Compositions for maintaining lentiviral vectors and their use

Abstract

The present disclosure provides improved aqueous formulations for storing viral vectors, as well as methods of preparing and using the same. The present disclosure generally relates to improved compositions for storing viral vectors, including but not limited in part to retroviral vectors or lentiviral vectors. 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. In another aspect, an aqueous viral composition is provided, the composition comprising a viral vector; a HEPES buffer; a carbohydrate; a salt; and a poloxamer.

Description

Technical Field

[0001] Compositions and Their Use Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 346,001, filed May 26, 2022. The entire content of the foregoing application is hereby expressly incorporated herein by reference.

[0002] The present disclosure relates to improved viral vector compositions having stability, cooling temperature, and freezing temperature at room temperature. More specifically, the present disclosure relates to improved aqueous and frozen liquid viral vector compositions that exhibit high titer recovery, particle integrity, and efficacy after storage under various conditions.

Background Art

[0003] Description of Related Fields Viral vectors have emerged as a prominent method for delivering therapeutic cargo to target cells. Such vectors include systems derived from adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), and lentivirus. These vectors are capable of introducing and / or integrating a gene of interest (e.g., a therapeutic gene) into the genome of a target cell. Thus, the target cell can theoretically be edited to express any gene of interest, which can also have therapeutic benefits. Such therapies are already in the clinic (see, e.g., Curr Gene Ther. 2015;15(1):64-81 and Blood Rev. 2022 Jan 21;100929), representing a novel approach for treating certain diseases. One factor limiting the use of viral vectors (including lentiviruses) in the clinical setting is the poor stability, aggregation, and loss of viral titers after storage at temperatures above and below freezing (see, e.g., Kumru et al., J Pharm Sci. 2018 Nov;107(11):2764-2774). In fact, viral vectors can be subjected to a wide range of temperatures during production, storage, and final research or commercial use. Viral vectors can be subjected to physiological temperatures (e.g., 25°C and / or 37°C) during production and transduction, low temperatures (e.g., 2 - 8°C) during purification and short-term storage, and freezing temperatures (e.g., below 0°C) during long-term storage. Furthermore, viral vectors can undergo several freeze-thaw cycles throughout their useful life and storage. Accordingly, there remains a need for improved compositions for the long-term storage of viral vectors.

PRIOR ART DOCUMENTS

NON-PATENT DOCUMENTS

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SUMMARY OF THE INVENTION

MEANS FOR SOLVING THE PROBLEM

[0005] The present disclosure generally relates, in part but not limited to, improved compositions for storing viral vectors, including retroviral vectors or lentiviral vectors.

[0006] In one aspect, an aqueous virus composition is provided, which comprises a virus vector, a HEPES or L - histidine buffer, a carbohydrate, and an amino acid.

[0007] In another aspect, an aqueous virus composition is provided, the composition comprising a virus vector; a HEPES buffer; a carbohydrate; a salt; and a poloxamer.

[0008] 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 certain embodiments, the buffer is a HEPES buffer. In certain embodiments, the buffer is an L - histidine buffer.

[0009] In various embodiments, the carbohydrate is present at a concentration of about 66 mM to about 80 mM, about 67 mM to about 79 mM, about 68 mM to about 78 mM, about 69 mM to about 77 mM, about 70 mM to about 76 mM, about 71 mM to about 75 mM, about 72 mM to about 74 mM, or about 73 mM. In various embodiments, the carbohydrate is present at a concentration of about 2.0 wt% to about 3.0 wt% per volume of the composition, about 2.1 wt% to about 2.9 wt% per volume of the composition, about 2.2 wt% to about 2.8 wt% per volume of the composition, about 2.3 wt% to about 2.7 wt% per volume of the composition, about 2.4 wt% to about 2.6 wt% per volume of the composition, or about 2.5 wt% 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 certain embodiments, the carbohydrate is sucrose. In certain embodiments, the carbohydrate is trehalose.

[0010] In various embodiments, the amino acid is present at a concentration of about 40 mM to about 60 mM, about 41 mM to about 59 mM, about 42 mM to about 58 mM, about 43 mM to about 57 mM, about 44 mM to about 56 mM, about 45 mM to about 55 mM, about 46 mM to about 54 mM, about 47 mM to about 53 mM, about 48 mM to about 52 mM, about 49 mM to about 51 mM, or about 50 mM. 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 certain embodiments, the amino acid is L-proline.

[0011] In various embodiments, the composition further comprises a salt. In some embodiments, the salt is present at a concentration of about 65 mM to about 85 mM, about 66 mM to about 84 mM, about 67 mM to about 83 mM, about 68 mM to about 82 mM, about 69 mM to about 81 mM, about 70 mM to about 80 mM, about 71 mM to about 79 mM, about 72 mM to about 78 mM, about 73 mM to about 77 mM, about 74 mM to about 76 mM, about 75 mM. In some embodiments, the salt is a chloride salt, KCl, or NaCl. In certain embodiments, the salt is NaCl.

[0012] 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 certain embodiments, the poloxamer is poloxamer 188 (P188).

[0013] In various embodiments, the composition has a pH of about 6.5 to about 8. In some embodiments, the composition has a pH of about 6.5. In some embodiments, the composition has a pH of about 7. In some embodiments, the composition has a pH of about 7.5. In some embodiments, the composition has a pH of about 8.

[0014] In certain embodiments, an aqueous viral composition is provided, the composition comprising 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 50 mM L-proline; the composition having a pH of about 7.

[0015] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM L-histidine; about 73 mM sucrose or about 2.5 wt% sucrose per volume of the composition; and about 50 mM L-proline, the composition having a pH of about 7.

[0016] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 wt% sucrose per volume of the composition; about 50 mM L-proline; and about 0.1 to about 0.8 mg / mL poloxamer 188, the composition having a pH of about 7.

[0017] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 wt% sucrose per volume of the composition; about 50 mM L-proline; and about 0.3 mg / mL poloxamer 188, the composition having a pH of about 7.

[0018] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 wt% sucrose per volume of the composition; about 50 mM L-proline; and about 75 mM NaCl, the composition having a pH of about 7.

[0019] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5 wt% 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, the composition having a pH of about 7.

[0020] In various embodiments, the composition further comprises a salt. In some embodiments, the salt is present at a concentration of about 65 mM to about 85 mM, about 66 mM to about 84 mM, about 67 mM to about 83 mM, about 68 mM to about 82 mM, about 69 mM to about 81 mM, about 70 mM to about 80 mM, about 71 mM to about 79 mM, about 72 mM to about 78 mM, about 73 mM to about 77 mM, about 74 mM to about 76 mM, about 75 mM. In certain embodiments, the salt is NaCl.

[0021] In certain embodiments, an aqueous virus composition is provided, the composition comprising 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 to about 0.08 wt% poloxamer 188 per volume of the composition, the composition having a pH of about 7.

[0022] In any of the aspects and embodiments contemplated herein, the viral vector is present at a titer of about 1×10 8 to about 2×10 9 TU / ml. In various embodiments, the viral vector is about 1×10 8 TU / ml, about 2×10 8 TU / ml, about 3×10 8 TU / ml, about 4×10 8 TU / ml, about 5×10 8 TU / ml, about 6×10 8 TU / ml, about 7×10 8 TU / ml, about 8×10 8 TU / ml, about 9×10 8 TU / ml, about 1×10 9 TU / ml, or about 2×10 9 TU / ml.

[0023] In various embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a vaccinia virus vector, or a retrovirus vector. In some embodiments, the viral vector is a lentivirus vector. In some embodiments, the lentivirus 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 lentivirus vector is derived from human immunodeficiency cirus-1 (HIV-1) or human immunodeficiency virus 2 (HIV-2). In certain embodiments, the lentivirus vector is derived from human immunodeficiency cirus-1 (HIV-1).

[0024] In any 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 certain embodiments, the viral vector is pseudotyped with vesicular stomatitis virus G (VSV-G) protein. In some embodiments, the viral vector is pseudotyped with an envelope derived from a measles envelope protein, a sindbis envelope protein, morbillivirus F and H proteins, Sendai F and HN proteins, or paramyxovirus F and H proteins.

[0025] 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 αβ T cell receptor (αβ-TCR), a γδ T cell receptor (γδ-TCR), a dimerizer-regulated immune receptor complex (DARIC), or a switch receptor. In some embodiments, the transgene or therapeutic protein is for the treatment of a monogenic disease, disorder, or condition. In some embodiments, the transgene or therapeutic protein is a therapeutic globin for the treatment of hemoglobinopathy or the ABCD1 gene for the treatment of CALD.

[0026] In certain embodiments, the compositions contemplated herein do not contain serum, human serum albumin (HSA), PIPES, sodium citrate, sodium phosphate, and / or Tris. In some embodiments, the compositions contemplated herein do not contain salts. In some embodiments, the compositions contemplated herein do not contain NaCl. In some embodiments, the compositions contemplated herein do not contain KCl. In some embodiments, the compositions contemplated herein do not contain trehalose.

[0027] In any of the aspects and embodiments contemplated herein, the viral vector maintains an infectivity titer recovery 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 after at least one freeze-thaw cycle, compared to the infectivity titer of the viral vector in the composition before storage or before at least one freeze-thaw cycle.

[0028] In any of the aspects and embodiments contemplated herein, the viral vector has a thermal unfolding temperature of about 56 °C to about 62 °C as measured by differential scanning fluorimetry (DSF).

[0029] 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).

[0030] In any of the aspects and embodiments contemplated herein, the viral vector maintains a hydrodynamic diameter of about 150 nm to about 170 nm and a viscosity value of 0.967 centipoise (cP) as measured by dynamic light scattering (DLS) at 25°C, compared to the hydrodynamic diameter of the viral vector in the composition before storage or at least one freeze-thaw cycle.

[0031] 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% efficacy as measured by transgene expression in PBMCs, compared to a reference standard, after storage.

[0032] In any of the aspects and embodiments contemplated herein, the composition does not have visible fibrous particles (whispy fibers) after storage. In any of the aspects and embodiments contemplated herein, the composition has 5 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition has 4 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition has 3 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition has 2 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition has 1 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition does not have visible particles after storage.

[0033] In various embodiments, storage is at 25°C, 2 - 8°C, or 37°C. In some embodiments, storage is for 24 hours, 48 hours, or 72, 96, 120, 144, or 168 hours or more.

[0034] In various embodiments, storage includes 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 include freezing the composition at about - 65°C or below for about 1.5 hours or more and thawing at 30°C for 1.5 hours.

[0035] In any of the aspects and embodiments contemplated herein, the composition is frozen.

[0036] In another aspect, a method for storing a viral vector is provided, comprising providing a viral vector, contacting the viral vector with any of the compositions contemplated herein, and storing the viral composition at a temperature of about 25°C or below.

[0037] In another aspect, a method for storing a viral vector is provided, comprising providing a viral vector, contacting the viral vector with any of the compositions contemplated herein, and storing the viral composition at a temperature of about 2 - 8°C or below for at least about 24 hours.

[0038] In another aspect, a method for cryopreserving a viral vector is provided, comprising providing a viral vector, contacting the viral vector with any one of the compositions contemplated herein, freezing the viral composition, and storing the viral composition at a temperature of about 0°C or below.

[0039] In various embodiments, the methods contemplated herein include 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.

[0040] In another aspect, methods are provided for expressing a transgene in a cell, including contacting the cell with any one 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 or progenitor cell. In some embodiments, the cell is a human CD34+ hematopoietic 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 + 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), tumor infiltrating lymphocyte (TIL), or helper T cell. In some embodiments, the cell is a natural killer (NK) cell or natural killer T (NKT) cell. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0042] A. Overview The present disclosure generally relates, in part, to viral vector (e.g., lentiviral vector) compositions that exhibit high titer recovery, thermal stability, particle integrity, no - low visible particle formation, and potency after storage under various conditions. Without wishing to be bound by any particular theory, many biopharmaceuticals (including viruses) lose protein structure and activity during the manufacturing process and storage when the optimal solution or formulation has not been determined. Viral vectors also lose their ability to efficiently transduce cells and change in structure when subjected to similar manufacturing and storage stresses in various solutions.

[0043] Thus, contemplated herein are formulations that minimize the loss of viral vectors surprisingly and maintain the integrity and activity of viral vectors under conditions known to affect the integrity of the virus. Specifically, the formulations contemplated herein exhibit high titer recovery, particle integrity, thermal stability, no to low visible particle formation (e.g., only 1, 2, or 3 visible particles), and potency after storage under various conditions including storage at 37°C, 25°C, 2 - 8°C, and / or after one or more freeze - thaw cycles. Also contemplated are methods of storage, cryopreservation, and transduction.

[0044] 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 are generally carried out 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 this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; 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, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); 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, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology(Academic Press, New York, 1991); Oligonucleotide Synthesis(N.Gait, Ed., 1984); Nucleic Acid The Hybridization(B.Hames & S.Higgins, Eds., 1985); Transcription and Translation(B.Hames & S.Higgins, Eds., 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 Edition, 2010 Humana Press); Immobilized Cells And Enzymes(IRL Press, 1986); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research papers in journals such as Advances in Immunology, etc. Please refer to them.

[0045] B. Definitions Before describing the present disclosure in more detail, it may be helpful to provide definitions of certain terms to be used herein.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, but preferred embodiments of compositions, methods, and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.

[0047] The articles "a", "an", and "the" are used herein to refer to one or more (i.e., at least one, or one or more) of the grammatical objects of the articles. By way of example, "an element" means one element, or one or more elements.

[0048] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives.

[0049] The term "and / or" is to be understood to mean either one, or both of the alternatives.

[0050] As used herein, the terms "about" or "approximately" refer to a quantity, level, value, number, 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% with respect to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the terms "about" or "approximately" refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% with respect to a reference quantity, level, value, number, 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% to 15% (e.g., ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%).

[0051] In one embodiment, a range such as, for example, 1 to 5, about 1 to 5, or about 1 to about 5 refers to each numerical value included in the range. For example, in one non-limiting, merely illustrative embodiment, the range of "1 to 5" is equivalent to the expression of 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 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.

[0052] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to all of the following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are necessary or obligatory and that no other elements can exist. "Consisting essentially of" means including any element listed after this phrase, and any element limited to those that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are essential or obligatory, but that no other elements substantially affect the activity or action of the listed elements.

[0053] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "a certain embodiment", "additional embodiments", or "further embodiments", or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that a positive recitation of a property in one embodiment does not serve as a basis for excluding that property in a particular embodiment.

[0054] As used herein, the term "buffer" refers to a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid.

[0055] As used herein, the term "freeze-thaw" or "freeze-thaw cycle" refers to the exposure of a liquid mixture, such as an aqueous solution or suspension, to a temperature below its freezing point until the mixture is frozen, followed by thawing the mixture at a temperature higher than its freezing point. The freezing step can be carried out, for example, by placing the mixture in an environment where the temperature is from about 0 °C to about -80 °C. In some embodiments, the freezing temperature is from about -20 °C to about -80 °C. In some embodiments, the freezing temperature is about -65 °C. The frozen aqueous solution or suspension may be stored for a period of 1 or more hours, days, weeks, months, or years prior to thawing. Thawing of the solution or suspension can be carried out by exposing it to conditions where the temperature is from 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.

[0056] As used herein, the term "weight percent per volume" or "% w / v" refers to the weight percentage (grams) of a single component relative to the total volume of a 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.

[0057] As used herein, the term "titer" or "viral titer" refers to the number of infectious vector particles that result in the production of a transgene product in a target cell, or "transducing units". Viral titers can be measured by functional assays such as those described in 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 titers can be measured by determining the amount of viral DNA integrated into the host cell genome, for example, using polymerase chain reaction (PCR) techniques known in the art.

[0058] Additional definitions are described throughout the present disclosure.

[0059] C. Formulations Disclosed herein are formulations that minimize surprisingly the loss of viral vectors and maintain activity under conditions known to affect the integrity of the virus. Formulations contemplated herein include at least a buffer and a carbohydrate.

[0060] In certain embodiments, the buffer is N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer. The chemical formula of HEPES is C8H 18It is N2O4S, which has a molecular weight of 238.3012 g / mol. The IUPAC ID for HEPES is 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid.

[0061] In various embodiments, the buffer is an L-histidine buffer. L-histidine is the L-enantiomer of the amino acid histidine. L-histidine has a chemical formula of C6H9N3O2 and a molecular weight of 155.157 g / mol.

[0062] 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.

[0063] In some embodiments, the buffer is present at a concentration of about 25 mM. In some embodiments, the buffer is present at a concentration of about 25.5 mM. In some embodiments, the buffer is present at a concentration of about 26 mM. In some embodiments, the buffer is present at a concentration of about 26.5 mM. In some embodiments, the buffer is present at a concentration of about 27 mM. In some embodiments, the buffer is present at a concentration of about 27.5 mM. In certain embodiments, the buffer is present at a concentration of 27.5 mM. In some embodiments, the buffer is present at a concentration of about 28 mM. In some embodiments, the buffer is present at a concentration of about 28.5 mM. In some embodiments, the buffer is present at a concentration of about 29 mM. In some embodiments, the buffer is present at a concentration of about 29.5 mM. In some embodiments, the buffer is present at a concentration of about 30 mM.

[0064] In various embodiments, the carbohydrate is present at a concentration of about 66 mM to about 80 mM. In some embodiments, the carbohydrate is present at a concentration of about 67 mM to about 79 mM. In some embodiments, the carbohydrate is present at a concentration of about 68 mM to about 78 mM. In some embodiments, the carbohydrate is present at a concentration of about 69 mM to about 77 mM. In some embodiments, the carbohydrate is present at a concentration of about 70 mM to about 76 mM. In some embodiments, the carbohydrate is present at a concentration of about 71 mM to about 75 mM. In some embodiments, the carbohydrate is present at a concentration of about 72 mM to about 74 mM.

[0065] In some embodiments, the carbohydrate is present at a concentration of about 66 mM. In some embodiments, the carbohydrate is present at a concentration of about 67 mM. In some embodiments, the carbohydrate is present at a concentration of about 68 mM. In some embodiments, the carbohydrate is present at a concentration of about 69 mM. In some embodiments, the carbohydrate is present at a concentration of about 70 mM. In some embodiments, the carbohydrate is present at a concentration of about 71 mM. In some embodiments, the carbohydrate is present at a concentration of about 72 mM. In some embodiments, the carbohydrate is present at a concentration of about 73 mM. In certain embodiments, the carbohydrate is present at a concentration of 73 mM. In some embodiments, the carbohydrate is present at a concentration of about 74 mM. In some embodiments, the carbohydrate is present at a concentration of about 75 mM. In some embodiments, the carbohydrate is present at a concentration of about 76 mM. In some embodiments, the carbohydrate is present at a concentration of about 77 mM. In some embodiments, the carbohydrate is present at a concentration of about 78 mM. In some embodiments, the carbohydrate is present at a concentration of about 79 mM. In some embodiments, the carbohydrate is present at a concentration of about 80 mM.

[0066] In various embodiments, the carbohydrate is present at a concentration of about 15 mM to about 45 mM. In some embodiments, the carbohydrate is present at a concentration of about 20 mM to about 40 mM. In some embodiments, the carbohydrate is present at a concentration of about 25 mM to about 35 mM. In some embodiments, the carbohydrate is present at a concentration of about 25 mM to about 34 mM. In some embodiments, the carbohydrate is present at a concentration of about 25 mM to about 33 mM. In some embodiments, the carbohydrate is present at a concentration of about 26 mM to about 32 mM. In some embodiments, the carbohydrate is present at a concentration of about 27 mM to about 31 mM. In some embodiments, the carbohydrate is present at a concentration of about 28 mM to about 30 mM.

[0067] In some embodiments, the carbohydrate is present at a concentration of about 15 mM. In some embodiments, the carbohydrate is present at a concentration of about 16 mM. In some embodiments, the carbohydrate is present at a concentration of about 17 mM. In some embodiments, the carbohydrate is present at a concentration of about 18 mM. In some embodiments, the carbohydrate is present at a concentration of about 19 mM. In some embodiments, the carbohydrate is present at a concentration of about 20 mM. In some embodiments, the carbohydrate is present at a concentration of about 21 mM. In some embodiments, the carbohydrate is present at a concentration of about 22 mM. In some embodiments, the carbohydrate is present at a concentration of about 23 mM. In some embodiments, the carbohydrate is present at a concentration of about 24 mM. In some embodiments, the carbohydrate is present at a concentration of about 25 mM. In some embodiments, the carbohydrate is present at a concentration of about 26 mM. In some embodiments, the carbohydrate is present at a concentration of about 27 mM. In some embodiments, the carbohydrate is present at a concentration of about 28 mM. In some embodiments, the carbohydrate is present at a concentration of about 29 mM. In some embodiments, the carbohydrate is present at a concentration of about 30 mM. In some embodiments, the carbohydrate is present at a concentration of about 31 mM. In some embodiments, the carbohydrate is present at a concentration of about 32 mM. In some embodiments, the carbohydrate is present at a concentration of about 33 mM. In some embodiments, the carbohydrate is present at a concentration of about 34 mM. In some embodiments, the carbohydrate is present at a concentration of about 35 mM. In some embodiments, the carbohydrate is present at a concentration of about 36 mM. In some embodiments, the carbohydrate is present at a concentration of about 37 mM. In some embodiments, the carbohydrate is present at a concentration of about 38 mM. In some embodiments, the carbohydrate is present at a concentration of about 39 mM. In some embodiments, the carbohydrate is present at a concentration of about 40 mM. In some embodiments, the carbohydrate is present at a concentration of about 41 mM. In some embodiments, the carbohydrate is present at a concentration of about 42 mM. In some embodiments, the carbohydrate is present at a concentration of about 43 mM. In some embodiments, the carbohydrate is present at a concentration of about 44 mM. In some embodiments, the carbohydrate is present at a concentration of about 45 mM.

[0068] In various embodiments, the carbohydrate is present at a concentration of about 2.0 wt% to about 3.0 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.1 wt% to about 2.9 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.2 wt% to about 2.8 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.3 wt% to about 2.7 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.4 wt% to about 2.6 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.5 wt% per volume of the composition. In certain embodiments, the carbohydrate is present at a concentration of 2.5 wt% per volume of the composition.

[0069] In various embodiments, the carbohydrate is present at a concentration of about 0.5 wt% to about 1.5 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.6 wt% to about 1.4 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.7 wt% to about 1.3 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.8 wt% to about 1.2 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.9 wt% to about 1.1 wt% per volume of the composition.

[0070] In some embodiments, the carbohydrate is present at a concentration of about 0.5 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.6 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.7 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.8 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 0.9 wt% 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 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.2 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.3 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.4 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.5 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.6 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.7 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.8 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 1.9 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.1 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.2 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.3 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.4 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.5 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.6 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.7 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 2.8 wt% per volume of the composition.In some embodiments, the carbohydrate is present at a concentration of about 2.9 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3 wt% per volume of the composition.

[0071] In various embodiments, the carbohydrate is present at a concentration of about 100 mM to about 135 mM. In some embodiments, the carbohydrate is present at a concentration of about 105 mM to about 130 mM. In some embodiments, the carbohydrate is present at a concentration of about 110 mM to about 125 mM. In some embodiments, the carbohydrate is present at a concentration of about 111 mM to about 124 mM. In some embodiments, the carbohydrate is present at a concentration of about 111 mM to about 123 mM. In some embodiments, the carbohydrate is present at a concentration of about 112 mM to about 122 mM. In some embodiments, the carbohydrate is present at a concentration of about 113 mM to about 121 mM. In some embodiments, the carbohydrate is present at a concentration of about 114 mM to about 120 mM. In some embodiments, the carbohydrate is present at a concentration of about 115 mM to about 119 mM. In some embodiments, the carbohydrate is present at a concentration of about 116 mM to about 118 mM.

[0072] 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 116 mM. In some embodiments, the carbohydrate is present at a concentration of about 117 mM. In some embodiments, the carbohydrate is present at a concentration of about 118 mM. In some embodiments, the carbohydrate is present at a concentration of about 119 mM. In some embodiments, the carbohydrate is present at a concentration of about 120 mM. In some embodiments, the carbohydrate is present at a concentration of about 121 mM. In some embodiments, the carbohydrate is present at a concentration of about 122 mM. In some embodiments, the carbohydrate is present at a concentration of about 123 mM. In some embodiments, the carbohydrate is present at a concentration of about 124 mM. In some embodiments, the carbohydrate is present at a concentration of about 125 mM. In some embodiments, the carbohydrate is present at a concentration of about 126 mM. In some embodiments, the carbohydrate is present at a concentration of about 127 mM. In some embodiments, the carbohydrate is present at a concentration of about 128 mM. In some embodiments, the carbohydrate is present at a concentration of about 129 mM. In some embodiments, the carbohydrate is present at a concentration of about 130 mM. In some embodiments, the carbohydrate is present at a concentration of about 131 mM.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 135 mM. In some embodiments, the carbohydrate is present at a concentration of about 136 mM. In some embodiments, the carbohydrate is present at a concentration of about 137 mM. In some embodiments, the carbohydrate is present at a concentration of about 138 mM. In some embodiments, the carbohydrate is present at a concentration of about 139 mM. In some embodiments, the carbohydrate is present at a concentration of about 140 mM. In some embodiments, the carbohydrate is present at a concentration of about 141 mM. In some embodiments, the carbohydrate is present at a concentration of about 142 mM. In some embodiments, the carbohydrate is present at a concentration of about 143 mM. In some embodiments, the carbohydrate is present at a concentration of about 144 mM. In some embodiments, the carbohydrate is present at a concentration of about 145 mM. In some embodiments, the carbohydrate is present at a concentration of about 146 mM. In some embodiments, the carbohydrate is present at a concentration of about 147 mM. In some embodiments, the carbohydrate is present at a concentration of about 148 mM. In some embodiments, the carbohydrate is present at a concentration of about 149 mM. In some embodiments, the carbohydrate is present at a concentration of about 150 mM.

[0073] In various embodiments, the carbohydrate is present at a concentration of about 265 mM to about 285 mM. In some embodiments, the carbohydrate is present at a concentration of about 266 mM to about 284 mM. In some embodiments, the carbohydrate is present at a concentration of about 267 mM to about 283 mM. In some embodiments, the carbohydrate is present at a concentration of about 268 mM to about 282 mM. In some embodiments, the carbohydrate is present at a concentration of about 269 mM to about 281 mM. In some embodiments, the carbohydrate is present at a concentration of about 270 mM to about 280 mM. In some embodiments, the carbohydrate is present at a concentration of about 271 mM to about 279 mM. In some embodiments, the carbohydrate is present at a concentration of about 272 mM to about 278 mM. In some embodiments, the carbohydrate is present at a concentration of about 273 mM to about 277 mM. In some embodiments, the carbohydrate is present at a concentration of about 274 mM to about 276 mM.

[0074] In some embodiments, the carbohydrate is present at a concentration of about 265 mM. In some embodiments, the carbohydrate is present at a concentration of about 266 mM. In some embodiments, the carbohydrate is present at a concentration of about 267 mM. In some embodiments, the carbohydrate is present at a concentration of about 268 mM. In some embodiments, the carbohydrate is present at a concentration of about 269 mM. In some embodiments, the carbohydrate is present at a concentration of about 270 mM. In some embodiments, the carbohydrate is present at a concentration of about 271 mM. In some embodiments, the carbohydrate is present at a concentration of about 272 mM. In some embodiments, the carbohydrate is present at a concentration of about 273 mM. In some embodiments, the carbohydrate is present at a concentration of about 274 mM. In some embodiments, the carbohydrate is present at a concentration of about 275 mM. In some embodiments, the carbohydrate is present at a concentration of about 276 mM. In some embodiments, the carbohydrate is present at a concentration of about 277 mM. In some embodiments, the carbohydrate is present at a concentration of about 278 mM. In some embodiments, the carbohydrate is present at a concentration of about 279 mM. In some embodiments, the carbohydrate is present at a concentration of about 280 mM. 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.

[0075] In various embodiments, the carbohydrate is present at a concentration of about 3.5 wt% to about 4.5 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.6 wt% to about 4.4 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.7 wt% to about 4.3 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.8 wt% to about 4.2 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.9 wt% to about 4.1 wt% per volume of the composition.

[0076] In some embodiments, the carbohydrate is present at a concentration of about 3.5 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.6 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.7 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.8 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 3.9 wt% 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 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.2 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.3 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.4 wt% per volume of the composition. In some embodiments, the carbohydrate is present at a concentration of about 4.5 wt% per volume of the composition.

[0077] In certain 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 certain embodiments, the carbohydrate is sucrose. In certain embodiments, the carbohydrate is trehalose.

[0078] In various embodiments, the composition comprises an amino acid. In some embodiments, the amino acid is present at a concentration of about 40 mM to about 60 mM. In some embodiments, the amino acid is present at a concentration of about 41 mM to about 59 mM. In some embodiments, the amino acid is present at a concentration of about 42 mM to about 58 mM. In some embodiments, the amino acid is present at a concentration of about 43 mM to about 57 mM. In some embodiments, the amino acid is present at a concentration of about 44 mM to about 56 mM. In some embodiments, the amino acid is present at a concentration of about 45 mM to about 55 mM. In some embodiments, the amino acid is present at a concentration of about 46 mM to about 54 mM. In some embodiments, the amino acid is present at a concentration of about 47 mM to about 53 mM. In some embodiments, the amino acid is present at a concentration of about 48 mM to about 52 mM. In some embodiments, the amino acid is present at a concentration of about 49 mM to about 51 mM.

[0079] In some embodiments, the amino acid is present at a concentration of about 40 mM. In some embodiments, the amino acid is present at a concentration of about 41 mM. In some embodiments, the amino acid is present at a concentration of about 42 mM. In some embodiments, the amino acid is present at a concentration of about 43 mM. In some embodiments, the amino acid is present at a concentration of about 44 mM. In some embodiments, the amino acid is present at a concentration of about 45 mM. In some embodiments, the amino acid is present at a concentration of about 46 mM. In some embodiments, the amino acid is present at a concentration of about 47 mM. In some embodiments, the amino acid is present at a concentration of about 48 mM. In some embodiments, the amino acid is present at a concentration of about 49 mM. In some embodiments, the amino acid is present at a concentration of about 50 mM. In certain embodiments, the amino acid is present at a concentration of 50 mM. In some embodiments, the amino acid is present at a concentration of about 51 mM. In some embodiments, the amino acid is present at a concentration of about 52 mM. In some embodiments, the amino acid is present at a concentration of about 53 mM. In some embodiments, the amino acid is present at a concentration of about 54 mM. In some embodiments, the amino acid is present at a concentration of about 55 mM. 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.

[0080] In various embodiments, the amino acids are present at a concentration of about 15 mM to about 35 mM. In some embodiments, the amino acids are present at a concentration of about 16 mM to about 34 mM. In some embodiments, the amino acids are present at a concentration of about 17 mM to about 33 mM. In some embodiments, the amino acids are present at a concentration of about 18 mM to about 32 mM. In some embodiments, the amino acids are present at a concentration of about 19 mM to about 31 mM. In some embodiments, the amino acids are present at a concentration of about 20 mM to about 30 mM. In some embodiments, the amino acids are present at a concentration of about 21 mM to about 29 mM. In some embodiments, the amino acids are present at a concentration of about 22 mM to about 28 mM. In some embodiments, the amino acids are present at a concentration of about 23 mM to about 27 mM. In some embodiments, the amino acids are present at a concentration of about 24 mM to about 26 mM.

[0081] In some embodiments, the amino acid is present at a concentration of about 15 mM. In some embodiments, the amino acid is present at a concentration of about 16 mM. In some embodiments, the amino acid is present at a concentration of about 17 mM. In some embodiments, the amino acid is present at a concentration of about 18 mM. In some embodiments, the amino acid is present at a concentration of about 19 mM. In some embodiments, the amino acid is present at a concentration of about 20 mM. In some embodiments, the amino acid is present at a concentration of about 21 mM. In some embodiments, the amino acid is present at a concentration of about 22 mM. In some embodiments, the amino acid is present at a concentration of about 23 mM. In some embodiments, the amino acid is present at a concentration of about 24 mM. In some embodiments, the amino acid is present at a concentration of about 25 mM. In some embodiments, the amino acid is present at a concentration of about 26 mM. In some embodiments, the amino acid is present at a concentration of about 27 mM. In some embodiments, the amino acid is present at a concentration of about 28 mM. In some embodiments, the amino acid is present at a concentration of about 29 mM. In some embodiments, the amino acid is present at a concentration of about 30 mM. In some embodiments, the amino acid is present at a concentration of about 31 mM. 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.

[0082] In various embodiments, the amino acids are present at a concentration of about 65 mM to about 85 mM. In some embodiments, the amino acids are present at a concentration of about 66 mM to about 84 mM. In some embodiments, the amino acids are present at a concentration of about 67 mM to about 83 mM. In some embodiments, the amino acids are present at a concentration of about 68 mM to about 82 mM. In some embodiments, the amino acids are present at a concentration of about 69 mM to about 81 mM. In some embodiments, the amino acids are present at a concentration of about 70 mM to about 80 mM. In some embodiments, the amino acids are present at a concentration of about 71 mM to about 79 mM. In some embodiments, the amino acids are present at a concentration of about 72 mM to about 78 mM. In some embodiments, the amino acids are present at a concentration of about 73 mM to about 77 mM. In some embodiments, the amino acids are present at a concentration of about 74 mM to about 76 mM.

[0083] In some embodiments, the amino acid is present at a concentration of about 65 mM. In some embodiments, the amino acid is present at a concentration of about 66 mM. In some embodiments, the amino acid is present at a concentration of about 67 mM. In some embodiments, the amino acid is present at a concentration of about 68 mM. In some embodiments, the amino acid is present at a concentration of about 69 mM. In some embodiments, the amino acid is present at a concentration of about 70 mM. In some embodiments, the amino acid is present at a concentration of about 71 mM. In some embodiments, the amino acid is present at a concentration of about 72 mM. In some embodiments, the amino acid is present at a concentration of about 73 mM. In some embodiments, the amino acid is present at a concentration of about 74 mM. In some embodiments, the amino acid is present at a concentration of about 75 mM. In some embodiments, the amino acid is present at a concentration of about 76 mM. In some embodiments, the amino acid is present at a concentration of about 77 mM. In some embodiments, the amino acid is present at a concentration of about 78 mM. In some embodiments, the amino acid is present at a concentration of about 79 mM. In some embodiments, the amino acid is present at a concentration of about 80 mM. In some embodiments, the amino acid is present at a concentration of about 81 mM. 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.

[0084] In some embodiments, the amino acid is a nonpolar 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 certain embodiments, the amino acid is L-proline.

[0085] In various embodiments, the composition further comprises a salt. In some embodiments, the salt is present at a concentration of from about 65 mM to about 85 mM. In some embodiments, the salt is present at a concentration of from about 66 mM to about 84 mM. In some embodiments, the salt is present at a concentration of from about 67 mM to about 83 mM. In some embodiments, the salt is present at a concentration of from about 68 mM to about 82 mM. In some embodiments, the salt is present at a concentration of from about 69 mM to about 81 mM. In some embodiments, the salt is present at a concentration of from about 70 mM to about 80 mM. In some embodiments, the salt is present at a concentration of from about 71 mM to about 79 mM. In some embodiments, the salt is present at a concentration of from about 72 mM to about 78 mM. In some embodiments, the salt is present at a concentration of from about 73 mM to about 77 mM. In some embodiments, the salt is present at a concentration of from about 74 mM to about 76 mM.

[0086] In some embodiments, the salt is present at a concentration of about 65 mM. In some embodiments, the salt is present at a concentration of about 66 mM. In some embodiments, the salt is present at a concentration of about 67 mM. In some embodiments, the salt is present at a concentration of about 68 mM. In some embodiments, the salt is present at a concentration of about 69 mM. In some embodiments, the salt is present at a concentration of about 70 mM. In some embodiments, the salt is present at a concentration of about 71 mM. In some embodiments, the salt is present at a concentration of about 72 mM. In some embodiments, the salt is present at a concentration of about 73 mM. In some embodiments, the salt is present at a concentration of about 74 mM. In some embodiments, the salt is present at a concentration of about 75 mM. In certain embodiments, the salt is present at a concentration of 75 mM. In some embodiments, the salt is present at a concentration of about 76 mM. In some embodiments, the salt is present at a concentration of about 77 mM. In some embodiments, the salt is present at a concentration of about 78 mM. In some embodiments, the salt is present at a concentration of about 79 mM. In some embodiments, the salt is present at a concentration of about 80 mM. 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 certain embodiments, the salt is KCl. In certain embodiments, the salt is NaCl.

[0087] In various embodiments, the composition further comprises a poloxamer. "Poloxamer" refers to a nonionic triblock copolymer composed 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(C2H40) x (C3H60) y (C2H40) z H.

[0088] The synthesis of the block copolymer results in a population of polymers having an average molecular weight. Thus, in certain embodiments, the term "poloxamer" as used herein can be used interchangeably with the term "poloxamer" (and represents the entity of several poloxamers, referred to as a mixture of poloxamers), unless otherwise explicitly stated. As used herein, (a) the term "average" in relation to the number of monomer units or the molecular weight of the poloxamer is the result of the technical inability to produce a composition all of which is the same, thus a poloxamer having the same molecular weight. Poloxamers produced according to state-of-the-art methods exist as a mixture of poloxamers, each showing variability with respect to their molecular weight, however, the mixture averages out to the molecular weight specified herein as a whole. BASF and Sigma Aldrich are sources of suitable poloxamers for use in the particular embodiments contemplated herein.

[0089] 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.

[0090] In some embodiments, the poloxamer is present at a concentration of from about 0.01 mg / ml to about 1 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.1 mg / ml to about 1 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.1 mg / ml to about 0.8 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.2 mg / ml to about 1 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.3 mg / ml to about 1 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.02 mg / ml to about 0.9 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.03 mg / ml to about 0.8 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.04 mg / ml to about 0.7 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.05 mg / ml to about 0.6 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.06 mg / ml to about 0.5 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.07 mg / ml to about 0.4 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.08 mg / ml to about 0.3 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.09 mg / ml to about 0.2 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.1 mg / ml to about 0.5 mg / ml. In some embodiments, the poloxamer is present at a concentration of from about 0.2 mg / ml to about 0.4 mg / ml.

[0091] 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, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, or poloxamer 407. In certain embodiments, the poloxamer is poloxamer 188 (P188). In some embodiments, the poloxamer is poloxamer 407.

[0092] In various embodiments, the composition has a pH of from about 6.5 to about 8.0. In some embodiments, the composition has a pH of about 6.5. In some embodiments, the composition has a pH of about 6.6. In some embodiments, the composition has a pH of about 6.7. In some embodiments, the composition has a pH of about 6.8. In some embodiments, the composition has a pH of about 6.9. In some embodiments, the composition has a pH of about 7.0. In some embodiments, the composition has a pH of about 7.1. In some embodiments, the composition has a pH of about 7.2. In some embodiments, the composition has a pH of about 7.3. In some embodiments, the composition has a pH of about 7.4. In some embodiments, the composition has a pH of about 7.5. In some embodiments, the composition has a pH of about 7.6. In some embodiments, the composition has a pH of about 7.7. In some embodiments, the composition has a pH of about 7.8. In some embodiments, the composition has a pH of about 7.9. In some embodiments, the composition has a pH of about 8.0.

[0093] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5% w / v sucrose; and about 50 mM L-proline; the composition having a pH of about 7.

[0094] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM L-histidine; about 73 mM sucrose or about 2.5% w / v sucrose; and about 50 mM L-proline; the composition having a pH of about 7.

[0095] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5% w / v sucrose; about 50 mM L-proline; and about 0.1 to about 0.8 mg / mL poloxamer 188; the composition having a pH of about 7.

[0096] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5% w / v sucrose; about 50 mM L-proline; and about 75 mM NaCl; the composition having a pH of about 7.

[0097] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5% w / v sucrose; about 50 mM L-proline; about 75 mM NaCl; and about 0.1 to about 0.8 mg / mL poloxamer 188; the composition having a pH of about 7.

[0098] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5% w / v 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% w / v poloxamer 188 per volume of the composition, the composition having a pH of about 7.

[0099] In certain embodiments, an aqueous viral composition is provided, the composition comprising a viral vector; about 27.5 mM HEPES; about 73 mM sucrose or about 2.5% w / v sucrose per volume of the composition; and about 75 mM NaCl, the composition having a pH of about 7.

[0100] In any of the embodiments contemplated herein, the viral vector is present at a titer or concentration of about 1×10 8 to about 2×10 9 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 1×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 2×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 3×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 4×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 5×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 6×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 7×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 8×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 9×10 8 TU / ml. In some embodiments, the viral vector is present at a titer or concentration of about 1×10 9It is present at a titer or concentration of TU / ml. In some embodiments, the viral vector is about 2×10 9 It is present at a titer or concentration of TU / ml.

[0101] In certain embodiments contemplated herein, the formulation does not contain certain components. Thus, in any of the embodiments contemplated herein, the composition does not contain PIPES. In any of the embodiments contemplated herein, the composition does not contain sodium citrate. In any of the embodiments contemplated herein, the composition does not contain sodium phosphate. In any of the embodiments contemplated herein, the composition does not contain Tris. In any of the embodiments contemplated herein, the composition does not contain salts. In any of the embodiments contemplated herein, the composition does not contain NaCl. In any of the embodiments contemplated herein, the composition does not contain KCl. In any of the embodiments contemplated herein, the composition does not contain serum. In any of the embodiments contemplated herein, the composition does not contain human serum albumin (HSA). In any of the embodiments contemplated herein, the compositions contemplated herein do not contain trehalose.

[0102] In any of the embodiments contemplated herein, the viral vector maintains a viral infectivity titer recovery of greater than about 75% in HOS cells after storage and / or freeze-thaw cycles as compared to the viral infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a viral infectivity titer recovery of greater than about 76% in HOS cells after storage and / or freeze-thaw cycles as compared to the viral infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a viral infectivity titer recovery of greater than about 77% in HOS cells after storage and / or freeze-thaw cycles as compared to the viral infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a viral infectivity titer recovery of greater than about 78% in HOS cells after storage and / or freeze-thaw cycles as compared to the viral infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a viral infectivity titer recovery of greater than about 79% in HOS cells after storage and / or freeze-thaw cycles as compared to the viral infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a viral infectivity titer recovery of greater than about 80% in HOS cells after storage and / or freeze-thaw cycles as compared to the viral infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a viral infectivity titer recovery of greater than about 81% in HOS cells after storage and / or freeze-thaw cycles as compared to the viral infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a viral infectivity titer recovery of greater than about 82% in HOS cells after storage and / or freeze-thaw cycles as compared to the viral infectivity titer of the viral vector in the composition before storage or freeze-thaw.In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 83% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 84% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 85% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 86% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 87% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 88% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 89% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 90% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw.In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 91% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 92% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 93% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 94% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw. In any of the embodiments contemplated herein, the viral vector maintains a infectivity titer recovery of greater than about 95% in HOS cells after storage and / or freeze-thaw cycles as compared to the infectivity titer of the viral vector in the composition before storage or freeze-thaw.

[0103] In any of the embodiments contemplated herein, the viral vector has a thermal unfolding temperature of from 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 from 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°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 57°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°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 59°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 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 61°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 62°C as measured by differential scanning fluorimetry (DSF).

[0104] In any of the embodiments contemplated herein, the viral vector maintains a hydrodynamic diameter of from about 150 nm to about 170 nm and a viscosity value of 0.967 centipoise (cP) as measured by dynamic light scattering (DLS) at 25°C after storage and / or at least one freeze-thaw cycle, compared to the hydrodynamic diameter of the viral vector in the composition before storage or at least one freeze-thaw cycle.

[0105] In any of the embodiments contemplated herein, the viral vector maintains at least 78% efficacy 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% efficacy 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% efficacy 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% efficacy 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% efficacy 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% efficacy 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% efficacy 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% efficacy 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% efficacy 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% efficacy 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% efficacy 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% efficacy as measured by transgene expression in PBMCs as compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 90% efficacy as measured by transgene expression in PBMCs as compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 91% efficacy as measured by transgene expression in PBMCs as compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 92% efficacy as measured by transgene expression in PBMCs as compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 93% efficacy as measured by transgene expression in PBMCs as compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 94% efficacy as measured by transgene expression in PBMCs as compared to a reference standard. In any of the embodiments contemplated herein, the viral vector maintains at least 95% efficacy as measured by transgene expression in PBMCs as compared to a reference standard.

[0106] In any of the aspects and embodiments contemplated herein, the composition does not have visible particles or visible fibrous particles (whispy fibers) after storage. In any of the aspects and embodiments contemplated herein, the composition has 5 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition has 4 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition has 3 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition has 2 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition has 1 or fewer visible particles or spots after storage. In any of the aspects and embodiments contemplated herein, the composition does not have visible particles after storage.

[0107] In any of the aspects and embodiments contemplated herein, the composition does not have visible particles or visible fibrous particles (whispy fibers) per 50 ml of the composition after storage. In any of the aspects and embodiments contemplated herein, the composition has 5 or fewer visible particles or spots per 50 ml of the composition after storage. In any of the aspects and embodiments contemplated herein, the composition has 4 or fewer visible particles or spots per 50 ml of the composition after storage. In any of the aspects and embodiments contemplated herein, the composition has 3 or fewer visible particles or spots per 50 ml of the composition after storage. In any of the aspects and embodiments contemplated herein, the composition has 2 or fewer visible particles or spots per 50 ml of the composition after storage. In any of the aspects and embodiments contemplated herein, the composition has 1 or fewer visible particles or spots per 50 ml of the composition after storage. In any of the aspects and embodiments contemplated herein, the composition does not have visible particles per 50 ml of the composition after storage.

[0108] In any of the aspects and embodiments contemplated herein, the composition has no visible particles or visible fibrous particles (whispy fibers) after storage per 36 ml of the composition. In any of the aspects and embodiments contemplated herein, the composition has 5 or fewer visible particles or spots after storage per 36 ml of the composition. In any of the aspects and embodiments contemplated herein, the composition has 4 or fewer visible particles or spots after storage per 36 ml of the composition. In any of the aspects and embodiments contemplated herein, the composition has 3 or fewer visible particles or spots after storage per 36 ml of the composition. In any of the aspects and embodiments contemplated herein, the composition has 2 or fewer visible particles or spots after storage per 36 ml of the composition. In any of the aspects and embodiments contemplated herein, the composition has 1 or fewer visible particles or spots after storage per 36 ml of the composition. In any of the aspects and embodiments contemplated herein, the composition has no visible particles after storage per 36 ml of the composition.

[0109] 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.

[0110] In various embodiments, the storage lasts for 24 hours, 48 hours, or 72 hours, 96 hours, 120 hours, 144 hours, or 168 hours or more. 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 one week).

[0111] In some embodiments, the storage lasts for at least one week. In some embodiments, the storage lasts for at least two weeks. In some embodiments, the storage lasts for at least three weeks. In some embodiments, the storage lasts for at least four weeks. In some embodiments, the storage lasts for at least one month. In some embodiments, the storage lasts for at least two months. In some embodiments, the storage lasts for at least three months. In some embodiments, the storage lasts for at least four months. In some embodiments, the storage lasts for at least five months. In some embodiments, the storage lasts for at least six months. In some embodiments, the storage lasts for at least seven months. In some embodiments, the storage lasts for at least eight months. In some embodiments, the storage lasts for at least nine months. In some embodiments, the storage lasts for at least ten months. In some embodiments, the storage lasts for at least eleven months. In some embodiments, the storage lasts for at least one year. In some embodiments, the storage lasts for at least two years. In some embodiments, the storage lasts for at least three years. In some embodiments, the storage lasts for at least four years. In some embodiments, the storage lasts for at least five years or more.

[0112] In various embodiments, storage includes one or more freeze-thaw cycles. In some embodiments, storage includes at least 1 freeze-thaw cycle. In some embodiments, storage includes at least 2 freeze-thaw cycles. In some embodiments, storage includes at least 3 freeze-thaw cycles. In some embodiments, storage includes at least 4 freeze-thaw cycles. In some embodiments, storage includes at least 5 freeze-thaw cycles. In some embodiments, one or more freeze-thaw cycles include freezing the composition at about -65 °C or below for about 1.5 hours or more and thawing at 30 °C for 1.5 hours.

[0113] In any of the embodiments contemplated herein, the composition is frozen.

[0114] D. Methods Also contemplated herein are methods of storage, cryopreservation, transduction, and expression of transgenes.

[0115] In one aspect, a method for storing a viral vector is provided, comprising providing a viral vector, contacting the viral vector with any of the compositions contemplated herein, and storing the viral composition at a temperature of about 25 °C or below. In some embodiments, the storage temperature is 2-8 °C or below. In some embodiments, the storage temperature is 0 °C or below.

[0116] In another aspect, a method for cryopreserving a viral vector is provided, comprising providing a viral vector, contacting the viral vector with any one of the compositions contemplated herein, freezing the viral composition, and storing the viral composition at a temperature of 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.

[0117] In various embodiments, the storage is for at least 24 hours. In some embodiments, the storage is for at least 48 hours. In some embodiments, the storage is for at least 72 hours. In some embodiments, the storage is for at least 96 hours. In some embodiments, the storage is for at least 120 hours. In some embodiments, the storage is for at least 144 hours. In some embodiments, the storage is for at least 168 hours. In some embodiments, the storage is for at least 1 week. In some embodiments, the storage is for at least 2 weeks. In some embodiments, the storage is for at least 3 weeks. In some embodiments, the storage is for at least 4 weeks. In some embodiments, the storage is for at least 1 month. In some embodiments, the storage is for at least 2 months. In some embodiments, the storage is for at least 3 months. In some embodiments, the storage is for at least 4 months. In some embodiments, the storage is for at least 5 months. In some embodiments, the storage is for at least 6 months. In some embodiments, the storage is for at least 7 months. In some embodiments, the storage is for at least 8 months. In some embodiments, the storage is for at least 9 months. In some embodiments, the storage is for at least 10 months. In some embodiments, the storage is for at least 11 months. In some embodiments, the storage is for at least 1 year. In some embodiments, the storage is for at least 2 years.

[0118] In another aspect, a method of transduction is provided, comprising contacting a cell with any one of the compositions comprising a viral vector contemplated herein, thereby transducing the cell.

[0119] In another aspect, a method of expressing a transgene in a cell is provided, comprising contacting the cell with any one of the compositions comprising a viral vector contemplated herein, wherein the viral vector comprises the transgene.

[0120] In various embodiments, the cells are mammalian cells. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are hematopoietic stem or progenitor cells. In some embodiments, the cells are CD34+ hematopoietic stem or progenitor cells. In some embodiments, the cells are human CD34+ hematopoietic stem or progenitor cells.

[0121] In some embodiments, the cells are peripheral blood mononuclear cells (PMBC). In some embodiments, the cells are T cells. In some embodiments, the cells are αβ T cells. In some embodiments, the cells are γδ T cells. In some embodiments, the cells are + CD3 + , CD4 + and / or CD8

[0122] "Immune effector cells" are any cells of the immune system having one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC, etc.). Exemplary immune effector cells contemplated herein are T lymphocytes, including but not limited to cytotoxic T cells (CTL; CD8 + T cells), TIL, and helper T cells (HTL; CD4 + T cells). In certain embodiments, the cells include αβ T cells. In certain embodiments, γδ T cells modified to express an αβ TCR are included. 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.

[0123] Immune effector cells can be self or non-self (e.g., allogeneic, syngeneic, or xenogeneic). "Autologous" as used herein refers to cells from the same subject. "Allogeneic" as used herein refers to cells of the same species that are genetically different from the cells being compared. "Syngeneic" as used herein refers to cells of different subjects that are genetically identical to the cells being compared. "Xenogeneic" as used herein refers to cells of a different species from the cells being compared. In a preferred embodiment, the cells are autologous.

[0124] Exemplary immune effector cells contemplated for use in certain embodiments include T lymphocytes. The terms "T cell" or "T lymphocyte" are recognized in the art and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 + T cells), cytotoxic T cells (CTL: CD8 + T cells), CD4 + T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain 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 ).

[0125] As will be appreciated by those skilled in the art, other cells may also be used as immune effector cells in the methods contemplated herein. In particular, immune effector cells include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include precursors of effector cells, such precursors being able to be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells are, for example, CD34 + precursors of immune effector cells, such as hematopoietic stem cells (HSCs) contained within a population of cells obtained from umbilical cord blood, bone marrow or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject or are induced in vitro to differentiate into mature immune effector cells.

[0126] As used herein, the term "CD34 + cells" refers to cells that express the CD34 protein on their cell surface. "CD34", as used herein, often refers to a cell-surface glycoprotein (e.g., sialomucin protein) that acts as a cell-cell adhesion factor and is involved in T cell entry into lymph nodes. CD34 + cell populations contain hematopoietic stem cells (HSCs) which, upon administration to a patient, differentiate and contribute to all hematopoietic lineages including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0127] Methods for generating immune effector cells that express a transgene or therapeutic protein as contemplated herein are provided in certain embodiments. In one embodiment, the method comprises transducing immune effector cells isolated from an individual with a viral vector composition (e.g., a lentiviral composition) contemplated herein such that the immune effector cells express a transgene or therapeutic protein. In certain embodiments, the transduced cells are subsequently cultured to expand and then administered to a subject.

[0128] In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells may then be re-administered directly to the individual. In further embodiments, immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a transgene, such as an engineered chimeric receptor (e.g., CAR, CCR, DARIC, or switch receptor), TCR, or other transgene (e.g., protein or cytokine). In this regard, immune effector cells may be cultured before and / or after being genetically modified.

[0129] In certain embodiments, the cell source is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein. In certain embodiments, the modified immune effector cells include T cells.

[0130] In certain embodiments, PBMCs may be directly genetically modified to express a transgene or therapeutic protein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be conserved in naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0131] For example, immune effector cells such as T cells may be genetically modified after being isolated using known methods, or the immune effector cells may be activated and expanded in vitro (or differentiated in the case of progenitor cells) and then genetically modified. In certain embodiments, immune effector cells, such as T cells, are activated, stimulated to proliferate, 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 proliferated before or after genetic modification using, for example, the methods described in U.S. Pat. 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.

[0132] In one embodiment, CD34 + hematopoietic stem or progenitor cells are transduced with a nucleic acid construct contemplated herein. In certain embodiments, the transduced CD34 + cells generally differentiate into mature immune effector cells in vivo after administration to the subject from which the cells were originally isolated. In another embodiment, CD34 + cells may be stimulated in vitro before exposure to one or more of the following cytokines: Flt-3 ligand (FLT3), stem cell factor (SCF), thrombopoietin (TPO), IL-3, and IL-6, using methods described previously (Asheuer et al., 2004; Imren, et al., 2004).

[0133] In certain embodiments, cells are transfected with the vectors contemplated herein in the presence of a polycationic polymer. In some embodiments, the polycationic polymer is polybrene, protamine sulfate, polyethyleneimine, or a polyethylene glycol / poly-L-lysine block copolymer. In some embodiments, cells are transfected in the presence of polybrene. In some embodiments, cells are transfected in the presence of about 2 μg / ml polybrene. In some embodiments, cells are transfected in the presence of about 3 μg / ml polybrene. In some embodiments, cells are transfected in the presence of about 4 μg / ml polybrene. In some embodiments, cells are transfected in the presence of about 5 μg / ml polybrene. In some embodiments, cells are transfected in the presence of about 6 μg / ml polybrene. In some embodiments, cells are transfected in the presence of about 7 μg / ml polybrene. In some embodiments, cells are transfected in the presence of about 8 μg / ml polybrene. In some embodiments, cells are transfected in the presence of from about 2 μg / ml to about 8 μg / ml polybrene. In some embodiments, cells are transfected in the presence of from about 3 μg / ml to about 8 μg / ml polybrene. In some embodiments, cells are transfected in the presence of from about 4 μg / ml to about 8 μg / ml polybrene. In some embodiments, cells are transfected in the presence of from about 5 μg / ml to about 8 μg / ml polybrene. In some embodiments, cells are transfected in the presence of from about 6 μg / ml to about 8 μg / ml polybrene. In some embodiments, cells are transfected in the presence of from about 7 μg / ml to about 8 μg / ml polybrene.

[0134] E. Viral Vectors The compositions and methods contemplated herein include viral vectors. The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally ligated to the vector nucleic acid molecule and inserted therein, for example. The vector may include sequences for self-replication in a cell or sequences sufficient to allow integration into the host cell DNA. In certain embodiments, the vector is a viral vector or a non-viral vector.

[0135] As will be apparent to those skilled in the art, the term "viral vector" is widely used to refer to either a nucleic acid molecule (e.g., a transfer plasmid) that contains viral-derived nucleic acid elements that typically facilitate the transfer of nucleic acid molecules or the integration of nucleic acid molecules into the genome of a cell, or a viral particle that mediates nucleic acid transfer. Viral particles typically contain, in addition to the nucleic acid, various viral components and sometimes also host cell components. The term "viral vector" or "lentiviral vector" can refer to either a virus or a viral particle capable of transferring nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses.

[0136] Exemplary examples of viral vector systems suitable for use in certain embodiments contemplated in certain embodiments include, but are not limited to, vectors of adeno-associated virus (AAV), retrovirus, herpes simplex virus (HSV), adenovirus, and vaccinia virus.

[0137] In various embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a vaccinia virus vector, or a retrovirus vector.

[0138] AAV is a small (about 26 nm), replication-deficient, mainly episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of the host cell. Recombinant AAV (rAAV) typically consists of a minimal transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequence is about 145 bp in length. In certain embodiments, rAAV contains ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0139] In some embodiments, chimeric rAAV is used. In some embodiments, rAAV contains one or more ITR sequences. In some embodiments, the ITR sequence is isolated from one AAV serotype and the capsid sequence is isolated from a different AAV serotype. For example, an rAAV containing an ITR sequence from AAV2 and a capsid sequence from AAV6 is referred to as AAV2 / AAV6. In certain embodiments, the rAAV vector can contain ITRs 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 contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6. In a preferred embodiment, rAAV contains an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

[0140] In some embodiments, methods of engineering and selection can be applied to AAV capsids to increase the probability that they transduce the cells of interest.

[0141] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799. Each of these is incorporated herein by reference in its entirety.

[0142] 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 that genomic DNA into the host genome. Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentivirus.

[0143] 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. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2), visna-maedi virus (VMV), 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.

[0144] In some embodiments, the lentiviral vector is derived from human immunodeficiency cirus-1 (HIV-1) or human immunodeficiency virus 2 (HIV-2). In certain embodiments, the lentiviral vector is derived from human immunodeficiency cirus-1 (HIV-1).

[0145] In various embodiments, the lentiviral vectors contemplated herein include 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 optionally, as otherwise discussed herein, WPRE or HPRE, insulator element, selection marker, and cell suicide gene.

[0146] In certain embodiments, the lentiviral vectors contemplated herein may be integrative, or non-integrative or integrase-deficient lentiviruses. As used herein, the term "integrase-deficient lentivirus" or "IDLV" refers to a lentivirus having an integrase that lacks the ability to integrate the viral genome into the genome of the host cell. Viral vectors lacking integrative ability are described in patent application WO2006 / 010834, which is incorporated herein by reference in its entirety.

[0147] Exemplary mutations in the appropriate HIV-1 pol gene to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D116I, D116A, N120G, N120I, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199C, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0148] In one embodiment, the HIV-1 integrase-defective pol gene comprises a D64V, D116I, D116A, E152G, or E152A mutation, a D64V, D116I, and E152G mutation, or a D64V, D116A, and E152A mutation.

[0149] In one embodiment, the HIV-1 integrase-defective pol gene comprises a D64V mutation.

[0150] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the ends of retroviral DNA, and in the native sequence the LTR is a direct repeat and includes U3, R, and U5 regions.

[0151] As used herein, the term "FLAP element", or "cPPT / FLAP", refers to a nucleic acid whose sequence includes a central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, such as HIV-1 and HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and in Zennou, et al., 2000, Cell, 101:173. In another embodiment, the lentiviral vector includes a FLAP element having one or more mutations in the cPPT and / or CTS elements. In yet another embodiment, the lentiviral vector includes either a cPPT or a CTS element. In yet another embodiment, the lentiviral vector does not include a cPPT or a CTS element.

[0152] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located within the retroviral genome and required for the insertion of viral RNA into the viral capsid or viral particle. See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0153] The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus of a cell to the cytoplasm. Examples of RNA export elements include, but are not limited to, the rev-responsive element (RRE) of human immunodeficiency virus (HIV) (see, e.g., Cullen 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).

[0154] In certain embodiments, expression of a heterologous sequence in a viral vector is increased by incorporating into the vector a post-transcriptional regulatory element, an efficient polyadenylation site, and, optionally, a transcription termination signal. A variety of post-transcriptional regulatory elements can increase the expression of a heterologous nucleic acid as protein. For example, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the hepatitis B virus post-transcriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766).

[0155] Lentiviral vectors preferably include several safety enhancements as a result of modifying the LTRs. “Self-inactivating” (SIN) vectors refer to replication-defective vectors in which, for example, the right (3′) LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5′ LTR with a heterologous promoter that induces transcription of the viral genome during production of viral particles. Examples of heterologous promoters that can be used include, for example, the promoters of 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).

[0156] As used herein, the term “pseudotype” or “pseudotyping” refers to a virus having a viral envelope protein replaced with an envelope protein of another virus having desirable properties. For example, the HIV envelope protein (encoded by the env gene) normally targets the virus to presenting cells, but pseudotyping HIV with the G-protein (VSV-G) envelope protein of vesicular stomatitis virus allows HIV to infect a wide range of cells. Other envelope proteins for pseudotyping include, but are not limited to, envelopes from other vesiculovirus strains (e.g., Indiana, Aragua, New Jersey, Isfahan, CoCal, Maraba, or Piry), measles envelope protein, sindbis envelope protein, morbillivirus proteins (e.g., F and H proteins), Sendai proteins (e.g., F and HN proteins), or paramyxovirus proteins (e.g., F and H proteins). + targets the virus to presenting cells, but pseudotyping HIV with the G-protein (VSV-G) envelope protein of vesicular stomatitis virus allows HIV to infect a wide range of cells. Other envelope proteins for pseudotyping include, but are not limited to, envelopes from other vesiculovirus strains (e.g., Indiana, Aragua, New Jersey, Isfahan, CoCal, Maraba, or Piry), measles envelope protein, sindbis envelope protein, morbillivirus proteins (e.g., F and H proteins), Sendai proteins (e.g., F and HN proteins), or paramyxovirus proteins (e.g., F and H proteins).

[0157] In certain embodiments, the lentiviral vector is produced by 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.

[0158] According to certain embodiments contemplated herein, the backbone sequences of most or all viral vectors are derived from lentiviruses, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, or that numerous substitutions and modifications of certain lentiviral sequences can be adapted without impairing the ability of the transfer vector to perform the functions described herein. Furthermore, a variety of 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 Nos. 6,013,516; and 5,994,136. Many of them can be adapted to produce the viral vectors or transfer plasmids contemplated herein.

[0159] In various embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is the AnkT9W vector Lenti-D vector. In some embodiments, the lentiviral vector is the AnkT9W vector, the T9Ank2W vector, the TNS9 vector, the TNS9.3 vector, the TNS9.3.55 vector, the lentiglobin HPV569 vector, the lentiglobin BB305 vector, the BG-1 vector, the BGM-1 vector, the GLOBE vector, the G-GLOBE vector, the βAS3-FB vector, or a derivative thereof. In some embodiments, the lentiviral vector is the AnkT9W vector or a derivative thereof. In some embodiments, the lentiviral vector is the T9Ank2W vector or a derivative thereof. In some embodiments, the lentiviral vector is the TNS9 vector or a derivative thereof. In some embodiments, the lentiviral vector is the TNS9.3 vector or a derivative thereof. In some embodiments, the lentiviral vector is the TNS9.3.55 vector or a derivative thereof. In some embodiments, the lentiviral vector is the lentiglobin HPV569 vector or a derivative thereof. In some embodiments, the lentiviral vector is the lentiglobin BB305 vector or a derivative thereof. In some embodiments, the lentiviral vector is the BG-1 vector or a derivative thereof. In some embodiments, the lentiviral vector is the BGM-1 vector or a derivative thereof. In some embodiments, the lentiviral vector is the GLOBE vector or a derivative thereof. In some embodiments, the lentiviral vector is the G-GLOBE vector or a derivative thereof. In some embodiments, the lentiviral vector is the βAS3-FB vector or a derivative thereof. In certain embodiments, the lentiviral vector is BB305.

[0160] F. Transgene 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.

[0161] As used herein, the term "transgene" refers to an exogenous nucleic acid sequence encoding a protein or functional nucleotide. A transgene can encode a non-natural or natural protein or polypeptide. As used herein, the term "therapeutic protein" refers to a protein or polypeptide encoded by a transgene that is useful for the treatment of a disease (e.g., cancer) in a patient.

[0162] In various embodiments, the transgene or therapeutic protein is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), an αβ T cell receptor (αβ-TCR), a γδ T cell receptor (γδ-TCR), a dimerizer-regulated immune receptor complex (DARIC), or a switch receptor that specifically binds to a target antigen. In some embodiments, the transgene or therapeutic protein is an exogenous costimulatory factor, an immunomodulatory factor, an agonist for a costimulatory factor, an antagonist for an immunosuppressive factor, an immune cell engager, or a fusion protein. In some embodiments, the transgene or therapeutic protein is a costimulatory factor. In some embodiments, the transgene or therapeutic protein is a cytokine.

[0163] As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" describe the binding of a binding domain to a target antigen with a binding affinity higher than background binding. The binding domain binds to or associates with an antigen, for example, with an affinity of about 10 5 M -1 or greater or a K a (i.e., the equilibrium binding constant of a particular binding interaction having units of 1 / M) for the antigen, when it "specifically binds" to the target antigen. In certain embodiments, the binding domain (or a fusion protein comprising the binding domain) binds to the target with an affinity of about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M-1 and 10 10 M -1 and 10 11 M -1 and 10 12 M -1 or 10 13 M -1 or K greater than or equal to 10 a are conjugated. The "high affinity" binding domain (or its single-chain fusion protein) has 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 K greater than or equal to 10 a and refers to a binding domain having the same.

[0164] The terms "binds selectively" or "selectively bound" or "selective binding" or "selectively targets" describe the preferential binding of one molecule to a target molecule (on-target binding) in the presence of multiple off-target molecules.

[0165] In various embodiments, the CAR, CCR, DARIC, or switch receptor is the alpha folate receptor (FRα), α 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 (EGFR), 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 acetylcholine esterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican 3 (GPC3), EGFR family including ErbB2 (HER2), IL-10Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), K-Ras, K-Ras G12C, K-Ras G12D, lambda, 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-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;It specifically binds to a target antigen selected from the group consisting of 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, X breakpoint 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).;

[0166] 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.

[0167] In various embodiments, the αβ-TCR or γδ-TCR specifically binds to a target antigen selected from the group consisting of: alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family member, brother of the regulator of imprinted sites (BORIS), cancer testis antigen, cancer testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, melanoma cytotoxic T lymphocyte (CTL) recognition antigen (CAMEL), 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) non-structural 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), preferentially expressed antigen in melanoma (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, 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).

[0168] In various embodiments, the αβ-TCR or γδ-TCR specifically binds to the MAGE-A4 peptide.

[0169] Exemplary transgenes, therapeutic proteins, including but not limited to CAR, CCR, αβ-TCR, γδ-TCR, DARIC, 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.

[0170] In various embodiments, the transgene or therapeutic protein is a therapeutic globin for the treatment of hemoglobinopathies or the ABCD1 gene for the treatment of CALD.

[0171] In certain embodiments, the transgene or therapeutic protein is a globin. In some embodiments, the globin is human β-globin, human δ-globin, anti-sickling globin, human γ-globin, human β A-T87Q -globin, human β A-G16D / E22A / T87Q -globin, or human β A-T87Q / K95E / K120E -globin protein. In certain embodiments, the globin is human β-globin protein. In certain embodiments, the globin is an anti-sickling globin protein. In certain embodiments, the globin is human γ-globin protein. In certain embodiments, the globin is human β A-T87Q -globin protein. In certain aspects, the globin is human β A-G16D / E22A / T87Q -globin protein. In certain aspects, the globin is human β A-T87Q / K95E / K120E -globin protein. In certain embodiments, β-globin is human β-globin. In certain embodiments, β-globin is β A-T87Q -globin.

[0172] In certain embodiments, polynucleotides encoding the transgenes or therapeutic proteins described herein are provided.

[0173] As used herein, the terms "polynucleotide" or "nucleic acid" refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded, and may be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, genomic RNA (gRNA), plus-strand RNA (RNA(+)), minus-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. A polynucleotide refers to a polymeric form 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, and includes ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, "intermediate length" will be readily understood to mean any length between the recited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, a 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.

[0174] As used herein, "isolated polynucleotide" refers to a polynucleotide purified from the sequences that flank it in nature, for example, a DNA fragment that has been removed from the sequences that normally flank the fragment. In certain embodiments, "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature but are man-made. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained from or derived from a recombinant source.

[0175] In various embodiments, the polynucleotide includes mRNA encoding a polypeptide contemplated herein. In certain embodiments, the mRNA includes a cap, one or more nucleotides, and a poly(A) tail.

[0176] In certain embodiments, a polynucleotide described herein that includes a polynucleotide encoding a transgene or therapeutic protein described herein may be codon-optimized. As used herein, the term "codon optimization" refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors that affect codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes, or a synthetically constructed bias table, (ii) variation in the degree of codon bias within an organism, gene, or gene set, (iii) systematic variation of codons including context, (iv) variation of codons by their decoding tRNAs, (v) variation of codons by GC% at either the overall or any one of the triplet positions, (vi) variation in the degree of similarity to a reference sequence, e.g., a naturally occurring sequence, (vii) variation in codon frequency cutoffs, (viii) structural properties of the mRNA transcribed from the DNA sequence, (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set should be based, (x) systematic variation of codon sets for each amino acid, and / or (xi) isolation and removal of spurious translation initiation sites.

[0177] As used herein, the term "nucleotide" refers to a heterocyclic nitrogen base linked to a phosphorylated sugar by an N-glycosidic bond. Nucleotides are understood to include natural bases and a wide range of modified bases recognized in the art. Such bases are generally located at the 1'-position of the nucleotide sugar moiety. Nucleotides generally include 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., deoxyribose is a sugar lacking the hydroxyl group present in ribose.

[0178] As used herein, terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes to the reference sequence under stringent conditions as defined herein. These terms include polynucleotides that are distinguishable from the 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 substituted with another nucleotide. In this regard, in the art, it is understood that a certain change including mutation, addition, deletion, and substitution can be made to a reference polynucleotide, and the modified polynucleotide may retain the biological function or bioactivity of the reference polynucleotide.

[0179] As used herein, a statement that includes "sequence identity," or for example "a sequence that is 50% identical to," refers to the degree to which the sequences are identical nucleotide by nucleotide or amino acid by amino acid over a comparison window. Thus, the "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100. Also 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.

[0180] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a gene sequence in a vector that can express RNA and then express a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, for example, a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, such as a promoter, enhancer, poly(A) sequence, and, for example, a gene of interest, for example, a polynucleotide of interest. The vector can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassettes are positioned and oriented contiguously within the vector such that the nucleic acids in the cassette can be transcribed into RNA and, if necessary, translated into a protein or polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, be targeted to the appropriate intracellular compartment and transferred to a compartment suitable for biological activity or secreted into the extracellular compartment. The cassette preferably has 3' and 5' ends that are compatible with immediate insertion into the vector, for example, having restriction endonuclease sites at each end. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.

[0181] The polynucleotide contains a polynucleotide of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide contemplated herein or serves as a template for transcription of an inhibitory polynucleotide.

[0182] The polynucleotides contemplated herein may 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, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., disclosed elsewhere herein or known in the art, regardless of the length of the coding sequence itself, and the overall length thereof may vary significantly. Thus, it is contemplated that polynucleotide fragments of almost all lengths can be employed, preferably with the overall length being limited by ease of preparation and intended use in recombinant DNA protocols.

[0183] Polynucleotides may be prepared, manipulated, expressed, and / or delivered using any of a variety of established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide may be inserted into an appropriate vector.

[0184] Exemplary examples of vectors include, but are not limited to, plasmids, self-replicating sequences, and transposable elements, such as Sleeping Beauty, PiggyBac.

[0185] Additional exemplary 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.

[0186] Exemplary examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0187] Exemplary examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of the polypeptides disclosed herein may be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.

[0188] The “expression control sequences,” “control elements,” or “regulatory sequences” present in an expression vector are the non-translated regions of the vector and include the origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), intron, polyadenylation sequence, 5′ and 3′ untranslated regions, all of which interact with host cell proteins to effect transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcriptional and translational elements may be used, including ubiquitous promoters and inducible promoters.

[0189] In certain embodiments, the polynucleotide comprises a vector, including, but not limited to, an expression vector and a viral vector. The vector may include one or more exogenous, endogenous, or heterologous control sequences, such as a promoter and / or an enhancer. An "endogenous control sequence" is a sequence that is naturally linked to a given gene in the genome. An "exogenous control sequence" is a sequence that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biology techniques), and the transcription of the gene is induced by the linked enhancer / promoter. A "heterologous control sequence" is an exogenous sequence from a species different from the cell being genetically engineered. A "synthetic" control sequence may further include one endogenous and / or exogenous sequence, and / or elements of sequences determined in vitro or in silico that provide optimal promoter activity and / or enhancer activity for a particular treatment.

[0190] As used herein, the term "promoter" refers to a recognition site on a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, a promoter that operates in mammalian cells includes an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or another sequence, the CNCAAT region, that is present 70-80 bases upstream from the transcription start site, where N can be any nucleotide.

[0191] The term "enhancer" refers to a DNA segment that includes sequences capable of providing enhanced transcription, and in some examples, can function independently of their orientation with respect to another control sequence. Enhancers can function cooperatively or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment that includes sequences capable of providing both promoter function and enhancer function.

[0192] The term "operatively linked" refers to an arrangement in which the recited components are in a relationship that enables them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer, etc.) and a second polynucleotide sequence, such as a polynucleotide of interest, wherein the expression control sequence directs the transcription of a nucleic acid corresponding to the second sequence.

[0193] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that enables the continuous or sequential transcription of an operatively linked sequence. The structural expression control sequence may be a "ubiquitous" promoter, enhancer, promoter / enhancer that enables expression in various cell types and tissue types, or may be a "cell-specific", "cell type-specific", "cell line-specific", or "tissue-specific" promoter, enhancer, or promoter / enhancer, respectively, that enables expression in limited cell types and tissue types.

[0194] Exemplary ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, viral 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, vaccinia virus - derived H5, P7.5 and P11 promoters, elongation factor 1 alpha (EF1a) promoter, early growth response protein 1 (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 ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477 - 1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase - 1 (PGK) promoter, cytomegalovirus enhancer / chicken beta - actin (CAG) promoter, beta - 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).

[0195] In one embodiment, the vector comprises an MNDU3 promoter. In one embodiment, the vector comprises an EF1a promoter comprising the first intron of the human EF1a gene. In one embodiment, the vector comprises an EF1a promoter lacking the first intron of the human EF1a gene.

[0196] In certain embodiments, it may be desirable to use cell, cell type, cell lineage, or tissue-specific expression control sequences to achieve cell type-specific, cell lineage-specific, or tissue-specific expression of a desired polynucleotide sequence (e.g., expressing a particular nucleic acid encoding a polypeptide only in a subset of cell types, cell lineages, or tissues, or during a particular stage of development).

[0197] In certain embodiments, it may be desirable to express a polynucleotide that is a T cell-specific promoter.

[0198] As used herein, "conditional expression" can refer to any type of conditional expression, including but not limited to inducible expression, repressive expression, expression in cells or tissues having a particular physiological, biological, or disease state. This definition is not intended to exclude cell type- or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, such that, for example, expression is controlled by exposing the cell, tissue, organism to a treatment or condition under which the polynucleotide is expressed, or a treatment or condition under which expression of the polypeptide encoded by the polynucleotide of interest is increased or decreased.

[0199] Exemplary examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as the promoters of genes encoding glucocorticoid receptor or estrogen receptor (inducible by treatment with the corresponding hormone), metallothionein promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), "GeneSwitch" mifepristone regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switch (WO2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducers include, but are not limited to, glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0200] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry to the start codon of a cistron (protein-coding region), such as ATG, etc., and leads to cap-independent gene 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 IRESs commonly employed by those skilled in the art include those described in U.S. Patent No. 6,692,736. Further examples of "IRES" known in the art include, but are not limited to, IRESs obtainable from picornaviruses (Jackson et al., 1990), and IRESs obtainable from viral mRNA or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF), etc. (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, as well as encephalomyocarditis virus (EMCV) (Duke et al., 1992. J. Virol 66(3):1602-9) and VEGF IRES (Huez et al., 1998. Mol Cell Biol 18(11):6178-90) commercially available from Novagen, and yeast transcription factors TFIID and HAP4. IRESs have also been reported in the viral genomes of species of the Picornaviridae, Dicistroviridae, and Flaviviridae families, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0201] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0202] In certain embodiments, the polynucleotide comprises a consensus Kozak sequence. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the ribosomal small subunit 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).

[0203] Elements for efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector comprises a 3' polyadenylation sequence of a polynucleotide encoding a polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both termination and polyadenylation of a nascent RNA transcript 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 thus contribute to improved translation efficiency. Cleavage and polyadenylation are directed by the poly(A) sequence in the RNA. The core poly(A) sequence of mammalian pre-mRNA has two recognition elements adjacent to the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of a more variable element rich in U residues or GU residues. Cleavage of the primary transcript occurs between these two elements, and up to 250 adenosines are added to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is the best polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 polyA sequence, the bovine growth hormone polyA sequence (BGHpA), the rabbit β-globin polyA sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art. In certain embodiments, the poly(A) sequence is synthetic.

[0204] A polynucleotide encoding one or more polypeptides, or a fusion polypeptide, may be introduced into immune effector cells, such as T cells, by both non-viral and viral methods. In certain embodiments, delivery of one or more polynucleotides may be provided by the same method or different methods, and / or by the same vector or different vectors.

[0205] All publications, patent applications, and registered patents cited in this specification are hereby incorporated by reference as if each individual publication, patent application, or registered patent was specifically and individually indicated to be incorporated by reference.

[0206] The foregoing embodiments have been described in detail by way of illustration and example for purposes of clarity of understanding, but it will be readily apparent to those skilled in the art that certain changes and modifications can be made without departing from the spirit or scope of the appended claims in light of the teachings contemplated herein. The following examples are provided for illustrative purposes only and not for purposes of limitation. Those skilled in the art will readily recognize various non-critical parameters that can be varied or modified to produce substantially similar results.

Example

[0207] Example 1 Preparation for Lentiviral Vector Purification and Screening The basic process for formulating a lentiviral vector (LVV) starts with ultrafiltration (UF) and diafiltration (DF) (together UFDF) via tangential flow filtration (TFF), followed by dilution in 2X / 1X stem cell growth medium (SCGM). A hollow fiber membrane with a 500 kDa cut-off is used to concentrate the purified LVV 4 - 5-fold. The product is then exchanged into a diafiltration buffer of 50 mM HEPES (pH 7.5) and 100 mM NaCl. The resulting material is the UFDF pool. To achieve a control formulated product, the UFDF pool is diluted in 2X / 1×SCGM at a 1:1 weight ratio. The formulated product is then filtered through a 0.22 micron polyethersulfone (PES) membrane to obtain an intermediate bulk. To achieve different formulated products for screening described herein, the process remained the same as described except that the diafiltration buffer was varied using different buffers and the UFDF pool was formulated at a 1:1 ratio with a 2-fold concentrated formulation solution. After production, approximately 1 mL of the intermediate bulk was filled into a 2 mL plastic vial sealed with a rubber serum stopper.

[0208] A broad overview of the tested screening process / steps and formulation components 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 at 2 - 8 °C for 48 or 168 hours. These conditions were used for screening. Stress conditions were also evaluated to assess the robustness of the formulation, including (i) up to 5 freeze - thaw cycles involving 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).

[0209] Example 2 Analysis of Lentiviral Cryopreserved Formulations Stability was evaluated by the infectivity titer measured by viral transduction of HOS cells, the hydrodynamic size by dynamic light scattering (DLS), and the number of visible particles observed under fluorescence light with a black or white background. The unfolding temperature of some formulations was evaluated by differential scanning fluorimetry (DSF). Formulations after storage at different temperatures and / or freeze - thaw conditions were compared to the initial value or baseline. Formulations that maintained the integrity of the LVV were further evaluated for CAR expression in PBMCs.

[0210] The infectivity titer was used as the main indicator of stability. The number of infectivity titers or transduction units was measured by viral transduction of HOS cells using standard techniques (see, e.g., GENE Therapy (2002), 9, 1155 - 1162). The selection criterion was a recovery of greater than 75% (infectivity titer after storage / starting infectivity titer × 100%).

[0211] Changes in the hydrodynamic size by dynamic light scattering (DLS) were observed when the size increased by approximately 10 nm or more after storage.

[0212] The number of visible particles was monitored by visual observation under light with an output exceeding 2000 Lux. The particles can be due to impurities such as host cell proteins (HCP) or lentivirus aggregation. The formulation was evaluated to determine whether a particle-free solution can be maintained over a long period. The appearance was monitored to distinguish between inherent particles, endogenous particles, and adventitious particles after vial filling.

[0213] DSF was used to characterize the conformational stability of lentivirus for formulation identification. A lower thermal unfolding temperature can indicate that the lentivirus is less stable in a particular formulation.

[0214] - Summary of experimental results on buffer species screening For the selection of buffers, the evaluated buffers were HEPES, sodium citrate, PIPES, L-histidine (L-His), sodium phosphate, and Tris at a concentration of 27.5 mM and pH 7. These formulations also contained 73 mM trehalose. Based on >75% infectivity titer recovery in transfected HOS cells, the HEPES, PIPES, and L-His formulations had the least change after storage at 25 °C for 24 hours and at 2 - 8 °C for 48 hours (Figure 3). By hydrodynamic size, no change in size was observed for any of the formulations after storage at 25 °C for 24 hours and at 2 - 8 °C for 48 hours. After storage at 37 °C for 48 hours, no change in size was observed for the HEPES, L-His, and Tris formulations, while the sodium citrate and sodium phosphate formulations had a change in size (Figure 4). By thermal unfolding measurements, all buffers had an equivalent onset of unfolding in the range between 45 - 47 °C (Figure 5). Sodium citrate, sodium phosphate, and PIPES appeared to have two unfolding transitions, while the L-His, HEPES, and Tris buffers appeared to have one unfolding transition. Based on the first unfolding transition, L-His, HEPES, and Tris had the highest thermal unfolding temperature of 54 - 57 °C, while sodium citrate, sodium phosphate, and PIPES had an unfolding temperature of 48 - 49 °C.

[0215] -Summary of experimental results for pH screening The target pH was evaluated by assessing LVV in 27.5 mM HEPES and 73 mM trehalose at pH 6.5, 7, or 8. In this study, LVV in 27.5 mM PIPES and 73 mM trehalose at pH 6.5 or 7 was also evaluated. Based on >75% infectivity titer recovery in transduced HOS cells, minimal changes were observed in HEPES (pH 7), HEPES (pH 8), and PIPES (pH 7) formulations after 25 °C storage for 24 hours and 2 - 8 °C storage for 48 hours (Figure 6). By hydrodynamic size, the HEPES (pH 8) formulation had changes observed after 25 °C storage for 24 hours and 2 - 8 °C storage for 48 hours. All formulations, except the HEPES (pH 7) formulation, had changes in size after 37 °C storage for 48 hours (Figure 7). By thermal unfolding measurements, HEPES (pH 6.5) and PIPES (pH 6.5) formulations had an earlier onset of unfolding at approximately 40 °C compared to PIPES (pH 7), HEPES (pH 7), and HEPES (pH 8) formulations that had an onset of unfolding between 45 - 48 °C (Figure 8). PIPES (pH 7) and HEPES (pH 8) formulations appeared to have two unfolding transitions, while the remaining formulations did not. Based on the first unfolding transition, PIPES (pH 6.5) and HEPES (pH 6.5) formulations had the highest unfolding temperature of approximately 60 °C, followed by the HEPES (pH 7) formulation with an unfolding temperature of 54 °C. Based on the overall evaluation, LVV HEPES (pH 7) best met many of the screening criteria.

[0216] - Summary of experimental results for disaccharide and salt screening The carbohydrates trehalose and sucrose were compared in HEPES buffer and PIPES buffer. Based on LVV stability (Figure 9), no differences were observed; thus, sucrose combinations were also further evaluated.

[0217] A 27.5 mM HEPES (pH 7) formulation with 73 mM sucrose was compared in combination with 73 mM sucrose and 75 mM NaCl or KCl. Based on the infectivity titer results, both the HEPES-based formulation and the salt formulation were equivalent after storage at 25 °C for 24 hours and at 2 - 8 °C for 48 hours (Figure 10). With freeze-thaw stress (5 cycles), formulations with 73 mM sucrose alone or with 75 mM NaCl had the least change by infectivity titer measurement (Figure 11). By hydrodynamic size, the HEPES-based formulation had no observed change after storage at 25 °C for 24 hours, at 2 - 8 °C for 48 hours, and at 37 °C for 48 hours (Figure 12). Changes in size were not observed for the HEPES-based formulation after 5 freeze-thaw cycles (Figure 13). From this test, the 27.5 mM HEPES (pH 7) formulations with and without 75 mM NaCl containing 73 mM sucrose were the top formulation choices.

[0218] PIPES-based formulations were further evaluated because they have been reported in the literature to be effective against lentivirus. 27.5 mM PIPES (pH 6.5) with 73 mM trehalose and 27.5 mM PIPES (pH 6.5) with 73 mM sucrose and 75 mM NaCl were compared. In addition, 27.5 mM PIPES (pH 7) with 73 mM sucrose in combination with 73 mM sucrose or 75 mM NaCl or 75 mM KCl was evaluated. Based on >80% infectivity titer recovery in transduced HOS cells, the PIPES (pH 6.5) formulation with 73 mM sucrose and 75 mM NaCl had the least change after storage at 25 °C for 24 hours and at 2-8 °C for 48 hours (Figure 10). After freeze-thaw (5 cycles), the PIPES (pH 6.5) formulation with 73 mM sucrose and 75 mM NaCl had the highest infectivity titer recovery (Figure 11). By hydrodynamic size, the PIPES-based formulations had no observed changes after storage at 25 °C for 24 hours and at 2-8 °C for 48 hours. Changes in size were observed for all PIPES-based formulations after storage at 37 °C for 48 hours (Figure 12). After 5 freeze-thaw cycles, only the PIPES (pH 6.5) formulation with 73 mM sucrose and 75 mM NaCl had an increase in size (Figure 13).

[0219] Based on the comparison of PIPES- and HEPES-based formulations at similar pH, LVV was surprisingly more stable in HEPES-based buffers, especially upon evaluation of particle size under various conditions and titer after freeze-thaw.

[0220] - Summary of experimental results for additional component screening Screening tests were completed using several additional components to buffer and sucrose, and whether the additional components improved LVV stability with a longer temperature holding period at 5 °C for 168 hours was seen by monitoring the number of visible particles. The formulations were 27.5 mM HEPES (pH 7) and: - 73 mM sucrose and 75 mM NaCl - 73 mM sucrose, 75 mM NaCl and 0.1 mg / mL P188 - 73 mM sucrose and 50 mM L-Pro - 146 mM sucrose - 146 mM sucrose and 75 mM NaCl - 146 mM sucrose and 50 mM L-Arg - 146 mM sucrose, 50 mM L-Arg and 50 mM L-Glu - 146 mM sucrose and 0.1 mg / mL P188 - composed of 146 mM sucrose and 16 mg / mL insulin The following formulations were also evaluated using 27.5 mM L-His (pH 7): - 146 mM sucrose - 146 mM sucrose and 50 mM L-Pro - 146 mM sucrose and 16 mg / mL insulin - 275 mM sorbitol - 73 mM sucrose and 75 mM NaCl - 73 mM sucrose and 50 mM L-Pro - 73 mM sucrose, 75 mM NaCl and 0.1 mg / mL P188

[0221] By infectivity titration, all formulations of 27.5 mM HEPES (pH 7) consisting of 146 mM sucrose showed poor performance after storage at 25°C for 24 hours and at 2-8°C for 168 hours (Figure 14). Formulations of 27.5 mM HEPES (pH 7) and 73 mM sucrose 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 (Figure 14). All formulations showed no change in hydrodynamic size after storage at 2-8°C for 168 hours (Figure 15). Finally, when comparing only the 73 mM sucrose-based formulations, the smallest number of visible particles was observed for the formulation containing 0.1 mg / mL Poloxamer 188 and 50 mM L-proline after storage at 2-8°C. Similarly, formulations of 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 a small number of visible particles after freezing at ≦-65°C for more than 1.5 hours and thawing at 30°C for 1.5 hours. (Figures 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 infectivity titer recovery, no change in hydrodynamic size, and the smallest number of visible particles after storage at 25°C for 24 hours, at 2-8°C for 168 hours, and after one freeze-thaw cycle (Figures 20-25).

[0222] A formulation of 27.5 mM HEPES (pH 7), 73 mM sucrose, and 50 mM L - Pro was evaluated across multiple lentiviral vectors (e.g., LVV1, LVV2, LVV3, LVV4, and LVV5) encoding various constructs targeting different genes. The titer recovery after storage at 2 - 8°C for up to 168 hours was evaluated by statistical analysis (Figure 26). Analyses were grouped by time at 24, 120, and 168 hours of retention. One - way ANOVA followed by Tukey - Kramer analysis was performed. The p - value comparing mean titer recoveries was not significant (greater than 0.05), indicating that formulation performance was similar for each LVV.

[0223] Additional evaluations were performed using poloxamer 188 to reduce the number of visible particles sometimes observed after manufacture. Formulations with only 27.5 mM HEPES (pH 7), 73 mM sucrose, and 50 mM L - Pro 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 (Figures 27 and 28). With an addition of at least 0.3 g / L, the appearance of particles decreased after 24 hours and the particle form was minimized after 120 hours of storage compared to formulations without P188 and with 0.08% P188. The infectivity 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 over 168 hours at 2 - 8°C and compared to a formulation without P188 (Figure 29). Both formulations had similar performance, indicating that P188 addition did not affect infectivity titer recovery.

[0224] Finally, it was interesting to store LVV at ≤ - 65°C for an extended period. Formulations with and without P188 were compared for LVV1, LVV2, and LVV3 at ≤ - 65°C for up to 6 months. All LVV and formulations maintained infectivity titer levels over the 6 - month storage (Figure 30).

[0225] Example 3 Analysis of Lentiviral Potency The efficacy of the lentivirus was measured in peripheral blood mononuclear cells (PBMCs). PBMCs were transduced with lentivirus at various multiplicities of infection (MOIs of 2.5, 5, 10, and 20), and the relative frequency of chimeric antigen receptor expression (% CAR+ cells) was measured. CAR+ was detected by a fluorescently labeled protein that binds to the CAR, and the cell population was evaluated by flow cytometry. Relative efficacy is reported as a relative measurement against a reference standard.

[0226] The following formulations had the highest infectious titer recovery after storage at 25°C, 2 - 8°C, and multiple freeze - thaw cycles associated with the lentivirus manufacturing process. As a confirmation of compatibility with the drug product process, relative potency measurements were performed and it was confirmed that they had no significant effect on potency (CAR+ expression) and the stabilized LVV (Figure 31). - 27.5 mM HEPES (pH 7), 73 mM sucrose, and 75 mM NaCl - 27.5 mM HEPES (pH 7), 73 mM sucrose, 75 mM NaCl, and 0.1 mg / mL P188 - 27.5 mM HEPES (pH 7), 73 mM sucrose, and 50 mM L - Pro - 27.5 mM L - His (pH 7), 73 mM sucrose, and 50 mM L - Pro

[0227] The top - ranked formulations evaluated here met the criteria required for the manufacturing process. The extended periods at 25°C and 2 - 8°C allow for flexibility in many manufacturing operations, and stability appeared to be maintained with combinations of HEPES (pH 7) and L - His (pH 7) and sucrose. Additionally, the freeze - thaw process significantly reduces the recovery of the lentiviral vector. The tests showed that in some instances, the addition of sodium chloride or L - proline improved recovery after a single freeze - thaw, and sometimes up to 5 freeze - thaw cycles. Finally, in the filling and finishing process, a visible decrease in particles may indicate aggregation of particles in solution. Poloxamer 188 and L - proline appeared to minimize these interactions.

[0228] Embodiment The present disclosure contemplates the following embodiments: 1. An aqueous virus composition comprising: a) a virus vector b) a HEPES or L-histidine buffer, c) a carbohydrate, and d) an amino acid. 2. The composition of Embodiment 1, wherein the buffer is present at a concentration of about 25 mM to about 30 mM. 3. The composition of any one of Embodiments 1 or 2, wherein the buffer is present at a concentration of about 26 mM to about 29 mM. 4. The composition of any one of Embodiments 1 to 3, wherein the buffer is present at a concentration of about 27 mM to about 28 mM. 5. The composition of any one of Embodiments 1 to 4, wherein the buffer is present at a concentration of about 27.5 mM. 6. The composition of any one of Embodiments 1 to 5, wherein the buffer is a HEPES buffer. 7. The composition of any one of Embodiments 1 to 5, wherein the buffer is an L-histidine buffer. 8. The composition of any one of Embodiments 1 to 7, wherein the carbohydrate is present at a concentration of about 66 mM to about 80 mM. 9. The composition of any one of Embodiments 1 to 8, wherein the carbohydrate is present at a concentration of about 67 mM to about 79 mM. 10. The composition of any one of Embodiments 1 to 9, wherein the carbohydrate is present at a concentration of about 68 mM to about 78 mM. 11. The composition of any one of Embodiments 1 to 10, wherein the carbohydrate is present at a concentration of about 69 mM to about 77 mM. 12. The composition of any one of Embodiments 1 to 11, wherein the carbohydrate is present at a concentration of about 70 mM to about 76 mM. 13. A composition according to any one of Embodiments 1 to 12, wherein the carbohydrate is present at a concentration of about 71 mM to about 75 mM. 14. A composition according to any one of Embodiments 1 to 13, wherein the carbohydrate is present at a concentration of about 72 mM to about 74 mM. 15. A composition according to any one of Embodiments 1 to 14, wherein the carbohydrate is present at a concentration of about 73 mM. 16. A composition according to any one of Embodiments 1 to 12, wherein the carbohydrate is present at a concentration of about 2.0 wt% to about 3.0 wt% per volume of the composition. 17. A composition according to any one of Embodiments 1 to 12, wherein the carbohydrate is present at a concentration of about 2.1 wt% to about 2.9 wt% per volume of the composition. 18. A composition according to any one of Embodiments 1 to 12, wherein the carbohydrate is present at a concentration of about 2.2 wt% to about 2.8 wt% per volume of the composition. 19. A composition according to any one of Embodiments 1 to 12, wherein the carbohydrate is present at a concentration of about 2.3 wt% to about 2.7 wt% per volume of the composition. 20. A composition according to any one of Embodiments 1 to 12, wherein the carbohydrate is present at a concentration of about 2.4 wt% to about 2.6 wt% per volume of the composition. 21. A composition according to any one of Embodiments 1 to 12, wherein the carbohydrate is present at a concentration of about 2.5 wt% per volume of the composition. 22. A composition according to any one of Embodiments 1 to 21, wherein the carbohydrate is a disaccharide. 23. A composition according to any one of Embodiments 1 to 21, wherein the carbohydrate is lactose, glucose, mannose, mannitol, sorbitol, sucrose, trehalose, and / or glycerol. 24. A composition according to any one of Embodiments 1 to 21, wherein the carbohydrate is sucrose and / or trehalose. 25. The composition according to any one of Embodiments 1 to 24, wherein the carbohydrate is sucrose. 26. The composition according to any one of Embodiments 1 to 24, wherein the carbohydrate is trehalose. 27. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 40 mM to about 60 mM. 28. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 41 mM to about 59 mM. 29. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 42 mM to about 58 mM. 30. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 43 mM to about 57 mM. 31. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 44 mM to about 56 mM. 32. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 45 mM to about 55 mM. 33. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 46 mM to about 54 mM. 34. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 47 mM to about 53 mM. 35. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 48 mM to about 52 mM. 36. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 49 mM to about 51 mM. 37. The composition according to any one of Embodiments 1 to 26, wherein the amino acid is present at a concentration of about 50 mM. 38. The composition according to any one of Embodiments 1 to 37, wherein the amino acid is a non-polar amino acid. 39. The composition according to 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. 40. The composition according to any one of Embodiments 1 to 37, wherein the amino acid is selected from the group consisting of phenylalanine, tyrosine, tryptophan, and proline. 41. The composition according to any one of Embodiments 1 to 37, wherein the amino acid is L-proline. 42. The composition according to any one of Embodiments 1 to 41, wherein the composition further comprises a salt. 43. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 65 mM to about 85 mM. 44. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 66 mM to about 84 mM. 45. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 67 mM to about 83 mM. 46. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 68 mM to about 82 mM. 47. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 69 mM to about 81 mM. 48. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 70 mM to about 80 mM. 49. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 71 mM to about 79 mM. 50. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 72 mM to about 78 mM. 51. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 73 mM to about 77 mM. 52. The composition according to Embodiment 42, wherein the salt is present at a concentration of about 74 mM to about 76 mM. 53. The composition of embodiment 42 in which the salt is present at a concentration of about 75 mM. 54. The composition of any one of embodiments 42 to 53 in which the salt is a chloride salt, KCl, or NaCl. 55. The composition of any one of embodiments 42 to 54 in which the salt is NaCl. 56. The composition of any one of embodiments 1 to 55 in which the composition further comprises a poloxamer. 57. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.01 mg / ml to about 1 mg / ml. 58. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.02 mg / ml to about 0.9 mg / ml. 59. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.03 mg / ml to about 0.8 mg / ml. 60. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.04 mg / ml to about 0.7 mg / ml. 61. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.05 mg / ml to about 0.6 mg / ml. 62. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.06 mg / ml to about 0.5 mg / ml. 63. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.07 mg / ml to about 0.4 mg / ml. 64. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.08 mg / ml to about 0.3 mg / ml. 65. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.09 mg / ml to about 0.2 mg / ml. 66. The composition of embodiment 56 in which the poloxamer is present at a concentration of about 0.1 mg / ml. 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. 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. 69. The composition of embodiment 56, wherein the poloxamer is present at a concentration of about 0.3 mg / ml. 70. The composition of any one of embodiments 56 to 69, wherein the poloxamer is poloxamer 188, poloxamer 288, poloxamer 335, poloxamer 338, or poloxamer 407. 71. The composition of any one of embodiments 56 to 70, wherein the poloxamer is poloxamer 188. 72. The composition of any one of embodiments 1 to 71, wherein the composition has a pH of about 6.5 to about 8. 73. The composition of any one of embodiments 1 to 72, wherein the composition has a pH of about 6.5. 74. The composition of any one of embodiments 1 to 73, wherein the composition has a pH of about 7. 75. The composition of any one of embodiments 1 to 74, wherein the composition has a pH of about 7.5. 76. The composition of any one of embodiments 1 to 75, wherein the composition has a pH of about 8. 77. An aqueous viral composition comprising: a) a viral vector, b) about 27.5 mM HEPES, c) about 73 mM sucrose or about 2.5% by weight sucrose per volume of the composition, and d) about 50 mM L-proline, wherein the composition has a pH of about 7. 78. An aqueous viral composition comprising: a) a viral vector, b) about 27.5 mM L-histidine, c) about 73 mM sucrose or about 2.5% by weight sucrose per volume of the composition, and d) contains about 50 mM L-proline, a composition, wherein the composition has a pH of about 7. 79. An aqueous viral composition comprising a) a viral vector, b) about 27.5 mM HEPES, c) about 73 mM sucrose or about 2.5% by weight sucrose per volume of the composition, d) about 50 mM L-proline, and e) contains about 0.1 mg / mL to about 0.8 mg / mL of poloxamer 188, a composition, wherein the composition has a pH of about 7. 80. An aqueous viral composition comprising a) a viral vector, b) about 27.5 mM HEPES, c) about 73 mM sucrose or about 2.5% by weight sucrose per volume of the composition, d) about 50 mM L-proline, and e) contains about 0.3 mg / mL of poloxamer 188, a composition, wherein the composition has a pH of about 7. 81. An aqueous viral composition comprising a) a viral vector, b) about 27.5 mM HEPES, c) about 73 mM sucrose or about 2.5% by weight sucrose per volume of the composition, d) about 50 mM L-proline, and e) contains about 75 mM of NaCl, a composition, wherein the composition has a pH of about 7. 82. An aqueous viral composition comprising a) a viral vector, b) about 27.5 mM HEPES, c) about 73 mM sucrose or about 2.5% by weight sucrose per volume of the composition, d) About 50 mM L-proline, e) About 75 mM NaCl, and f) Containing about 0.1 to about 0.8 mg / mL of poloxamer 188, A composition having a pH of about 7. 83. An aqueous virus composition comprising: a) A virus vector, b) HEPES buffer, c) A carbohydrate, d) A salt, and e) A poloxamer. 84. The composition of embodiment 83, wherein the composition does not contain L-proline. 85. The composition of embodiment 83 or 84, wherein the HEPES buffer is present at a concentration of about 25 mM to about 30 mM. 86. The composition of embodiment 83 or 84, wherein the HEPES buffer is present at a concentration of about 26 mM to about 29 mM. 87. The composition of embodiment 83 or 84, wherein the HEPES buffer is present at a concentration of about 27 mM to about 28 mM. 88. The composition of embodiment 83 or 84, wherein the HEPES buffer is present at a concentration of about 27.5 mM. 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. 90. The composition of any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 67 mM to about 79 mM. 91. The composition of any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 68 mM to about 78 mM. 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. 93. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 70 mM to about 76 mM. 94. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 71 mM to about 75 mM. 95. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 72 mM to about 74 mM. 96. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 73 mM. 97. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 2.0 wt% to about 3.0 wt% per volume of the composition. 98. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 2.1 wt% to about 2.9 wt% per volume of the composition. 99. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 2.2 wt% to about 2.8 wt% per volume of the composition. 100. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 2.3 wt% to about 2.7 wt% per volume of the composition. 101. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 2.4 wt% to about 2.6 wt% per volume of the composition. 102. A composition according to any one of embodiments 83 to 88, wherein the carbohydrate is present at a concentration of about 2.5 wt% per volume of the composition. 103. A composition according to any one of embodiments 83 to 102, wherein the carbohydrate is a disaccharide. 104. A composition according to any one of embodiments 83 to 102, wherein the carbohydrate is lactose, glucose, mannose, mannitol, sorbitol, sucrose, trehalose, and / or glycerol. 105. A composition according to any one of Embodiments 83 to 102, wherein the carbohydrate is sucrose and / or trehalose. 106. A composition according to any one of Embodiments 83 to 102, wherein the carbohydrate is sucrose. 107. A composition according to any one of Embodiments 83 to 102, wherein the carbohydrate is trehalose. 108. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 65 mM to about 85 mM. 109. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 66 mM to about 84 mM. 110. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 67 mM to about 83 mM. 111. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 68 mM to about 82 mM. 112. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 69 mM to about 81 mM. 113. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 70 mM to about 80 mM. 114. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 71 mM to about 79 mM. 115. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 72 mM to about 78 mM. 116. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 73 mM to about 77 mM. 117. A composition according to any one of Embodiments 83 to 107, wherein the salt is present at a concentration of about 74 mM to about 76 mM. 118. A composition according to any one of embodiments 83 to 107, wherein the salt is present at a concentration of about 75 mM. 119. A composition according to any one of embodiments 83 to 118, wherein the salt is KCl or NaCl. 120. A composition according to embodiment 119, wherein the salt is NaCl. 121. A composition according to 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. 122. A composition according to 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. 123. A composition according to 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. 124. A composition according to 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. 125. A composition according to 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. 126. A composition according to 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. 127. A composition according to 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. 128. A composition according to 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. 129. A composition according to 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. 130. A composition according to any one of embodiments 83 to 120, wherein the poloxamer is present at a concentration of about 0.1 mg / ml. 131. A composition according to any one of embodiments 83 to 130, wherein the poloxamer is poloxamer 188, poloxamer 288, poloxamer 335, poloxamer 338, or poloxamer 407. 132. A composition according to any one of embodiments 83 to 130, wherein the poloxamer is poloxamer 188 (P188). 133. A composition according to any one of embodiments 83 to 132, wherein the composition has a pH of about 7 to about 8. 134. A composition according to any one of embodiments 83 to 132, wherein the composition has a pH of about 7. 135. A composition according to any one of embodiments 83 to 132, wherein the composition has a pH of about 7.5. 136. A composition according to any one of embodiments 83 to 132, wherein the composition has a pH of about 8. 137. An aqueous virus composition comprising: a) a virus vector, b) about 27.5 mM HEPES, c) about 73 mM sucrose per volume of the composition, d) about 75 mM NaCl, and e) about 0.1 to about 0.8 mg / ml of poloxamer 188, wherein the composition has a pH of about 7. 138. The virus vector is present at a titer of about 1×10 8 ~about 2×10 9 TU / ml in a composition according to any one of embodiments 1 to 137. 139. The virus vector is present at a titer of about 1×10 8 TU / ml in a composition according to any one of embodiments 1 to 138. 140. The virus vector is present at a titer of about 2×108 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 141. The viral vector is present at a titer of about 3×10 8 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 142. The viral vector is present at a titer of about 4×10 8 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 143. The viral vector is present at a titer of about 5×10 8 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 144. The viral vector is present at a titer of about 6×10 8 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 145. The viral vector is present at a titer of about 7×10 8 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 146. The viral vector is present at a titer of about 8×10 8 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 147. The viral vector is present at a titer of about 9×10 8 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 148. The viral vector is present at a titer of about 1×10 9 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 149. The viral vector is present at a titer of about 2×10 9 A composition according to any one of Embodiments 1 to 139, present at a titer of TU / ml. 150. The composition of any one of Embodiments 1 to 149, wherein the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a vaccinia virus vector, or a retrovirus vector. 151. The composition of any one of Embodiments 1 to 150, wherein the viral vector is a lentivirus vector. 152. The composition of Embodiment 151, wherein the lentivirus 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). 153. The composition of Embodiment 151 or Embodiment 152, wherein the lentivirus vector is derived from human immunodeficiency cirus-1 (HIV-1) or human immunodeficiency virus 2 (HIV-2). 154. The composition of any one of Embodiments 151 to 153, wherein the lentivirus vector is derived from human immunodeficiency cirus-1 (HIV-1). 155. The composition of any one of Embodiments 1 to 154, wherein the viral vector is pseudotyped. 156. The composition of any one of Embodiments 1 to 155, wherein the viral vector is pseudotyped with an envelope protein from a strain of vesicular stomatitis virus. 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, CoCal, Maraba, or Piry. 158. The composition of any one of Embodiments 1 to 157, wherein the viral vector is pseudotyped with vesicular stomatitis virus G (VSV-G) protein. 159. A composition according to any one of Embodiments 1 to 158, wherein the viral vector is pseudotyped with an envelope derived from a measles envelope protein, a Sindbis envelope protein, a morbillivirus F and H protein, a Sendai F and HN protein, or a paramyxovirus F and H protein. 160. A composition according to any one of Embodiments 1 to 159, wherein the viral vector comprises a polynucleotide containing a transgene. 161. The composition according to Embodiment 160, wherein the transgene encodes a therapeutic protein. 162. The composition according to Embodiment 160 or 161, wherein the transgene or the therapeutic protein is for the treatment of a monogenic disease, disorder, or condition. 163. The composition according to any one of Embodiments 160 to 162, wherein the transgene or the therapeutic protein is a chimeric antigen receptor (CAR), a chimeric co-stimulatory receptor (CCR), an αβ T cell receptor (αβ-TCR), a γδ T cell receptor (γδ-TCR), a dimerizer-regulated immune receptor complex (DARIC), or a switch receptor. 164. The composition according to any one of Embodiments 160 to 162, wherein the transgene or the therapeutic protein is a therapeutic globin for the treatment of hemoglobinopathy or the ABCD1 gene for the treatment of CALD. 165. A composition according to any one of Embodiments 1 to 164, wherein the composition does not contain serum. 166. A composition according to any one of Embodiments 1 to 165, wherein the composition does not contain PIPES. 167. A composition according to any one of Embodiments 1 to 166, wherein the composition does not contain sodium citrate. 168. A composition according to any one of Embodiments 1 to 167, wherein the composition does not contain sodium phosphate. 169. A composition according to any one of Embodiments 1 to 168, wherein the composition does not contain Tris. 170. A composition according to any one of Embodiments 1 to 169, wherein the composition does not contain a salt. 171. A composition according to any one of Embodiments 1 to 170, wherein the composition does not contain a chloride salt. 172. A composition according to any one of Embodiments 1 to 171, wherein the composition does not contain NaCl. 173. A composition according to any one of Embodiments 1 to 172, wherein the composition does not contain KCl. 174. A composition according to any one of Embodiments 1 to 173, wherein the composition does not contain trehalose. 175. A composition according to any one of Embodiments 1 to 174, wherein the viral vector maintains a viral infectivity titer recovery of more than about 75% in HOS cells after storage as compared to the viral infectivity titer of the viral vector in the composition before storage or at least one freeze-thaw cycle. 176. A composition according to any one of Embodiments 1 to 175, wherein the viral vector maintains a viral infectivity titer recovery of more than about 80% in HOS cells after storage as compared to the viral infectivity titer of the viral vector in the composition before storage or at least one freeze-thaw cycle. 177. A composition according to any one of Embodiments 1 to 176, wherein the viral vector maintains a viral infectivity titer recovery of more than about 85% in HOS cells after storage as compared to the viral infectivity titer of the viral vector in the composition before storage or at least one freeze-thaw cycle. 178. A composition according to any one of Embodiments 1 to 177, wherein the viral vector maintains a viral infectivity titer recovery of more than about 90% in HOS cells after storage as compared to the viral infectivity titer of the viral vector in the composition before storage or at least one freeze-thaw cycle. 179. The composition according to any one of Embodiments 1 to 178, wherein the viral vector maintains an infectivity titer recovery of more than about 95% in HOS cells after storage, compared to the infectivity titer of the viral vector in the composition before storage or at least one freeze-thaw cycle; compared to the infectivity titer of the viral vector in the composition before storage or at least one freeze-thaw cycle. 180. The composition according to any one of Embodiments 1 to 179, wherein the viral vector has a thermal unfolding temperature of about 56° to about 62°C when measured by differential scanning fluorimetry (DSF). 181. The composition according to any one of Embodiments 1 to 180, wherein the viral vector has a thermal unfolding temperature of about 58° to about 60°C when measured by differential scanning fluorimetry (DSF). 182. The composition according to any one of Embodiments 1 to 181, wherein the viral vector maintains a hydrodynamic diameter of about 150 nm to about 170 nm and a viscosity value of 0.967 centipoise (cP) as measured by dynamic light scattering (DLS) at 25°C after storage, compared to the hydrodynamic diameter of the viral vector in the composition before storage or at least one freeze-thaw cycle. 183. The composition according to any one of Embodiments 1 to 182, wherein the viral vector maintains at least about 78% efficacy as measured by transgene expression in PBMC after storage, compared to a reference standard. 184. The composition according to any one of Embodiments 1 to 183, wherein the viral vector maintains at least about 80% efficacy as measured by transgene expression in PBMC after storage, compared to a reference standard. 185. The composition according to any one of Embodiments 1 to 184, wherein the viral vector maintains at least about 85% efficacy as measured by transgene expression in PBMC after storage, compared to a reference standard. 186. A composition according to any one of embodiments 1 to 185, wherein the viral vector maintains at least about 90% potency as measured by transgene expression in PBMCs after storage, compared to a reference standard. 187. A composition according to any one of embodiments 1 to 186, wherein the viral vector maintains at least about 95% potency as measured by transgene expression in PBMCs after storage, compared to a reference standard. 188. A composition according to any one of embodiments 1 to 187, having no visible fibrous particles (whispy fibers) after storage. 189. A composition according to any one of embodiments 1 to 188, having 5 or fewer visible particles (or spots) after storage. 190. A composition according to any one of embodiments 1 to 189, having 4 or fewer visible particles (or spots) after storage. 191. A composition according to any one of embodiments 1 to 190, having 3 or fewer visible particles (or spots) after storage. 192. A composition according to any one of embodiments 1 to 191, having 2 or fewer visible particles (or spots) after storage. 193. A composition according to any one of embodiments 1 to 192, having 1 or fewer visible particles (or spots) after storage. 194. A composition according to any one of embodiments 1 to 193, having no visible particles in the composition after storage. 195. A composition according to any one of embodiments 1 to 194, having no visible fibrous particles (whispy fibers) per 50 ml of the composition after storage. 196. A composition according to any one of embodiments 1 to 195, having 5 or fewer visible particles (or spots) per 50 ml of the composition after storage. 197. A composition according to any one of Embodiments 1 to 196, having 4 or fewer visible particles (or spots) per 50 ml of the composition after storage. 198. A composition according to any one of Embodiments 1 to 197, having 3 or fewer visible particles (or spots) per 50 ml of the composition after storage. 199. A composition according to any one of Embodiments 1 to 198, having 2 or fewer visible particles (or spots) per 50 ml of the composition after storage. 200. A composition according to any one of Embodiments 1 to 199, having 1 or fewer visible particles (or spots) per 50 ml of the composition after storage. 201. A composition according to any one of Embodiments 1 to 200, having no visible particles per 50 ml of the composition after storage. 202. A composition according to any one of Embodiments 1 to 201, having no visible fibrous particles (whispy fibers) per 36 ml of the composition after storage. 203. A composition according to any one of Embodiments 1 to 202, having 5 or fewer visible particles (or spots) per 36 ml of the composition after storage. 204. A composition according to any one of Embodiments 1 to 203, having 4 or fewer visible particles (or spots) per 36 ml of the composition after storage. 205. A composition according to any one of Embodiments 1 to 204, having 3 or fewer visible particles (or spots) per 36 ml of the composition after storage. 206. A composition according to any one of Embodiments 1 to 205, having 2 or fewer visible particles (or spots) per 36 ml of the composition after storage. 207. A composition according to any one of Embodiments 1 to 206, having 1 or fewer visible particles (or spots) per 36 ml of the composition after storage. 208. A composition according to any one of Embodiments 1 to 207, having no visible particles per 36 ml of the composition after storage. 209. A composition according to any one of Embodiments 175 to 208, wherein the storage is at 25°C, 2 - 8°C, or 37°C. 210. A composition according to any one of Embodiments 175 to 209, wherein the storage is for 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, or longer. 211. A composition according to any one of Embodiments 175 to 210, wherein the storage includes at least one freeze - thaw cycle. 212. A composition according to Embodiment 211, wherein the at least one freeze - thaw cycle is one freeze - thaw cycle. 213. A composition according to Embodiment 211, wherein the at least one freeze - thaw cycle is two freeze - thaw cycles. 214. A composition according to Embodiment 211, wherein the at least one freeze - thaw cycle is three freeze - thaw cycles. 215. A composition according to Embodiment 211, wherein the at least one freeze - thaw cycle is four freeze - thaw cycles. 216. A composition according to Embodiment 211, wherein the at least one freeze - thaw cycle is five freeze - thaw cycles. 217. A composition according to any one of Embodiments 211 to 216, wherein the one or more freeze - thaw cycles include freezing the composition at about - 65°C or lower for about 1.5 hours or longer and thawing at 30°C for 1.5 hours. 218. A composition according to any one of Embodiments 1 to 217, wherein the composition is frozen. 219. A method for storing a viral vector, comprising: a) providing a composition according to any one of Embodiments 1 to 218, and b) A method comprising storing the viral composition at a temperature of about 25°C or lower. 220. A method for storing a viral vector, comprising: a) providing a composition according to any one of embodiments 1 to 218, and b) storing the viral composition at a temperature of about 2 to 8°C or lower. 221. A method for cryopreserving a viral vector, comprising: a) providing a composition according to any one of embodiments 1 to 218, b) freezing the viral composition, and c) storing the viral composition at a temperature of about 0°C or lower. 222. Any one of methods 219 to 221, wherein the method comprises storing the viral composition for at least about 24 hours. 223. Any one of methods 219 to 222, wherein the method comprises storing the viral composition for at least about 48 hours. 224. Any one of methods 219 to 223, wherein the method comprises storing the viral composition for at least about 72 hours. 225. Any one of methods 219 to 224, wherein the method comprises storing the viral composition for at least about 96 hours. 226. Any one of methods 219 to 225, wherein the method comprises storing the viral composition for at least about 120 hours. 227. Any one of methods 219 to 226, wherein the method comprises storing the viral composition for at least about 148 hours. 228. Any one of methods 219 to 227, wherein the method comprises storing the viral composition for at least about 168 hours. 229. A method for expressing a transgene in a cell, comprising contacting the cell with a composition according to any one of Embodiments 1 to 217. 230. The method according to Embodiment 229, wherein the cell is a mammalian cell. 231. The method according to Embodiment 229 or Embodiment 230, wherein the cell is a hematopoietic cell. 232. The method according to any one of Embodiments 229 to 231, wherein the cell is an immune effector cell. 233. The cell is CD3 + , CD4 + , and / or CD8 + The method according to any one of Embodiments 229 to 232, wherein the cell is a cell. 234. The method according to any one of Embodiments 229 to 233, wherein the cell is a T cell. 235. The method according to any one of Embodiments 229 to 234, wherein the cell is an αβ T cell. 236. The method according to any one of Embodiments 229 to 234, wherein the cell is a γδ T cell. 237. The 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. 238. The method according to any one of Embodiments 229 to 234, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. 239. The method according to any one of Embodiments 229 to 231, wherein the cell is a hematopoietic stem or progenitor cell. 240. The method according to any one of Embodiments 229 to 231, wherein the cell is a human CD34+ hematopoietic stem or progenitor cell.

[0229] Generally, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments contemplated in this specification and the claims, but rather the claims should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Claims

1. Aqueous virus composition, a) Lentiviral vectors b) HEPES buffer; c) Sucrose, and d) A composition comprising L-proline.

2. The composition according to claim 1, wherein the buffer solution is present at a concentration of 25 mM (±1 to 15%) to 30 mM (±1 to 15%), or the buffer solution is present at a concentration of 27.5 mM (±1 to 15%).

3. The aforementioned sucrose, (a) 66mM (±1 to 15%) to 80mM (±1 to 15%), (b) 73mM (±1-15%), (c) 2.0% by weight (±1 to 15%) to 3.0% by weight (±1 to 15%) per volume of the composition, and / or (d) 2.5% by weight (±1 to 15%) of the volume of the composition The composition according to claim 1, which is present at the concentration of [a certain value].

4. The composition according to claim 1, wherein the L-proline is present at a concentration of 40 mM (±1 to 15%) to 60 mM (±1 to 15%), or the L-proline is present at a concentration of 50 mM (±1 to 15%).

5. The composition according to claim 1, further comprising a salt.

6. The composition according to claim 5, wherein the salt is present at a concentration of 65 mM (±1 to 15%) to 85 mM (±1 to 15%), or the salt is present at a concentration of 75 mM (±1 to 15%).

7. The composition according to claim 5, wherein the salt is a chloride, KCl, or NaCl.

8. The composition according to claim 1, further comprising poloxamer 188.

9. The composition according to claim 8, wherein the poloxamer 188 is present at a concentration of 0.01 mg / ml (±1 to 15%) to 1 mg / ml (±1 to 15%), or the poloxamer 188 is present at a concentration of 0.1 mg / ml (±1 to 15%) or 0.3 mg / ml (±1 to 15%).

10. The composition according to claim 1, wherein the composition comprises a pH of 6.5 (±1 to 15%) to 8 (±1 to 15%).

11. Aqueous virus composition, a) Lentiviral vector, b) 27.5mM (±1-15%) HEPES, c) 73 mM (±1-15%) sucrose or 2.5% by weight (±1-15%) sucrose per volume of the composition, and d) A composition comprising 50 mM (±1 to 15%) L-proline, wherein the composition has a pH of 7 (±1 to 15%).

12. Aqueous virus composition, a) Lentiviral vector, b) 27.5mM (±1-15%) HEPES, c) 73 mM (±1-15%) sucrose or 2.5% by weight (±1-15%) sucrose per volume of the composition. d) 50 mM (±1-15%) L-proline, and e) Contains 0.3 mg / mL (±1-15%) of poloxamer 188, The composition comprises a pH of 7 (±1 to 15%).

13. The aforementioned lentiviral vector (a) Human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Visner-Maedivirus (VMV) virus, canine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV), (b) Human immunodeficiency virus-1 (HIV-1) or human immunodeficiency virus-2 (HIV-2), (c) Human immunodeficiency virus-1 (HIV-1) A composition according to any one of claims 1, 11, and 12, selected from the group consisting of the following.

14. The aforementioned lentiviral vector is pseudotyped. The aforementioned lentiviral vector is pseudotyped with an envelope protein from a strain of vesicular stomatitis virus. The aforementioned strain of vesicular stomatitis virus is selected from the group consisting of Indiana, Alagoas, New Jersey, Isfahan, CoCal, Malaba, or Piri. The lentiviral vector is pseudotyped with vesicular stomatitis virus G (VSV-G) protein, and / or The aforementioned 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 paramyxovirus F and H proteins. The composition according to any one of claims 1, 11, and 12.

15. The composition is (a) serum; (b) PIPES, (c) Sodium citrate, (d) Sodium phosphate, (e) Tris, and / or (f) Trehalose A composition according to any one of claims 1, 11, and 12, which does not contain [the specified substance].

16. The composition according to any one of claims 1, 11, and 12, wherein the composition does not contain salt.

17. The composition according to any one of claims 1, 11, and 12, wherein the composition does not contain chloride, KCl, and / or NaCl.

18. An aqueous virus composition, a) Lentiviral vector, b) 27.5mM (±1-15%) HEPES, c) 73 mM (±1-15%) sucrose or 2.5% by weight (±1-15%) sucrose per volume of the composition, and d) 50 mM (±1-15%) L-proline Includes, The aforementioned lentiviral vector is derived from HIV-1, The aforementioned lentiviral vector is pseudotyped with vesicular stomatitis virus G (VSV-G) protein. The composition does not contain salt, The composition contains a pH of 7 (±1 to 15%). composition.

19. An aqueous virus composition, a) Lentiviral vector, b) 27.5mM (±1-15%) HEPES, c) 73 mM (±1-15%) sucrose or 2.5% by weight (±1-15%) sucrose per volume of the composition. d) 50 mM (±1-15%) L-proline, and e) 0.3 mg / mL (±1-15%) poloxamer 188 Includes, The aforementioned lentiviral vector is derived from HIV-1, The aforementioned lentiviral vector is pseudotyped with vesicular stomatitis virus G (VSV-G) protein. The composition does not contain salt, The composition contains a pH of 7 (±1 to 15%). composition.

20. The composition according to any one of claims 1, 11-12, and 18-19, wherein the viral lentivector is present at a titer of 1 × 10⁸ (±1-15%) to 2 × 10⁹ (±1-15%) TU / ml.

21. The aforementioned lentiviral vector contains a polynucleotide including the transgene, The aforementioned transgene encodes a therapeutic protein. The aforementioned transgene or therapeutic protein is for the treatment of a monogenic disease, disorder, or condition. The transgene or therapeutic protein is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), an αβ T cell receptor (αβ-TCR), a γδ T cell receptor (γδ-TCR), a dimerizing agent-regulated immune receptor complex (DARIC), or a switch receptor, and / or The aforementioned transgene or therapeutic protein is a therapeutic globin for the treatment of hemoglobin disorders, or the ABCD1 gene for the treatment of CALD. The composition according to any one of claims 1, 11-12, and 18-19.

22. The lentiviral vector maintains an infectivity titer recovery of more than 75% (±1-15%) in HOS cells after storage compared to the infectivity titer of the lentiviral vector in the composition before storage or at least one freeze-thaw cycle, or The lentiviral vector maintains a recovery of infectivity titer of more than 95% (±1-15%) in HOS cells after storage, compared to the infectivity titer of the lentiviral vector in the composition before storage or at least one freeze-thaw cycle. The composition according to any one of claims 1, 11-12, and 18-19.

23. The composition according to any one of claims 1, 11-12, and 18-19, wherein the lentiviral vector has a thermal unfolding temperature of 56° (±1-15%) to 62°C (±1-15%) when measured by differential scanning fluorescence (DSF).

24. The composition according to any one of claims 1, 11-12 and 18-19, wherein the lentiviral vector maintains, after storage, a hydrodynamic diameter of 150 nm (±1-15%) to 170 nm (±1-15%) and a viscosity of 0.967 centipoise (cP), as measured by dynamic light scattering (DLS) at 25°C, compared to the lentiviral vector in the composition before storage or at least one freeze-thaw cycle.

25. The composition according to any one of claims 1, 11-12, and 18-19, wherein the lentiviral vector maintains at least 78% (±1-15%) efficacy or at least 95% (±1-15%) efficacy when measured by transgene expression in a PBMC after storage, compared to a reference standard.

26. (a) No visible fibrous particles (whispy fibers) after storage, (b) After storage, there are five or fewer visible particles or spots, and / or (c) No visible particles are present in the composition after storage. The composition according to any one of claims 1, 11-12, and 18-19.

27. The storage temperature is 25°C, 2-8°C, or 37°C. The aforementioned preservation lasts for 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, or longer. The aforementioned preservation includes one or more freeze-thaw cycles, and / or The one or more freeze-thaw cycles include freezing the composition at -65°C (±1 to 15%) or lower for 1.5 hours (±1 to 15%) or more, and thawing it at 30°C for 1.5 hours. The composition according to claim 22.

28. The composition according to any one of claims 1, 11-12 and 18-19, wherein the composition is frozen.

29. A method for storing viral vectors, a) To provide the composition according to any one of claims 1, 11-12 and 18-19, and b) A method comprising storing the composition at a temperature of 25°C (±1 to 15%) or less, or at a temperature of 2 to 8°C (±1 to 15%) or less.

30. A method for cryopreserving viral vectors, a) To provide the composition according to any one of claims 1, 11-12 and 18-19, b) Freezing the composition, and c) A method comprising storing the composition at a temperature of 0°C (±1 to 15%) or lower.

31. The method according to claim 29, wherein the method comprises storing the lentivirus composition for at least 24 hours (±1 to 15%) or at least 168 hours (±1 to 15%).

32. The method according to claim 30, wherein the method comprises storing the lentivirus composition for at least 24 hours (±1 to 15%) or at least 168 hours (±1 to 15%).

33. A method for expressing an introduced gene in cells, comprising contacting cells with the composition described in any one of claims 1, 11-12, and 18-19.

34. The aforementioned cells, (a) mammalian cells; (b) hematopoietic cells, (c) Immune effector cells, (d) CD3+, CD4+, and / or CD8+ cells, (e) T cells, (f) αβ T cells, (g) γδT cells, (h) Cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), or helper T cells, (i) Natural killer (NK) cells or natural killer T (NKT) cells, (j) Hematopoietic stem cells or progenitor cells, (k) Human CD34+ hematopoietic stem cells or progenitor cells The method according to claim 33.

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