[0328] 24. The composition of any one of embodiments 1 to 21, wherein the carbohydrate is sucrose and / or trehalose.

[0329] 25. The composition of any one of embodiments 1 to 24, wherein the carbohydrate is sucrose.

[0330] 26. The composition of any one of embodiments 1 to 24, wherein the carbohydrate is trehalose.

[0331] 27. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 40 mM to about 60 mM.

[0332] 28. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 41 mM to about 59 mM.

[0333] 29. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 42 mM to about 58 mM.

[0334] 30. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 43 mM to about 57 mM.

[0335] 31. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 44 mM to about 56 mM.

[0336] 32. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 45 mM to about 55 mM.

[0337] 33. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 46 mM to about 54 mM.

[0338] 34. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 47 mM to about 53 mM.

[0339] 35. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 48 mM to about 52 mM.

[0340] 36. The composition of any one of embodiments 1 to 26, wherein the amino acid is present at a concentration of about 49 mM to about 51 mM.

[0341] 37. The composition of any one of embodiments 1 to 26, wherein said amino acid is present at a concentration of about 50 mM.

[0342] 38. The composition of any one of embodiments 1 to 37, wherein the amino acid is a non-polar amino acid.

[0343] 39. The composition of any one of embodiments 1 to 37, wherein the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, methionine, isoleucine phenylalanine, tyrosine, and tryptophan.

[0344] 40. The composition of any one of embodiments 1 to 37, wherein said amino acid is selected from the group consisting of phenylalanine, tyrosine, tryptophan, and proline.

[0345] 41. The composition of any one of embodiments 1-37, wherein the amino acid is L-proline.

[0346] 42. The composition of any preceding embodiment, wherein the composition further comprises a salt.

[0347] 43. The composition of embodiment 42, wherein the salt is present at a concentration of about 65 mM to about 85 mM.

[0348] 44. The composition of embodiment 42, wherein the salt is present at a concentration of about 66 mM to about 84 mM.

[0349] 45. The composition of embodiment 42, wherein the salt is present at a concentration of about 67 mM to about 83 mM.

[0350] 46. ​​The composition of embodiment 42, wherein the salt is present at a concentration of about 68 mM to about 82 mM.

[0351] 47. The composition of embodiment 42, wherein the salt is present at a concentration of about 69 mM to about 81 mM.

[0352] 48. The composition of embodiment 42, wherein the salt is present at a concentration of about 70 mM to about 80 mM.

[0353] 49. The composition of embodiment 42, wherein the salt is present at a concentration of about 71 mM to about 79 mM.

[0354] 50. The composition of embodiment 42, wherein the salt is present at a concentration of about 72 mM to about 78 mM.

[0355] 51. The composition of embodiment 42, wherein the salt is present at a concentration of about 73 mM to about 77 mM.

[0356] 52. The composition of embodiment 42, wherein the salt is present at a concentration of about 74 mM to about 76 mM.

[0357] 53. The composition of embodiment 42, wherein the salt is present at a concentration of about 75 mM.

[0358] 54. The composition of any one of embodiments 42-53, wherein the salt is a chloride salt, KCl, or NaCl.

[0359] 55. The composition of any one of embodiments 42-54, wherein the salt is NaCl.

[0360] 56. The composition of any preceding embodiment, wherein the composition further comprises a poloxamer.

[0361] 57. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.01 mg / ml to about 1 mg / ml.

[0362] 58. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.02 mg / ml to about 0.9 mg / ml.

[0363] 59. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.03 mg / ml to about 0.8 mg / ml.

[0364] 60. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.04 mg / ml to about 0.7 mg / ml.

[0365] 61. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.05 mg / ml to about 0.6 mg / ml.

[0366] 62. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.06 mg / ml to about 0.5 mg / ml.

[0367] 63. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.07 mg / ml to about 0.4 mg / ml.

[0368] 64. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.08 mg / ml to about 0.3 mg / ml.

[0369] 65. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.09 mg / ml to about 0.2 mg / ml.

[0370] 66. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.1 mg / ml.

[0371] 67. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.1 mg / ml to about 0.5 mg / ml.

[0372] 68. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.2 mg / ml to about 0.4 mg / ml.

[0373] 69. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.3 mg / ml.

[0374] 70. The composition of any one of embodiments 56 to 69, wherein the poloxamer is poloxamer 188, poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407.

[0375] 71. The composition of any one of embodiments 56 to 70, wherein the poloxamer is poloxamer 188.

[0376] 72. The composition of any preceding embodiment, wherein the composition comprises a pH of about 6.5 to about 8.

[0377] 73. The composition of any preceding embodiment, wherein the composition comprises a pH of about 6.5.

[0378] 74. The composition of any preceding embodiment, wherein the composition comprises a pH of about 7.

[0379] 75. The composition of any preceding embodiment, wherein the composition comprises a pH of about 7.5.

[0380] 76. The composition of any preceding embodiment, wherein the composition comprises a pH of about 8.

[0381] 77. An aqueous viral composition comprising:

[0382] a) viral vectors,

[0383] b) about 27.5 mM HEPES,

[0384] c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition, and

[0385] d) about 50 mM L-proline; wherein the composition comprises a pH of about 7.

[0386] 78. An aqueous viral composition comprising:

[0387] a) viral vectors,

[0388] b) about 27.5 mM L-histidine,

[0389] c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition, and

[0390] d) about 50 mM L-proline;

[0391] wherein the composition comprises a pH of about 7.

[0392] 79. An aqueous viral composition comprising:

[0393] a) viral vectors,

[0394] b) about 27.5 mM HEPES,

[0395] c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition,

[0396] d) about 50 mM L-proline, and

[0397] e) about 0.1 mg / mL to about 0.8 mg / mL poloxamer 188;

[0398] wherein the composition comprises a pH of about 7.

[0399] 80. An aqueous viral composition comprising:

[0400] a) viral vectors,

[0401] b) about 27.5 mM HEPES,

[0402] c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition,

[0403] d) about 50 mM L-proline, and

[0404] e) about 0.3 mg / mL poloxamer 188;

[0405] wherein the composition comprises a pH of about 7.

[0406] 81. An aqueous viral composition comprising:

[0407] a) viral vectors,

[0408] b) about 27.5 mM HEPES,

[0409] c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition,

[0410] d) about 50 mM L-proline, and

[0411] e) about 75 mM NaCl;

[0412] wherein the composition comprises a pH of about 7.

[0413] 82. An aqueous viral composition comprising:

[0414] a) viral vectors,

[0415] b) about 27.5 mM HEPES,

[0416] c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition,

[0417] d) about 50 mM L-proline,

[0418] e) about 75 mM NaCl, and

[0419] f) about 0.1 to about 0.8 mg / mL Poloxamer 188;

[0420] wherein the composition comprises a pH of about 7.

[0421] 83. An aqueous viral composition comprising:

[0422] a) viral vectors,

[0423] b) HEPES buffer,

[0424] c) carbohydrates,

[0425] d) salt; and

[0426] e) Poloxamers.

[0427] 84. The composition of embodiment 83, wherein the composition does not comprise L-proline.

[0428] 85. The composition of Embodiment 83 or Embodiment 84, wherein the HEPES buffer is present at a concentration of about 25 mM to about 30 mM.

[0429] 86. The composition of Embodiment 83 or Embodiment 84, wherein the HEPES buffer is present at a concentration of about 26 mM to about 29 mM.

[0430] 87. The composition of Embodiment 83 or Embodiment 84, wherein the HEPES buffer is present at a concentration of about 27 mM to about 28 mM.

[0431] 88. The composition of Embodiment 83 or Embodiment 84, wherein the HEPES buffer is present at a concentration of about 27.5 mM.

[0432] 89. The composition of any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 66 mM to about 80 mM.

[0433] 90. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 67 mM to about 79 mM.

[0434] 91. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 68 mM to about 78 mM.

[0435] 92. The composition of any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 69 mM to about 77 mM.

[0436] 93. The composition of any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 70 mM to about 76 mM.

[0437] 94. The composition of any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 71 mM to about 75 mM.

[0438] 95. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 72 mM to about 74 mM.

[0439] 96. The composition of any one of embodiments 83-88, wherein said carbohydrate is present at a concentration of about 73 mM.

[0440] 97. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 2.0% to about 3.0% by weight per volume of the composition.

[0441] 98. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 2.1% to about 2.9% by weight per volume of the composition.

[0442] 99. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 2.2% to about 2.8% by weight per volume of the composition.

[0443] 100. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 2.3% to about 2.7% by weight per volume of the composition.

[0444] 101. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 2.4% to about 2.6% by weight per volume of the composition.

[0445] 102. The composition of any one of embodiments 83-88, wherein the carbohydrate is present at a concentration of about 2.5% by weight per volume of the composition.

[0446] 103. The composition of any one of embodiments 83 to 102, wherein the carbohydrate is a disaccharide.

[0447] 104. The composition of any one of embodiments 83 to 102, wherein the carbohydrate is lactose, glucose, mannose, mannitol, sorbitol, sucrose, trehalose and / or glycerol.

[0448] 105. The composition of any one of embodiments 83 to 102, wherein the carbohydrate is sucrose and / or trehalose.

[0449] 106. The composition of any one of embodiments 83-102, wherein said carbohydrate is sucrose.

[0450] 107. The composition of any one of embodiments 83-102, wherein said carbohydrate is trehalose.

[0451] 108. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 65 mM to about 85 mM.

[0452] 109. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 66 mM to about 84 mM.

[0453] 110. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 67 mM to about 83 mM.

[0454] 111. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 68 mM to about 82 mM.

[0455] 112. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 69 mM to about 81 mM.

[0456] 113. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 70 mM to about 80 mM.

[0457] 114. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 71 mM to about 79 mM.

[0458] 115. The composition of any one of embodiments 83-107, wherein said salt is present at a concentration of about 72 mM to about 78 mM.

[0459] 116. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 73 mM to about 77 mM.

[0460] 117. The composition of any one of embodiments 83 to 107, wherein said salt is present at a concentration of about 74 mM to about 76 mM.

[0461] 118. The composition of any one of embodiments 83-107, wherein said salt is present at a concentration of about 75 mM.

[0462] 119. The composition of any one of embodiments 83-118, wherein the salt is KCl or NaCl.

[0463] 120. The composition of embodiment 119, wherein the salt is NaCl.

[0464] 121. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.01 mg / ml to about 1 mg / ml.

[0465] 122. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.02 mg / ml to about 0.9 mg / ml.

[0466] 123. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.03 mg / ml to about 0.8 mg / ml.

[0467] 124. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.04 mg / ml to about 0.7 mg / ml.

[0468] 125. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.05 mg / ml to about 0.6 mg / ml.

[0469] 126. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.06 mg / ml to about 0.5 mg / ml.

[0470] 127. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.07 mg / ml to about 0.4 mg / ml.

[0471] 128. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.08 mg / ml to about 0.3 mg / ml.

[0472] 129. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.09 mg / ml to about 0.2 mg / ml.

[0473] 130. The composition of any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.1 mg / ml.

[0474] 131. The composition of any one of embodiments 83 to 130, wherein the poloxamer is poloxamer 188, poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407.

[0475] 132. The composition of any one of embodiments 83 to 130, wherein the poloxamer is poloxamer 188 (P188).

[0476] 133. The composition of any one of embodiments 83 to 132, wherein the composition comprises a pH of about 7 to about 8.

[0477] 134. The composition of any one of embodiments 83-132, wherein the composition comprises a pH of about 7.

[0478] 135. The composition of any one of embodiments 83-132, wherein said composition comprises a pH of about 7.5.

[0479] 136. The composition of any one of embodiments 83-132, wherein said composition comprises a pH of about 8.

[0480] 137. An aqueous viral composition comprising:

[0481] a) viral vectors,

[0482] b) about 27.5 mM HEPES,

[0483] c) about 73 mM sucrose by weight per volume of the composition,

[0484] d) about 75 mM NaCl, and

[0485] e) about 0.1 to about 0.8 mg / ml Poloxamer 188;

[0486] wherein the composition comprises a pH of about 7.

[0487] 138. The composition according to any one of the preceding embodiments, wherein the viral vector is present in an amount of about 1 x 10 8 About 2x 10 9 Titers of TU / ml are present.

[0488] 139. The composition according to any one of the preceding embodiments, wherein the viral vector is present in an amount of about 1 x 10 8 Titers of TU / ml are present.

[0489] 140. The composition according to any one of the preceding embodiments, wherein the viral vector is present in an amount of about 2 x 10 8Titers of TU / ml are present.

[0490] 141. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 3 x 10 8 Titers of TU / ml are present.

[0491] 142. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 4 x 10 8 Titers of TU / ml are present.

[0492] 143. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 5 x 10 8 Titers of TU / ml are present.

[0493] 144. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 6 x 10 8 Titers of TU / ml are present.

[0494] 145. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 7 x 10 8 Titers of TU / ml are present.

[0495] 146. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 8 x 10 8 Titers of TU / ml are present.

[0496] 147. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 9 x 10 8 Titers of TU / ml are present.

[0497] 148. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 1 x 10 9 Titers of TU / ml are present.

[0498] 149. The composition of any one of embodiments 1 to 139, wherein the viral vector is present in an amount of about 2 x 10 9 Titers of TU / ml are present.

[0499] 150. The composition of any one of the preceding embodiments, wherein the vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral vector.

[0500] 151. The composition of any preceding embodiment, wherein the viral vector is a lentiviral vector.

[0501] 152. A composition according to embodiment 151, wherein the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), Vesnamedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0502] 153. The composition of embodiment 151 or embodiment 152, wherein the lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1) or human immunodeficiency virus 2 (HIV-2).

[0503] 154. The composition of any one of embodiments 151 to 153, wherein the lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1).

[0504] 155. The composition of any preceding embodiment, wherein the viral vector is pseudotyped.

[0505] 156. The composition of any preceding embodiment, wherein the viral vector is pseudotyped with an envelope protein from a strain of vesicular stomatitis virus.

[0506] 157. The composition of embodiment 156, wherein the strain of vesicular stomatitis virus is selected from the group consisting of Indiana, Alagoas, New Jersey, Isfahan, Kokar, Malaba, or Pirie.

[0507] 158. The composition of any preceding embodiment, wherein the viral vector is pseudotyped with vesicular stomatitis virus G (VSV-G) protein.

[0508] 159. The composition of any of the preceding embodiments, wherein the viral vector is pseudotyped with an envelope derived from: measles envelope protein, Sindbis envelope protein, measles virus F and H proteins, Sendai virus F and HN proteins, or Paramyxoviridae F and H proteins.

[0509] 160. The composition of any preceding embodiment, wherein the viral vector comprises a polynucleotide comprising a transgene.

[0510] 161. The composition of embodiment 160, wherein said transgene encodes a therapeutic protein.

[0511] 162. The composition of embodiment 160 or embodiment 161, wherein the transgene or therapeutic protein is used to treat a monogenic disease, disorder, or condition.

[0512] 163. A composition according to any one of embodiments 160 to 162, wherein the transgene or therapeutic protein is a chimeric antigen receptor (CAR), a chimeric co-stimulatory receptor (CCR), an αβT cell receptor (αβ-TCR), a γδT cell receptor (γδ-TCR), a dimerizer-regulated immune receptor complex (DARIC), or a switch receptor.

[0513] 164. The composition of any one of embodiments 160 to 162, wherein the transgene or therapeutic protein is a therapeutic globin for treating a hemoglobinopathy or an ABCD1 gene for treating CALD.

[0514] 165. The composition of any preceding embodiment, wherein the composition does not comprise serum.

[0515] 166. The composition of any preceding embodiment, wherein the composition does not comprise PIPES.

[0516] 167. The composition of any preceding embodiment, wherein the composition does not comprise sodium citrate.

[0517] 168. The composition of any preceding embodiment, wherein the composition does not comprise sodium phosphate.

[0518] 169. The composition of any preceding embodiment, wherein the composition does not comprise Tris.

[0519] 170. The composition of any preceding embodiment, wherein the composition does not comprise salt.

[0520] 171. The composition of any preceding embodiment, wherein the composition does not comprise chloride salts.

[0521] 172. The composition of any preceding embodiment, wherein the composition does not comprise NaCl.

[0522] 173. The composition of any preceding embodiment, wherein the composition does not comprise KCl.

[0523] 174. The composition of any preceding embodiment, wherein the composition does not comprise trehalose.

[0524] 175. A composition according to any of the preceding embodiments, wherein the viral vector maintains an infectious titer recovery rate of greater than about 75% in HOS cells after storage relative to the infectious titer of the viral vector in the composition before storage or at least one freeze-thaw cycle.

[0525] 176. A composition according to any of the preceding embodiments, wherein the viral vector maintains an infectious titer recovery rate of greater than about 80% in HOS cells after storage relative to the infectious titer of the viral vector in the composition before storage or at least one freeze-thaw cycle.

[0526] 177. A composition according to any of the preceding embodiments, wherein the viral vector maintains an infectious titer recovery rate of greater than about 85% in HOS cells after storage relative to the infectious titer of the viral vector in the composition before storage or at least one freeze-thaw cycle.

[0527] 178. A composition according to any of the preceding embodiments, wherein the viral vector maintains an infectious titer recovery rate of greater than about 90% in HOS cells after storage relative to the infectious titer of the viral vector in the composition before storage or at least one freeze-thaw cycle.

[0528] 179. A composition according to any of the preceding embodiments, wherein the viral vector maintains an infectious titer recovery rate of greater than about 95% in HOS cells after storage relative to the infectious titer of the viral vector in the composition before storage or at least one freeze-thaw cycle; relative to the infectious titer of the viral vector in the composition before storage or at least one freeze-thaw cycle.

[0529] 180. The composition of any one of the preceding embodiments, wherein the viral vector has a thermal unfolding temperature of about 56° to about 62°C as measured by differential scanning fluorimetry (DSF).

[0530] 181. The composition of any of the preceding embodiments, wherein the viral vector has a thermal unfolding temperature of about 58° to about 60°C as measured by differential scanning fluorimetry (DSF).

[0531] 182. A composition according to any of the preceding embodiments, wherein the viral vector maintains a hydrodynamic diameter of about 150 nm to about 170 nm after storage relative to the hydrodynamic diameter of the viral vector in the composition before storage or at least one freeze-thaw cycle, as measured by dynamic light scattering (DLS) at 25°C and a viscosity value of 0.967 centipoise (cP).

[0532] 183. The composition of any of the preceding embodiments, wherein the viral vector maintains at least about 78% potency after storage compared to a reference standard as measured by transgene expression in PBMCs.

[0533] 184. The composition of any of the preceding embodiments, wherein the viral vector maintains at least about 80% potency after storage compared to a reference standard as measured by transgene expression in PBMCs.

[0534] 185. The composition of any of the preceding embodiments, wherein the viral vector maintains at least about 85% potency after storage compared to a reference standard as measured by transgene expression in PBMCs.

[0535] 186. The composition of any of the preceding embodiments, wherein the viral vector maintains at least about 90% potency after storage compared to a reference standard as measured by transgene expression in PBMCs.

[0536] 187. The composition of any of the preceding embodiments, wherein the viral vector maintains at least about 95% potency after storage compared to a reference standard as measured by transgene expression in PBMCs.

[0537] 188. The composition of any preceding embodiment, wherein no fiber particles (bundled fibers) are visible after storage.

[0538] 189. The composition of any preceding embodiment, wherein 5 or fewer visible particles (or microparticles) are present after storage.

[0539] 190. The composition of any preceding embodiment, wherein 4 or fewer visible particles (or microparticles) are present after storage.

[0540] 191. The composition of any preceding embodiment, wherein there are 3 or fewer visible particles (or microparticles) after storage.

[0541] 192. The composition of any preceding embodiment, wherein 2 or fewer visible particles (or microparticles) are present after storage.

[0542] 193. The composition of any preceding embodiment, wherein 1 or fewer visible particles (or microparticles) are present after storage.

[0543] 194. The composition of any preceding embodiment, wherein no visible particles are present in the composition after storage.

[0544] 195. The composition of any preceding embodiment, wherein no visible fiber particles (bundled fibers) are present per 50 ml of the composition after storage.

[0545] 196. The composition of any preceding embodiment, wherein there are 5 or fewer visible particles (or microparticles) per 50 ml of the composition after storage.

[0546] 197. The composition of any preceding embodiment, wherein there are 4 or fewer visible particles (or microparticles) per 50 ml of the composition after storage.

[0547] 198. The composition of any preceding embodiment, wherein there are 3 or fewer visible particles (or microparticles) per 50 ml of the composition after storage.

[0548] 199. The composition of any preceding embodiment, wherein after storage there are 2 or fewer visible particles (or microparticles) per 50 ml of the composition.

[0549] 200. The composition of any preceding embodiment, wherein after storage there are 1 or fewer visible particles (or microparticles) per 50 ml of the composition.

[0550] 201. The composition of any preceding embodiment, wherein no visible particles are present per 50 ml of the composition after storage.

[0551] 202. The composition of any preceding embodiment, wherein no visible fiber particles (bundled fibers) are present per 36 ml of the composition after storage.

[0552] 203. The composition of any preceding embodiment, wherein after storage there are 5 or fewer visible particles (or microparticles) per 36 ml of the composition.

[0553] 204. The composition of any preceding embodiment, wherein after storage there are 4 or fewer visible particles (or microparticles) per 36 ml of the composition.

[0554] 205. The composition of any preceding embodiment, wherein after storage there are 3 or fewer visible particles (or microparticles) per 36 ml of the composition.

[0555] 206. The composition of any preceding embodiment, wherein after storage there are 2 or fewer visible particles (or microparticles) per 36 ml of the composition.

[0556] 207. The composition of any preceding embodiment, wherein after storage there are 1 or fewer visible particles (or microparticles) per 36 ml of the composition.

[0557] 208. The composition of any preceding embodiment, wherein no visible particles are present per 36 ml of the composition after storage.

[0558] 209. The composition of any one of embodiments 175 to 208, wherein the storage is at 25°C, 2-8°C, or 37°C.

[0559] 210. The composition of any one of embodiments 175-209, wherein the storage is for 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours or longer.

[0560] 211. The composition of any one of embodiments 175 to 210, wherein said storage comprises at least 1 freeze-thaw cycle.

[0561] 212. The composition of embodiment 211, wherein the at least 1 freeze-thaw cycle is 1 freeze-thaw cycle.

[0562] 213. The composition of embodiment 211, wherein the at least 1 freeze-thaw cycle is 2 freeze-thaw cycles.

[0563] 214. The composition of embodiment 211, wherein the at least 1 freeze-thaw cycle is 3 freeze-thaw cycles.

[0564] 215. The composition of embodiment 211, wherein the at least 1 freeze-thaw cycle is 4 freeze-thaw cycles.

[0565] 216. The composition of embodiment 211, wherein the at least 1 freeze-thaw cycle is 5 freeze-thaw cycles.

[0566] 217. The composition of any one of embodiments 211 to 216, wherein the one or more freeze-thaw cycles comprises freezing the composition at about -65°C or less for about 1.5 hours or more, and thawing at 30°C for 1.5 hours.

[0567] 218. The composition of any preceding embodiment, wherein the composition is frozen.

[0568] 219. A method for storing a viral vector, the method comprising:

[0569] a) providing a composition according to any one of embodiments 1 to 218, and

[0570] b) storing the viral composition at about 25°C or below.

[0571] 220. A method for storing a viral vector, the method comprising:

[0572] a) providing a composition according to any one of embodiments 1 to 218, and

[0573] b) storing the viral composition at about 2-8°C or lower.

[0574] 221. A method for cryopreservation of a viral vector, the method comprising:

[0575] a) providing a composition according to any one of embodiments 1 to 218,

[0576] b) freezing the virus composition, and

[0577] c) storing the viral composition at a temperature of about 0°C or below.

[0578] 222. The method of any one of embodiments 219 to 221, wherein said method comprises storing said viral composition for at least about 24 hours.

[0579] 223. The method of any one of embodiments 219 to 222, wherein said method comprises storing said viral composition for at least about 48 hours.

[0580] 224. The method of any one of embodiments 219 to 223, wherein said method comprises storing said viral composition for at least about 72 hours.

[0581] 225. The method of any one of embodiments 219 to 224, wherein said method comprises storing said viral composition for at least about 96 hours.

[0582] 226. The method of any one of embodiments 219 to 225, wherein said method comprises storing said viral composition for at least about 120 hours.

[0583] 227. The method of any one of embodiments 219 to 226, wherein said method comprises storing said viral composition for at least about 148 hours.

[0584] 228. The method of any one of embodiments 219 to 227, wherein said method comprises storing said viral composition for at least about 168 hours.

[0585] 229. A method of expressing a transgene in a cell, the method comprising contacting the cell with the composition of any one of embodiments 1-217.

[0586] 230. The method of embodiment 229, wherein the cell is a mammalian cell.

[0587] 231. The method of embodiment 229 or embodiment 230, wherein the cells are hematopoietic cells.

[0588] 232. The method of any one of embodiments 229 to 231, wherein the cell is an immune effector cell.

[0589] 233. The method of any one of embodiments 229 to 232, wherein the cell is a CD3 + 、CD4 + and / or CD8 + cell.

[0590] 234. The method of any one of embodiments 229 to 233, wherein the cell is a T cell.

[0591] 235. A method according to any one of embodiments 229 to 234, wherein the cell is an αβT cell.

[0592] 236. A method according to any one of embodiments 229 to 234, wherein the cell is a γδT cell.

[0593] 237. A method according to any one of embodiments 229 to 234, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

[0594] 238. The method of any one of embodiments 229 to 234, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0595] 239. A method according to any one of embodiments 229 to 231, wherein the cell is a hematopoietic stem cell or progenitor cell.

[0596] 240. According to the method according to any one of embodiments 229 to 231, the cell is a human CD34+ hematopoietic stem cell or progenitor cell.

[0597] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments contemplated in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by the present disclosure.

Claims

1. An aqueous virus composition comprising: a) Lentiviral vector; b) HEPES or L-histidine buffer; c) sucrose; and d) L-proline.

2. The composition of claim 1, wherein the buffer is present at a concentration of about 25 mM to about 30 mM.

3. The composition of any one of the preceding claims, wherein the buffer is present at a concentration of about 27.5 mM.

4. The composition according to any one of the preceding claims, wherein the buffer is a HEPES buffer.

5. The composition according to any one of the preceding claims, wherein the buffer is an L-histidine buffer.

6. The composition of any one of the preceding claims, wherein the sucrose is present at a concentration of about 66 mM to about 80 mM.

7. The composition of any one of the preceding claims, wherein the sucrose is present at a concentration of about 73 mM.

8. The composition of any one of claims 1 to 6, wherein the sucrose is present at a concentration of about 2.0% to about 3.0% by weight per volume of the composition.

9. The composition of any one of claims 1 to 6, wherein the sucrose is present at a concentration of about 2.5% by weight per volume of the composition.

10. The composition of any one of the preceding claims, wherein the L-proline is present at a concentration of about 40 mM to about 60 mM.

11. The composition of any one of the preceding claims, wherein the L-proline is present at a concentration of about 50 mM.

12. The composition according to any one of the preceding claims, wherein the composition further comprises a salt.

13. The composition of claim 12, wherein the salt is present at a concentration of about 65 mM to about 85 mM.

14. The composition of claim 13, wherein the salt is present at a concentration of about 75 mM.

15. The composition according to any one of claims 12 to 14, wherein the salt is a chloride salt, KCl or NaCl.

16. The composition of any one of the preceding claims, wherein the composition further comprises poloxamer 188.

17. The composition of claim 16, wherein the poloxamer 188 is present at a concentration of about 0.01 mg / ml to about 1 mg / ml.

18. The composition of claim 17, wherein the poloxamer 188 is present at a concentration of about 0.1 mg / ml or about 0.3 mg / ml.

19. The composition of any one of the preceding claims, wherein the composition comprises a pH of about 6.5 to about 8.

20. An aqueous viral composition comprising: a) Lentiviral vectors, b) about 27.5 mM HEPES, c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition, and d) about 50 mM L-proline; wherein the composition comprises a pH of about 7.

21. An aqueous viral composition comprising: a) Lentiviral vectors, b) about 27.5 mM HEPES, c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition, d) about 50 mM L-proline, and e) about 0.1 mg / mL to about 0.8 mg / mL poloxamer 188; wherein the composition comprises a pH of about 7.

22. An aqueous viral composition comprising: a) Lentiviral vectors, b) about 27.5 mM HEPES, c) about 73 mM sucrose or about 2.5% sucrose by weight per volume of the composition, d) about 50 mM L-proline, and e) about 0.3 mg / mL poloxamer 188; wherein the composition comprises a pH of about 7.

23. An aqueous viral composition comprising: a) Lentiviral vectors, b) HEPES buffer, c) sucrose, d) salt; and e) Poloxamers.

24. The composition of claim 23, wherein the composition does not comprise L-proline.

25. The composition of claim 23 or claim 24, wherein the HEPES buffer is present at a concentration of about 25 mM to about 30 mM.

26. The composition of any one of claims 23 to 25, wherein the HEPES buffer is present at a concentration of about 27.5 mM.

27. The composition of any one of claims 23 to 26, wherein the sucrose is present at a concentration of about 66 mM to about 80 mM.

28. The composition of any one of claims 23 to 27, wherein the sucrose is present at a concentration of about 73 mM.

29. The composition of any one of claims 23 to 28, wherein the sucrose is present at a concentration of about 2.0% to about 3.0% by weight per volume of the composition.

30. The composition of any one of claims 23 to 29, wherein the sucrose is present at a concentration of about 2.5% by weight per volume of the composition.

31. The composition of any one of claims 23 to 30, wherein the salt is present at a concentration of about 65 mM to about 85 mM.

32. The composition of any one of claims 23 to 31, wherein the salt is present at a concentration of about 75 mM.

33. The composition of any one of claims 23 to 32, wherein the salt is KCl or NaCl.

34. The composition of any one of claims 23 to 33, wherein the poloxamer is present at a concentration of about 0.01 mg / ml to about 1 mg / ml.

35. The composition of any one of claims 23 to 34, wherein the poloxamer is present at a concentration of about 0.1 mg / ml.

36. The composition of any one of claims 23 to 35, wherein the poloxamer is poloxamer 188 (P188).

37. The composition of any one of claims 23 to 36, wherein the composition comprises a pH of about 7 to about 8.

38. An aqueous viral composition comprising: a) Lentiviral vectors, b) about 27.5 mM HEPES, c) about 73 mM sucrose by weight per volume of the composition, d) about 75 mM NaCl, and e) about 0.1 to about 0.8 mg / ml Poloxamer 188; wherein the composition comprises a pH of about 7.

39. The composition according to any of the preceding claims, wherein the viral lentiviral vector is present in an amount of about 1×10 8 About 2x10 9 Titers of TU / ml are present.

40. The composition of any of the preceding claims, wherein the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

41. The composition of any of the preceding claims, wherein the lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1) or human immunodeficiency virus 2 (HIV-2).

42. The composition of any of the preceding claims, wherein the lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1).

43. The composition of any of the preceding claims, wherein the lentiviral vector is pseudotyped.

44. The composition of any of the preceding claims, wherein the lentiviral vector is pseudotyped with an envelope protein from a strain of vesicular stomatitis virus.

45. The composition of claim 44, wherein the strain of vesicular stomatitis virus is selected from the group consisting of Indiana, Alagoas, New Jersey, Isfahan, CoCal, Maraba, or Piry.

46. ​​The composition of any of the preceding claims, wherein the lentiviral vector is pseudotyped with vesicular stomatitis virus G (VSV-G) protein.

47. The composition of any of the preceding claims, wherein the lentiviral vector is pseudotyped with an envelope derived from Measles envelope protein, Sindbis envelope protein, Morbillivirus F and H proteins, Sendai F and HN proteins, or Paramyxoviridae F and H proteins.

48. The composition of any of the preceding claims, wherein the lentiviral vector comprises a polynucleotide comprising a transgene.

49. The composition of claim 48, wherein the transgene encodes a therapeutic protein.

50. The composition of claim 48 or claim 49, wherein the transgene or therapeutic protein is used to treat a monogenic disease, disorder or condition.

51. A composition according to any one of claims 48 to 50, wherein the transgene or therapeutic protein is a chimeric antigen receptor (CAR), a chimeric co-stimulatory receptor (CCR), an αβT cell receptor (αβ-TCR), a γδT cell receptor (γδ-TCR), a dimerizer-regulated immune receptor complex (DARIC) or a switch receptor.

52. The composition of any one of claims 48 to 51, wherein the transgene or therapeutic protein is a therapeutic globin for treating a hemoglobinopathy or the ABCD1 gene for treating CALD.

53. The composition of any of the preceding claims, wherein the composition does not comprise serum.

54. A composition according to any one of the preceding claims, wherein the composition does not comprise PIPES.

55. A composition according to any one of the preceding claims, wherein the composition does not comprise sodium citrate.

56. A composition according to any one of the preceding claims, wherein the composition does not comprise sodium phosphate.

57. A composition according to any one of the preceding claims, wherein the composition does not comprise Tris.

58. A composition according to any one of the preceding claims, wherein the composition does not comprise a salt.

59. A composition according to any one of the preceding claims, wherein the composition does not comprise chloride salts.

60. A composition according to any one of the preceding claims, wherein the composition does not comprise NaCl.

61. The composition of any of the preceding claims, wherein the composition does not comprise KCl.

62. A composition according to any one of the preceding claims, wherein the composition does not comprise trehalose.

63. The composition of any of the preceding claims, wherein the lentiviral vector maintains greater than about 75% recovery of infectious titer in HOS cells after storage relative to the infectious titer of the lentiviral vector in the composition prior to storage or at least one freeze-thaw cycle.

64. The composition of any of the preceding claims, wherein the lentiviral vector maintains greater than about 95% recovery of the infectious titer in HOS cells after storage relative to the infectious titer of the lentiviral vector in the composition prior to storage or at least one freeze-thaw cycle; relative to the infectious titer of the lentiviral vector in the composition prior to storage or at least one freeze-thaw cycle.

65. The composition of any of the preceding claims, wherein the lentiviral vector has a thermal unfolding temperature of about 56° to about 62°C as measured by differential scanning fluorimetry (DSF).

66. The composition of any of the preceding claims, wherein the lentiviral vector maintains a hydrodynamic diameter of about 150 nm to about 170 nm after storage relative to the hydrodynamic diameter of the lentiviral vector in the composition before storage or at least one freeze-thaw cycle as measured by dynamic light scattering (DLS) at 25°C and a viscosity value of 0.967 centipoise (cP).

67. The composition of any of the preceding claims, wherein the lentiviral vector maintains at least about 78% potency after storage compared to a reference standard as measured by transgene expression in PBMCs.

68. The composition of any of the preceding claims, wherein the lentiviral vector maintains at least about 95% potency after storage as compared to a reference standard as measured by transgene expression in PBMCs.

69. A composition according to any one of the preceding claims, wherein no visible fiber particles (bundled fibers) are present after storage.

70. A composition according to any of the preceding claims, wherein there are 5 or fewer visible particles or microparticles after storage.

71. A composition according to any of the preceding claims, wherein no visible particles are present in the composition after storage.

72. The composition of any one of claims 63 to 71, wherein the storage is at 25°C, 2-8°C or 37°C.

73. according to the composition described in any one of claims 63 to 72, wherein said storage continues 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours or longer.

74. The composition of any one of claims 63 to 73, wherein the storage comprises one or more freeze-thaw cycles.

75. The composition of any claim 74, wherein the one or more freeze-thaw cycles comprises freezing the composition at about -65°C or less for about 1.5 hours or more and thawing at 30°C for 1.5 hours.

76. A composition according to any of the preceding claims, wherein the composition is frozen.

77. A method for storing a viral vector, the method comprising: a) providing a composition according to any one of claims 1 to 76; and b) storing the composition at about 25°C or below.

78. A method for storing a viral vector, the method comprising: a) providing a composition according to any one of claims 1 to 76; and b) storing the composition at about 2-8°C or lower.

79. A method for cryopreservation of a viral vector, the method comprising: a) providing a composition according to any one of claims 1 to 76, b) freezing the composition, and c) storing the composition at about 0°C or below.

80. The method of any one of claims 77 to 79, wherein the method comprises storing the lentiviral composition for at least about 24 hours.

81. The method of any one of claims 77 to 79, wherein the method comprises storing the lentiviral composition for at least about 168 hours.

82. A method of expressing a transgene in a cell, the method comprising contacting the cell with a composition according to any one of claims 1 to 76.

83. The method of claim 82, wherein the cell is a mammalian cell.

84. The method of claim 82 or claim 83, wherein the cell is a hematopoietic cell.

85. The method of any one of claims 82 to 84, wherein the cell is an immune effector cell.

86. The method according to any one of claims 82 to 85, wherein the cell is a CD3 + 、CD4 + and / or CD8 + cell.

87. The method of any one of claims 82 to 86, wherein the cell is a T cell.

88. The method of any one of claims 82 to 87, wherein the cell is an αβ T cell.

89. The method of any one of claims 82 to 87, wherein the cell is a γδ T cell.

90. The method of any one of claims 82 to 87, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

91. The method of any one of claims 82 to 85, wherein the cell is a Natural Killer (NK) cell or a Natural Killer T (NKT) cell.

92. The method of any one of claims 82 to 85, wherein the cell is a hematopoietic stem or progenitor cell.

93. The method of any one of claims 82 to 85, wherein the cells are human CD34+ hematopoietic stem or progenitor cells.

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