Processes for filtration or purification of viral particles

Abstract

Provided herein are methods for purifying and / or concentrating viral vectors, using them to treat diseases such as cancer or autoimmune diseases, and preparing sterile compositions, such as sterile pharmaceutical compositions, in which the viral vectors are passed through a sterile filter coated with a protein before the steps are performed.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 482,617, filed February 1, 2023, and U.S. Provisional Application No. 63 / 510,391, filed June 27, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Reference to electronically filed sequence listings

[0001] This application contains a Sequence Listing that has been filed electronically in XML format. The Sequence Listing is incorporated herein by reference in its entirety. The XML copy created on January 20, 2024 is titled INH-024WO_SL and is 97,699 bytes in size.

[0003] The embodiments presented herein relate to a process for producing viral vectors that involves filtration. [Background technology]

[0004] Sterile filtration is typically performed at the end of processing large biomolecules (proteins, monoclonal antibodies, viral vectors) to remove biological contaminants and produce sterile drug substances. Many sterile filters are commercially available, with a variety of pore sizes, construction materials, membrane surface charges, pore morphologies, and filtration membrane forms. Any of these factors can affect the recovery rate of the desired product through the filter. Product losses during sterile filtration have been reported for viruses, and these losses are due to physical retention of the product, either related to size and / or charge. Research has been conducted on the type of sterile filter and the concentration of the feed solution to maximize the recovery rate of a specific desired product. The present invention addresses these needs and others. Summary of the Invention

[0005] In some embodiments, a method for purifying a viral vector or preparing a concentrated sterile solution is provided, comprising: clarifying a solution comprising a cell culture medium and a viral vector; filtering the clarified solution comprising the viral vector through a first chromatography filter to prepare a filtered clarified solution comprising the viral vector; passing the filtered clarified solution comprising the viral vector through a protein-coated sterile membrane to prepare a sterile solution comprising the viral vector; and concentrating the sterile solution comprising the viral vector to prepare a concentrated sterile solution comprising the viral vector.

[0006] In some embodiments, the protein solution comprises one or more components selected from a protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride, or a combination thereof.

[0007] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride.

[0008] In some embodiments, the protein-coated sterile membrane has a positive charge prior to coating with the protein.

[0009] In some embodiments, the membrane is selected from a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane.

[0010] In some embodiments, the method comprises digesting DNA in a solution comprising the viral vector to prepare a first digested solution comprising the viral vector; clarifying the digested solution comprising the viral vector; filtering the clarified digested solution comprising the viral vector through a first chromatography filter to prepare a filtered clarified digested solution comprising the viral vector; digesting DNA in the filtered clarified digested solution comprising the viral vector to prepare a filtered clarified second-digested solution comprising the viral vector; filtering the filtered clarified second-digested solution comprising the viral vector through a second chromatography filter to prepare a twice-filtered clarified second-digested solution comprising the viral vector; passing the twice-filtered clarified second-digested solution comprising the viral vector through a protein-coated sterile membrane to prepare a sterile solution comprising the viral vector; and concentrating the sterile solution comprising the viral vector to prepare a concentrated sterile solution comprising the viral vector.

[0011] In some embodiments, the protein solution comprises one or more components selected from a protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride, or a combination thereof.

[0012] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0.

[0013] In some embodiments, the protein-coated sterile membrane has a positive charge prior to coating with the protein.

[0014] In some embodiments, the membrane is selected from a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the viral vector production process. [Figure 2] FIG. 1 shows the recovery rate of viral vectors from PES with and without pre-use washing with protein. [Figure 3] FIG. 1 shows the recovery rate of viral vectors from uncharged sterile filters. [Figure 4] FIG. 1 shows the effect of pre-coating on charged sterilizing filters. [Figure 5] FIG. 1 shows the effect of pre-coating on neutral sterilizing filters. [Figure 6] FIG. 1 shows that both HSA and recombinant HSA can be used as sterile filter pre-coating protein solutions. [Figure 7A] FIG. 1 shows the crystal structure of VSV-G bound to LDL-R. [Figure 7B] FIG. 1 shows the crystal structure of VSV-G bound to LDL-R. [Figure 8A] FIG. 1 shows the effect of adding negatively charged amino acids to the VSV-G:LDL-R binding interface on the original tropism and membrane fusogenicity. [Figure 8B] FIG. 1 shows the effect of adding negatively charged amino acids to the VSV-G:LDL-R binding interface on the original tropism and membrane fusogenicity. [Figure 9-1] FIG. 1 shows an alignment of the ectodomains of different VSV-G proteins from different strains. [Figure 9-2] Continued from Figure 9-1. [Figure 9-3] Continued from Figure 9-2. [Figure 9-4] Continued from Figure 9-3. DETAILED DESCRIPTION OF THE INVENTION

[0016] Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. Where any ambiguity may exist, the definitions set forth herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless specifically stated otherwise. The use of the term "including" and its variants (e.g., "includes" and "included") means non-limiting.

[0017] Generally, the terms used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The methods and techniques described herein are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and set forth throughout the specification, unless otherwise indicated. Enzymatic reactions are performed according to manufacturer's specifications, as commonly practiced in the art, or as described herein. The terms used in connection with, and the laboratory procedures and techniques therefor, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.

[0018] In order that this disclosure may be more readily understood, certain terms are defined below.

[0019] As used herein, the terms "a" or "an" mean "at least one" or "one or more" unless the context clearly indicates otherwise.

[0020] As used herein, the term "about" means that a numerical value is approximate and that small variations do not significantly affect the practice of the disclosed embodiments. When a numerical limitation is used, unless the context indicates otherwise, "about" means that the numerical value can vary by ±10% and still remain within the scope of the disclosed embodiments. Furthermore, in the expression "about x to y," the term "about" modifies both x and y and can be used synonymously with the expression "about x to about y," unless the context indicates otherwise.

[0021] As used herein, the terms "individual" or "subject" or "patient" are used interchangeably and refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, such as humans.

[0022] As used herein, the terms "comprising" (and any conjugation of "comprising," such as "comprise," "comprises," and "comprised"), "having" (and any conjugation of "having," such as "have" and "has"), "including" (and any conjugation of "including," such as "includes" and "include"), or "containing" (and any conjugation of "containing," such as "contains" and "contain") are open-ended or open-ended and do not exclude additional, unrecited elements or method steps. Any step or composition using the transitional phrase "comprise" or "comprising" can be said to describe the same thing as using the transitional phrase "consisting of" or "consists."

[0023] As used herein, the terms "pre-washed" or "pre-coated" or any variations thereof (e.g., "pre-wash" or "pre-coat") describe a filter that has a solution passed through it before the filter is used with a viral vector solution.

[0024] As used herein, the expression "viral vector recovery" is defined as the amount of viral vector after filtration relative to the amount of viral vector before filtration, expressed as a percentage.

[0025] As used herein, the phrase "purified viral vector" is defined as a viral vector that has been purified from a cell culture harvest using at least one chromatography step.

[0026] As used herein, a "non-concentrated" viral vector is a viral vector in a solution that has not undergone a concentration step during its preparation. As used herein, a "non-concentrated" viral vector is not limited to the concentration of viral particles therein, but rather simply refers to whether or not the solution containing the viral vector has undergone a concentration step, unless explicitly stated otherwise.

[0027] As used herein, the term "contacting" refers to bringing two elements together in an in vitro system or an in vivo system. For example, "contacting" a virus or vector described herein with an individual or patient or cell includes administering the virus to an individual or patient, such as a human, and introducing a compound into a sample, including, for example, a cell preparation or purified preparation, that contains the cell.

[0028] As used herein, the terms "fused" or "linked" when used with reference to proteins having different domains or heterologous sequences means that such protein domains are connected to one another by either peptide bonds or other covalent bonds and are part of the same peptide chain. The domains or sections may be directly linked or fused to one another, or another domain or peptide sequence may be between the two domains or sequences and such sequences would still be considered fused or linked to one another. In some embodiments, the various domains or proteins shown herein are directly linked or fused to one another, or a linker sequence, such as the glycine / serine sequences described herein, links the two domains.

[0029] An animal "disease" refers to a health condition in which the animal is unable to maintain homeostasis and, if the disease is not corrected, the animal's health continues to deteriorate. An animal "disorder" refers to a health condition in which the animal is able to maintain homeostasis but is less favorable than if the disorder were absent. If left untreated, a disorder does not necessarily result in a further decline in the animal's health.

[0030] The terms "effective amount" and "therapeutically effective amount," used interchangeably herein, refer to an amount of a compound, formulation, substance, or composition described herein that is effective to achieve a particular biological result or to produce a therapeutic or prophylactic effect. Such a result includes, but is not limited to, that administration of an amount of the composition to a mammal results in a detectable level of immune cell activation compared to immune cell activation detected in the absence of the composition. The immune response can be readily assessed by a number of art-recognized methods. As will be apparent to one of skill in the art, the amount of the composition administered herein will vary and can be readily determined based on many factors, including the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, and the particular compound being administered.

[0031] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template in biological processes for the synthesis of other polymers and macromolecules having either a particular sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a particular sequence of amino acids, as well as the biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to the gene. Both the coding strand (the one whose nucleotide sequence is identical to the mRNA sequence, usually shown in a sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be referred to as encoding the protein or other product of the gene or cDNA.

[0032] An "expression vector" refers to a vector containing a recombinant polynucleotide, the recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art. Examples of such expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate the recombinant polynucleotide.

[0033] As used herein, the term "ex vivo" with respect to cells that have been transduced, transfected, or transformed ex vivo refers to cells that are transduced, transfected, or transformed outside of a subject, i.e., cells that have been removed from a subject before being transduced, transfected, or transformed.

[0034] As used herein, a "filter" refers to any object through which a solution or composition passes to remove a portion (for example) of the solution or composition. Thus, "filter" is not intended to be limited to membrane filters, but encompasses any object of any thickness that allows a solution or composition to pass through. Thus, the term "filter" encompasses, without limitation, membranes such as PES, nylon, or PVDF membranes; membrane chromatography units such as Sartobind Q and Mustang Q; or stationary phases such as ion-exchange stationary phases (e.g., cross-linked polymer resins such as divinylbenzene-crosslinked polystyrene); affinity stationary phases (e.g., nickel resins, streptavidin resins, glutathione-bound resins, Protein A- or Protein G-bound resins); hydrophobic stationary phases (e.g., silica resins bound with butyl, phenyl, ether, amide, or propyl ligands); size-exclusion stationary phases (e.g., silica resins with appropriate diameters and pore sizes); or any combination thereof (i.e., multimodal chromatography). In some embodiments, a "filter" is a resin, such as the chromatography resins described herein. Similarly, the term "chromatography filter" refers to any object that can be used for chromatographic separation of a solution or composition. Thus, "chromatography filter" is not intended to be limited to membrane filters, but encompasses any body of any thickness that allows a solution or composition to pass through.Thus, the term "chromatographic filter" includes, but is not limited to, membranes such as PES, nylon, or PVDF membranes; membrane chromatography units such as Sartobind Q, Mustang Q, or stationary phases such as ion exchange stationary phases (e.g., cross-linked polymer resins such as divinylbenzene cross-linked polystyrene), affinity stationary phases (e.g., nickel resins, streptavidin resins, glutathione-bound resins, protein A- or protein G-bound resins, etc.); hydrophobic stationary phases (e.g., silica resins bound with butyl, phenyl, ether, amide, or propyl ligands); size exclusion stationary phases (e.g., silica resins with appropriate diameters and pore sizes), or any combination thereof (i.e., multimodal chromatography).

[0035] As used herein, "identity" refers to the identity of the subunit sequences between two polymer molecules (e.g., between two nucleic acid molecules or amino acid molecules, e.g., between two polynucleotide molecules or polypeptide molecules). If two amino acid sequences have the same residue at the same position, for example, if each position in two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences or two nucleic acid sequences have the same residue at the same position in alignment is often expressed as a percentage. The identity between two amino acid sequences or two nucleic acid sequences directly depends on the number of matching or identical positions; for example, if half of the positions in the two sequences are identical, the two sequences are 50% identical; if 90% (e.g., 9 out of 10) of the positions are matched or identical, the two amino acid sequences are 90% identical.

[0036] By "substantially identical" is meant that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In some embodiments, such a sequence is at least 60%, 80%, or 85%, or 90%, 95%, or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison. Other identity percentages for specific sequences are described herein.

[0037] Sequence identity can be measured / determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the group of glycine and alanine; substitutions within the group of valine, isoleucine, and leucine; substitutions within the group of aspartic acid, glutamic acid, asparagine, and glutamine; substitutions within the group of serine and threonine; substitutions within the group of lysine and arginine; and substitutions within the group of phenylalanine and tyrosine. An exemplary method for determining the degree of identity is to use the BLAST program, in which a probability score of e3 to e100 indicates closely related sequences. In some embodiments, sequence identity is determined using BLAST with default settings.

[0038] To the extent that embodiments provided herein encompass compositions comprising various proteins, such proteins may comprise amino acid sequences having sequence identity to the amino acid sequences disclosed herein. Thus, in certain embodiments, depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the SEQ ID NOs disclosed herein. In addition to these percentages, other identity percentages are also provided herein. Identity between polypeptides can be determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular) using an affine gap search with parameters gap opening penalty = -12 and gap extension penalty = 1. Such proteins contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid substitutions (i.e., replacement of one amino acid with another amino acid having a homologous side chain) compared to the proteins of the present disclosure. Genetically encoded amino acids are generally classified into four families: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. Typically, single amino acid substitutions within these families do not significantly affect biological activity. The protein may have one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to the disclosed protein sequences.The proteins may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g., of 1, 2, 3, 4, or 5 amino acids, respectively) relative to the disclosed protein sequences.

[0039] As used herein, the term "in vivo" with respect to cells that have been transduced, transfected, or transformed in vivo refers to cells that have been transduced, transfected, or transformed within a subject, and that have not been removed from the subject prior to being transduced, transfected, or transformed.

[0040] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide would be "isolated" if it was partially or completely separated from the coexisting materials in its natural state. An isolated nucleic acid or protein can exist in a substantially purified form or in a non-native environment, such as, for example, a host cell.

[0041] As used herein, "lentivirus" refers to a genus of the Retroviridae family that can infect non-dividing cells. Non-limiting examples of lentiviruses are HIV, SIV, and FIV. Lentivirus-derived vectors or virus-like particles can be used to transduce cells to deliver genes or other molecules and express them in cells in vitro, ex vivo, or in vivo.

[0042] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell as described herein. Molecules can be modified in a variety of ways, including chemical, structural, and functional, such as mutation, substitution, insertion, or deletion (e.g., internal deletion, truncation). Cells can be modified through the introduction of a nucleic acid or the expression of a heterologous protein.

[0043] As used herein, the term "modulation" means mediating an increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of the treatment or compound and / or compared to the level of the response in an otherwise identical subject not receiving the treatment. This term encompasses perturbing and / or affecting the natural signal or response, thereby mediating a beneficial therapeutic response in a subject, such as a human.

[0044] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate and encode the same amino acid sequence. A reference to a nucleotide sequence encoding a protein or RNA can include introns, to the extent that in some cases the nucleotide sequence encoding the protein may contain intron(s).

[0045] The term "oligonucleotide" generally refers to a short polynucleotide. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also refers to the corresponding RNA sequence in which "T" is replaced by "U" (i.e., A, U, C, G).

[0046] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection (infusion), or intrathecal injection techniques.

[0047] The term "polynucleotide," as used herein, is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, the terms "nucleic acid" and "polynucleotide" are interchangeable. As used herein, polynucleotide encompasses, without limitation, all nucleic acid sequences obtained by any method available in the art. Such methods include, but are not limited to, recombinant methods, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using cloning techniques and PCR, and by synthetic means.

[0048] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of multiple amino acid residues covalently joined by peptide bonds. As used herein, the term refers to both short chains (e.g., also commonly referred to in the art as peptides, oligopeptides, and oligomers) and longer chains (commonly referred to in the art as proteins), of which there are many varieties. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0049] As used herein, the term "pseudotype" or "pseudotyped virus particle" refers to a viral particle having glycoproteins derived from other enveloped viruses, or to a viral vector encoding an envelope glycoprotein from a virus different from the parent virus. Thus, the host range of the vector particle can be expanded or altered depending on the type of cell surface receptor used by the glycoprotein. For example, as shown herein, a virus can be pseudotyped using a VSV-G mutant protein.

[0050] As used herein with respect to antibodies, the term "specifically binds" means that the antibody recognizes a specific antigen while not substantially recognizing or binding to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in and of itself, change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in and of itself, change the specific classification of the antibody. In some cases, the terms "specific binding" or "specifically binds" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species (e.g., an antibody recognizes and binds to a specific protein structure, rather than proteins in general). If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or unlabeled, free A) in a reaction involving labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody. In some embodiments, the targeting moieties described herein can be used to target viral particles containing mutant VSV-G proteins, or viral particles containing other viral structural proteins used to pseudotype the virus, and such targeting moieties can specifically bind to their targets.

[0051] As used herein, "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is achieved by suppression, amelioration, or eradication of a disease condition.

[0052] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny. In some embodiments, transfection, transformation, or transduction is performed or occurs in vivo.

[0053] A "vector" is a composition comprising an isolated nucleic acid encoding a protein or peptide. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, plasmids, DNA, and RNA. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

[0054] "Carriers" or "delivery vehicles" include viral particles, viruses, polylysine compounds, and liposomes, which facilitate the transfer of nucleic acids into cells. Carriers or delivery vehicles can also be used to deliver proteins or peptides to cells.

[0055] Ranges: Throughout this disclosure, various aspects of the embodiments may be expressed in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as inflexible limitations. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range. Unless expressly stated to the contrary, a disclosed range includes the endpoints of the range.

[0056] Without being bound by any particular theory, standard virus purification techniques tend to result in loss of viral product during filtration. This occurs particularly when concentrating the viral product before sterile filtration, as is typically done. Passing a pre-wash protein solution through a sterile filter before filtering the viral product increases the virus yield. This is believed to be because the protein solution prevents the viral product from being trapped on the filter due to charge interactions. Furthermore, it has been shown that performing sterile filtration before concentrating the viral product increases the product yield.

[0057] Method for purifying viral vectors and / or preparing concentrated sterile solutions containing viral vectors In some embodiments, methods for purifying a viral vector and / or preparing a concentrated sterile solution comprising a viral vector are provided. In some embodiments, the method includes clarifying a solution comprising a cell culture medium and the viral vector, filtering the clarified solution comprising the viral vector through a first chromatography filter to prepare a filtered clarified solution comprising the viral vector, passing the filtered clarified solution comprising the viral vector through a protein-coated sterile membrane to prepare a sterile solution comprising the viral vector, and concentrating the sterile solution comprising the viral vector to prepare a concentrated sterile solution comprising the viral vector.

[0058] In some embodiments, the method includes clarifying a solution comprising the cell culture medium and the viral vector; filtering the clarified solution comprising the viral vector through a first chromatography filter to prepare a filtered clarified solution comprising the viral vector; concentrating the filtered clarified solution comprising the viral vector to prepare a concentrated solution comprising the viral vector; and passing the concentrated solution comprising the viral vector through a protein-coated sterile membrane to prepare a concentrated sterile solution comprising the viral vector.

[0059] In some embodiments, the method further comprises combining the concentrated sterile solution comprising the viral vector with one or more sterile, pyrogen-free buffers and / or additives to prepare a sterile composition comprising the viral vector. In some embodiments, the concentrated sterile solution comprising the viral vector is combined with one pyrogen-free buffer and / or additive. In some embodiments, the concentrated sterile solution comprising the viral vector is combined with two or more pyrogen-free buffers and / or additives. In some embodiments, the method of purifying a viral vector comprises clarifying a solution comprising a cell culture medium and the viral vector, filtering the clarified solution comprising the viral vector through a first chromatography filter to prepare a filtered clarified solution comprising the viral vector, passing the filtered clarified solution comprising the viral vector through a protein-coated sterile membrane to prepare a sterile solution comprising the viral vector, concentrating the sterile solution comprising the viral vector to prepare a concentrated sterile solution comprising the viral vector, and combining the concentrated sterile solution comprising the viral vector with one or more sterile, pyrogen-free buffers and / or additives to prepare a sterile composition comprising the viral vector. In some embodiments, the concentrated sterile solution containing the viral vector is mixed with one pyrogen-free buffer and / or additive, hi some embodiments, the concentrated sterile solution containing the viral vector is mixed with two or more pyrogen-free buffers and / or additives.

[0060] In some embodiments, the method further comprises recovering the medium from the cell culture producing the viral vector prior to clarifying the solution, hi some embodiments, the medium recovered from the cell culture producing the viral vector comprises the viral vector.

[0061] In some embodiments, filtering the clarified solution through a first chromatography filter comprises filtering the clarified solution by capture chromatography. In some embodiments, the capture chromatography is ion exchange chromatography, affinity chromatography, hydrophobic chromatography, size exclusion chromatography, or multimodal chromatography. In some embodiments, the capture chromatography is ion exchange chromatography. In some embodiments, the capture chromatography is affinity chromatography. In some embodiments, the capture chromatography is hydrophobic chromatography. In some embodiments, the capture chromatography is size exclusion chromatography. In some embodiments, the capture chromatography is multimodal chromatography.

[0062] In some embodiments, the method further comprises filtering the filtered clarified solution containing the viral vector through a second chromatography filter to prepare a twice-filtered clarified solution containing the viral vector. In some embodiments, the second filtration step is performed before passing the filtered clarified solution through a protein-coated sterile membrane. In some embodiments, the twice-filtered clarified solution containing the viral vector is passed through a protein-coated sterile membrane.

[0063] In some embodiments, filtering the first filtered solution through a second chromatography filter comprises filtering the first filtered solution through polishing chromatography. In some embodiments, the polishing chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimodal chromatography. In some embodiments, the polishing chromatography is ion exchange chromatography. In some embodiments, the polishing chromatography is hydrophobic interaction chromatography. In some embodiments, the polishing chromatography is size exclusion chromatography. In some embodiments, the polishing chromatography is multimodal chromatography.

[0064] In some embodiments, the protein-coated sterile membrane comprises a sterile membrane that has been pre-washed with a protein solution. In some embodiments, the sterile membrane is pre-washed with a protein solution to create a protein-coated sterile membrane. In some embodiments, the method further comprises pre-washing a sterile membrane with a protein solution to create a protein-coated sterile membrane. In some embodiments, the method further comprises coating a sterile membrane with a protein solution to create a protein-coated sterile membrane.

[0065] In some embodiments, the method includes passing a filtered clarified solution comprising the viral vector through a protein-coated sterile membrane to prepare a sterile solution comprising the viral vector, wherein the protein-coated sterile membrane has a pore size of about 0.01 μm to about 0.45 μm. In some embodiments, the pore size of the protein coated sterile membrane is about 0.01 μm, about 0.10 μm, about 0.11 μm, about 0.12 μm, about 0.13 μm, about 0.14 μm, about 0.15 μm, about 0.16 μm, about 0.17 μm, about 0.18 μm, about 0.19 μm, about 0.2 μm, about 0.21 μm, about 0.22 μm, about 0.23 μm, about 0.24 μm, about 0.25 μm, about 0.26 μm, about 0.27 μm, about 0.28 μm, about 0.29 μm, about 0.30 μm, about 0.31 μm, about 0.32 μm, about 0.33 μm, about 0.34 μm, about 0.35 μm, about 0.36 μm, about 0.37 μm, about 0.38 μm, about 0.39 μm, about 0.40 μm, about 0.41 μm, about 0.42 μm, about 0.43 μm, about 0.44 μm, about 0.45 μm, about 0.46 μm, about 0.47 μm, about 0.48 μm, about 0.49 μm, about 10 ...

[0023] In some embodiments, the pore size of the protein-coated sterile membrane is about 0.01 μm. In some embodiments, the pore size of the protein-coated sterile membrane is about 0.10 μm. In some embodiments, the pore size of the protein-coated sterile membrane is about 0.11 μm. In some embodiments, the pore size of the protein-coated sterile membrane is about 0.12 μm. In some embodiments, the pore size of the protein-coated sterile membrane is about 0.13 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.14 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.15 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.16 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.17 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.18 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.19 μm.In some embodiments, the protein-coated sterile membrane has a pore size of 0.20 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.21 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.22 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.23 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.24 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.25 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.26 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.27 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.28 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.29 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.30 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.31 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.32 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.33 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.34 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.35 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.36 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.37 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.38 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.39 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.40 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.41 μm.In some embodiments, the protein-coated sterile membrane has a pore size of 0.42 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.43 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.44 μm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.45 μm.

[0066] In some embodiments, the protein-coated sterile membrane does not significantly retain the viral vector, such as when a solution containing the viral vector is passed through the membrane. In some embodiments, the recovery of the viral vector from a solution containing the viral vector is at least about 60%, 61%, 62%, 63%, 64%, 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%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the recovery of the solution containing the viral vector is at least about 60%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 61%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 62%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 63%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 64%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 65%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 66%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 67%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 68%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 69%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 70%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 71%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 72%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 73%. In some embodiments, the recovery rate of the solution containing the viral vector is at least about 74%.In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 75%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 76%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 77%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 78%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 79%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 80%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 81%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 82%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 83%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 84%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 85%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 86%. In some embodiments, the recovery rate of a solution comprising a viral vector is at least about 87%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 88%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 89%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 90%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 91%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 92%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 93%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 94%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 95%. In some embodiments, the recovery rate of the solution comprising the viral vector is at least about 96%.In some embodiments, the recovery rate of the solution containing the viral vector is at least about 97%. In some embodiments, the recovery rate of the solution containing the viral vector is at least about 98%. In some embodiments, the recovery rate of the solution containing the viral vector is at least about 99%. In some embodiments, the recovery rate of the solution containing the viral vector is about 100%.

[0067] In some embodiments, the protein solution comprises one or more components selected from a protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride, or a combination thereof. In some embodiments, the protein solution comprises a protein. In some embodiments, the protein solution comprises 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS). In some embodiments, the protein solution comprises histidine, phosphate. In some embodiments, the protein solution comprises HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the protein solution comprises sodium chloride. In some embodiments, the protein solution comprises a protein and one or more components selected from 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride, or combinations thereof. In some embodiments, the protein solution comprises a protein and 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS). In some embodiments, the protein solution comprises a protein and histidine, phosphate. In some embodiments, the protein solution comprises a protein and HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the protein solution comprises a protein and sodium chloride.

[0068] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v protein, or any value or range therein. In some embodiments, the protein solution comprises about 0.2% to about 3% w / v protein. In some embodiments, the protein solution comprises about 0.3% to about 3% w / v protein. In some embodiments, the protein solution comprises about 0.4% to about 3% w / v protein. In some embodiments, the protein solution comprises about 0.5% to about 3% w / v protein. In some embodiments, the protein solution comprises about 0.6% to about 3% w / v protein. In some embodiments, the protein solution comprises about 0.7% to about 3% w / v protein. In some embodiments, the protein solution comprises about 0.8% to about 3% w / v protein. In some embodiments, the protein solution comprises about 0.9% to about 3% w / v protein. In some embodiments, the protein solution comprises about 1% to about 3% w / v protein. In some embodiments, the protein solution comprises about 1.2% to about 3% w / v protein. In some embodiments, the protein solution comprises about 1.4% to about 3% w / v protein. In some embodiments, the protein solution comprises about 1.6% to about 3% w / v protein. In some embodiments, the protein solution comprises about 1.8% to about 3% w / v protein. In some embodiments, the protein solution comprises about 2% to about 3% w / v protein. In some embodiments, the protein solution comprises about 2.2% to about 3% w / v protein. In some embodiments, the protein solution comprises about 2.4% to about 3% w / v protein. In some embodiments, the protein solution comprises about 2.6% to about 3% w / v protein. In some embodiments, the protein solution comprises about 2.8% to about 3% w / v protein.

[0069] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 2.8% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 2.6% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 2.4% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 2.2% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 2% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 1.8% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 1.6% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 1.4% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 1.2% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 1.0% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 0.9% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 0.8% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 0.7% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 0.6% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 0.5% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 0.4% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 0.3% w / v protein. In some embodiments, the protein solution comprises about 0.1% to about 0.2% w / v protein.

[0070] In some embodiments, the protein solution comprises about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0.8% w / v, about 0.9% w / v, about 1.0% w / v, about 1.2% w / v, about 1.4% w / v, about 1.6% w / v, 1.8% w / v, about 2.0% w / v, about 2.2% w / v, about 2.4% w / v, about 2.6% w / v, about 2.8% w / v, or about 3.0% w / v of protein, or any value therein. In some embodiments, the protein solution comprises about 0.1% w / v of protein. In some embodiments, the protein solution comprises about 0.2% w / v of protein. In some embodiments, the protein solution comprises about 0.3% w / v protein. In some embodiments, the protein solution comprises about 0.4% w / v protein. In some embodiments, the protein solution comprises about 0.5% w / v protein. In some embodiments, the protein solution comprises about 0.6% w / v protein. In some embodiments, the protein solution comprises about 0.7% w / v protein. In some embodiments, the protein solution comprises about 0.8% w / v protein. In some embodiments, the protein solution comprises about 0.9% w / v protein. In some embodiments, the protein solution comprises about 1.0% w / v protein. In some embodiments, the protein solution comprises about 1.2% w / v protein. In some embodiments, the protein solution comprises about 1.4% w / v protein. In some embodiments, the protein solution comprises about 1.6% w / v protein. In some embodiments, the protein solution comprises about 1.8% w / v protein. In some embodiments, the protein solution comprises about 2.0% w / v protein. In some embodiments, the protein solution comprises about 2.2% w / v protein. In some embodiments, the protein solution comprises about 2.4% w / v protein. In some embodiments, the protein solution comprises about 2.6% w / v protein. In some embodiments, the protein solution comprises about 2.8% w / v protein.In some embodiments, the protein solution comprises about 3.0% w / v protein.

[0071] In some embodiments, the protein solution comprises about 100 mM to about 400 mM sodium chloride, or any value or range therein. In some embodiments, the protein solution comprises about 110 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 120 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 130 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 140 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 150 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 160 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 170 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 180 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 190 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 200 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 210 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 220 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 230 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 240 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 250 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 260 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 270 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 280 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 290 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 300 mM to about 400 mM sodium chloride.In some embodiments, the protein solution comprises about 310 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 320 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 330 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 340 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 350 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 360 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 370 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 380 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 390 mM to about 400 mM sodium chloride.

[0072] In some embodiments, the protein solution comprises about 100 mM to about 400 mM sodium chloride, or any value or range therein. In some embodiments, the protein solution comprises about 100 mM to about 390 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 380 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 370 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 360 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 350 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 340 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 330 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 320 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 310 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 300 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 290 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 280 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 270 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 260 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 250 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 240 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 230 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 220 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 210 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 200 mM sodium chloride.In some embodiments, the protein solution comprises about 100 mM to about 190 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 180 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 170 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 160 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 150 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 140 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 130 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 120 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM to about 110 mM sodium chloride.

[0073] In some embodiments, the protein solution comprises about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, or about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 100 mM sodium chloride. In some embodiments, the protein solution comprises about 110 mM sodium chloride. In some embodiments, the protein solution comprises about 120 mM sodium chloride. In some embodiments, the protein solution comprises about 130 mM sodium chloride. In some embodiments, the protein solution comprises about 140 mM sodium chloride. In some embodiments, the protein solution comprises about 150 mM sodium chloride. In some embodiments, the protein solution comprises about 160 mM sodium chloride. In some embodiments, the protein solution comprises about 170 mM sodium chloride. In some embodiments, the protein solution comprises about 180 mM sodium chloride. In some embodiments, the protein solution comprises about 190 mM sodium chloride. In some embodiments, the protein solution comprises about 200 mM sodium chloride. In some embodiments, the protein solution comprises about 210 mM sodium chloride. In some embodiments, the protein solution comprises about 220 mM sodium chloride. In some embodiments, the protein solution comprises about 230 mM sodium chloride. In some embodiments, the protein solution comprises about 240 mM sodium chloride. In some embodiments, the protein solution comprises about 250 mM sodium chloride. In some embodiments, the protein solution comprises about 260 mM sodium chloride. In some embodiments, the protein solution comprises about 270 mM sodium chloride. In some embodiments, the protein solution comprises about 280 mM sodium chloride.In some embodiments, the protein solution comprises about 290 mM sodium chloride. In some embodiments, the protein solution comprises about 300 mM sodium chloride. In some embodiments, the protein solution comprises about 310 mM sodium chloride. In some embodiments, the protein solution comprises about 320 mM sodium chloride. In some embodiments, the protein solution comprises about 330 mM sodium chloride. In some embodiments, the protein solution comprises about 340 mM sodium chloride. In some embodiments, the protein solution comprises about 350 mM sodium chloride. In some embodiments, the protein solution comprises about 360 mM sodium chloride. In some embodiments, the protein solution comprises about 370 mM sodium chloride. In some embodiments, the protein solution comprises about 380 mM sodium chloride. In some embodiments, the protein solution comprises about 390 mM sodium chloride. In some embodiments, the protein solution comprises about 300 mM sodium chloride.

[0074] In some embodiments, the protein solution comprises about 5 mM to about 50 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), or any value or range therein. In some embodiments, the protein solution comprises about 10 mM to about 50 mM TRIS. In some embodiments, the protein solution comprises about 15 mM to about 50 mM TRIS. In some embodiments, the protein solution comprises about 20 mM to about 50 mM TRIS. In some embodiments, the protein solution comprises about 25 mM to about 50 mM TRIS. In some embodiments, the protein solution comprises about 30 mM to about 50 mM TRIS. In some embodiments, the protein solution comprises about 35 mM to about 50 mM TRIS. In some embodiments, the protein solution comprises about 40 mM to about 50 mM TRIS. In some embodiments, the protein solution comprises about 45 mM to about 50 mM TRIS.

[0075] In some embodiments, the protein solution comprises about 5 mM to about 50 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), or any value or range therein. In some embodiments, the protein solution comprises about 10 mM to about 45 mM TRIS. In some embodiments, the protein solution comprises about 10 mM to about 40 mM TRIS. In some embodiments, the protein solution comprises about 10 mM to about 35 mM TRIS. In some embodiments, the protein solution comprises about 10 mM to about 30 mM TRIS. In some embodiments, the protein solution comprises about 10 mM to about 25 mM TRIS. In some embodiments, the protein solution comprises about 10 mM to about 20 mM TRIS. In some embodiments, the protein solution comprises about 10 mM to about 15 mM TRIS.

[0076] In some embodiments, the protein solution comprises about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS). In some embodiments, the protein solution comprises about 5 mM TRIS. In some embodiments, the protein solution comprises about 10 mM TRIS. In some embodiments, the protein solution comprises about 15 mM TRIS. In some embodiments, the protein solution comprises about 20 mM TRIS. In some embodiments, the protein solution comprises about 25 mM TRIS. In some embodiments, the protein solution comprises about 30 mM TRIS. In some embodiments, the protein solution comprises about 35 mM TRIS. In some embodiments, the protein solution comprises about 40 mM TRIS. In some embodiments, the protein solution comprises about 45 mM TRIS. In some embodiments, the protein solution comprises about 50 mM TRIS.

[0077] In some embodiments, the protein solution comprises about 5 mM to about 100 mM histidine buffer, or any value or range therein. In some embodiments, the protein solution comprises about 10 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 15 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 20 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 25 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 30 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 35 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 40 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 45 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 50 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 55 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 60 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 65 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 70 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 75 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 80 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 85 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 90 mM to about 100 mM histidine buffer. In some embodiments, the protein solution comprises about 95 mM to about 100 mM histidine buffer.

[0078] In some embodiments, the protein solution comprises about 5 mM to about 100 mM histidine buffer, or any value or range therein. In some embodiments, the protein solution comprises about 5 mM to about 95 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 90 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 85 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 80 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 75 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 70 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 65 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 60 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 55 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 50 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 45 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 40 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 35 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 30 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 25 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 20 mM histidine buffer. In some embodiments, the protein solution comprises about 5 mM to about 15 mM histidine buffer, hi some embodiments, the protein solution comprises about 5 mM to about 10 mM histidine buffer.

[0079] In some embodiments, the protein solution comprises about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM histidine buffer, or any value therein. In some embodiments, the protein solution comprises about 5 mM histidine buffer. In some embodiments, the protein solution comprises about 10 mM histidine buffer. In some embodiments, the protein solution comprises about 15 mM histidine buffer. In some embodiments, the protein solution comprises about 20 mM histidine buffer. In some embodiments, the protein solution comprises about 25 mM histidine buffer. In some embodiments, the protein solution comprises about 30 mM histidine buffer. In some embodiments, the protein solution comprises about 35 mM histidine buffer. In some embodiments, the protein solution comprises about 40 mM histidine buffer. In some embodiments, the protein solution comprises about 45 mM histidine buffer. In some embodiments, the protein solution comprises about 50 mM histidine buffer. In some embodiments, the protein solution comprises about 55 mM histidine buffer. In some embodiments, the protein solution comprises about 60 mM histidine buffer. In some embodiments, the protein solution comprises about 65 mM histidine buffer. In some embodiments, the protein solution comprises about 70 mM histidine buffer. In some embodiments, the protein solution comprises about 75 mM histidine buffer. In some embodiments, the protein solution comprises about 80 mM histidine buffer. In some embodiments, the protein solution comprises about 85 mM histidine buffer. In some embodiments, the protein solution comprises about 90 mM histidine buffer. In some embodiments, the protein solution comprises about 95 mM histidine buffer. In some embodiments, the protein solution comprises about 100 mM histidine buffer.

[0080] In some embodiments, the protein solution has a pH of about 5.5 to about 8.5, or any value or range therein. In some embodiments, the protein solution has a pH of about 6.0 to about 8.5. In some embodiments, the protein solution has a pH of about 6.5 to about 8.5. In some embodiments, the protein solution has a pH of about 7.0 to about 8.5. In some embodiments, the protein solution has a pH of about 7.5 to about 8.5. In some embodiments, the protein solution has a pH of about 8.0 to about 8.5. In some embodiments, the protein solution has a pH of about 6.0 to about 8.0.

[0081] In some embodiments, the protein solution has a pH of about 5.5 to about 8.5. In some embodiments, the protein solution has a pH of about 5.5 to about 8.0. In some embodiments, the protein solution has a pH of about 5.5 to about 7.5. In some embodiments, the protein solution has a pH of about 5.5 to about 7.0. In some embodiments, the protein solution has a pH of about 5.5 to about 6.5. In some embodiments, the protein solution has a pH of about 5.5 to about 6.0.

[0082] In some embodiments, the protein solution has a pH of about 5.5, about 6.0, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or about 8.5, or any value therein. In some embodiments, the protein solution has a pH of about 5.5. In some embodiments, the protein solution has a pH of about 6.0. In some embodiments, the protein solution has a pH of about 6.5. In some embodiments, the protein solution has a pH of about 6.6. In some embodiments, the protein solution has a pH of about 6.7. In some embodiments, the protein solution has a pH of about 6.8. In some embodiments, the protein solution has a pH of about 6.9. In some embodiments, the protein solution has a pH of about 7.0. In some embodiments, the protein solution has a pH of about 7.1. In some embodiments, the protein solution has a pH of about 7.2. In some embodiments, the protein solution has a pH of about 7.3. In some embodiments, the protein solution has a pH of about 7.4. In some embodiments, the protein solution has a pH of about 7.5. In some embodiments, the protein solution has a pH of about 7.6. In some embodiments, the protein solution has a pH of about 7.7. In some embodiments, the protein solution has a pH of about 7.8. In some embodiments, the protein solution has a pH of about 7.9. In some embodiments, the protein solution has a pH of about 8.0. In some embodiments, the protein solution has a pH of about 8.5.

[0083] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v protein, about 5 mM to about 50 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 0.1% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 1% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride.

[0084] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v protein, about 5 mM to about 50 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0. In some embodiments, the protein solution comprises about 0.1% protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0. In some embodiments, the protein solution comprises about 1% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0.

[0085] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v protein, about 5 mM to about 100 mM histidine buffer, and about 100 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 0.1% w / v protein, about 5 mM to about 100 mM histidine buffer, and about 100 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 1% w / v protein, about 5 mM to about 100 mM histidine buffer, and about 100 mM to about 400 mM sodium chloride.

[0086] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v protein, about 5 mM to about 100 mM histidine buffer, and about 100 mM to about 400 mM sodium chloride. In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v protein, about 5 mM to about 100 mM histidine buffer, and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0. In some embodiments, the protein solution comprises about 0.1% w / v protein, about 5 mM to about 100 mM histidine buffer, and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0. In some embodiments, the protein solution comprises about 1% w / v protein, about 5 mM to about 100 mM histidine buffer, and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0.

[0087] In some embodiments, the protein solution comprises any protein having an isoelectric point of about 5.0 to about 8.0, or any value or range therein. In some embodiments, the protein has an isoelectric point of about 5.0 to about 8.0. In some embodiments, the protein has an isoelectric point of about 5.2 to about 8.0. In some embodiments, the protein has an isoelectric point of about 5.4 to about 8.0. In some embodiments, the protein has an isoelectric point of about 5.6 to about 8.0. In some embodiments, the protein has an isoelectric point of about 5.8 to about 8.0. In some embodiments, the protein has an isoelectric point of about 6.0 to about 8.0. In some embodiments, the protein has an isoelectric point of about 6.2 to about 8.0. In some embodiments, the protein has an isoelectric point of about 6.4 to about 8.0. In some embodiments, the protein has an isoelectric point of about 6.6 to about 8.0. In some embodiments, the protein has an isoelectric point of about 6.8 to about 8.0. In some embodiments, the protein has an isoelectric point of about 7.0 to about 8.0. In some embodiments, the protein has an isoelectric point of about 7.2 to about 8.0. In some embodiments, the protein has an isoelectric point of about 7.4 to about 8.0. In some embodiments, the protein has an isoelectric point of about 7.6 to about 8.0. In some embodiments, the protein has an isoelectric point of about 7.8 to about 8.0. In some embodiments, the protein has an isoelectric point of about 5.0 to about 7.8. In some embodiments, the protein has an isoelectric point of about 5.0 to about 7.6. In some embodiments, the protein has an isoelectric point of about 5.0 to about 7.4. In some embodiments, the protein has an isoelectric point of about 5.0 to about 7.2. In some embodiments, the protein has an isoelectric point of about 5.0 to about 7.0. In some embodiments, the protein has an isoelectric point of about 5.0 to about 6.8. In some embodiments, the protein has an isoelectric point of about 5.0 to about 6.6. In some embodiments, the protein has an isoelectric point of about 5.0 to about 6.4. In some embodiments, the protein has an isoelectric point of about 5.0 to about 6.2. In some embodiments, the protein has an isoelectric point of about 5.0 to about 6.0.In some embodiments, the protein has an isoelectric point of about 5.0 to about 5.8. In some embodiments, the protein has an isoelectric point of about 5.0 to about 5.6. In some embodiments, the protein has an isoelectric point of about 5.0 to about 5.4. In some embodiments, the protein has an isoelectric point of about 5.0 to about 5.2. In some embodiments, the protein has an isoelectric point of about 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, or any value therebetween. In some embodiments, the protein has an isoelectric point of about 5.0. In some embodiments, the protein has an isoelectric point of about 5.1. In some embodiments, the protein has an isoelectric point of about 5.2. In some embodiments, the protein has an isoelectric point of about 5.3. In some embodiments, the protein has an isoelectric point of about 5.4. In some embodiments, the protein has an isoelectric point of about 5.5. In some embodiments, the protein has an isoelectric point of about 5.6. In some embodiments, the protein has an isoelectric point of about 5.7. In some embodiments, the protein has an isoelectric point of about 5.8. In some embodiments, the protein has an isoelectric point of about 5.9. In some embodiments, the protein has an isoelectric point of about 6.0. In some embodiments, the protein has an isoelectric point of about 6.1. In some embodiments, the protein has an isoelectric point of about 6.2. In some embodiments, the protein has an isoelectric point of about 6.3. In some embodiments, the protein has an isoelectric point of about 6.4. In some embodiments, the protein has an isoelectric point of about 6.5. In some embodiments, the protein has an isoelectric point of about 6.6. In some embodiments, the protein has an isoelectric point of about 6.7. In some embodiments, the protein has an isoelectric point of about 6.8. In some embodiments, the protein has an isoelectric point of about 6.9. In some embodiments, the protein has an isoelectric point of about 7.0. In some embodiments, the protein has an isoelectric point of about 7.1. In some embodiments, the protein has an isoelectric point of about 7.2.In some embodiments, the protein has an isoelectric point of about 7.3. In some embodiments, the protein has an isoelectric point of about 7.4. In some embodiments, the protein has an isoelectric point of about 7.5. In some embodiments, the protein has an isoelectric point of about 7.6. In some embodiments, the protein has an isoelectric point of about 7.7. In some embodiments, the protein has an isoelectric point of about 7.8. In some embodiments, the protein has an isoelectric point of about 7.9. In some embodiments, the protein has an isoelectric point of about 8.0.

[0088] In some embodiments, the protein solution comprises a protein selected from, but not limited to, myoglobulin, serum albumin, bovine serum albumin, human serum albumin, immunoglobulins, immunoglobulin fragments, fibronectin, vitronectin, or combinations thereof. In some embodiments, the protein solution comprises serum albumin. In some embodiments, the protein solution comprises human serum albumin. In some embodiments, the protein solution comprises human serum albumin. In some embodiments, the human serum albumin is recombinant human serum albumin. In some embodiments, the human serum albumin is non-recombinant human serum albumin. In some embodiments, the non-recombinant human serum albumin is United States Pharmacopeia (USP) grade non-recombinant human serum albumin. In some embodiments, the non-recombinant human serum albumin is FDA-approved non-recombinant human serum albumin. Human serum albumin can be produced from any source. Methods for preparing human serum albumin are known in the art, and any such method is within the scope of the present application. In some embodiments, the human serum albumin is prepared from a bacterial source (e.g., bacterial cells). In some embodiments, the human serum albumin is prepared from an insect source (e.g., insect cells). In some embodiments, the human serum albumin is prepared from a mammalian source (e.g., mammalian cells). In some embodiments, the human serum albumin is prepared from a plant source. Similarly, recombinant human serum albumin can be recombinantly prepared from any source. Methods for preparing recombinant human serum albumin are known in the art, and all such methods are within the scope of the present application. In some embodiments, the human serum albumin is recombinantly prepared from a bacterial source (e.g., bacterial cells). In some embodiments, the recombinant human serum albumin is recombinantly prepared from an insect source (e.g., insect cells). In some embodiments, the recombinant human serum albumin is recombinantly prepared from a mammalian source (e.g., mammalian cells). In some embodiments, the recombinant human serum albumin is recombinantly prepared from a plant source.

[0089] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v human serum albumin, or any value or range therein. In some embodiments, the protein solution comprises about 0.2% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.3% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.4% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.5% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.6% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.7% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.8% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.9% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 1% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.2% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.4% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.6% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.8% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 2% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 2.2% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 2.4% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 2.6% to about 3% w / v human serum albumin, hi some embodiments, the protein solution comprises about 2.8% to about 3% w / v human serum albumin.

[0090] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.8% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.6% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.4% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.2% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.8% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.6% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.4% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.2% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.0% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.9% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.8% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.7% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.6% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.5% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.4% w / v human serum albumin, in some embodiments, about 0.1% to about 0.3% w / v human serum albumin, in some embodiments, about 0.1% to about 0.2% w / v human serum albumin.

[0091] In some embodiments, the protein solution comprises about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0.8% w / v, about 0.9% w / v, about 1.0% w / v, about 1.2% w / v, about 1.4% w / v, about 1.6% w / v, 1.8% w / v, about 2.0% w / v, about 2.2% w / v, about 2.4% w / v, about 2.6% w / v, about 2.8% w / v, or about 3.0% w / v of human serum albumin, or any value therein. In some embodiments, the protein solution comprises about 0.1% w / v of human serum albumin. In some embodiments, the protein solution comprises about 0.2% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.4% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.5% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.6% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.7% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.8% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.9% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.0% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.2% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.4% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.6% w / v human serum albumin. In some embodiments, the protein solution comprises about 1.8% w / v human serum albumin. In some embodiments, the protein solution comprises about 2.0% w / v human serum albumin. In some embodiments, the protein solution comprises about 2.2% w / v human serum albumin. In some embodiments, the protein solution comprises about 2.4% w / v human serum albumin.In some embodiments, the protein solution comprises about 2.6% w / v human serum albumin, in some embodiments, the protein solution comprises about 2.8% w / v human serum albumin, in some embodiments, the protein solution comprises about 3.0% w / v human serum albumin.

[0092] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v recombinant human serum albumin, or any value or range therein. In some embodiments, the protein solution comprises about 0.2% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.3% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.4% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.5% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.6% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.7% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.8% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.9% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.2% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.4% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.6% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.8% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.2% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.4% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.6% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.8% to about 3% w / v recombinant human serum albumin.

[0093] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.8% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.6% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.4% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.2% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.8% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.6% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.4% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.2% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.0% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.9% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.8% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.7% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.6% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.5% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.4% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.2% w / v recombinant human serum albumin.

[0094] In some embodiments, the protein solution comprises about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0.8% w / v, about 0.9% w / v, about 1.0% w / v, about 1.2% w / v, about 1.4% w / v, about 1.6% w / v, about 1.8% w / v, about 2.0% w / v, about 2.2% w / v, about 2.4% w / v, about 2.6% w / v, about 2.8% w / v, or about 3.0% w / v of recombinant human serum albumin, or any value therein. In some embodiments, the protein solution comprises about 0.1% w / v of recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.2% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.3% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.4% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.5% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.6% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.7% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.8% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.9% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.0% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.2% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.4% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.6% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.8% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.0% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.2% w / v recombinant human serum albumin.In some embodiments, the protein solution comprises about 2.4% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.6% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.8% w / v recombinant human serum albumin. In some embodiments, the protein solution comprises about 3.0% w / v recombinant human serum albumin.

[0095] In some embodiments, the recombinant human serum albumin is any recombinant human serum albumin. In some embodiments, the recombinant human serum albumin is Exbumin, Cellastim S, Albagen, Recombumin, or Optibumin. In some embodiments, the recombinant human serum albumin is Exbumin. In some embodiments, the recombinant human serum albumin is Cellastim S. In some embodiments, the recombinant human serum albumin is Albagen. In some embodiments, the recombinant human serum albumin is Recombumin. In some embodiments, the recombinant human serum albumin is Optibumin.

[0096] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v non-recombinant human serum albumin, or any value therein. In some embodiments, the protein solution comprises about 0.2% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.3% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.4% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.5% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.6% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.7% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.8% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.9% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.2% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.4% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.6% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.8% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.2% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.4% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.6% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.8% to about 3% w / v non-recombinant human serum albumin.

[0097] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.8% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.6% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.4% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2.2% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 2% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.8% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.6% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.4% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.2% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 1.0% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.9% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.8% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.7% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.6% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.5% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.4% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.3% w / v non-recombinant human serum albumin.In some embodiments, the protein solution comprises about 0.1% to about 0.2% w / v non-recombinant human serum albumin.

[0098] In some embodiments, the protein solution comprises about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0.8% w / v, about 0.9% w / v, about 1.0% w / v, about 1.2% w / v, about 1.4% w / v, about 1.6% w / v, about 1.8% w / v, about 2.0% w / v, about 2.2% w / v, about 2.4% w / v, about 2.6% w / v, about 2.8% w / v, or about 3.0% w / v of non-recombinant human serum albumin, or any value therein. In some embodiments, the protein solution comprises about 0.1% w / v of non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.2% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.3% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.4% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.5% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.6% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.7% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.8% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 0.9% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.0% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.2% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.4% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.6% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 1.8% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.0% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.2% w / v non-recombinant human serum albumin.In some embodiments, the protein solution comprises about 2.4% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.6% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 2.8% w / v non-recombinant human serum albumin. In some embodiments, the protein solution comprises about 3.0% w / v non-recombinant human serum albumin.

[0099] In some embodiments, the non-recombinant human serum albumin is any non-recombinant human serum albumin. In some embodiments, the non-recombinant human serum albumin is FDA-approved USP-grade non-recombinant human serum albumin. FDA-approved USP-grade non-recombinant human serum albumin can be obtained from any suitable commercial supplier, such as, but not limited to, Nova, Octapharma, Grifols Bio Supplies.

[0100] In some embodiments, the method for purifying a viral vector further comprises utilizing a protein-coated sterile membrane that was positively charged prior to coating with the protein. In some embodiments, the membrane is a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane. In some embodiments, the membrane is a polyethersulfone (PES) membrane. In some embodiments, the membrane is a nylon membrane. In some embodiments, the membrane is a polyvinylidene fluoride (PVDF) membrane.

[0101] In some embodiments, a method for purifying a viral vector or a method for preparing a concentrated sterile solution containing a viral vector comprises digesting DNA in a solution containing a cell culture medium and a viral vector before clarifying the solution. In some embodiments, a method for purifying a viral vector comprises digesting DNA in a solution containing a viral vector after filtering the clarified solution. In some embodiments, a method for purifying a viral vector comprises a first DNA digestion step comprising digesting DNA in a solution containing a cell culture medium and a viral vector before clarifying the solution, and a second DNA digestion step comprising digesting DNA in a solution containing a viral vector after filtering the clarified solution.

[0102] In some embodiments, the method comprises combining a concentrated sterile solution comprising the viral vector with one or more sterile, pyrogen-free buffers and / or additives in a sterile environment to prepare a sterile pharmaceutical composition comprising the viral vector. In some embodiments, the method comprises combining a concentrated sterile solution comprising the viral vector with one or more sterile, pyrogen-free buffers and / or additives to prepare a sterile composition comprising the viral vector. In some embodiments, the sterile, pyrogen-free buffers and / or additives include, but are not limited to, TRIS, HEPES, histidine buffer, phosphate buffer, sucrose, trehalose, polyethylene glycol, or any combination thereof.

[0103] In some embodiments, the method comprises digesting DNA in a solution comprising the viral vector to prepare a first digested solution comprising the viral vector; clarifying the digested solution comprising the viral vector; filtering the clarified digested solution comprising the viral vector through a first chromatography filter to prepare a filtered clarified digested solution comprising the viral vector; digesting DNA in the filtered clarified digested solution comprising the viral vector to prepare a filtered clarified second-digested solution comprising the viral vector; filtering the filtered clarified second-digested solution comprising the viral vector through a second chromatography filter to prepare a twice-filtered clarified second-digested solution comprising the viral vector; passing the twice-filtered clarified second-digested solution comprising the viral vector through a protein-coated sterile membrane to prepare a sterile solution comprising the viral vector; and concentrating the sterile solution comprising the viral vector to prepare a concentrated sterile solution comprising the viral vector.

[0104] In some embodiments, the method comprises digesting DNA in a solution comprising the viral vector to prepare a first digested solution comprising the viral vector; clarifying the digested solution comprising the viral vector; filtering the clarified digested solution comprising the viral vector through a first chromatography filter to prepare a filtered clarified digested solution comprising the viral vector; digesting DNA in the filtered clarified digested solution comprising the viral vector to prepare a filtered clarified double-digested solution comprising the viral vector; filtering the filtered clarified double-digested solution comprising the viral vector through a second chromatography filter to prepare a twice-filtered clarified double-digested solution comprising the viral vector; concentrating the twice-filtered clarified double-digested solution comprising the viral vector to prepare a concentrated twice-filtered clarified double-digested solution comprising the viral vector; and passing the concentrated twice-filtered clarified double-digested solution comprising the viral vector through a protein-coated sterile membrane to prepare a concentrated sterile solution comprising the viral vector.

[0105] In some embodiments, digestion of DNA is performed using an endonuclease. In some embodiments, the endonuclease is a DNAase. In some embodiments, the endonuclease has both DNAase and RNAase activity. In some embodiments, the endonuclease is selected from the group including, but not limited to, DNase I, DENARASE, Cryonase, or a combination thereof. In some embodiments, the endonuclease is DNase I. In some embodiments, the endonuclease is DENARASE. In some embodiments, the endonuclease is Cryonase.

[0106] In some embodiments, filtering the clarified digestion solution through a first chromatography filter comprises filtering the clarified digestion solution by capture chromatography. In some embodiments, the capture chromatography is ion exchange chromatography, affinity chromatography, hydrophobic chromatography, size exclusion chromatography, or multimodal chromatography. In some embodiments, the capture chromatography is ion exchange chromatography. In some embodiments, the capture chromatography is affinity chromatography. In some embodiments, the capture chromatography is hydrophobic chromatography. In some embodiments, the capture chromatography is size exclusion chromatography. In some embodiments, the capture chromatography is multimodal chromatography.

[0107] In some embodiments, the second chromatography filter is a filter as defined herein. In some embodiments, the second chromatography step is a membrane as defined herein, a membrane filter unit as defined herein, or a stationary phase as defined herein. In some embodiments, the stationary phase is a membrane filter unit. In some embodiments, the stationary phase is a resin.

[0108] In some embodiments, the second chromatography filter is a resin.

[0109] In some embodiments, filtering the filtered clarified double-digested solution through a second chromatography filter comprises filtering the filtered clarified double-digested solution through polishing chromatography. In some embodiments, the polishing chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimodal chromatography. In some embodiments, the polishing chromatography is ion exchange chromatography. In some embodiments, the polishing chromatography is hydrophobic interaction chromatography. In some embodiments, the polishing chromatography is size exclusion chromatography. In some embodiments, the polishing chromatography is multimodal chromatography.

[0110] In some embodiments, the method for purifying a viral vector further comprises recovering the medium from the cell culture that produces the viral vector. In some embodiments, the method for purifying a viral vector further comprises recovering the medium from the cell culture that produces the viral vector before digesting the DNA. In some embodiments, the method for purifying a viral vector further comprises recovering the medium from the cell culture that produces the viral vector before clarifying the digestion solution.

[0111] In some embodiments, the protein-coated sterile membrane comprises a sterile membrane that has been pre-washed with a protein solution. In some embodiments, the sterile membrane is pre-washed with a protein solution to create a protein-coated sterile membrane. In some embodiments, the method further comprises pre-washing a sterile membrane with a protein solution to create a protein-coated sterile membrane. In some embodiments, the method further comprises coating a sterile membrane with a protein solution to create a protein-coated sterile membrane.

[0112] In some embodiments, the membrane is a charged sterile membrane. In some embodiments, the protein-coated sterile membrane has a charge prior to coating with the protein solution. In some embodiments, the membrane has a positive charge. In some embodiments, the protein-coated sterile membrane has a positive charge prior to coating with the protein solution. In some embodiments, the membrane is a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane. In some embodiments, the membrane is a polyethersulfone (PES) membrane. In some embodiments, the membrane is a nylon membrane. In some embodiments, the membrane is a polyvinylidene fluoride (PVDF) membrane.

[0113] In some embodiments, the protein-coated sterile membrane has a pore size as set forth herein. In some embodiments, the protein-coated sterile membrane has a pore size of about 0.01 μm to 0.45 μm, as set forth herein, or any value or range therein. In some embodiments, the protein-coated sterile membrane has a pore size of 0.01 μm, 0.10 μm, 0.11 μm, 0.12 μm, 0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.2 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, as set forth herein. m, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, or 0.45 μm.

[0114] In some embodiments, the protein-coated sterile membrane does not significantly retain the viral vector. In some embodiments, the recovery of the solution comprising the viral vector is as set forth herein. In some embodiments, the recovery of the solution comprising the viral vector is at least about 60%, 61%, 62%, 63%, 64%, 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%, 96%, 97%, 98%, 99%, or 100% as set forth herein.

[0115] In some embodiments, the method of purifying a viral vector further comprises freezing the concentrated sterile solution comprising the viral vector, hi some embodiments, the freezing is carried out by controlled rate freezing.

[0116] In some embodiments, the method of purifying a viral vector further comprises diluting the concentrated sterile solution containing the viral vector prior to one or more of the above steps.

[0117] In some embodiments, the viral vector is present in a concentration of about 1 x 10 before passing the solution through a sterile filter. 3 ~Approx. 1×10 7 In some embodiments, the viral vector is at a concentration of about 1 x 10 transducing units (TU) / mL before passing the solution through a sterile filter. 4 ~Approx. 1×10 6 The concentration is in transducing units (TU) / mL. In some embodiments, the viral vector is at a concentration of 1 x 10 6 ~1×10 10 In some embodiments, the viral vector is at a concentration of 1 x 10 viral particles (vp) / mL before filtering the solution. 7 ~1×10 9 Concentration is in virus particles (vp) / mL.

[0118] In some embodiments, the methods provided herein can be used to purify any viral vector. In some embodiments, the viral vector is an adenovirus, an adeno-associated virus, a lentivirus, or a retrovirus. In some embodiments, the viral vector is an adenovirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus. In some embodiments, the viral vector is a retrovirus. In some embodiments, the viral vector is a pseudotyped viral vector. In some embodiments, the viral vector is a pseudotyped lentivirus. In some embodiments, the viral vector is a pseudotyped retrovirus.

[0119] Exemplary Methods Figure 1 illustrates a non-limiting, exemplary method for purifying a viral vector and / or preparing a concentrated, sterile solution containing a viral vector as described herein. It should be understood that the method illustrated in Figure 1 is illustrative only and is not intended to be limiting in any way.

[0120] As shown in Figure 1, step 110 involves thawing a vial of cells. The cells can be any suitable cell line capable of producing a viral vector (e.g., a viral vector disclosed herein). In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293T cells. In some embodiments, the cells are adherent. In some embodiments, the cells are cultured in suspension.

[0121] The cells are passaged (step 115) to return the cells to an appropriate growth rate and then transfected with one or more nucleic acid molecules encoding one or more viral vectors (step 120). In some embodiments, the one or more nucleic acid molecules encode viral vectors as described herein. In some embodiments, the one or more nucleic acid molecules or nucleic acid molecules further encode additional elements for viral production, such as, but not limited to, Gag-Pol and Rev accessory elements. As will be readily recognized by one of skill in the art, additional elements for viral production will vary depending on the viral vector being generated, and thus, any such elements are within the scope of the present disclosure.

[0122] In step 125, transfection is allowed to proceed for a predetermined period of time. In various embodiments, transfection is allowed to proceed for 1 hour to 120 hours. In various embodiments, transfection is allowed to proceed for 2 hours to 90 hours, 3 hours to 80 hours, 5 hours to 70 hours, 10 hours to 60 hours, 20 hours to 55 hours, or 30 hours to 50 hours. In various embodiments, transfection is allowed to proceed for 40 to 50 hours, e.g., 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, or 50 hours. In one non-limiting aspect, transfection is allowed to proceed for 48 hours. After transfection, the cell supernatant is collected. The cell supernatant is then treated with an endonuclease to digest the DNA in the supernatant (step 130). In some embodiments, DNA digestion is performed using an endonuclease as described herein.

[0123] After DNA digestion, the digested cell culture medium is clarified (step 135). In some embodiments, the clarification step comprises passing the digested cell culture medium through a filter. In some embodiments, the clarification step comprises passing the digested cell culture medium through a series of multiple filters. In some embodiments, the clarification step comprises passing the digested cell culture medium through a first filter and then passing the digested cell culture medium through a second filter, where the first filter has a larger pore size than the second filter.

[0124] The clarified solution is then filtered using capture chromatography (step 140). Further details of capture chromatography are provided herein. After chromatographic filtration, the filtered solution is subjected to a second round of DNA digestion (step 145). In various embodiments, DNA digestion is carried out using an endonuclease. Further details of specific endonucleases are provided herein. In some embodiments, the endonuclease used in step 145 is the same as the endonuclease used in step 130.

[0125] The twice-digested solution is then filtered using polishing chromatography (step 150). Further details of polishing chromatography are provided herein. After polishing filtration, the solution is subjected to sterile filtration (step 155). In some embodiments, the sterile filtration step includes a first step of pre-washing a sterile filter membrane with a protein solution before passing the digested solution through the sterile filter. Further details of specific protein solutions are provided herein. In some embodiments, the protein in the protein solution is human serum albumin (HSA), e.g., recombinant HSA or non-recombinant HSA. Further details of specific proteins in the protein solution are provided herein.

[0126] The sterile-filtered solution is then concentrated and a buffer exchange is performed on the product preparation (step 160). In some embodiments, the concentration step is performed by ultracentrifugation. In some embodiments, the concentration step is performed by dialysis. In some embodiments, the concentration step is performed by centrifugation. In some embodiments, the concentration step is performed by a chromatographic method. In some embodiments, the concentration step is performed by tangential flow filtration. In some embodiments, the tangential flow filtration is a two-stage tangential flow filtration. Once the solution is appropriately concentrated, a buffer exchange is performed to obtain the final concentrated viral particle formulation. After buffer exchange, the product may be subjected to a further concentration step according to methods provided herein. Once the final product is prepared, the formulation is subjected to controlled-rate freezing for long-term storage (step 165).

[0127] viral vectors In some embodiments, the pseudotyped viral vector comprises a VSV-G polypeptide.

[0128] In some embodiments, a VSV-G protein is provided, the VSV-G protein comprising a mutation at position 198 compared to SEQ ID NO:1 or a mutation at position 182 compared to SEQ ID NO:2. SEQ ID NO:1 is the full-length protein, and SEQ ID NO:2 is the ectodomain of the VSV-G protein. Cleavage of the 16-amino acid signal peptide of MKCLLYLAFLFIGVNC (SEQ ID NO:65) at the N-terminus of SEQ ID NO:1 leaves the protein of SEQ ID NO:2. Thus, mutations can be designated with respect to SEQ ID NO:2, but of course, can also be designated with respect to SEQ ID NO:1. SEQ ID NO:1 includes this leader sequence and therefore has 16 higher position numbers than the positions shown for SEQ ID NO:2. In some embodiments, the mutation inhibits or reduces binding of the VSV-G protein to the LDL receptor (LDL-R). In some embodiments, the mutation is an I182D mutation compared to SEQ ID NO:2. In some embodiments, the mutation is an I182E mutation compared to SEQ ID NO:2.

[0129] In some embodiments, a VSV-G protein is provided, the VSV-G protein comprising a mutation at position 198 compared to SEQ ID NO: 10, or a mutation at position 182 compared to SEQ ID NO: 11. SEQ ID NO: 10 is the full-length protein, and SEQ ID NO: 11 is the ectodomain of the VSV-G protein. Cleavage of the 16-amino acid signal peptide of MLSYLIFALVVSPILG (SEQ ID NO: 66) at the N-terminus of SEQ ID NO: 10 leaves the protein of SEQ ID NO: 11. Thus, mutations can be designated with respect to SEQ ID NO: 11, but of course, can also be designated with respect to SEQ ID NO: 10. SEQ ID NO: 10 includes this leader sequence and therefore has 16 more position numbers than the positions shown for SEQ ID NO: 11. In some embodiments, the mutations inhibit or reduce binding of the VSV-G protein to the LDL receptor (LDL-R). In some embodiments, the mutation is a T182D mutation compared to SEQ ID NO: 11. In some embodiments, the mutation is a T182E mutation compared to SEQ ID NO: 11.

[0130] In some embodiments, a VSV-G protein is provided, the VSV-G protein comprising a mutation at position 198 compared to SEQ ID NO: 12, or a mutation at position 182 compared to SEQ ID NO: 13. SEQ ID NO: 12 is the full-length protein, and SEQ ID NO: 13 is the ectodomain of the VSV-G protein. Cleavage of the 16-amino acid signal peptide of MLRLFLFCFLALGAHS (SEQ ID NO: 67) at the N-terminus of SEQ ID NO: 12 leaves the protein of SEQ ID NO: 13. Thus, mutations can be designated with respect to SEQ ID NO: 13, but of course, can also be designated with respect to SEQ ID NO: 12. SEQ ID NO: 12 includes this leader sequence and therefore has 16 more position numbers than the positions shown for SEQ ID NO: 13. In some embodiments, the mutation inhibits or reduces binding of the VSV-G protein to the LDL receptor (LDL-R). In some embodiments, the mutation is an A182D mutation compared to SEQ ID NO: 13. In some embodiments, the mutation is an A182E mutation compared to SEQ ID NO: 13.

[0131] In some embodiments, a VSV-G protein is provided, the VSV-G protein comprising a mutation at position 203 compared to SEQ ID NO: 14, or a mutation at position 182 compared to SEQ ID NO: 15. SEQ ID NO: 14 is the full-length protein, and SEQ ID NO: 15 is the ectodomain of the VSV-G protein. Cleavage of the 21 amino acid signal peptide of MKMKMVIAGLILCIGILPAIG (SEQ ID NO: 68) at the N-terminus of SEQ ID NO: 14 leaves the protein of SEQ ID NO: 15. Thus, mutations can be designated with respect to SEQ ID NO: 15, but of course, can also be designated with respect to SEQ ID NO: 14. SEQ ID NO: 14 includes this leader sequence and therefore has 21 more position numbers than the positions shown for SEQ ID NO: 15. In some embodiments, the mutation inhibits or reduces binding of the VSV-G protein to the LDL receptor (LDL-R). In some embodiments, the mutation is a V182D mutation compared to SEQ ID NO: 15. In some embodiments, the mutation is a V182E mutation compared to SEQ ID NO: 15.

[0132] In some embodiments, a VSV-G protein is provided, the VSV-G protein comprising a mutation at position 199 compared to SEQ ID NO: 16, or a mutation at position 182 compared to SEQ ID NO: 17. SEQ ID NO: 16 is the full-length protein, and SEQ ID NO: 17 is the ectodomain of the VSV-G protein. Cleavage of the 17-amino acid signal peptide of MTPAFILCMLLAGSSWA (SEQ ID NO: 69) at the N-terminus of SEQ ID NO: 16 leaves the protein of SEQ ID NO: 17. Thus, mutations can be designated with respect to SEQ ID NO: 17, but of course, can also be designated with respect to SEQ ID NO: 16. SEQ ID NO: 16 includes this leader sequence and therefore has 17 more position numbers than the positions shown for SEQ ID NO: 17. In some embodiments, the mutation inhibits or reduces binding of the VSV-G protein to the LDL receptor (LDL-R). In some embodiments, the mutation is a V182D mutation compared to SEQ ID NO: 17. In some embodiments, the mutation is a V182E mutation compared to SEQ ID NO: 17.

[0133] In some embodiments, a VSV-G protein is provided, the VSV-G protein comprising a mutation at position 199 compared to SEQ ID NO: 18, or a mutation at position 182 compared to SEQ ID NO: 19. SEQ ID NO: 18 is the full-length protein, and SEQ ID NO: 19 is the ectodomain of the VSV-G protein. Cleavage of the 17-amino acid signal peptide of MNFLLLTFIVLPLCSHA (SEQ ID NO: 70) at the N-terminus of SEQ ID NO: 18 leaves the protein of SEQ ID NO: 19. Thus, mutations can be designated with respect to SEQ ID NO: 19, but of course, can also be designated with respect to SEQ ID NO: 18. SEQ ID NO: 18 includes this leader sequence and therefore has 17 more position numbers than the positions shown for SEQ ID NO: 19. In some embodiments, the mutation inhibits or reduces binding of the VSV-G protein to the LDL receptor (LDL-R). In some embodiments, the mutation is a V182D mutation compared to SEQ ID NO: 19. In some embodiments, the mutation is a V182E mutation compared to SEQ ID NO: 19.

[0134] In some embodiments, a VSV-G protein is provided, the VSV-G protein comprising a mutation at position 199 compared to SEQ ID NO:20, or a mutation at position 182 compared to SEQ ID NO:21. SEQ ID NO:20 is the full-length protein, and SEQ ID NO:21 is the ectodomain of the VSV-G protein. Cleavage of the 17-amino acid signal peptide of MLVLYLLLSLLALGAQC (SEQ ID NO:71) at the N-terminus of SEQ ID NO:20 leaves the protein of SEQ ID NO:21. Thus, mutations can be designated with respect to SEQ ID NO:21, but of course, can also be designated with respect to SEQ ID NO:20. SEQ ID NO:20 includes this leader sequence and therefore has 17 more position numbers than the positions shown for SEQ ID NO:21. In some embodiments, the mutation inhibits or reduces binding of the VSV-G protein to the LDL receptor (LDL-R). In some embodiments, the mutation is a I182D mutation compared to SEQ ID NO:21. In some embodiments, the mutation is a I182E mutation compared to SEQ ID NO:21.

[0135] As used herein, when a polypeptide is said to have a mutation compared to a reference sequence, such comparison is based on an alignment (e.g., an alignment using BlastP, ClustalW, or ClutalOmega alignment software using default parameters). For example, position 182 can be found in SEQ ID NO: 2 and further compared to other strains as shown in the figure. The figure shows a Clustal alignment of the wild-type sequences of the ectodomains of various strains of VSV-G protein. The bold and underlined residues are those that align to position 182 of SEQ ID NO: 2 of various strains. SEQ ID NO: 2 shows the ectodomain of the VSV-G protein of the Indiana strain. SEQ ID NO: 11 shows the ectodomain of the VSV-G protein of the New Jersey strain. SEQ ID NO: 13 shows the ectodomain of the VSV-G protein of the Marraba strain. SEQ ID NO: 15 shows the ectodomain of the VSV-G protein of the Carajas strain. SEQ ID NO: 17 shows the ectodomain of the VSV-G protein of the Alagoa strain. SEQ ID NO: 19 shows the ectodomain of the VSV-G protein of the Cocal strain. SEQ ID NO: 21 shows the ectodomain of the VSV-G protein of the Morreton strain. Thus, the residue that aligns with residue 182 compared to SEQ ID NO: 2 can also be mutated as shown herein.

[0136] In some embodiments, the mutation at position 182 compared to SEQ ID NO: 2 is not an alanine. In some embodiments, the mutation at position 182 compared to SEQ ID NO: 2 is not a valine.

[0137] In some embodiments, the mutation at position 182 compared to SEQ ID NO:2 is I182S, I182H, I182T, I182Q, or I182N. In some embodiments, the mutation at position 182 compared to SEQ ID NO:11 is T182S, T182H, T182Q, or T182N. In some embodiments, the mutation at position 182 compared to SEQ ID NO:13 is A182S, A182H, A182T, A182Q, or A182N. In some embodiments, the mutation at position 182 compared to SEQ ID NO:15 is V182S, V182H, V182T, V182Q, or V182N. In some embodiments, the mutation at position 182 compared to SEQ ID NO:17 is V182S, V182H, V182T, V182Q, or V182N. In some embodiments, the mutation at position 182 relative to SEQ ID NO: 19 is V182S, V182H, V182T, V182Q, or V182N. In some embodiments, the mutation at position 182 relative to SEQ ID NO: 21 is I182S, I182H, I182T, I182Q, or I182N. In some embodiments, the mutation at position 182 is not a hydrophobic residue. In some embodiments, the mutation at position 182 is a charged residue. In some embodiments, the mutation at position 182 is a negatively charged residue.

[0138] Mutations can be described with reference to SEQ ID NO: 1 or SEQ ID NO: 2, which are VSV-G proteins from the Indiana strain, but mutations can also be used in other strains of VSV-G proteins. For example, mutations can be made in the New Jersey strain of VSV-G, the Marraba strain of VSV-G, the Carajas strain of VSV-G, the Alagoa strain of VSV-G, the Cocal strain of VSV-G, or the Morreton strain of VSV-G. In some embodiments, the respective sequences are as set forth herein. Examples of these can be found, for example, in U.S. Patent Application Publication No. 20200216502, which is incorporated herein by reference. For example, the wild-type full-length or ectodomain of the New Jersey strain of VSV-G is SEQ ID NO: 10 and SEQ ID NO: 11, respectively; the wild-type full-length or ectodomain of the Marraba strain of VSV-G is SEQ ID NO: 12 and SEQ ID NO: 13, respectively; the wild-type full-length or ectodomain of the Carajas strain of VSV-G is SEQ ID NO: 14 and SEQ ID NO: 15, respectively; the wild-type full-length or ectodomain of the Alagoa strain of VSV-G is SEQ ID NO: 16 and SEQ ID NO: 17, respectively; the wild-type full-length or ectodomain of the Cocal strain of VSV-G is SEQ ID NO: 18 and SEQ ID NO: 19, respectively; or the wild-type full-length or ectodomain of the Morreton strain of VSV-G is SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0139] A VSV-G protein comprising a mutation at position 182 compared to SEQ ID NO:2 can also comprise other mutations, such as those described in U.S. Patent Application Publication No. 20200216502, which is incorporated by reference in its entirety. For example, the VSV-G protein can comprise mutations at positions corresponding to positions 8, 47, 209, and / or 354 of SEQ ID NO:2.

[0140] In some embodiments, the substitution at position 8 is with any amino acid different from the amino acid at that position in the sequence of SEQ ID NO: 2, except for Y. In some embodiments, the substitution at position 209 is with any amino acid different from the amino acid at that position in the sequence of SEQ ID NO: 2, except for H. In some embodiments, the substitution at position 47 is with any amino acid different from the amino acid at that position in SEQ ID NO: 2, except for K or R. In some embodiments, the substitution at position 354 is with any amino acid different from the amino acid at that position in the sequence of SEQ ID NO: 2, except for K or R.

[0141] In some embodiments, the substitution is with A, G, F, or Q at position 47 or 354, or at both positions 47 and 354. In some embodiments, the substitution is A or Q.

[0142] In some embodiments, the substitution at position 8 is an alanine, ie, H8A.

[0143] In some embodiments, the substitution at position 47 is Q or N, ie, K47Q or K47N.

[0144] In some embodiments, the protein comprises a mutation (substitution) at position 10. In some embodiments, the substitution / mutation is Q10A, Q10R, or Q10K.

[0145] In some embodiments, a protein comprising a mutation at position 182 relative to SEQ ID NO:2 comprises a mutation at position 182 and has a nucleotide sequence similar to that of SEQ ID NO:2 (or SEQ ID NO:1 when the full length protein is used) of at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%. 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises an I182D or I182E mutation. In some embodiments, the VSV-G protein comprises an I182S, I182H, I182T, I182Q, or I182N mutation.

[0146] In some embodiments, the protein comprises a mutation at position 182 relative to SEQ ID NO: 11 and has a mutation at position 182 that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 11109, 11209, 11309, 11409, 11509, 11609, 11709, 11809, 11909, 119109, 119209, 119309, 119409, 119509, 119609, 119709, 119809, 119909, 120009, 1210009, 1220009, 1230009, 1240009, 1250009, 1260009, 1270009, 1280009, 1290009, 1300009, 1310009, 1320009, 1330009, 1340009, 1350009, 1360009, 1370009, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises a T182D or T182E mutation. In some embodiments, the VSV-G protein comprises a T182S, T182H, T182Q, or T182N mutation.

[0147] In some embodiments, the protein comprises a mutation at position 182 relative to SEQ ID NO: 13 and has a mutation at position 182 that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises an A182D or A182E mutation. In some embodiments, the VSV-G protein comprises an A182S, A182H, A182T, A182Q, or A182N mutation.

[0148] In some embodiments, the protein comprises a mutation at position 182 relative to SEQ ID NO: 15 and has a mutation at position 182 that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises a V182D or V182E mutation. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0149] In some embodiments, the protein comprises a mutation at position 182 relative to SEQ ID NO: 17 and has a mutation at position 182 that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises a V182D or V182E mutation. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0150] In some embodiments, the protein comprises a mutation at position 182 relative to SEQ ID NO: 19 and has a sequence identity that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises a V182D or V182E mutation. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0151] In some embodiments, the protein comprises a mutation at position 182 relative to SEQ ID NO:21 and has a mutation at position 182 that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1610, 1620, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises an I182D or I182E mutation. In some embodiments, the VSV-G protein comprises an I182S, I182H, I182T, I182Q, or I182N mutation.

[0152] virus particles Mutant VSV-G proteins can be used, for example, to pseudotype viruses, such as, but not limited to, lentiviruses. Thus, in some embodiments, viral particles are provided that include the mutant VSV-G proteins provided herein. In some embodiments, the viral particles include a VSV-G protein that includes a mutation at position 198 relative to SEQ ID NO: 1. In some embodiments, the viral particle includes a protein that includes a mutation at position 182 relative to SEQ ID NO: 2, and has a mutation at position 182 that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises an I182D or I182E mutation compared to SEQ ID NO: 2. In some embodiments, the VSV-G protein comprises an I182S, I182H, I182T, I182Q, or I182N mutation.

[0153] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 198 relative to SEQ ID NO: 10. In some embodiments, the protein comprising a mutation at position 182 relative to SEQ ID NO: 11 comprises a mutation at position 182 and has a sequence identity that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 11109, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises a T182D or T182E mutation compared to SEQ ID NO: 11. In some embodiments, the VSV-G protein comprises a T182S, T182H, T182Q, or T182N mutation.

[0154] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 198 relative to SEQ ID NO: 12. In some embodiments, the protein comprising a mutation at position 182 relative to SEQ ID NO: 13 comprises a mutation at position 182 and has a mutation at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises an A182D or A182E mutation compared to SEQ ID NO: 13. In some embodiments, the VSV-G protein comprises an A182S, A182H, A182T, A182Q, or A182N mutation.

[0155] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 203 relative to SEQ ID NO: 14. In some embodiments, the protein comprising a mutation at position 182 relative to SEQ ID NO: 15 comprises a mutation at position 182 and has a mutation at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises a V182D or V182E mutation compared to SEQ ID NO: 15. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0156] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 199 relative to SEQ ID NO: 16. In some embodiments, the protein comprising a mutation at position 182 relative to SEQ ID NO: 17 comprises a mutation at position 182 and has a sequence identity that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% or higher relative to SEQ ID NO: 17 (or SEQ ID NO: 16 if the full-length protein is used). 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises a V182D or V182E mutation compared to SEQ ID NO: 17. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0157] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 199 relative to SEQ ID NO: 18. In some embodiments, the protein comprising a mutation at position 182 relative to SEQ ID NO: 19 comprises a mutation at position 182 and has a sequence identity that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% or higher relative to SEQ ID NO: 19 (or SEQ ID NO: 18 if the full-length protein is used). 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises a V182D or V182E mutation compared to SEQ ID NO: 19. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0158] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 199 relative to SEQ ID NO: 20. In some embodiments, the protein comprising a mutation at position 182 relative to SEQ ID NO: 21 comprises a mutation at position 182 and has a sequence identity that is at least 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% or higher relative to SEQ ID NO: 21 (or SEQ ID NO: 20 if the full-length protein is used). 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or about 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% identical. In some embodiments, the polypeptide comprises an I182D or I182E mutation compared to SEQ ID NO: 21. In some embodiments, the VSV-G protein comprises an I182S, I182H, I182T, I182Q, or I182N mutation.

[0159] In some embodiments, the VSV-G protein further comprises a mutation at a position corresponding to position 214 and / or 352 of SEQ ID NO:2. In some embodiments, the residue corresponding to position 214 of SEQ ID NO:2 is T214. In some embodiments, the residue corresponding to position 352 of SEQ ID NO:2 is T352. In some embodiments, the VSV-G protein comprises a mutation equivalent to the T214N mutation compared to SEQ ID NO:2. In some embodiments, the VSV-G protein comprises a mutation equivalent to the T352A mutation compared to SEQ ID NO:2. In some embodiments, the VSV-G protein comprises the T214N and T352A mutations compared to SEQ ID NO:2. These mutations can be combined with any other mutations set forth herein. In some embodiments, the T214N and / or T352A mutations are combined with the I182E or I182D mutation. In some embodiments, the VSV-G protein comprises the amino acid sequences of SEQ ID NO:22 and SEQ ID NO:23, which combine I182D or I182E with T214N and T352A mutations, respectively. Such sequences are also shown below, along with the leader sequence that is removed during protein processing.

[0160] VSV-G protein_I196D, T230N, and T368A mutations (including leader sequence and adjusted numbering) MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQS CGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLDSMDITFFSEDGELSSLGKEGTGFRSNYFAYENGGKACKMQYCKHWGVRLPSGVWFEMADKD LFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTAERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 24)

[0161] VSV-G protein_I182D, T214N, and T352A mutations (not including leader sequence) KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAV IVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLDSMDITFFSEDGELSSLGKEGTGFRSNYFAYENGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFP ECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTAERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 22)

[0162] VSV-G protein containing the I196D, T230N, and T368A mutations (including leader sequence and adjusted numbering) MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQS CGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLESMDITFFSEDGELSSLGKEGTGFRSNYFAYENGGKACKMQYCKHWGVRLPSGVWFEMADKD LFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTAERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 25)

[0163] VSV-G protein (without leader sequence) containing the mutations I182E, T214N, and T352A KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAV IVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLESMDITFFSEDGELSSLGKEGTGFRSNYFAYENGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFP ECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTAERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 23)

[0164] In some embodiments, the VSV-G protein comprising a mutation at position 182 relative to SEQ ID NO:2 further comprises mutations at positions corresponding to positions 38 and / or 320 of SEQ ID NO:2. In some embodiments, the residue corresponding to position 38 of SEQ ID NO:2 is T38. In some embodiments, the residue corresponding to position 320 of SEQ ID NO:2 is T320. In some embodiments, the VSV-G protein comprises a mutation equivalent to the T38A mutation relative to SEQ ID NO:2. In some embodiments, the VSV-G protein comprises a mutation equivalent to the T320A mutation relative to SEQ ID NO:2. In some embodiments, the VSV-G protein comprises the T38A and T320A mutations relative to SEQ ID NO:2. These mutations can be combined with any other mutations set forth herein.

[0165] In some embodiments, other strains of the VSV-G protein described herein can further include one or more mutations compared to SEQ ID NO: 2 that correspond to any of the other mutations set forth herein. For example, other strains of the VSV-G protein described herein can also include mutations corresponding to T38A, T214N, T320A, and / or T352A in SEQ ID NO: 2. In some embodiments, other strains of the VSV-G protein described herein can also include mutations corresponding to T214N and / or T352A in SEQ ID NO: 2, as set forth in SEQ ID NO: 22 and SEQ ID NO: 23.

[0166] In some embodiments, the composition comprises a mutation as described in Hwang et al., Gene Ther 2013 Aug;20(8):807-15. (Epub 2013 Jan 31), the entire contents of which are incorporated herein by reference. For example, the mutation can be at positions 230, 368, 66, and / or 162, corresponding to SEQ ID NO: 1. These positions are reduced by 16 compared to SEQ ID NO: 2 when the leader sequence is removed. In some embodiments, the mutation at these positions is, for example, T230N, T368A, K66T, S162T, or any combination thereof. In some embodiments, the VSV-G protein comprises the mutations T230N and T368A. In some embodiments, the VSV-G polypeptide comprises K66T, S162T, T230N, and T368A. These positions correspond to those in the full-length protein (SEQ ID NO: 1). In some embodiments, the VSV-G protein comprises a T230N mutation, a T368A mutation, a K66T mutation, an S162T mutation, or any combination thereof. In some embodiments, the VSV-G protein further comprises one or more mutations in addition to the mutation corresponding to position 182 of SEQ ID NO:2, such as those described in U.S. Patent Application Publication No. 20200216502, which is incorporated herein by reference in its entirety. For example, the VSV-G protein can further comprise mutations at positions corresponding to positions 8, 47, 209, and / or 354 of SEQ ID NO:2.

[0167] In some embodiments, the substitution at position 8 is with any amino acid different from the amino acid at that position in the sequence of SEQ ID NO:2, except for Y. In some embodiments, the substitution at position 209 is with any amino acid different from the amino acid at that position in the sequence of SEQ ID NO:2, except for H. In some embodiments, the substitution at position 47 is with any amino acid different from the amino acid at that position in the sequence of SEQ ID NO:2, except for K or R. In some embodiments, the substitution at position 354 is with any amino acid different from the amino acid at that position in the sequence of SEQ ID NO:2, except for K or R. In some embodiments, the substitution is with A, G, F, or Q at position 47 or 354, or at both positions 47 and 354. In some embodiments, the substitution is A or Q. In some embodiments, the substitution at position 8 is alanine, i.e., H8A. In some embodiments, the substitution at position 47 is Q or N, i.e., K47Q or K47N. In some embodiments, the protein comprises a mutation (substitution) at position 10. In some embodiments, the substitution / mutation is Q10A, Q10R, or Q10K.

[0168] Additionally, in some embodiments, instead of the VSV-G protein or a variant thereof, the virus can be pseudotyped with other viral structural proteins. Suitable examples of other viral structural proteins can be found in at least WO2023 / 064884, WO2023 / 114698, WO2023 / 114884, and WO2023 / 154858, each of which is incorporated by reference in its entirety.

[0169] Targeting part In some embodiments, the viral particle comprises a targeting moiety. The targeting moiety can be used to target viral particles comprising a mutant VSV-G protein to cells expressing the target to which the targeting moiety binds. In some embodiments, the targeting moiety is an antibody, an scFv antibody, an antigen-binding domain, ankyrin repeats (e.g., DARPIN), a VHH domain antibody, a nanobody, a single-domain antibody, an FN3 domain, or any combination thereof. The targeting moiety can be attached to the viral surface via the Fc stalk of IgG. In some embodiments, the stalk comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the targeting moiety is bound (fused or linked) to the envelope glycoprotein G or H of a Paramyxoviridae virus (e.g., a morbillivirus such as measles virus, or a henipavirus such as Nipah virus, Cedar virus, or Hendra virus). In some embodiments, the targeting moiety can be attached (fused or linked) to a glycoprotein of a Rhabdoviridae virus (e.g., vesicular stomatitis New Jersey virus, vesicular stomatitis Indiana virus, vesicular stomatitis Alagoas virus, vesicular stomatitis Maraba virus, vesicular stomatitis Carajas virus, parainfluenza virus, Spodoptera frugiperda rhabdovirus isolate Sf G, Drosophila obscura sigma virus 10A, Wuhan insect virus 7, Perch virus, or Spring viremia of carp virus). In some embodiments, the VSV protein is a mutant protein, such as those shown herein. In some embodiments, the targeting moiety is attached to a glycoprotein of a Filoviridae virus, such as Ebola virus, or a glycoprotein of an Arenaviridae virus, such as Machupo virus.

[0170] In some embodiments, the targeting moiety is an scFv. In some embodiments, the targeting moiety is a single domain antibody. In some embodiments, the targeting moiety is a VHH.

[0171] In some embodiments, the targeting moiety binds to: CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, a glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, kinase anchor protein 4 (AKAP-4), adrenoceptor beta 3 (ADRB3), AFP, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin-binding cell surface receptor 2 (Tie 2), autoantibodies to desmoglein 1 (Dsgl), autoantibodies to desmoglein 3 (Dsg3), B7H3 (CD276), biotin, bone marrow stromal cell antigen 2 (BST2), BST1 / CD157, cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-la), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CCCTC-binding factor (zinc finger protein)-like (analogue of BORIS or imprinted region regulator), CCR4, CD5, CD19, CD20, CD22, CD24, CD30, CD3 2 (FCGR2A), CD33, CD34, CD38, CD44v6, CD72, CD79a, CD79b, CD97, CD99, CD123, CD171, CD179a, CD179b-IGLll, CD200R, CD276 / B7H3, CD300 molecule-like family member f (CD300LF), CDH1-CD324, CDH6, CDH17, CDH19, chromosome X open reading frame 61 (CXORF61), claudin 6 (CLDN6), claudin l8.2 (CLD18A2 or CLDN18A.2), CMVpp65, C-MYC epitope tag, Cripto, CS1 (also known as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24), CSF2RA (GM-CSFR alpha), C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule 1 (CLL-1 or CLECL1), cyclin B1, cytochrome P450 IB 1 (CYP1B 1), DLL3, EBV-EBNA3c, EGF-bke module-containing mucin-like hormone receptor-like 2 (EMR2), mutant elongation factor 2 (ELF2M), ephrin B2, ephrin type A receptor 2 (EphA2), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRviii), epithelial cell adhesion molecule (EPCAM), ERG, ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fibroblast growth factor receptor (EFGR), and fibroblast growth factor receptor (FCR). Blast activation protein alpha (FAP), FITC, Fms-like tyrosine kinase 3 (FLT3), folate receptor alpha (FRa or FR1), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), Fos-related antigen 1, fucosyl-GMl, G protein-coupled receptor class C group 5 member D (GPRC5D), G protein-coupled receptor 20 (GPR20), GAD, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4) )bDGlcp(ll)Cer), ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l-4)bDGlcp(ll)Cer), GD3, GFR alpha 4, glycoprotein 100 (gplOO), glypican 3 (GPC3), gonadotropin hormone receptor (CGHR or GR), GpA33, GpNMB, GPRC5D, guanylate cyclase C (GCC), heat shock protein 70-2 mutant (muthsp70-2), hepatitis A virus cellular receptor 1 (HAVCR1), hexasaccharide moiety of globoH glycoceramide (GloboH), high molecular weight melanoma-associated antigen (HMWMAA), HIV-1 envelope glycoprotein, HLA, HLA-DOA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DM, HLA-DOB, HLA-DP, HLA-DQ, HLA-DR, HLA-G, HTLV-1-Tax, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human telomerase reverse transcriptase (hTERT), IgE, IL13Ra2, IL1 lRa, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin (HA), insulin-like growth factor 1 receptor (IGF-I receptor), interleukin-11 receptor alpha (IL-llRa), interleukin-13 receptor subunit alpha 2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, KIT (CD117), KSHV K8.1, KSHV-gH, LAMP1, legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), luteinizing hormone receptor (LHR), Lewis (Y) antigen, Lewis Ag, Livl, Locus K9 (LY6K), low-conductance chloride channel, lymphocyte antigen 6 complex, lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), mammary differentiation antigen (NY-BR-1), melanoma antigen 1 recognized by T cells (MelanA or MARTI), melanoma-associated antigen 1 (MAGE-A1), melanoma cancer-testis antigen 1 (MAD-CT-1), melanoma cancer-testis antigen 2 (MAD-CT-2), melanoma inhibitor of apoptosis (ML-IAP), mesothelin, MPL, mucin 1 cell surface-associated (MUC1), N-acetylglucosaminyltransferase V (NA17), nectin-4, neural cell adhesion molecule (NCAM), NKG2D, NYBR1, O-acetylglucosaminyltransferase V (NA17), and O-acetylglucosaminyltransferase V (O-acetylglucosaminyltransferase V). Cetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), P53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), PDL1, P-glycoprotein, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor beta (PDGFR-beta), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), prostase, prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostein, protease serine 21 (testisin or PRSS21), proteasome (prosomal macropain) subunit beta type 9 (LMP2), PTK7, Ras G12V, Ras homologFamily member C (RhoC), rat sarcoma (Ras) mutant, receptor for advanced glycation end products (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine protein kinase ERBB2 or Her-22 / neu, kidney ubiquitous 1 (RU1), kidney ubiquitous 2 (RU2), sarcoma translocation breakpoint, serine 2 (TMPRSS2) ETS fusion gene, sialyl Lewis adhesion molecule (sLe), SLAMF4, SLAMF6, Slea (CA19.9 or sialyl Lewis antigen), sperm protein 17 (SPA 17), squamous cell carcinoma antigen 3 recognized by T cells (SART3), stage-specific embryonic antigen 4 (SSEA-4), STEAP1, survivin, synovial sarcoma X breakpoint 2 (SSX2), TCR gamma alternative reading frame protein (TARP), TCR-beta 1 chain, TCR-beta 2 chain, TCR-delta chain, TCR-gamma chain, TCR-gamma delta, telomerase, TGF-beta R2, antigen recognized by TNT antibodies, thyroid-stimulating hormone receptor (TSHR), Timl- / HVCR1, tissue factor 1 (TF1), Tn ag, Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), TNF receptor family member B-cell maturation (BCMA), transglutaminase 5 (TGS5), transmembrane protease, TROP2, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor-associated glycoprotein 72 (TAG72), tyrosinase, tyrosinase-related protein 2 (TRP-2), uroplakin 2 (UPK2), vascular endothelial growth factor receptor 2 (VEGFR2), V-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Wilms tumor protein (WT1), or X antigen family member 1A (XAGE1). In some embodiments, the targeting moiety binds to CD7. In some embodiments, the targeting moiety binds to CD8.

[0172] In some embodiments, the targeting moiety binds to a target present on a cell, such as an immune cell. In some embodiments, the cell is an immune cell. Such immune cells include, but are not limited to, T cells, B cells, NK cells, dendritic cells, neutrophils, macrophages, cancer cells, or CD3+ T cells, CD4+ T cells, CD7+ T cells, CD8+ T cells, CD19+ B cells, CD19+ cancer cells, CD20+ B cells, CD20+ cancer cells, CD30+ lung epithelial cells, CD34+ hematopoietic stem cells, CD105+ endothelial cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD133+ cancer cells, EpCAM+ cancer cells, GluA2+ neurons, GluA4+ neurons, hematopoietic stem cells, hepatocytes, Her2 / Neu+ cancer cells, NKG2D+ natural killer cells, SLC1A3+ astrocytes, SLC7A10+ adipocytes, etc. In some embodiments, the cell is a T cell. In some embodiments, the cells are B cells. In some embodiments, the cells are CD7+ T cells and / or CD8+ T cells.

[0173] In some embodiments, the targeting moiety (polypeptide) is capable of binding to CD7.

[0174] In some embodiments, the polypeptide binds to CD7. In some embodiments, the polypeptide that binds to CD7 is an antibody that binds to non-human primate CD7. In some embodiments, the polypeptide that binds to CD7 is an antibody that binds to human CD7. The sequence of human CD7 (UniProtKB P09564) is as follows (SEQ ID NO: 29): MAGPPRLLLLPLLLALARGLPGALAAQEVQQSPHCTTVPVGASVNITCSTSGGLRGIYLRQLGPQPQDIIYYEDGVVPTTDRRFRGRIDFSGSQDNLTITMHRLQLSDTGTYTCQAITEVNVYGSGTLVLVTEEQSQGWHRCSDAPPRASALPAPPTGSALPDPQTASALPDPPAASALPAALAVISFLLGLGLGVACVLARTQIKKLCSWRDKNSAACVVYEDMSHSRCNTLSSPNQYQ (SEQ ID NO: 29)

[0175] In some embodiments, the CD7 antibody comprises an Fc region. The Fc region can be attached to the heavy or light chain of the antibody. The Fc region can be directly fused to the heavy or light chain of the antibody, or indirectly fused to the heavy or light chain of the antibody via, for example, a peptide linker as described herein. In some embodiments, the Fc region is an IgG Fc. In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IgG Fc is an IgG1 Fc. In some embodiments, the antibody comprises the Fc constant region of SEQ ID NO: 26, as shown below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26)

[0176] In some embodiments, the IgG fc is an IgG2 Fc. In some embodiments, the antibody comprises the Fc constant region of SEQ ID NO: 27, as shown below. STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27)

[0177] In some embodiments, the IgG fc is an IgG4 Fc. In some embodiments, the antibody comprises the Fc constant region of SEQ ID NO: 28, as shown below. STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 28)

[0178] In some embodiments, the IgG1 Fc is a variant of the IgG1 Fc protein (SEQ ID NO: 26). In some embodiments, the IgG1 Fc protein variant comprises one or more mutations corresponding to those selected from the group consisting of L234A, L235A, N297A, P329G, I253A, H310A, and H435A of SEQ ID NO: 26. Any of the mutations L234A, L235A, N297A, P329G, I253A, H310A, and H435A of SEQ ID NO: 26 may be present or absent, and these mutations may be present in any combination. In some embodiments, the IgG1 Fc protein variant comprises mutations corresponding to L234A and L235A of SEQ ID NO: 26. In some embodiments, the IgG1 Fc protein variant comprises a mutation corresponding to N297A of SEQ ID NO: 26. In some embodiments, the IgG1 Fc protein variant comprises a mutation corresponding to P329G of SEQ ID NO: 26. In some embodiments, the IgG1 Fc protein variant comprises mutations corresponding to L234A, L235A, N297A, and P329G of SEQ ID NO: 83. In some embodiments, the IgG1 Fc protein variant comprises a mutation corresponding to I253A of SEQ ID NO: 26. In some embodiments, the IgG1 Fc protein variant comprises a mutation corresponding to H310A of SEQ ID NO: 26. In some embodiments, the IgG1 Fc protein variant comprises a mutation corresponding to H435A of SEQ ID NO: 26. In some embodiments, the IgG1 Fc protein variant comprises mutations corresponding to I253A, H310A, and H435A of SEQ ID NO: 26.

[0179] In some embodiments, the IgG2 Fc is a variant of an IgG2 Fc protein (SEQ ID NO: 27). In some embodiments, the IgG2 Fc protein variant comprises one or more mutations selected from the group consisting of N297A, P329G, I253A, H310A, and H435A at positions corresponding to SEQ ID NO: 27. Any of the mutations N297A, P329G, I253A, H310A, and H435A of SEQ ID NO: 27 may be present or absent, and any combination of these mutations may be present. In some embodiments, the IgG2 Fc protein variant comprises a mutation corresponding to N297A of SEQ ID NO: 27. In some embodiments, the IgG2 Fc protein variant comprises a mutation corresponding to P329G of SEQ ID NO: 27. In some embodiments, the IgG2 Fc protein variant comprises mutations corresponding to N297A and P329G of SEQ ID NO: 27. In some embodiments, the IgG2 Fc protein variant comprises a mutation corresponding to I253A of SEQ ID NO: 27. In some embodiments, the IgG2 Fc protein variant comprises a mutation corresponding to H310A of SEQ ID NO: 27. In some embodiments, the IgG2 Fc protein variant comprises a mutation corresponding to H435A of SEQ ID NO: 27. In some embodiments, the IgG2 Fc protein variant comprises mutations corresponding to I253A, H310A, and H435A of SEQ ID NO: 27.

[0180] In some embodiments, the IgG4 Fc protein is a variant of the IgG4 Fc protein (SEQ ID NO: 28). In some embodiments, the IgG4 Fc protein variant comprises one or more mutations selected from the group consisting of S228P, L235E, N297A, P329G, 1253A, H310A, and H435A at positions corresponding to SEQ ID NO: 28. Any of the mutations S228P, L235E, N297A, P329G, 1253A, H310A, and H435A of SEQ ID NO: 28 may be present or absent, and these mutations may be present in any combination. In some embodiments, the IgG4 Fc protein variant comprises a mutation corresponding to S228P in SEQ ID NO: 28. In some embodiments, the IgG4 Fc protein variant comprises a mutation corresponding to L235E in SEQ ID NO: 28. In some embodiments, the IgG4 Fc protein variant comprises a mutation corresponding to N297A in SEQ ID NO: 28. In some embodiments, the IgG4 Fc protein variant comprises a mutation corresponding to P329G of SEQ ID NO: 28. In some embodiments, the IgG4 Fc protein variant comprises mutations corresponding to S228P, L235E, N297A, and P329G of SEQ ID NO: 28. In some embodiments, the IgG4 Fc protein variant comprises a mutation corresponding to I253A of SEQ ID NO: 28. In some embodiments, the IgG4 Fc protein variant comprises a mutation corresponding to H310A of SEQ ID NO: 28. In some embodiments, the IgG4 Fc protein variant comprises a mutation corresponding to H435A of SEQ ID NO: 28. In some embodiments, the IgG4 Fc protein variant comprises mutations corresponding to I253A, H310A, and H435A of SEQ ID NO: 28.

[0181] In some embodiments, the Fc region comprises an Fc polypeptide variant. In some embodiments, the Fc polypeptide variant is an Fc polypeptide variant set forth in PCT Publication No. WO2024026284 (incorporated herein by reference in its entirety). In some embodiments, the Fc polypeptide variant comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 82 below. EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK (SEQ ID NO: 82) In some embodiments, the Fc polypeptide variant comprises the amino acid sequence of SEQ ID NO:82.

[0182] In some embodiments, the targeting moiety binds to CD7 and comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, and VL as shown in Tables 1 and 2 below. [Table 1] [Table 2]

[0183] The VH and VL sequences may be in any structure, including, but not limited to, an scFv structure in which the VH and VL regions are linked by a peptide linker. An example of a peptide linker that can be used to link the various peptides shown herein is (GGGGS). n(SEQ ID NO: 64) (each n is independently 1 to 4). In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, the variable regions are not linked by a peptide linker. In some embodiments, the polypeptide comprises SEQ ID NO: 42 and SEQ ID NO: 43. In some embodiments, the targeting moiety is represented by Formula V L -ZV H where Z is a peptide linker. In some embodiments, the targeting moiety comprises a heavy chain variable region set forth in SEQ ID NO: 42 linked to a light chain variable region set forth in SEQ ID NO: 43 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the targeting moiety comprises a V H V connected to L The targeting moiety comprising has the sequence shown below: DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNDSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPTTFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKLSCKASGYPFTSYWIHWVKQRPGRGLEWLGRIDPNSGDTKYNEKFKNKATLTVDKSSTTAYMQLSSLTSEDSAVYYCARSPYYSNDNSMDYWGQGTSVTVSS (SEQ ID NO: 44)

[0184] In some embodiments, the targeting moiety has formula V H -ZV L where Z is a peptide linker. In some embodiments, the targeting moiety comprises a heavy chain variable region set forth in SEQ ID NO: 42 linked to a light chain variable region set forth in SEQ ID NO: 43 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the targeting moiety comprises a V LV connected to H The targeting moiety comprising has the sequence shown below: QVQLQQPGAELVKPGASVKLSCKASGYPFTSYWIHWVKQRPGRGLEWLGRIDPNSGDTKYNEKFKNKATLTVDKSSTTAYMQLSSLTSEDSAVYYCARSPYYSNDNSMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNDSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPTTFGGGTKLEIKR (SEQ ID NO: 45)

[0185] In some embodiments, the targeting moiety (polypeptide) is capable of binding to CD8.

[0186] In some embodiments, the polypeptide binds to CD8. In some embodiments, the polypeptide binds to CD8 alpha. In some embodiments, the polypeptide binds to CD8 beta. In some embodiments, the polypeptide binds to a CD8 heterodimer. In some embodiments, the CD8 heterodimer comprises a CD8 alpha subunit and a CD8 beta subunit. In some embodiments, the polypeptide binds to a CD8 alpha homodimer. In some embodiments, the polypeptide that binds to CD8 is an antibody that binds to non-human primate CD8. In some embodiments, the antibody that binds to non-human primate CD8 is an antibody that binds to non-human primate CD8 alpha. In some embodiments, the antibody that binds to non-human primate CD8 is an antibody that binds to non-human primate CD8 beta. In some embodiments, the antibody that binds to non-human primate CD8 is an antibody that binds to non-human primate CD8 alpha homodimer. In some embodiments, the antibody that binds to non-human primate CD8 is an antibody that binds to non-human primate CD8 heterodimer. In some embodiments, the polypeptide that binds to CD8 is an antibody that binds to human CD8. In some embodiments, the antibody that binds to human CD8 is an antibody that binds to human CD8 alpha. In some embodiments, the antibody that binds to human CD8 is an antibody that binds to human CD8 beta. In some embodiments, the antibody that binds to human CD8 is an antibody that binds to human CD8 alpha homodimer. In some embodiments, the antibody that binds to human CD8 is an antibody that binds to human CD8 heterodimer. The sequence of human CD8 alpha (UniProtKB Q8TAW8) is as follows (SEQ ID NO: 46): MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGCYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 46)

[0187] The sequence of human CD8 beta (UniProtKB Q8TD28) is as follows (SEQ ID NO: 47): MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQLYK (SEQ ID NO: 47)

[0188] In some embodiments, the CD8 antibody comprises an Fc region. The Fc region can be attached to the heavy or light chain of the antibody. The Fc region can be directly fused to the heavy or light chain of the antibody, or indirectly fused to the heavy or light chain of the antibody via, for example, a peptide linker as described herein. In some embodiments, the Fc region is an IgG Fc as described herein. In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IgG fc is an IgG1 Fc as described herein. In some embodiments, the antibody comprises an Fc constant region of SEQ ID NO: 26. In some embodiments, the IgG fc is an IgG2 Fc as described herein. In some embodiments, the antibody comprises an Fc constant region of SEQ ID NO: 27. In some embodiments, the IgG fc is an IgG4 Fc as described herein. In some embodiments, the antibody comprises an Fc constant region of SEQ ID NO: 28.

[0189] In some embodiments, the targeting moiety binds to CD8 and comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, and VL as shown in Tables 3 and 4 below. [Table 3] [Table 4]

[0190] The VH and VL sequences may be in any structure, including, but not limited to, an scFv structure in which the VH and VL regions are linked by a peptide linker. An example of a peptide linker that can be used to link the various peptides shown herein is (GGGGS). n(SEQ ID NO: 64) (each n is independently 1 to 4). In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, the variable regions are not linked by a peptide linker. In some examples, the polypeptide comprises SEQ ID NO: 60 and SEQ ID NO: 61.

[0191] In some embodiments, the targeting moiety has formula V L -ZV H where Z is a peptide linker. In some embodiments, the targeting moiety comprises a heavy chain variable region set forth in SEQ ID NO: 60 linked to a light chain variable region set forth in SEQ ID NO: 61 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the targeting moiety comprises a V H V connected to L The targeting moiety comprising has the sequence shown below: NIVLTQSPASLAVSLGQRATISCRASESVDGFGNSFMNWYQQKPGQSPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPYTFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKASRYTFTDYNLHWVKLSHEKSLEWIGFIYPYNGGTGYNQKFKNKAKLTVDYSSSTAYMELRSLTSVDAAVYYCARDHRYNEGVSFDYWGQGTTLTVSS (SEQ ID NO: 62)

[0192] In some embodiments, the targeting moiety has formula V H -ZV Lwhere Z is a peptide linker. In some embodiments, the targeting moiety comprises a heavy chain variable region set forth in SEQ ID NO: 60 linked to a light chain variable region set forth in SEQ ID NO: 61 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the targeting moiety comprises a V L V connected to H The targeting moiety comprising has the sequence shown below: EVQLQQSGPELVKPGASVKISCKASRYTFTDYNLHWVKLSHEKSLEWIGFIYPYNGGTGYNQKFKNKAKLTVDYSSSTAYMELRSLTSVDAAVYYCARDHRYNEGVSFDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSNIVLTQSPASLAVSLGQRATISCRASESVDGFGNSFMNWYQQKPGQSPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPYTFGGGTKLEIKR (SEQ ID NO: 63)

[0193] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 44, or is substantially similar to SEQ ID NO: 44, or is an active fragment of SEQ ID NO: 44. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 44. In some embodiments, the targeting moiety comprises the amino acid sequence of SEQ ID NO:44.

[0194] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 45, or is substantially similar to SEQ ID NO: 45, or is an active fragment of SEQ ID NO: 45. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 45. In some embodiments, the targeting moiety comprises the amino acid sequence of SEQ ID NO:45.

[0195] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 62, or is substantially similar to SEQ ID NO: 62, or is an active fragment of SEQ ID NO: 62. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 62. In some embodiments, the targeting moiety comprises the amino acid sequence of SEQ ID NO: 62.

[0196] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 63, or is substantially similar to SEQ ID NO: 63, or is an active fragment of SEQ ID NO: 63. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 63. In some embodiments, the targeting moiety comprises the amino acid sequence of SEQ ID NO: 63.

[0197] In some embodiments, the targeting moiety described herein is attached to the surface of the virus via the stalk protein S1. In some embodiments, the targeting moiety is represented by the formula T-S1, where T is a targeting moiety described herein and S1 is a stalk protein. In some embodiments, the stalk protein S1 is as described in PCT Publication No. WO2024026284 (incorporated herein by reference in its entirety). In some embodiments, the stalk protein S1 comprises an Fc protein variant described herein represented by the formula L1-Fc-L2-X1, where L1 is a linker or absent, Fc is an Fc protein variant, L2 is a linker or absent, and X1 is a polypeptide comprising a transmembrane domain. Thus, in some embodiments, the formula representing the targeting moiety can also be written as T-L1-Fc-L2-X1. In some embodiments, the stalk protein S1 does not include an Fc region variant and is represented by the formula L3-X1, where L3 is a flexible peptide linker and X1 is a polypeptide comprising a transmembrane domain. Thus, in some embodiments, the formula representing the targeting moiety can also be written as T-L3-X1. In some embodiments, the polypeptide comprising the transmembrane domain X1 is represented by the formula ECD-T M-ICD polypeptide, wherein the ECD is an extracellular domain of a cell surface protein or a fragment thereof, or is absent, M is the transmembrane domain of a transmembrane protein, and ICD is a protein that facilitates incorporation of the targeting moiety into the intracellular domain of the protein or the envelope of the viral particle, or is absent. Thus, the formula for a targeting moiety linked to a stalk protein is: T-L1-Fc-L2-ECD-T M -ICD or T-L3-ECD-T M -Can also be written as ICD. L1, L2, L3, Fc, ECD, T M , and specific examples of ICDs can be found in PCT Publication No. WO2024026284, which is incorporated by reference in its entirety.

[0198] In some embodiments, the targeting moiety is T-L1-Fc-L2-ECD-T M -ICD. In some embodiments, T comprises an amino acid sequence having at least 90% identity to SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, L1 is present and comprises the amino acid sequence of SEQ ID NO:72. In some embodiments, Fc comprises an amino acid sequence having at least 90% identity to SEQ ID NO:82. In some embodiments, L2 is absent. In some embodiments, ECD comprises an amino acid sequence having at least 90% identity to SEQ ID NO:83 below. KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 83)

[0199] In some embodiments, T M comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 84 below. FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 84)

[0200] In some embodiments, the ICD comprises an amino acid sequence comprising an env uptake motif, wherein the env uptake motif comprises the amino acid sequence of SEQ ID NO:85 below. NRVRQGYS (SEQ ID NO: 85)

[0201] In some embodiments, the targeting moiety is T-L1-Fc-L2-ECD-T M In some embodiments, T comprises the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 62, or SEQ ID NO: 63. In some embodiments, L1 is present and comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, Fc comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, L2 is absent. In some embodiments, ECD comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, T M comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the ICD comprises an amino acid sequence comprising an env uptake motif, wherein the env uptake motif comprises the amino acid sequence of SEQ ID NO:85.

[0202] In some embodiments, the targeting moiety is T-L1-Fc-L2-ECD-T M In some embodiments, T comprises the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 45. In some embodiments, L1 is present and comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, Fc comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, L2 is absent. In some embodiments, ECD comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, T M comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the ICD comprises an amino acid sequence comprising an env uptake motif, wherein the env uptake motif comprises the amino acid sequence of SEQ ID NO:85.

[0203] In some embodiments, the targeting moiety is T-L1-Fc-L2-ECD-T MIn some embodiments, T comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, L1 is present and comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, Fc comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, L2 is absent. In some embodiments, ECD comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, T M comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the ICD comprises an amino acid sequence comprising an env uptake motif, wherein the env uptake motif comprises the amino acid sequence of SEQ ID NO:85.

[0204] In some embodiments, the targeting moiety is T-L3-ECD-T M -ICD. In some embodiments, T comprises an amino acid sequence having at least 90% identity to SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, L3 comprises the amino acid sequence of SEQ ID NO:64, where n is 1, 2, or 4. In some embodiments, ECD comprises an amino acid sequence having at least 90% identity to SEQ ID NO:86 below. FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 86)

[0205] In some embodiments, V M comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 87 below. IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 87)

[0206] In some embodiments, the ICD comprises an amino acid sequence comprising an env uptake motif, wherein the env uptake motif comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 88 below. GGTETSQVAPA (SEQ ID NO: 88)

[0207] In some embodiments, the targeting moiety is T-L3-ECD-T M In some embodiments, T has the formula of -ICD. In some embodiments, T comprises the amino acid sequence of SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, L3 comprises the amino acid sequence of SEQ ID NO:64, where n is 1, 2, or 4. In some embodiments, ECD comprises the amino acid sequence of SEQ ID NO:86. In some embodiments, T M comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the ICD comprises an amino acid sequence comprising an env uptake motif, wherein the env uptake motif comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO:88.

[0208] In some embodiments, the targeting moiety is T-L3-ECD-T M In some embodiments, T has the formula of -ICD. In some embodiments, T comprises the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 45. In some embodiments, L3 comprises the amino acid sequence of SEQ ID NO: 64, where n is 1, 2, or 4. In some embodiments, ECD comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, T M comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the ICD comprises an amino acid sequence comprising an env uptake motif, wherein the env uptake motif comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO:88.

[0209] In some embodiments, the targeting moiety is T-L3-ECD-T M In some embodiments, T has the formula of -ICD. In some embodiments, T comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, L3 comprises the amino acid sequence of SEQ ID NO: 64, where n is 1, 2, or 4. In some embodiments, ECD comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, T Mcomprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the ICD comprises an amino acid sequence comprising an env uptake motif, wherein the env uptake motif comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO:88.

[0210] In some embodiments, the targeting moiety having the formula T-S1 comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:89. (SEQ ID NO: 89)

[0211] In some embodiments, a targeting moiety having the formula of T-S1 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 89. In some embodiments, a targeting moiety having the formula of T-S1 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 89. In some embodiments, a targeting moiety having the formula of T-S1 comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 89. In some embodiments, a targeting moiety having the formula of T-S1 comprises the amino acid sequence of SEQ ID NO: 89.

[0212] In some embodiments, a viral particle comprising a mutant VSV-G protein as described herein and comprising a targeting moiety as described herein comprises a nucleic acid molecule encoding a heterologous molecule of interest or "cargo." For example, a heterologous molecule of interest is intended to refer to any product that can be encoded by a nucleic acid molecule. As non-limiting examples, a "cargo" or "heterologous molecule of interest" may refer to an siRNA, shRNA, a peptide, a polypeptide, a protein, a viral payload, a viral genome, or a combination thereof. In some embodiments, the polypeptide is a chimeric antigen receptor ("CAR").

[0213] As used herein, "chimeric antigen receptor" or "CAR" refers to an antigen-binding domain fused directly or indirectly (e.g., via a hinge or transmembrane domain) to an intracellular signaling domain capable of activating or stimulating immune cells. Most commonly, the extracellular binding domain of a CAR is composed of a single-chain variable fragment (scFv) obtained by fusing the variable heavy and variable light chain regions of a murine or humanized monoclonal antibody. Alternatively, scFvs derived from Fabs (not derived from antibodies, e.g., from a Fab library) can be used. In various embodiments, the scFvs are fused to a transmembrane domain and then to an intracellular signaling domain. However, the antigen-binding domain can be any molecule capable of binding to a target on a cell. For example, the antigen-binding domain of a CAR can be an antibody, an scFv antibody, an antigen-binding domain, an ankyrin repeat (e.g., DARPIN), a VHH domain antibody, a nanobody, a single-domain antibody, an FN3 domain, or any combination thereof. In some embodiments, a CAR includes one that provides only a CD3ζ signal upon antigen binding. In some embodiments, CARs include those that provide both costimulation (e.g., CD28 or CD137) and activation (CD3ζ). In some embodiments, CARs include those that provide multiple costimulations (e.g., CD28 and CD137) and activation (CD3ζ). In various embodiments, the CAR is selected to have high affinity or avidity for the antigen. In some embodiments, the CAR also includes a 4-1BB domain. These are merely exemplary and not limiting to this embodiment; any chimeric antigen receptor can be delivered with the viral particles and vectors provided herein. These are non-limiting examples of CARs; any CAR construct can be encoded by a nucleic acid molecule.

[0214] In some embodiments, the antigen binding domain of the CAR is V H Domain or V L Contains domains or V H Domains and VL In some embodiments, the V H A domain includes an amino acid sequence having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 73 below, or any value or range of identity therebetween. EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS (SEQ ID NO: 73)

[0215] In some embodiments, V H The V domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 73. In some embodiments, the V H The V domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 73. In some embodiments, the V H The V domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 73. In some embodiments, the V H The V domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 73. In some embodiments, the V H The domain comprises an amino acid sequence having the sequence of SEQ ID NO:73.

[0216] In some embodiments, V L A domain includes an amino acid sequence having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 74 below, or any value or range of identity therebetween. EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK (SEQ ID NO: 74)

[0217] In some embodiments, V L The V domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 74. In some embodiments, the V L The V domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 74. In some embodiments, the V L The V domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 74. In some embodiments, the V L The V domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 74. In some embodiments, the V L The domain comprises an amino acid sequence having the sequence of SEQ ID NO:74.

[0218] In some embodiments, the antigen binding domain of the CAR is V H Domain and V L In some embodiments, the V H Domains and V L The domains are not linked by a linker peptide. H Domains and V L The domains are linked by a linker peptide, for example as shown herein. Such linker peptides include (GGGGS) n Examples include, but are not limited to, (SEQ ID NO: 64) (where each n is independently 1 to 5). In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0219] In some embodiments, V H Domain and V LThe antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 73. H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 74. L In some embodiments, V H Domain and V L The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 73. H domain and further having at least 90% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 73. H domain and further having at least 95% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 73. H domain and further having at least 98% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and VL The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 73. H domain and further having at least 99% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 73. H domain and further having the sequence of SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 90% identity to SEQ ID NO: 73 H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 74. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 95% identity to SEQ ID NO: 73. H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 74. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 98% identity to SEQ ID NO: 73. Hdomain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 74. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 99% identity to SEQ ID NO: 73. H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 74. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having the amino acid sequence of SEQ ID NO: 73. H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 74. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 90% identity to SEQ ID NO: 73 H domain and further having at least 90% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 95% identity to SEQ ID NO: 73. H domain and further having at least 90% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V LThe antigen-binding domain of the CAR comprises a V domain having at least 90% identity to SEQ ID NO: 73 H domain and further having at least 95% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 95% identity to SEQ ID NO: 73. H domain and further having at least 95% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 98% identity to SEQ ID NO: 73. H domain and further having at least 98% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 99% identity to SEQ ID NO: 73. H domain and further having at least 99% identity to SEQ ID NO: 74 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having the amino acid sequence of SEQ ID NO: 73. H domain and further having the amino acid sequence of SEQ ID NO: 74 L Includes.

[0220] In some embodiments, the antigen binding domain of the CAR is V H -ZV L wherein V H is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 73, Z is a linker comprising the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 77), and V L is a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, VH -ZV L The antigen-binding domain of a CAR having the formula has the amino acid sequence shown below. EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK (SEQ ID NO: 75)

[0221] In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 75. In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 75. In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 95% identity to the sequence of SEQ ID NO: 75. In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 98% identity to the sequence of SEQ ID NO: 75. In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 99% identity to the sequence of SEQ ID NO: 75. In some embodiments, the antigen binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 75.

[0222] In some embodiments, the antigen binding domain of the CAR is V L -ZV H wherein V L is a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74, Z is a linker comprising the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 77), and VH is a variable region comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, V L -ZV H The antigen-binding domain of a CAR having the formula has the amino acid sequence shown below. EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS (SEQ ID NO: 76)

[0223] In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 76. In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 76. In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 95% identity to the sequence of SEQ ID NO: 76. In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 98% identity to the sequence of SEQ ID NO: 76. In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 99% identity to the sequence of SEQ ID NO: 76. In some embodiments, the antigen binding domain of a CAR comprises the amino acid sequence of SEQ ID NO: 76.

[0224] In some embodiments, the antigen binding domain of the CAR is V H Domain or V L Contains domains or V H Domains and V LIn some embodiments, the V H A domain comprises an amino acid sequence having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 78 below, or any value or range of identity therebetween. DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTS (SEQ ID NO: 78)

[0225] In some embodiments, V H The V domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 78. In some embodiments, the V H The V domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 78. In some embodiments, the V H The V domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 78. In some embodiments, the V H The V domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 78. In some embodiments, the V H The domain comprises an amino acid sequence having the sequence of SEQ ID NO:78.

[0226] In some embodiments, V L A domain includes an amino acid sequence having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 79 below, or any value or range of identity therebetween. EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS (SEQ ID NO: 79)

[0227] In some embodiments, V L The V domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 79. In some embodiments, the V L The V domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 79. In some embodiments, the V L The V domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 79. In some embodiments, the V L The V domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 79. In some embodiments, the V L The domain comprises an amino acid sequence having the sequence of SEQ ID NO:79.

[0228] In some embodiments, V H Domain and V L The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 78. H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 79. L In some embodiments, V H Domain and V LThe antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 78. H domain and further having at least 90% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 78. H domain and further having at least 95% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 78. H domain and further having at least 98% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 78. H domain and further having at least 99% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V LThe antigen binding domain of the CAR comprises a V domain having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 78. H domain and further having the sequence of SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 90% identity to SEQ ID NO: 78 H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 79. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 95% identity to SEQ ID NO: 78 H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 79. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 98% identity to SEQ ID NO: 78 H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 79. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 99% identity to SEQ ID NO: 78 Hdomain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 79. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having the amino acid sequence of SEQ ID NO: 78. H domain and further having at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 79. L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 90% identity to SEQ ID NO: 78 H domain and further having at least 90% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 95% identity to SEQ ID NO: 78 H domain and further having at least 90% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 90% identity to SEQ ID NO: 78 H domain and further having at least 95% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 95% identity to SEQ ID NO: 78 H domain and further having at least 95% identity to SEQ ID NO: 79 LIn some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 98% identity to SEQ ID NO: 78 H domain and further having at least 98% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having at least 99% identity to SEQ ID NO: 78 H domain and further having at least 99% identity to SEQ ID NO: 79 L In some embodiments, V H Domain and V L The antigen-binding domain of the CAR comprises a V domain having the amino acid sequence of SEQ ID NO: 78. H domain and further having the amino acid sequence of SEQ ID NO: 79 L Includes.

[0229] In some embodiments, the antigen binding domain of the CAR is V H -ZV L wherein V H is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78, Z is a linker comprising the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 77), and V L is a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, V H -ZV L The antigen-binding domain of a CAR having the formula has the amino acid sequence shown below. DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGGSGGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS (SEQ ID NO: 80)

[0230] In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 80. In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 80. In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 95% identity to the sequence of SEQ ID NO: 80. In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 98% identity to the sequence of SEQ ID NO: 80. In some embodiments, the antigen binding domain of the CAR comprises an amino acid sequence having at least 99% identity to the sequence of SEQ ID NO: 80. In some embodiments, the antigen binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 80.

[0231] In some embodiments, the antigen binding domain of the CAR is V L -ZV H wherein V L is a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79, Z is a linker comprising the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 77), and V H is a variable region comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, V L -ZV HThe antigen-binding domain of a CAR having the formula has the amino acid sequence shown below. SEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTS (SEQ ID NO: 81)

[0232] In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 81. In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 81. In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 95% identity to the sequence of SEQ ID NO: 81. In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 98% identity to the sequence of SEQ ID NO: 81. In some embodiments, the antigen binding domain of a CAR comprises an amino acid sequence having at least 99% identity to the sequence of SEQ ID NO: 81. In some embodiments, the antigen binding domain of a CAR comprises the amino acid sequence of SEQ ID NO: 81.

[0233] In some embodiments, the antigen-binding domain of the CAR comprises rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, or ublituximab. In some embodiments, the antigen-binding domain comprises rituximab. In some embodiments, the antigen-binding domain comprises ofatumumab. In some embodiments, the CAR also comprises a 4-1BB domain. These are merely exemplary and not limiting to this embodiment; any chimeric antigen receptor can be delivered with the viral particles and vectors provided herein. These are non-limiting examples of CARs; any CAR construct can be encoded by a nucleic acid molecule.

[0234] In some embodiments, the CAR comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 90 below; MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYY YGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITF GQGTRLEIKSGLDFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 90), or is substantially similar to, or an active fragment of, SEQ ID NO: 90. In some embodiments, the CAR comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 90. In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 90. In some embodiments, the CAR comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 90. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the subdomains of the CAR (e.g., antigen binding domain, hinge domain, transmembrane domain, costimulatory domain, signaling domain) are those set forth in PCT Publication No. WO2024026284, which is incorporated herein by reference in its entirety.

[0235] In some embodiments, the pseudotyped virus particle further comprises a heterologous nucleic acid molecule encoding a cargo of interest. The nucleic acid molecule may be useful for regulating the expression of a target gene. In some embodiments, the cargo can be used to regulate cellular activity or express a protein that is transported to the surface of a target cell. Thus, in some embodiments, the nucleic acid may comprise an siRNA or shRNA. The nucleic acid may also encode a cargo of interest. Thus, in some embodiments, the cargo of interest may comprise a polypeptide or portion thereof, a protein or portion thereof, a chimeric antigen receptor or portion thereof, or a tumor antigen or portion thereof. In some embodiments, the cargo of interest is an antibody produced by the virus, which can then be secreted by cells infected with the virus. The term "protein" can refer to any polypeptide that performs its native function within a cellular environment. Thus, in some embodiments, the protein encoded by the nucleic acid cargo of interest may comprise an enzyme, a nuclear receptor, a transporter, a ribosomal protein, a membrane-bound protein, a cytoplasmic protein, a G-protein-coupled receptor, a voltage-gated ion channel, a secreted protein, a mitochondrial protein, a cytokine, a chimeric antigen receptor, a tumor antigen, or portions or chimeric species thereof.

[0236] Without being bound by any particular theory, viral particles comprising the mutant VSV-G proteins described herein that include a targeting moiety can be used to express a heterologous molecule of interest in target cells. Thus, for example, a CAR can be expressed in a T cell, and viral particles pseudotyped with the VSV-G proteins described herein will target the T cell. If a T cell is the intended target, the viral particle can include a targeting moiety that binds to a target on the surface of the T cell (e.g., but not limited to, CD2, CD3, CD4, CD5, CD7, or CD8). In some embodiments, the target is CD2. In some embodiments, the target is CD3. In some embodiments, the target is CD4. In some embodiments, the target is CD5. In some embodiments, the target is CD6. In some embodiments, the target is CD7. In some embodiments, the target is CD8.

[0237] In some embodiments, the pseudotyped viral particles are recombinant lentivirus. In some embodiments, the recombinant pseudotyped viral particles are replication competent. In some embodiments, the recombinant pseudotyped viral particles are replication incompetent.

[0238] Exemplary Virus Particles In some embodiments, a viral particle is provided, the viral particle comprising a heterologous viral glycoprotein and a targeting moiety, wherein the heterologous viral glycoprotein comprises an amino acid sequence selected from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, and the targeting moiety comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the viral particle further comprises a nucleic acid molecule encoding a heterologous molecule of interest. In some embodiments, the heterologous molecule of interest is as described herein. In some embodiments, the heterologous molecule of interest is a CAR described herein. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO:90, at least 90% identity to SEQ ID NO:90, at least 95% identity to SEQ ID NO:90, at least 99% identity to SEQ ID NO:90, or at least 100% identity to SEQ ID NO:90.

[0239] In some embodiments, a viral particle is provided, the viral particle comprising a heterologous viral glycoprotein and a targeting moiety, wherein the heterologous viral glycoprotein comprises an amino acid sequence selected from SEQ ID NO:22 or SEQ ID NO:23, and the targeting moiety comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the viral particle further comprises a nucleic acid molecule encoding a heterologous molecule of interest. In some embodiments, the heterologous molecule of interest is as described herein. In some embodiments, the heterologous molecule of interest is a CAR described herein. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO:90, at least 90% identity to SEQ ID NO:90, at least 95% identity to SEQ ID NO:90, at least 99% identity to SEQ ID NO:90, or at least 100% identity to SEQ ID NO:90.

[0240] In some embodiments, a viral particle is provided, the viral particle comprising a heterologous viral glycoprotein and a targeting moiety, wherein the heterologous viral glycoprotein comprises the amino acid sequence of SEQ ID NO:22 and the targeting moiety comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the viral particle further comprises a nucleic acid molecule encoding a heterologous molecule of interest. In some embodiments, the heterologous molecule of interest is as described herein. In some embodiments, the heterologous molecule of interest is a CAR described herein. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO:90, at least 90% identity to SEQ ID NO:90, at least 95% identity to SEQ ID NO:90, at least 99% identity to SEQ ID NO:90, or at least 100% identity to SEQ ID NO:90.

[0241] In some embodiments, a viral particle is provided, the viral particle comprising a heterologous viral glycoprotein and a targeting moiety, wherein the heterologous viral glycoprotein comprises the amino acid sequence of SEQ ID NO:23 and the targeting moiety comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the viral particle further comprises a nucleic acid molecule encoding a heterologous molecule of interest. In some embodiments, the heterologous molecule of interest is as described herein. In some embodiments, the heterologous molecule of interest is a CAR described herein. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO:90, at least 90% identity to SEQ ID NO:90, at least 95% identity to SEQ ID NO:90, at least 99% identity to SEQ ID NO:90, or at least 100% identity to SEQ ID NO:90.

[0242] Enumeration of Embodiments The following examples are illustrative, but not limiting, of the compounds, compositions, and methods described herein. Other suitable modifications and variations known to those skilled in the art are within the scope of the embodiments set forth below. 1. A method for preparing a concentrated sterile solution containing a viral vector, comprising: Clarifying the solution containing the cell culture medium and the viral vector; filtering the clarified solution containing the viral vector through a first chromatography filter to prepare a filtered clarified solution containing the viral vector; passing the filtered clarified solution containing the viral vector through a protein-coated sterile membrane to produce a sterile solution containing the viral vector; concentrating the sterile solution containing the viral vector to prepare the concentrated sterile solution containing the viral vector. 2. The method of embodiment 1, further comprising mixing said concentrated sterile solution comprising said viral vector with one or more sterile, pyrogen-free buffers and / or additives to prepare a sterile pharmaceutical composition comprising said viral vector. 3. The method of embodiment 1, further comprising, prior to clarifying the solution, recovering the cell culture medium from a cell culture producing the viral vector. 4. The method of any one of embodiments 1-3, further comprising filtering the filtered clarified solution containing the viral vector through a second chromatography filter before passing the filtered clarified solution through the protein-coated sterile membrane to prepare a twice-filtered clarified solution containing the viral vector. 5. The method of embodiment 1, wherein said protein-coated sterile membrane comprises a sterile membrane that has been pre-washed with a protein solution. 6. The method of embodiment 1, wherein said method further comprises coating a sterile membrane with a protein solution to produce a sterile membrane coated with said protein. 7. The method of embodiment 5 or 6, wherein said protein solution comprises one or more ingredients selected from protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride, or a combination thereof. 8. The method of embodiment 7, wherein the protein solution comprises about 1% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0. 9. The method of embodiment 7, wherein said protein comprises human serum albumin, myoglobulin, bovine serum albumin, immunoglobulin, immunoglobulin fragment, fibronectin, vitronectin, or any combination thereof. 10. The method of embodiment 9, wherein said human serum albumin is recombinant human serum albumin, such as that produced from a plant. 11. The method of embodiment 9, wherein said human serum albumin is produced from a plant. 12. The method of embodiment 9, wherein said human serum albumin is non-recombinant human serum albumin. 13. The method of embodiment 12, wherein said non-recombinant human serum albumin is United States Pharmacopeia (USP) grade non-recombinant human serum albumin. 14. The method of embodiment 6, wherein said protein-coated sterile membrane has a positive charge prior to coating with protein. 15. The method of embodiment 6, wherein the membrane is a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane. 16. The method of any one of embodiments 1-15, wherein the clarified solution is filtered through the first chromatography filter by capture chromatography. 17. The method of embodiment 16, wherein said capture chromatography is ion exchange chromatography, affinity chromatography, hydrophobic chromatography, size exclusion chromatography, or multimodal chromatography. 18. The method of embodiment 4, wherein the filtered solution is filtered through the second chromatography filter by polishing chromatography. 19. The method of embodiment 18, wherein said polishing chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimodal chromatography. 20. The method of any one of embodiments 1-19, further comprising digesting DNA in the solution containing the cell culture medium and the viral vector before clarifying the solution. 21. The method of any one of embodiments 1-20, further comprising, after filtering the clarified solution, digesting DNA in the solution containing the viral vector. 22. The method of any one of embodiments 1-21, wherein said concentrating step comprises concentrating said viral vector by tangential flow filtration. 23. The method of embodiment 22, wherein said tangential flow filtration comprises two stages of tangential flow filtration. 24. The method of embodiment 22 or 23, wherein said tangential flow filtration utilizes fibers with an inner diameter of 1.0 mm and a pore size of 750 kDa cutoff. 25. The method of any one of embodiments 1 to 24, wherein the concentrated sterile solution comprising the viral vector is mixed with the one or more sterile, pyrogen-free buffers and / or additives in a sterile environment to prepare the sterile pharmaceutical composition comprising the viral vector. 26. The method of embodiment 25, wherein said mixing with said one or more sterile, pyrogen-free buffers and / or additives comprises a buffer exchange step. 27. The method comprises: Before clarifying the solution, i) obtaining a solution comprising a cell culture medium and the viral vector; ii) digesting DNA in the solution containing the cell culture medium and the viral vector to prepare a first digestion solution containing the viral vector; Clarifying the solution comprises clarifying the digested solution containing the viral vector to prepare a clarified digested solution containing the viral vector; After filtering the clarified solution and before passing the filtered clarified solution through a protein-coated sterile membrane, the method comprises: i) digesting DNA in the filtered clarified digested solution containing the viral vector to prepare a filtered clarified twice-digested solution containing the viral vector; and ii) filtering the filtered clarified twice-digested solution containing the viral vector through a second chromatography filter to prepare a twice-filtered clarified twice-digested solution containing the viral vector; the protein-coated sterile membrane comprises a membrane pre-washed with a protein solution; 2. The method of embodiment 1, wherein the protein solution comprises about 0.1% w / v to 2.0% w / v protein, about 10 mM to about 30 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0. 28. A method for preparing a concentrated sterile solution containing a viral vector, comprising: digesting DNA in a solution containing the viral vector to prepare a first digested solution containing the viral vector; Clarifying the digested solution containing the viral vector to prepare a clarified digested solution containing the viral vector; filtering the clarified digested solution containing the viral vector through a first chromatography filter to produce a filtered clarified digested solution containing the viral vector; digesting DNA in the filtered clarified digested solution containing the viral vector to prepare a filtered clarified twice-digested solution containing the viral vector; and filtering the filtered clarified twice-digested solution containing the viral vector through a second chromatography filter to prepare a twice-filtered clarified twice-digested solution containing the viral vector; passing the twice-filtered clarified twice-digested solution containing the viral vector through a protein-coated sterile membrane to prepare a sterile solution containing the viral vector; concentrating the sterile solution containing the viral vector to prepare the concentrated sterile solution containing the viral vector. 29. The method of embodiment 28, wherein the digestion of the DNA is carried out using an endonuclease having DNAase activity, RNAase activity, such as deoxyribonuclease I, DENARASE, Cryonase, or a combination thereof. 30. The method of embodiment 28, wherein said second chromatography filter is a resin. 31. The method of embodiment 28, further comprising mixing the concentrated sterile solution comprising the viral vector with one or more sterile, pyrogen-free buffers and / or additives to prepare a sterile pharmaceutical composition comprising the viral vector. 32. The method of embodiment 28, further comprising collecting the medium from the cell culture producing the viral vector. 33. The method of embodiment 28, further comprising, prior to digesting the DNA, collecting the medium from the cell culture producing said viral vector. 34. The method of embodiment 28, further comprising, prior to clarifying the digestion solution, recovering the medium from the cell culture producing the viral vector. 35. The method of embodiment 28, wherein said protein-coated sterile membrane comprises a sterile membrane that has been pre-washed with a protein solution. 36. The method of embodiment 35, wherein the method further comprises coating a sterile membrane with a protein solution to produce a sterile membrane coated with the protein. 37. The method of embodiment 36, wherein the protein solution comprises one or more of a protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride, or a combination thereof. 38. The method of embodiment 37, wherein the protein solution comprises about 1% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0. 39. The method of any one of embodiments 37-38, wherein the protein is human serum albumin. 40. The method of embodiment 39, wherein said human serum albumin is recombinant human serum albumin. 41. The method of embodiment 39, wherein said human serum albumin is produced from a plant. 42. The method of embodiment 39, wherein said human serum albumin is non-recombinant human serum albumin. 43. The method of embodiment 42, wherein said non-recombinant human serum albumin is United States Pharmacopeia (USP) grade non-recombinant human serum albumin. 44. The method of any one of embodiments 35-43, wherein the protein-coated sterile membrane has a positive charge before being coated with the protein. 45. The method of any one of embodiments 35-44, wherein the membrane is a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane. 46. ​​The pore size of the protein-coated sterile membrane is about 0.01 μm, about 0.10 μm, about 0.11 μm, about 0.12 μm, about 0.13 μm, about 0.14 μm, about 0.15 μm, about 0.16 μm, about 0.17 μm, about 0.18 μm, about 0.19 μm, about 0.2 μm, about 0.21 μm, about 0.22 μm, about 0.23 μm, about 0.24 μm, about 0.25 μm, about 0.26 μm, about 0.27 μm, about 0.28 μm, about 0.29 μm, about 0.30 μm, about 0.31 μm, about 0.32 μm, about 0.33 μm, about 0.34 μm, about 0.35 μm, about 0.36 μm, about 0.37 μm, about 0.38 μm, about 0.39 μm, about 0.40 μm, about 0.41 μm, about 0.42 μm, about 0.43 μm, about 0.44 μm, about 0.45 μm, about 0.46 μm, about 0.47 μm, about 0.48 μm, about 0.49 μm, about 0.50 μm, about 0.51 μm, about 0.52 μm, about 0.53 μm, about 0.54 μm, about 0.55 μm, about 0.56 μm, about 0.57 μm, about 0.58 μm, about 0.59 μm, about 100 μm, about 100 μm, about 100 μm, about 100 μm, about 10 46. ​​The method of any one of embodiments 28-45, wherein the thickness of the nanotube is about 0.31 μm, about 0.32 μm, about 0.33 μm, about 0.34 μm, about 0.35 μm, about 0.36 μm, about 0.37 μm, about 0.38 μm, about 0.39 μm, about 0.40 μm, about 0.41 μm, about 0.42 μm, about 0.43 μm, about 0.44 μm, or about 0.45 μm. 47. The method of any one of embodiments 28-46, wherein the protein-coated sterile membrane does not significantly retain viral vectors. 48. The method of embodiment 47, wherein the recovery rate of the viral vector from the solution is at least about 60%, 61%, 62%, 63%, 64%, 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%, 96%, 97%, 98%, 99%, or about 100%. 49. The method of any one of embodiments 28-48, wherein the clarified solution is filtered through the first chromatography filter by capture chromatography. 50. The method of embodiment 49, wherein said capture chromatography is ion exchange chromatography, affinity chromatography, hydrophobic chromatography, size exclusion chromatography, or multimodal chromatography. 51. The method of embodiment 28, wherein the first filtered solution is filtered through the second filter by polishing chromatography. 52. The method of embodiment 51, wherein said polishing chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimodal chromatography. 53. The method of any one of embodiments 28-52, wherein the concentrating step comprises concentrating the viral vector by tangential flow filtration. 54. The method of embodiment 53, wherein said tangential flow filtration comprises two-stage tangential flow filtration. 55. The method of embodiment 53 or embodiment 54, wherein said tangential flow filtration utilizes fibers with an inner diameter of 1.0 mm and a pore size of 750 kDa cutoff. 56. The method of any one of embodiments 28 to 55, wherein the concentrated sterile solution containing the viral vector is mixed with an excipient in a sterile environment to prepare the sterile pharmaceutical composition containing the viral vector. 57. The method of embodiment 56, wherein said mixing with an additive comprises a buffer exchange step. 58. The method of any one of embodiments 1-57, further comprising freezing the concentrated sterile solution comprising the viral vector or the sterile pharmaceutical composition comprising the viral vector. 59. The method of embodiment 58, wherein said freezing is performed by controlled rate freezing. 60. The method of any one of embodiments 1-59, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector. 61. The method of any one of embodiments 1-60, wherein the concentrated sterile solution containing the viral vector is diluted prior to one or more of the steps. 62. The viral vector is at a concentration of about 1 x 10 before passing the solution through the sterile filter. 4 ~Approx. 1×10 6 62. The method of any one of embodiments 1-61, wherein the concentration is in transducing units (TU) / mL. 63. The viral vector is diluted to 1 x 10 7 ~1×10 9 The method of any one of embodiments 1-62, wherein the concentration is in viral particles (vp) / mL. 64. The method of any one of the preceding embodiments, wherein the viral vector comprises a VSV-G polypeptide. 65. The method of any one of the preceding embodiments, wherein said viral vector, such as a lentiviral vector, is pseudotyped with a VSV-G polypeptide comprising a mutation corresponding to the mutation at position 182 of SEQ ID NO:2. 66. The method of embodiment 64 or 65, wherein the VSV-G polypeptide comprises the amino acid sequence of SEQ ID NO: 2 with a mutation at position 182 and has at least 95% identity to SEQ ID NO: 2. 67. The method of embodiments 64 to 66, wherein the VSV-G polypeptide comprises the mutation I182E or I182D compared to SEQ ID NO: 2. 68. The method of any one of embodiments 64 to 67, wherein the VSV-G polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with a mutation at position 198 and has at least 70% identity to SEQ ID NO: 2. 69. The method of any one of embodiments 64 to 68, wherein the VSV-G polypeptide comprises a mutation corresponding to I182D or I182E compared to the sequence of SEQ ID NO: 2. 70. The method of any one of embodiments 64 to 69, wherein the VSV-G polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO:4. 71. The method of any one of embodiments 64 to 70, wherein the VSV-G polypeptide comprises the amino acid sequence of SEQ ID NO: 4. 72. The method of any one of embodiments 64 to 69, wherein the VSV-G polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 5. 73. The method of any one of embodiments 64 to 72, wherein the VSV-G polypeptide comprises the amino acid sequence of SEQ ID NO: 5. 74. The method of any one of embodiments 64 to 73, wherein the VSV-G polypeptide further comprises mutations in the VSV-G protein corresponding to positions 8, 10, 47, 209, and / or 354 compared to SEQ ID NO: 2. 75. The method of any one of embodiments 64 to 74, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 8 of SEQ ID NO: 2, wherein the mutation is any amino acid different from the amino acid at that position of SEQ ID NO: 2, except for Y. 76. The method of any one of embodiments 64 to 75, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 209 of SEQ ID NO: 2, wherein the mutation is any amino acid different from the amino acid at that position in SEQ ID NO: 2, except for H. 77. The method of any one of embodiments 64 to 76, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 47 of SEQ ID NO: 2, and the mutation is any amino acid different from the amino acid at that position in SEQ ID NO: 2, except for K or R. 78. The method of any one of embodiments 64 to 77, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 354 of SEQ ID NO: 2, and the mutation is any amino acid different from the amino acid at that position in SEQ ID NO: 2, except for K or R. 79. The method of any one of embodiments 64 to 78, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 10 of SEQ ID NO: 2, and the mutation is any amino acid different from the amino acid at that position of SEQ ID NO: 2, except for Q or N. 80. The method of any one of embodiments 64 to 79, wherein the VSV-G polypeptide comprises a substitution at position 47 or 354, or at both positions 47 and 354, each of which is independently substituted by A, G, F, Q, or N. 81. The method of any one of embodiments 64 to 80, wherein the VSV-G polypeptide comprises a substitution at position 8, wherein the substitution is H8A, H8I, H8V, H8L, etc. 82. The method of any one of embodiments 64 to 81, wherein the VSV-G polypeptide comprises a substitution at position 47, wherein the substitution is K47Q or K47N. 83. The method of any one of embodiments 64 to 82, wherein the VSV-G polypeptide comprises the following mutations: H8A and / or K47Q, which are substitutions. 84. The method of any one of embodiments 64-83, wherein the VSV-G polypeptide comprises a Q10A, Q10R, or Q10K substitution. 85. The method of any one of embodiments 64 to 84, wherein the VSV-G polypeptide further comprises a mutation corresponding to the mutation at position 214 and / or 352 of SEQ ID NO:2. 86. The method of embodiment 85, wherein the VSV-G polypeptide comprises a T214N and / or T352A mutation. 87. The method of any one of embodiments 64 to 86, wherein the viral particle comprises a VSV-G polypeptide having a substitution at position I182 and at least one of T214 and T352 of SEQ ID NO:2. 88. The method of embodiment 87, wherein the VSV-G polypeptide comprises substitutions at positions I182, T214, and T352 of SEQ ID NO:2. 89. The method of embodiment 87 or 88, wherein said substitution at position 182 is I182D or I182E, said substitution at position 214 is T214N, and said substitution at position 352 is T352A. 90. The method of any one of embodiments 87-89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO: 23, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 25. 91. The method of any one of embodiments 87 to 89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO: 23. 92. The method of any one of embodiments 87 to 89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO: 22. 93. The method of any one of embodiments 87 to 89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO: 24. 94. The method of any one of embodiments 87 to 89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO: 25. 95. The method of any one of embodiments 1-94, wherein the viral vector comprises a targeting moiety. 96. The method of embodiment 95, wherein the targeting moiety binds to CD7. 97. The method of embodiment 96, wherein the targeting moiety comprises: (i) a heavy chain variable region comprising a heavy chain CDR1 sequence, a heavy chain CDR2 sequence, and a heavy chain CDR3 sequence, or a variant of any of them, wherein the heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 30, the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 31, and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 32; and (ii) a light chain variable region comprising a light chain CDR1 sequence, a light chain CDR2 sequence, and a light chain CDR3 sequence, or a variant of any of them, wherein the light chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 33, the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 34, and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 35. 98. The method of embodiment 96 or 97, wherein the targeting moiety comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 43. 99. The method of any one of embodiments 96-98, wherein the targeting moiety comprises an antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 45. 100. The method of embodiment 95, wherein the targeting moiety binds to CD8. 101. The method of embodiment 100, wherein the targeting moiety comprises: (i) a heavy chain variable region comprising a heavy chain CDR1 sequence, a heavy chain CDR2 sequence, and a heavy chain CDR3 sequence, or a variant of any of them, wherein the heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 48, the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 49, and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 50; and (ii) a light chain variable region comprising a light chain CDR1 sequence, a light chain CDR2 sequence, and a light chain CDR3 sequence, or a variant of any of them, wherein the light chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 51, the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 52, and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 53. 102. The method of embodiment 100 or 101, wherein the targeting moiety comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 60, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 61. 103. The method of any one of embodiments 100-102, wherein the targeting moiety comprises an antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63. 104. The method of any one of embodiments 95 to 103, wherein the targeting moiety has the formula T-S1, where T is the target binding domain and S1 is a stalk portion, and the targeting moiety is attached to the surface of the viral vector via the stalk portion S1. 105. The method of embodiment 104, wherein the stalk portion S1 comprises an Fc protein variant. 106. The stem portion S1 has the formula L1-Fc-L2-X1, wherein: L1 is a linker or is absent, Fc is an Fc protein variant, L2 is a linker or is absent, X1 is a polypeptide containing a transmembrane domain, 106. The method of embodiment 105, wherein said targeting moiety having the formula T-S1 has the formula T-L1-Fc-L2-X1. 107.X1 is ECD-T M -ICD, wherein the ECD is the extracellular domain of a cell surface protein or a fragment thereof, or is absent; T M is the transmembrane domain of a transmembrane protein, the ICD is a protein or intracellular domain that facilitates incorporation of the targeting moiety into the envelope of the viral particle, or is absent; The targeting moiety having the formula T-L1-Fc-L2-X1 is T-L1-Fc-L2-ECD-T M - The method of embodiment 106, having an ICD formula. 108.T comprises the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 62, or SEQ ID NO: 63; L1 comprises the amino acid sequence of SEQ ID NO: 72; Fc comprises the amino acid sequence of SEQ ID NO: 82; L2 does not exist, the ECD comprises the amino acid sequence of SEQ ID NO: 83; T M comprises the amino acid sequence of SEQ ID NO: 84; The method of embodiment 107, wherein the ICD comprises an amino acid sequence comprising an env uptake motif, and the env uptake motif comprises the amino acid sequence of SEQ ID NO: 85. 109. The method of embodiment 104, wherein the stalk portion S1 does not comprise an Fc protein variant. 110. The handle portion S1 has a formula of L3-X1, wherein: L3 is a flexible peptide linker, X1 is a polypeptide containing a transmembrane domain, 110. The method of embodiment 109, wherein said targeting moiety having the formula T-S1 has the formula T-L3-X1. 111.X1 is ECD-T M -ICD, wherein the ECD is the extracellular domain of a cell surface protein or a fragment thereof, or is absent; T M is the transmembrane domain of a transmembrane protein, the ICD is a protein or intracellular domain that facilitates incorporation of the targeting moiety into the envelope of the viral particle, or is absent; The targeting moiety having the formula T-L3-X1 is T-L3-ECD-T M - The method of embodiment 110, having an ICD formula. 112.T comprises the amino acid sequence of SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:62, or SEQ ID NO:63; L3 comprises the amino acid sequence of SEQ ID NO: 64, and n is 1, 2, or 4; the ECD comprises the amino acid sequence of SEQ ID NO: 86; T M comprises the amino acid sequence of SEQ ID NO: 87; The method of embodiment 111, wherein the ICD comprises an amino acid sequence comprising an env uptake motif, and the env uptake motif comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 88. 113. The method of embodiment 104, wherein said targeting moiety having the formula T-S1 comprises an amino acid sequence having at least 90% identity to SEQ ID NO:89. 114. The method of embodiment 104, wherein said targeting moiety having the formula T-S1 comprises an amino acid sequence having at least 95% identity to SEQ ID NO:89. 115. The method of embodiment 104, wherein said targeting moiety having the formula T-S1 comprises the amino acid sequence of SEQ ID NO: 89. 116. The method of any one of embodiments 1 to 115, wherein the viral vector further comprises a nucleic acid molecule encoding a heterologous molecule of interest. 117. The method of embodiment 116, wherein the heterologous molecule of interest is an siRNA, shRNA, non-coding RNA (e.g., a guide RNA for a CRISPR system), a peptide, a polypeptide, a protein, a viral payload, a viral genome, or a combination thereof. 118. The method of embodiment 116 or 117, wherein the heterologous molecule of interest is a chimeric antigen receptor (CAR). 119. The method of embodiment 118, wherein the CAR comprises an antigen-binding domain comprising an antibody or a fragment thereof. 120. The method of embodiment 119, wherein the antibody or fragment thereof is an antibody, an scFv antibody, an antigen-binding domain, ankyrin repeat, a VHH domain antibody, a nanobody, a single domain antibody, or an FN3 antibody. 121. The method of embodiment 119 or 120, wherein the antigen-binding domain of the CAR binds to CD20. 122. The method of embodiment 121, wherein the antigen-binding domain that binds to CD20 comprises a polypeptide having a light chain and a heavy chain, wherein the light chain and the heavy chain comprise a heavy chain variable region of the heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 73, and a light chain variable region of the light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 74. 123. The method of embodiment 121 or 122, wherein said antigen-binding domain that binds to CD20 comprises the amino acid sequence of SEQ ID NO: 75 or SEQ ID NO: 76. 124. The method of embodiment 121, wherein the antigen-binding domain that binds to CD20 comprises a polypeptide having a light chain and a heavy chain, wherein the light chain and the heavy chain comprise a heavy chain variable region of the heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 78, and a light chain variable region of the light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 79. 125. The method of embodiment 123 or 124, wherein said antigen-binding domain that binds to CD20 comprises the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 81. 126. The method of any one of embodiments 118-121, wherein the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 90. 127. The method of any one of embodiments 118-121, wherein the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 90. 128. The method of any one of embodiments 118-121, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 90. [Example]

[0243] Various embodiments are further described in detail with reference to the following examples. These examples are provided for illustrative purposes only and, unless otherwise specified, are not intended to be limiting. Therefore, the embodiments should in no way be construed as being limited to the following examples, but rather should be construed to encompass any variations that become apparent as a result of the teachings herein.

[0244] Example 1: Viral vector production process

[0245] Viral vector production: Viral vectors were produced in 293T cells. For transfection, self-inactivating lentivirus (LV) pseudotyped with VSV-G protein containing mutations corresponding to I182E, T214N, and T352A and expressing GFP or other genes of interest (e.g., chimeric antigen receptor (CAR20, a chimeric antigen receptor with an antigen-binding domain that binds to CD20)) under the EF1A promoter was produced using a five-plasmid system (modified VSVg expression plasmid (pModified-VSVg), Gag-Pol expression plasmid (pGag-Pol), Rev expression plasmid (pRev), binder expression plasmid (pBinder), and an LV plasmid expressing GFP or CAR20). The LV was transfected at 300 ng / cm2 in a DNA:polyethyleneimine (PEI) ratio of 1 mcg:1 mcL in T175 and CellSTACK™ cell culture vessels. 2 The plasmids were used for transfection by PEIpro-mediated transfection. The cell culture medium used during transfection was DMEM supplemented with either 10% or 3% fetal bovine serum (FBS).

[0246] DNase digestion: 48 hours after transfection, 2 mM MgCl2 and 150 units of DNase / mL were added to the cell culture for 2 hours. The cell culture was incubated at 37°C in an incubator. After the incubation was completed, the cell culture was cooled to room temperature.

[0247] Clarification: A peristaltic pump containing Sartopore® PP3 1.2 micron and Sartopore® PP3 0.45 micron in series was used at 250 L / m 2 The digested cell culture was clarified using a clarification filter with a flux of 1000 kJ / hr (LMH). 2Flow 80-200 L of digested cell culture per filter. Pre-condition the filter with water and then fill with DMEM + 3% FBS (10 L / ml). 2 ), followed by DMEM (5 L / m 2 After filtration, the filter was washed with a minimum filter void volume of DMEM and emptied to recover the product.

[0248] Ion exchange chromatography: Mustang® Q ion exchange membranes were used for the post-clarification LV capture and purification step. The membranes were conditioned using 20 mM Tris, 100 mM NaCl, pH 7.5, with or without 4% sucrose, for 25 membrane volumes (MV) at a flow rate of 5 MV / min. After conditioning, the clarified cell culture was pumped through the Mustang® Q membrane using an AKTA Avant25 or Avant150 chromatography system at a flow rate of 5 MV / min. After loading, the membrane was washed with 25 MV of 20 mM Tris, 100 mM NaCl, pH 7.5, with or without 4% sucrose to remove residual sediments and weakly bound molecules. LVs were then recovered by elution with 20 mM Tris, 1000 mM NaCl, pH 7.5, with or without 4% sucrose. After the start of the elution step, the eluted LV was collected at 1 MV until the UV signal at 280 nm flattened. Immediately after completing the elution, the LV eluate was diluted with 20 mM Tris, pH 7.5, with or without 4% sucrose to stabilize the LV function. The ratio of LV eluate to dilution buffer during dilution was 1:2 or 1:6. A dilution ratio of 1:2 resulted in a higher conductivity (approximately 50 mS / cm), while a dilution ratio of 1:6 resulted in a conductivity of approximately 12.5 mS / cm.

[0249] Post-chromatography DNase: DNase digestion of the diluted chromatography product was performed using the same type of DNase as the initial digestion after harvest. The diluted chromatography product was supplemented with 2 mM MgCl2 and 100 units of DNase / mL and stored in a 4°C cold storage for 16-24 hours. After this incubation, the product was allowed to warm to room temperature.

[0250] Polishing chromatography: Capto Core 700 resin was packed to a resin bed height of 10–20 cm. After packing, the packed Capto Core 700 column was conditioned with 10 column volumes (CV) of 20 mM Tris, 100 mM NaCl, pH 7.5, with or without 4% sucrose. The Mustang™ Q chromatography product was loaded onto the Capto Core 700 column at a column load volume of 10–20 mL per mL of resin volume. LV flowed through the Capto Core 700, and after loading was complete, the column was washed and the remaining LV flow-through fraction was collected. Product collection was based on UV absorbance at 280 nm; collection began when UV280 began to increase and ended when UV280 decreased after washing.

[0251] Sterile Filtration: Two types of sterile filtration filters were used to generate the positively charged sterile filter data presented in this disclosure: Sartopore® 2XLG with a 0.8 micron / 0.2 micron bilayer and Pall Supor® with a 0.8 micron / 0.2 micron bilayer membrane. The sterile filters were washed with water followed by 1% w / v Exbumin® (20 mL wash / cm). 2 It can be used without or with pre-washing depending on the filter area, and Capto Core 700 product can be used at a flow rate of 50-200 L / m 2After product introduction, the sterile filter was washed with 20 mM Tris, 100 mM NaCl, pH 7.5 to recover the hold-up volume in the filter and tubing.

[0252] Final Concentration, Buffer Exchange, and Controlled-Rate Freezing: For the final concentration step and buffer exchange, a two-stage tangential flow filtration (TFF) was utilized to achieve a high concentration ratio. Both stages utilized modified PES (mPES) hollow fiber modules with 1.0 mm internal diameter (ID) fibers and a 750 kDa cutoff pore size. However, the filter area in the first stage was approximately 20 times larger than that in the second stage. Prior to use, the hollow fibers were washed with water and 20 mM Tris, 100 mM NaCl, pH 7.5. In the first stage, the sterile-filtered product was concentrated approximately 20-fold. The 20-fold concentrated product was then further concentrated approximately 15-fold using a second stage of TFF. At that point, the product was buffer exchanged against 20 mM Tris, 100 mM NaCl, 4% sucrose, pH 7.5, for 6–8 diafiltration volumes (DV). After buffer exchange was complete, the product was further concentrated 2-fold and human serum albumin was added to a final concentration of 0.5% v / v. The product was frozen at -80°C.

[0253] Example 2: Viral Vector Recovery with or without Pre-Washing of Charged Sterile Filters

[0254] In the initial development of the sterile filtration process, unconcentrated purified lentiviral vector (approximately 2 × 10 ) was filtered through three types of sterile filters: Pall Supor® 0.8 / 0.2 micron, Sartopore® 2XLG 0.8 / 0.2 micron, and Millipore Multimedia Durapore® 1.2 / 0.5 / 0.22 micron. 8The recovery of 1000kJ / mL (vp / mL) was less than 50% in a 20 mM Tris, 100 mM NaCl, pH 7.5 buffer system. The sterile filters tested were composed of modified polyethersulfone (PES) or polyvinylidene fluoride (PVDF), which have been reported to have a positive charge at approximately pH 6 to 8 (Basic Aspects of Membrane Science and Engineering, N. Hilal, D. Johnson, in Comprehensive Membrane Science and Engineering, 2010).

[0255] Vector recovery was compared between non-prewashed PES sterile filters and PES sterile filters prewashed with 1% recombinant human albumin for two feed conductivities (12.5 mS / cm and 41.0 mS / cm). For low conductivity, prewashing with a 1% recombinant human albumin solution prior to sterile filtration resulted in a two-fold increase in recovery compared to no prewashing of the PES filter. For high conductivity feeds, prewashing the sterile filter slightly increased yield compared to no prewashing. The results are shown in Figure 2.

[0256] In contrast, pre-washing Sartobran P sterile filters with recombinant human albumin solution slightly increased virus recovery (Figure 3). Sartobran P is made from cellulose acetate, a material reported to have a negative charge (Li, Nuncy et al. “Synthesis and Characterization of a High Flux Nanocellulose-Celluloseacetate Nanocomposite Membrane.” Membranes vol. 9, 6, 70. 6 Jun. 2019, doi:10.3390 / membranes9060070). This reveals that viral vector loss during sterile filtration is primarily due to charge-induced adsorption to the filter. This loss can be avoided by coating the filter with a protein such as recombinant human serum albumin.

[0257] Example 3: Initial pre-coating of sterile filters

[0258] The effect of pre-coating sterile filters with protein to reduce vector loss was investigated. For unconcentrated purified viral vectors, viral vector recovery was evaluated at conductivities of 12.5 mS / cm and 30 mS / cm using sterile filters washed with water and 20 mM Tris, 100 mM NaCl, pH 7.5, or with water followed by 20 mM Tris, 100 mM NaCl, and 1% Exbumin®, pH 7.5, prior to lentiviral filtration. The sterile filters used in this study were Pall Super® 0.8 / 0.2 micron filters composed of modified polyethersulfone (PES). The pre-coating protein was recombinant human serum albumin (e.g., Exbumin® (Invitria)). The results are shown in Figure 4. In Figure 4, experimental filtration results are indicated by Exb(+), and control filtration results without recombinant human serum albumin wash are indicated by Exb(-). Pre-coating sterile filters with recombinant human serum albumin solution improved lentiviral vector recovery by up to two-fold compared to sterile filtration, as demonstrated by three orthogonal analytical methods (Figure 4). This therefore demonstrates that viral vector loss during sterile filtration is primarily due to adsorption to the filter and that this loss can be avoided by coating with a protein such as recombinant human serum albumin.

[0259] Example 4: Pre-coating of a negatively charged sterile filter

[0260] The effect of protein pre-coating on negatively charged sterile filters was also investigated. Protein pre-coating was performed on Sartobran® P sterile filters. This sterile filter is reported to be composed of cellulose acetate material and has a negative charge (Synthesis and Characterization of a High Flux Nanocellulose-Cellulose Acetate Nanocomposite Membrane; Nancy Li, Jackie Zheng, Pejman Hadi, Mengying Yang, Xiangyu Huang, Hongyang Ma, Harold W. Walker, & Bejamin S. Hsiao; Membranes volume 9(6), 70, (2019)). A smaller degree of improvement was observed compared to that observed with positively charged sterile filters (Figure 5). Control experiments without protein pre-coating yielded approximately 80% vector recovery, while experiments with protein pre-coating yielded approximately 70% recovery. These results indicated that lentiviral vectors are less susceptible to loss due to charge interactions with Sartobran P filters (see Figure 5). Lentiviral vectors themselves are negatively charged at neutral pH (see Perry C, Rayat ACME. Lentiviral Vector Bioprocessing. Viruses. 2021 Feb 9;13(2):268. doi:10.3390 / v13020268. PMID:33572347; PMCID:PMC7916122, and / or Rodrigues, Teresa et al. “Removal of envelope protein-free retroviral vectors by anion-exchange chromatography to improve product quality.” Journal of separation science vol.31,20(2008):3509-18. doi:10.1002 / jssc.200800195).Therefore, when negatively charged filters are used, the recovery rate of the viral vectors may not be affected by pre-washing the filters, since the lentiviral vectors have the same charge as the filters.

[0261] Example 5: Comparison of the Effect of Pre-Coating Sterile Filters with HSA and Recombinant HSA

[0262] The effect of pre-coating sterile filters with human serum albumin (HSA) to reduce vector loss was compared to recombinant human serum albumin (Exbumin®). In the first condition (Figure 6, left-most data), viral vector recovery was assessed for unconcentrated purified viral vectors to 12.5 mS / cm using sterile filters washed with water and 20 mM Tris, 100 mM NaCl, pH 7.5, or sterile filters washed with water followed by 20 mM Tris, 100 mM NaCl + 1% Exbumin®, pH 7.5, prior to lentiviral filtration. In the second condition (Fig. 6, middle data), viral vector recovery was assessed to 12.5 mS / cm for unconcentrated purified viral vectors using sterile filters washed with water and 20 mM Tris, 100 M NaCl, pH 7.5 or sterile filters washed with water followed by 20 mM Tris, 100 mM NaCl + 1% HSA, pH 7.5 prior to lentiviral filtration. In the third condition (Fig. 6, right-most data), viral vector recovery was assessed to 12.5 mS / cm for unconcentrated purified viral vectors using sterile filters washed with water and 20 mM Tris, 100 M NaCl, pH 7.5 or sterile filters washed with water followed by 20 mM Tris, 100 mM NaCl + 0.1% HSA, pH 7.5 prior to lentiviral filtration. The sterile filters used in this experiment were Sartopore® 2XLG 0.8 / 0.2 microns, composed of modified polyethersulfone (PES). Pre-coating the sterile filters with either 1% or 0.1% HSA or Exbumin® solution resulted in comparable lentiviral vector recovery rates, as assayed by viral particle count or genome copies by p24. This demonstrates that both HSA (e.g., non-recombinant HSA) and recombinant HSA can be used as pre-coating protein solutions for sterile filters.

[0263] Example 6: As shown in Figures 7A-8B, mutations at position 182 of VSV-G abolish LDL-R interaction but retain fusion properties.

[0264] Plasmids / Sequences: All VSV-G plasmids were derived from the pCMV-VSV-G envelope vector (Cell Bio Labs, catalog RV-110). Point mutations and their combinations were introduced using site-directed mutagenesis (New England Biolabs). The individual mutations H8A and K47Q were previously shown to partially "blind" (inactivate) VSV-G and reduce binding to LDL-R, VSV's natural cellular receptor (PMID: 29531262, DOI: 10.1038 / s41467-018-03432-4). In this experiment, a single binder molecule consisting of a CD7-directed scFv (clone MT701) fused to an IgG "stalk" carrying the CD28 transmembrane domain was used.

[0265] Cells: HEK293T cells were grown in DMEM containing 10% FBS. SupT1 cells were maintained in RPMI medium containing 10% FBS. Human PBMCs were purchased from AllCells and cultured in X-Vivo 10 (Lonza) supplemented with 20 ng / mL IL-2 (Peprotech). PBMCs were activated with anti-CD3 / CD28 Dynabeads (Cell Therapy Systems) 48 hours prior to transduction.

[0266] Generation of lentiviral particles: Recombinant lentiviral particles co-expressing VSV-G glycoprotein and binder molecules were generated by introducing plasmids into HEK293T cells using Lipofectamine 3000 (ThermoFisher Scientific). A total of five plasmids were transfected: (1) a plasmid expressing VSV-G glycoprotein, (2) a plasmid expressing a binder protein, (3) a plasmid expressing a lentiviral transfer genome encoding eGFP, (4) a plasmid expressing gag-pol, and (5) a plasmid expressing rev. Supernatants from transfected cells were collected 48 hours later. Virus in the cell supernatant was concentrated by centrifugation through a sucrose cushion and resuspended in PBS. Lentiviral particle titers were measured using the Lenti-X p24 Rapid Titer Kit (Takara Bio, San Jose, CA).

[0267] Lentiviral transduction assay: Serial 10-fold dilutions of concentrated lentivirus (in cell culture medium) were prepared and used to infect SupT1 and activated human PBMCs. The medium was changed after 6 hours, and transduced cells were analyzed by flow cytometry on days 4 and 7 post-transduction. Cells were stained with a viability dye and an anti-CD7 antibody to detect CD7-positive cells (PeCy7 mouse anti-human CD7, clone CD7-6B7, BD Biosciences). Transduction efficiency was calculated by measuring eGFP expression.

[0268] Structurally Informed Design of Novel Loss-of-Binding Mutations: Using the published crystal structures of VSV-G bound to CR2 and CR3 of LDL-R (pdb 50YL and 50Y9, respectively), we identified two putative positions of VSV-G with side chains oriented toward the binding interface on LDL-R (Figures 7A and 7B). Residue Q10 (SEQ ID NO: 2) was thought to form several interactions with residues in both CR2 and CR3. In CR3, this included interactions with a positively charged arginine residue. Therefore, three substitutions were tested: Q10A, which reduces side chain interactions potentially stabilizing LDL-R binding, and Q10R and Q10K, which create electrostatic repulsion.

[0269] Residue I182 (SEQ ID NO: 2) also appeared to contact several residues in both CR2 and CR3. Three substitutions were tested: I182A, which reduces side chain interactions that potentially stabilize LDL-R binding, and I182D and I182E, which create electrostatic repulsion for the primary binding interface on the LDL-R.

[0270] Addition of a negative charge at the binding interface abolishes intrinsic targeting without altering membrane fusion activity: We validated the structural prediction of residue I182 by titrating viral supernatants against the CD7+ T cell line SupT1. In the absence of any compensatory binder molecules, wild-type VSV-G titers reached 3.0e8, whereas both I182D and I182E resulted in titers approximately three orders of magnitude lower (Figure 8A). Substitution at residue I182 restored titers to 1e8 in the presence of binders that redirected virions to CD7, thereby maintaining membrane fusion activity.

[0271] The data are shown in Figures 8A and 8B. Figures 8A and 8B demonstrate that adding a negative charge at the binding interface abolishes intrinsic tropism without altering membrane fusion ability. Figure 8A (top) shows titration of VSV-G constructs in SupT1 cells. The plot is the percentage of SupT1 cells expressing GFP at each viral input dose, relative to the p24 antigen. The dashed line / open circle indicates the VSV-G construct alone, while the solid line / filled circle indicates the same construct with a CD7 tropism molecule expressed in trans. Figure 8B shows the functional titer (TU / mL) of each construct calculated from the titration of A, expressed as transducing units per mL of concentrated viral supernatant.

[0272] Thus, these examples demonstrate that mutation at position 182 is sufficient to abolish LDL-R interaction while retaining membrane fusion ability when combined with a targeting moiety that binds to a target on a target cell.

[0273] Thus, the examples presented herein demonstrate that pre-coating a sterile filter, such as a positively charged sterile filter, with a protein such as recombinant human serum albumin can significantly increase virus recovery. This effect appears to be greater with positively charged filters. This is surprising and unexpected. Furthermore, the methods presented herein can lead to increased recovery even if steps are performed after passing the solution through the sterile filter, while still allowing the preparation of a sterile composition. This is in contrast to other methods in which the sterile filtration step is the final step. Thus, the present methods and embodiments lead to increased recovery without sacrificing product sterility, which is important for pharmaceutical compositions. Using such methods, high-dose pharmaceutical compositions containing viral vectors can be administered in smaller volumes, which is advantageous for patients undergoing viral vector-based therapy.

[0274] This specification contains numerous citations to patents, patent applications, and publications, each of which is incorporated herein by reference for all purposes.

[0275] The specification also refers to various sequences, such as those shown herein and below: An alignment of the ectodomains of different VSV-G proteins from different strains is shown in FIG.

[0276] [ka] VSV-G Indiana ectodomain wild type: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAV IVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFP ECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 2) VSV-G Indiana ectodomain I182A: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLASMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 3) VSV-G Indiana ectodomain I182D: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLDSMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 4) VSV-G Indiana ectodomain I182E: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLESMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO:5) VSV-G Indiana ectodomain H8A + K47Q: KFTIVFPANQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 6) VSV-G Indiana ectodomain Q10A: KFTIVFPHNAKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:7) VSV-G Indiana ectodomain Q10R: KFTIVFPHNRKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:8) VSV-G Indiana ectodomain Q10K: KFTIVFPHNKKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO:9)

Chem.

Claims

1. 1. A method for preparing a concentrated sterile solution comprising a viral vector, comprising: Clarifying the solution containing the cell culture medium and the viral vector; filtering the clarified solution containing the viral vector through a first chromatography filter to prepare a filtered clarified solution containing the viral vector; passing the filtered clarified solution containing the viral vector through a protein-coated sterile membrane to produce a sterile solution containing the viral vector; concentrating the sterile solution containing the viral vector to prepare the concentrated sterile solution containing the viral vector.

2. 10. The method of claim 1, further comprising mixing the concentrated sterile solution containing the viral vector with one or more sterile, pyrogen-free buffers and / or additives to prepare a sterile pharmaceutical composition containing the viral vector.

3. 10. The method of claim 1, further comprising collecting the cell culture medium from the cell culture producing the viral vector prior to clarifying the solution.

4. 4. The method of any one of claims 1 to 3, further comprising filtering the filtered clarified solution containing the viral vector through a second chromatography filter before passing the filtered clarified solution through the protein-coated sterile membrane to prepare a twice-filtered clarified solution containing the viral vector.

5. 10. The method of claim 1, wherein the protein-coated sterile membrane comprises a membrane that has been pre-washed with a protein solution.

6. 10. The method of claim 1, further comprising coating a sterile membrane with a protein solution to produce a sterile membrane coated with said protein.

7. 7. The method of claim 5 or 6, wherein the protein solution comprises one or more components selected from a protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride, or a combination thereof.

8. 8. The method of claim 7, wherein the protein solution comprises about 1% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.

0.

9. 8. The method of claim 7, wherein the protein comprises human serum albumin, myoglobulin, bovine serum albumin, immunoglobulin, immunoglobulin fragment, fibronectin, vitronectin, or any combination thereof.

10. 10. The method of claim 9, wherein the human serum albumin is recombinant human serum albumin, human serum albumin produced from a plant, or non-recombinant human serum albumin.

11. 11. The method of claim 10, wherein the human serum albumin is non-recombinant human serum albumin.

12. 12. The method of claim 10 or 11, wherein the non-recombinant human serum albumin is United States Pharmacopeia (USP) grade non-recombinant human serum albumin.

13. 7. The method of claim 6, wherein the protein-coated sterile membrane has a positive charge prior to coating with the protein.

14. 7. The method of claim 6, wherein the membrane is a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane.

15. 15. The method of any one of claims 1 to 14, wherein the clarified solution is filtered through the first chromatographic filter by capture chromatography.

16. 16. The method of claim 15, wherein the capture chromatography is ion exchange chromatography, affinity chromatography, hydrophobic chromatography, size exclusion chromatography, or multimodal chromatography.

17. 5. The method of claim 4, wherein the filtered solution is filtered through the second chromatographic filter by polishing chromatography.

18. 18. The method of claim 17, wherein the polishing chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimodal chromatography.

19. 19. The method of any one of claims 1 to 18, further comprising digesting DNA in the solution containing the cell culture medium and the viral vector before clarifying the solution.

20. 20. The method of any one of claims 1 to 19, further comprising, after filtering the clarified solution, digesting DNA in the solution containing the viral vector.

21. The method of claim 1 , wherein the concentration step comprises tangential flow filtration.

22. 22. The method of claim 21, wherein the tangential flow filtration is a two-stage tangential flow filtration.

23. 23. The method of claim 21 or claim 22, wherein the tangential flow filtration utilizes a 1.0 mm internal diameter fiber and a 750 kDa cutoff pore size.

24. 3. The method of claim 2, wherein the concentrated sterile solution containing the viral vector is mixed with the one or more sterile, pyrogen-free buffers and / or additives in a sterile environment to prepare the sterile pharmaceutical composition containing the viral vector.

25. 25. The method of claim 24, wherein said mixing with one or more sterile, pyrogen-free buffers and / or additives comprises a buffer exchange step.

26. Prior to clarifying the solution, the method comprises: i) obtaining a solution comprising a cell culture medium and the viral vector; ii) digesting DNA in the solution containing the cell culture medium and the viral vector to prepare a first digested solution containing the viral vector; Clarifying the solution includes clarifying the first digested solution containing the viral vector to prepare a clarified digested solution containing the viral vector; After filtering the clarified solution and before passing the filtered clarified solution through a protein-coated sterile membrane, the method comprises: i) digesting DNA in the filtered clarified digested solution containing the viral vector to prepare a filtered clarified twice-digested solution containing the viral vector; and ii) filtering the filtered clarified twice-digested solution containing the viral vector through a second chromatography filter to prepare a twice-filtered clarified twice-digested solution containing the viral vector; the protein-coated sterile membrane comprises a membrane pre-washed with a protein solution; 10. The method of claim 1, wherein the protein solution comprises about 0.1% w / v to 2.0% w / v protein, about 10 mM to about 30 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.

0.

27. 1. A method for preparing a concentrated sterile solution comprising a viral vector, comprising: digesting DNA in a solution containing the viral vector to prepare a first digested solution containing the viral vector; Clarifying the digested solution containing the viral vector to prepare a clarified digested solution containing the viral vector; filtering the clarified digested solution containing the viral vector through a first chromatography filter to produce a filtered clarified digested solution containing the viral vector; digesting DNA in the filtered clarified digested solution containing the viral vector to prepare a filtered clarified double-digested solution containing the viral vector; filtering the filtered clarified double-digested solution containing the viral vector through a second chromatography filter to prepare a twice-filtered clarified double-digested solution containing the viral vector; passing the twice-filtered clarified twice-digested solution containing the viral vector through a protein-coated sterile membrane to prepare a sterile solution containing the viral vector; concentrating the sterile solution containing the viral vector to prepare the concentrated sterile solution containing the viral vector.

28. 28. The method of claim 27, wherein the digestion of the DNA is carried out using an endonuclease having DNAase activity, RNAase activity, or a combination thereof.

29. 28. The method of claim 27, wherein the second chromatography filter is a resin.

30. 28. The method of claim 27, further comprising mixing the concentrated sterile solution comprising the viral vector with one or more sterile, pyrogen-free buffers and / or additives to prepare a sterile pharmaceutical composition comprising the viral vector.

31. 28. The method of claim 27, further comprising collecting the medium from the cell culture producing the viral vector prior to digesting the DNA.

32. 28. The method of claim 27, further comprising recovering the medium from the cell culture producing the viral vector prior to clarifying the digestion solution.

33. 28. The method of claim 27, wherein the protein-coated sterile membrane comprises a membrane that has been pre-washed with a protein solution.

34. 28. The method of claim 27, further comprising coating a sterile membrane with a protein solution to produce a sterile membrane coated with said protein.

35. 35. The method of claim 34, wherein the protein solution comprises one or more of a protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride, or a combination thereof.

36. 36. The method of claim 35, wherein the protein solution comprises about 1% w / v protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.

0.

37. 37. The method of claim 35 or 36, wherein the protein is human serum albumin.

38. 38. The method of claim 37, wherein the human serum albumin is recombinant human serum albumin, human serum albumin produced from a plant, or non-recombinant human serum albumin.

39. 39. The method of claim 38, wherein the human serum albumin is non-recombinant human serum albumin.

40. 40. The method of claim 38 or 39, wherein the non-recombinant human serum albumin is United States Pharmacopeia (USP) grade non-recombinant human serum albumin.

41. 41. The method of any one of claims 27 to 40, wherein the protein-coated sterile membrane has a positive charge before being coated with the protein.

42. 42. The method of any one of claims 27 to 41, wherein the membrane is a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane.

43. The pore size of the protein-coated sterile membrane is about 0.01 μm, about 0.10 μm, about 0.11 μm, about 0.12 μm, about 0.13 μm, about 0.14 μm, about 0.15 μm, about 0.16 μm, about 0.17 μm, about 0.18 μm, about 0.19 μm, about 0.2 μm, about 0.21 μm, about 0.22 μm, about 0.23 μm, about 0.24 μm, about 0.25 μm, about 0.26 μm, about 0.27 μm, about 0.28 μm, about 0.29 μm, about 0.30 μm, about 0.31 μm, about 0.32 μm, about 0.33 μm, about 0.34 μm, about 0.35 μm, about 0.36 μm, about 0.37 μm, about 0.38 μm, about 0.39 μm, about 0.40 μm, about 0.41 μm, about 0.42 μm, about 0.43 μm, about 0.44 μm, about 0.45 μm, about 0.46 μm, about 0.47 μm, about 0.48 μm, about 0.49 μm, about 10 ...

43. The method of claim 27, wherein the thickness of the nanotube is about 0.31 μm, about 0.32 μm, about 0.33 μm, about 0.34 μm, about 0.35 μm, about 0.36 μm, about 0.37 μm, about 0.38 μm, about 0.39 μm, about 0.40 μm, about 0.41 μm, about 0.42 μm, about 0.43 μm, about 0.44 μm, or about 0.45 μm.

44. 44. The method of any one of claims 27 to 43, wherein the protein-coated sterile membrane does not significantly retain the viral vector.

45. 45. The method of claim 44, wherein the recovery of the viral vector from the solution is at least about 60%, 61%, 62%, 63%, 64%, 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%, 96%, 97%, 98%, 99%, or about 100%.

46. 46. ​​The method of any one of claims 27 to 45, wherein the clarified solution is filtered through the first chromatographic filter by capture chromatography.

47. 47. The method of claim 46, wherein the capture chromatography is ion exchange chromatography, affinity chromatography, hydrophobic chromatography, size exclusion chromatography, or multimodal chromatography.

48. 28. The method of claim 27, wherein the first filtered solution is filtered through the second filter by polishing chromatography.

49. 49. The method of claim 48, wherein the polishing chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimodal chromatography.

50. 50. The method of any one of claims 27 to 49, wherein the concentrating step comprises concentrating the viral vector by tangential flow filtration.

51. 51. The method of claim 50, wherein the tangential flow filtration is a two-stage tangential flow filtration.

52. 50. The method of claim 51, wherein the tangential flow filtration utilizes fibers with an inner diameter of 1.0 mm and a pore size cutoff of 750 kDa.

53. 31. The method of claim 30, wherein the concentrated sterile solution containing the viral vector is mixed with an excipient in a sterile environment to prepare the sterile pharmaceutical composition containing the viral vector.

54. 54. The method of claim 53, wherein said mixing with an additive comprises a buffer exchange step.

55. 56. The method of any one of claims 1 to 55, further comprising freezing the concentrated sterile solution comprising the viral vector or the sterile pharmaceutical composition comprising the viral vector.

56. 2. The method of claim 1, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

57. 28. The method of claim 27, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

58. 58. The method of any one of claims 1 to 57, wherein prior to one or more of the steps, the concentrated sterile solution containing the viral vector is diluted.

59. The viral vectors were diluted to about 1 x 10 before the solution was passed through the sterile filter. ４ ~Approx. 1×10 ６ 59. The method of any one of claims 1 to 58, at a concentration of transducing units (TU) / mL.

60. The viral vector was diluted to 1×10 ７ ~1 x 10 ９ 60. The method of any one of claims 1 to 59, wherein the concentration is in virus particles (vp) / mL.

61. The method of claim 1, wherein the viral vector is a lentiviral vector pseudotyped with a VSV-G polypeptide containing a mutation corresponding to the mutation at position 182 of SEQ ID NO:

2.

62. 28. The method of claim 27, wherein the viral vector is a lentiviral vector pseudotyped with a VSV-G polypeptide containing a mutation corresponding to the mutation at position 182 of SEQ ID NO:

2.

63. 61. The method of any one of claims 2 to 26 or 28 to 60, wherein the viral vector, such as a lentiviral vector, is pseudotyped with a VSV-G polypeptide comprising a mutation corresponding to the mutation at position 182 of SEQ ID NO:2.

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