Method for filtering or purifying viral particles

Abstract

Provided herein are methods of purifying and / or concentrating a viral vector, methods of treating a disease, such as cancer or an autoimmune disease, using the viral vector, and methods of producing a sterile composition, such as a sterile pharmaceutical composition, wherein the method may perform the steps after passing the viral vector through a protein-coated sterile filter.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 482,617, filed February 1, 2023, and U.S. Provisional Application Serial No. 63 / 510,391, filed June 27, 2023, each of which is hereby incorporated by reference in its entirety. Incorporation by Reference of Electronically Submitted Sequence Listing

[0003] This application contains a Sequence Listing which has been filed electronically in XML format and which is hereby incorporated by reference in its entirety. The XML copy, created on January 20, 2024, is named “INH-024WO_SL” and is 97,699 bytes in size. TECHNICAL FIELD

[0004] Embodiments provided herein relate to methods of producing viral vectors, the methods comprising filtration. BACKGROUND

[0005] Sterile filtration is typically performed at the end of large biomolecule (protein, monoclonal antibody, viral vector) processing to remove bioburden and produce a sterile drug product. A number of sterile filters are commercially available, which vary in pore size, structure material, surface charge of the membrane, pore morphology, and filter membrane form. Any of the variables can affect the recovery of the product of interest through the filter. For viruses, loss of product during sterile filtration has been reported to be due to physical retention of the product, which is related to size and / or charge. Studies on sterile filter type and feed concentration have been reported to maximize recovery of a particular product of interest. The present invention addresses these and other needs. SUMMARY

[0006] In some embodiments, a method of purifying a viral vector or a method of producing a concentrated sterilized solution is provided, the method comprising the steps of: clarifying a solution comprising cell culture media and a viral vector; filtering the clarified solution comprising the viral vector through a first chromatography filter to produce a filtered clarified solution comprising the viral vector; passing the filtered clarified solution comprising the viral vector through a protein-coated sterile membrane to produce a sterilized solution comprising the viral vector; and concentrating the sterilized solution comprising the viral vector to produce a concentrated sterilized solution comprising the viral vector.

[0007] In some embodiments, the protein solution comprises one or more components selected from the group consisting of a protein, 2-amino-2-(hydroxymethyl)propane-l,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), sodium chloride, or a combination thereof.

[0008] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v of a protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-l,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride.

[0009] In some embodiments, the protein-coated sterile membrane is positively charged prior to being coated with the protein.

[0010] In some embodiments, the membrane is selected from a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane.

[0011] In some embodiments, the method comprises the steps of: digesting DNA in a solution comprising a viral vector to produce 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 produce a filtered, clarified, digested solution comprising the viral vector; digesting DNA in the filtered, clarified, digested solution comprising the viral vector to produce a twice-digested solution comprising the viral vector; filtering the twice-digested solution comprising the viral vector through a second chromatography filter to produce a twice-filtered, clarified, twice-digested solution comprising the viral vector; passing the twice-filtered, clarified, twice-digested solution comprising the viral vector through a protein-coated sterile membrane to produce a sterilized solution comprising the viral vector; concentrating the sterilized solution comprising the viral vector to produce a concentrated sterilized solution comprising the viral vector.

[0012] In some embodiments, the protein solution comprises one or more components selected from a protein, 2-amino-2-(hydroxymethyl)propane-l,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), sodium chloride, or a combination thereof.

[0013] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v of a protein, about 20 mM 2-amino-2-(hydroxymethyl)propane-l,3-diol (TRIS), about 100 mM to about 400 mM sodium chloride, and a pH of about 6.0 to about 8.0.

[0014] In some embodiments, the protein-coated sterile membrane is positively charged prior to being coated with the protein.

[0015] In some embodiments, the membrane is selected from a polyethersulfone (PES), a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1Virus vector production process is shown.

[0017] Figure 2 Virus vector recovery from PES is shown with and without pre-rinse with protein before use.

[0018] Figure 3 Virus vector recovery from uncharged sterile filters is shown.

[0019] Figure 4 Effect of pre-coating on charged sterile filters is shown.

[0020] Figure 5 Effect of pre-coating on neutral sterile filters is shown.

[0021] Figure 6 Both HSA and recombinant HSA can be used as sterile filter pre-coat protein solutions is shown.

[0022] Figure 7A and Figure 7B Crystal structure of VSV-G bound to LDL-R is shown.

[0023] Figure 8A and Figure 8B Effect of adding negatively charged amino acids to the VSV-G:LDL-R binding interface on tropism and fusogenicity is shown.

[0024] Figure 9 Alignment of ectodomains of different VSV-G proteins from different strains is shown. DETAILED DESCRIPTION

[0025] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by a person of ordinary skill in the art. If there is an inherent discrepancy between the definitions provided herein and any dictionary or other extrinsic definition, the definition provided herein controls. Singular terms shall include plural and plural terms shall include singular, unless otherwise expressly stated by context. The use of “or” means “and / or” unless otherwise stated. The use of the term “including,” and other forms thereof, such as “includes” and “included,” is not limiting.

[0026] Generally, the nomenclature used in connection with, the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well- known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited throughout this disclosure. Enzymatic reactions are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art.

[0027] To enable a clearer understanding of the present disclosure, selected terms are defined below.

[0028] As used herein, the terms "a" and "an" mean "at least one" or "one or more" unless otherwise indicated by context.

[0029] As used herein, the term "about" means that a value is approximate, and small variations would not significantly impact the implementation of the disclosed embodiments. In the case of using numerical limits, "about" means that the value can vary by ±10%, unless otherwise indicated by context. Additionally, in the case of a phrase reciting "about x to y," the term "about" modifies both x and y, and is used interchangeably with the phrase "about x to about y," unless the context indicates otherwise.

[0030] As used herein, the terms "individual" or "subject" or "patient" are used interchangeably and mean any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine; cattle, sheep, horses, or primates (such as humans).

[0031] As used herein, the terms "comprising" (and any form of comprising, such as "comprise," "comprises," and "comprised"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive 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 also be described using the transitional phrase "consist of" or "consisting of."

[0032] As used herein, the term "pre-flushed" or "pre-coated" or any variation of these terms such as "pre-flushing" or "pre-coating" describes a filter where the solution has been passed through the filter prior to use of the filter with a viral vector solution.

[0033] As used herein, the phrase "viral vector recovery" is defined as the amount of viral vector after filtration compared to the amount of viral vector prior to filtration, expressed as a percentage.

[0034] 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.

[0035] As used herein, a "non-concentrated" viral vector is a viral vector in solution that has not undergone a concentration step during its production. As used herein, a "non-concentrated" viral vector has no limitation on the concentration of viral particles in it, but rather only indicates whether the solution comprising the viral vector has undergone a concentration step, unless explicitly stated otherwise.

[0036] As used herein, the term "contacting" means bringing two elements together in an in vitro system or in 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, for example, introducing a compound into a sample containing a cell preparation or cell-containing purification preparation.

[0037] As used herein, the term "fusion" or "linked" when used in reference to proteins having different domains or heterologous sequences means that the protein domains are part of the same peptide chain linked to one another with a peptide bond or other covalent linkage. Domains or segments can be linked or fused directly to one another, or another domain or peptide sequence can be between two domains or sequences and such sequences will still be considered to be fused or linked to one another. In some embodiments, the various domains or proteins provided herein are linked or fused directly to one another, or a linker sequence, such as a glycine / serine sequence described herein, links the two domains together.

[0038] A "disease" in an animal is a state of health in which the animal cannot maintain homeostasis and in which the health of the animal continues to worsen if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis but in which the state of health of the animal is less favorable than it would be in the absence of the disorder. A disorder does not necessarily cause the state of health of the animal to decline further if left untreated.

[0039] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition, as described herein, effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results can include, but are not limited to, an amount that causes a detectable level of immune cell activation when administered to a mammal as compared to immune cell activation detected in the absence of the composition. Immune responses can be readily assessed by a variety of art- recognized methods. The skilled artisan will appreciate that the amount of a composition administered herein will vary and can be readily determined based on a variety of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound administered, and the like.

[0040] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having the specific sequences of nucleotides, i.e., rRNA, tRNA, and mRNA, or the specific sequences of amino acids and proteins, or as a template for the synthesis of a complementary,, or "coding" strand sequence. Thus, if a gene encodes a protein, that gene's nucleotide sequence, which is identical to the mRNA sequence and is provided in the sequence listing, as well as the non-coding strand sequence - which is complementary to the mRNA sequence - are said to encode the protein or other product.

[0041] An “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; additional elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all of those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) into which a recombinant polynucleotide is incorporated.

[0042] As used herein, the phrase “ex vivo” with respect to a cell that is transduced, transfected, or transformed refers to a cell that is transduced, transfected, or transformed outside of a subject, i.e., the cell is removed from the subject prior to such cell being transduced, transfected, or transformed.

[0043] As used herein, a “filter” refers to any substance through which a solution or composition passes to remove a portion of the solution or composition, e.g. As such, a “filter” is not meant to be limited to a membrane filter, but also includes any substance of any thickness through which a solution or composition passes. Thus, the term “filter” can include, but is not limited to, a membrane, such as a PES, nylon, or PVDF membrane; a membrane chromatography unit, such as a Sartobind Q, Mustang Q, etc.; or a stationary phase, such as an ion exchange stationary phase (e.g., a cross-linked polymer resin, such as a divinylbenzene cross-linked polystyrene), an affinity stationary phase (e.g., a nickel resin, a streptavidin resin, a glutathione conjugated resin, a protein A or protein G conjugated resin, etc.), a hydrophobic stationary phase (e.g., a silica resin bonded with butyl, phenyl, ether, amide, or propyl ligands), a size exclusion stationary phase (e.g., a silica resin with appropriate diameter and pore size), or any combination thereof (i.e., a multimodal chromatography). In some embodiments, a “filter” is a resin, such as a chromatography resin provided herein. Similarly, the term “chromatography filter” refers to any substance that can be used for the chromatographic separation of a solution or composition. As such, a “chromatography filter” is not meant to be limited to a membrane filter, but also includes any substance of any thickness through which a solution or composition passes. Thus, the term “chromatography filter” can include, but is not limited to, a membrane, such as a PES, nylon, or PVDF membrane; a membrane chromatography unit, such as a Sartobind Q, Mustang Q, etc.; or a stationary phase, such as an ion exchange stationary phase (e.g., a cross-linked polymer resin, such as a divinylbenzene cross-linked polystyrene), an affinity stationary phase (e.g., a nickel resin, a streptavidin resin, a glutathione conjugated resin, a protein A or protein G conjugated resin, etc.), a hydrophobic stationary phase (e.g., a silica resin bonded with butyl, phenyl, ether, amide, or propyl ligands), a size exclusion stationary phase (e.g., a silica resin with appropriate diameter and pore size), or any combination thereof (i.e., a multimodal chromatography).

[0044] As used herein, "identity" refers to the subunit sequence identity between two polymer molecules, such as between two nucleic acid or amino acid molecules, such as between two polynucleotide or polypeptide molecules. When two amino acid sequences have the same residue at the same position; for example, if a position in each of two polypeptide molecules is occupied by an arginine, then the two are identical at that position. Identity or the degree of identity between two amino acid or two nucleic acid sequences is often measured at the level of best fit between two sequences aligned for maximum correspondence. Two amino acid or two nucleic acid sequences are "identical" if they have a specified percentage of amino acid residues or nucleic acid bases that are the same (e.g., 50% identity), when compared and aligned for maximum correspondence over a specified comparison window. The term "comparison window" refers to a conceptual window of comparison along the backbone of the polypeptide sequence, e.g., of 20, 50 or 100 or more amino acid positions. Optimal alignment of sequences for comparison can be conducted using those known in the art, such as those described in the aforementioned publications.

[0045] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any 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 level or nucleic acid level to the sequence used for comparison. Other percent identities are described herein with respect to particular sequences.

[0046] Sequence identity can be measured / determined using sequence analysis software (e.g., the Sequence Analysis Software Package of the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs) of the Genetics Computer Group. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining degrees of identity, the BLAST program can be used, wherein a probability score between e3 and e100 indicates closely related sequences. In some embodiments, sequence identity is determined by using BLAST with default settings.

[0047] The embodiments provided herein include, to some extent, compositions comprising various proteins, which in some cases can comprise an amino acid sequence having sequence identity to an amino acid sequence disclosed herein. Thus, in certain embodiments, depending on the particular sequence, the degree of sequence identity to a SEQ ID NO disclosed herein is preferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In addition to these percentages, other percentages of identity are 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. These proteins can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid substitutions compared to the disclosed proteins, i.e., the substitution of one amino acid for another amino acid with an associated side chain. Genetically encoded amino acids are generally divided into four families: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, 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 referred to as aromatic amino acids. Generally, substitution of a single amino acid within these families has little effect on biological activity. The proteins can have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to the disclosed protein sequences. The proteins can also include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g., 1, 2, 3, 4, or 5 amino acids each) relative to the disclosed protein sequences.

[0048] As used herein, the phrase “in vivo” with respect to cells transduced, transfected, or transformed, refers to cells that are transduced, transfected, or transformed within a subject, without removing the cells from the subject prior to the cells being transduced, transfected, or transformed.

[0049] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely removed from its natural environment is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as a host cell.

[0050] “Lentivirus” as used herein refers to the genus of the Retroviridae family that is capable of infecting non-dividing cells. Non-limiting examples of lentiviruses are HIV, SIV, and FIV. Vectors or viral-like particles derived from lentiviruses can be used to transduce cells and deliver genes or other molecules and have them expressed in cells in vitro, ex vivo, or in vivo.

[0051] The term “modified” as used herein means that the state or structure of a molecule or cell as provided herein is altered. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally, such as mutation, substitution, insertion, or deletion (e.g., internal deletion truncation). Cells can be modified by the introduction of nucleic acids or expression of heterologous proteins.

[0052] The term “modulate” as used herein means to mediate an increase or decrease in the level of a response in a subject as compared to the level of the response in the subject in the absence of treatment or a compound, and / or as compared to the level of the response in a subject that is otherwise identical but is not treated. The term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, such as a human.

[0053] Unless otherwise indicated, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences which are degenerate variations of each other, and which encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or an RNA can also include introns to the extent that the nucleotide sequence encoding the protein can contain one or more introns.

[0054] The term “oligonucleotide” generally refers to a short polynucleotide. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also provides a corresponding RNA sequence (i.e., A, U, C, G) in which “U” is substituted for “T.”

[0055] “Parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intrathecal, or infusion techniques.

[0056] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, the terms "nucleic acid" and "polynucleotide" as used herein are interchangeable. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any method available in the art, including but not limited to recombinant methods (i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using cloning techniques and PCR, etc.) as well as by synthetic means.

[0057] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of a plurality of amino acid residues connected by peptide bonds. As used herein, the terms refer to short chains and longer chains, the short chains also being commonly referred to in the art as peptides, oligopeptides, and oligomers, and the longer chains being commonly referred to in the art as proteins, which have many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural, recombinant, synthetic, or combinations thereof.

[0058] As used herein, the term "pseudotyped" or "pseudotyped viral particles" refers to viral particles bearing a glycoprotein derived from another virus with an envelope or viral vectors encoding an envelope glycoprotein from a virus different from the parental 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, viruses can be pseudotyped with VSV-G mutant proteins as provided herein.

[0059] The term "specifically binds" as used herein in reference to an antibody means an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species can also bind to the antigen from one or more species. However, such species cross-reactivity does not alter the specific classification of the antibody per se. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, such cross-reactivity does not alter the specific classification of the antibody per se. In some instances, the term "specifically binds" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to indicate that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure rather than binding broadly to proteins. If an antibody has specificity for an epitope "A", the presence of a molecule containing epitope A (or free, unlabelled A) will decrease the amount of labelled A bound to the antibody in a reaction containing labelled "A" and the antibody. In some embodiments, the targeting moieties described herein can specifically bind their target, which can be used to target viral particles comprising a mutant VSV-G protein or other viral structural proteins used to pseudotype viruses.

[0060] The term "therapeutic" as used herein means treatment and / or prevention. A therapeutic effect is obtained by suppressing, alleviating, or eradicating a disease state.

[0061] The term "transfection" or "transformation" or "transduction" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into a cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the original recipient cell and its progeny. In some embodiments, transfection, transformation, or transduction is performed or occurs in vivo.

[0062] A "vector" is a composition of matter comprising an isolated nucleic acid that encodes a protein or peptide. Many 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.

[0063] A "carrier" or "delivery vehicle" includes viral particles, viruses, polylysine compounds, and liposomes that facilitate the transfer of nucleic acid into a cell. A carrier or delivery vehicle can also be used to deliver a protein or peptide to a cell.

[0064] Ranges: Throughout this disclosure, various aspects of the embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and does not dictate a strict limitation on the scope of an implementation. Unless otherwise specified, a range format description shall be taken to include any numerical values implicit to the range, as well as explicitly recited values. For example, a description of a range such as from 1 to 6 shall be interpreted to include not only the explicitly recited values of 1, 2, 3, 4, 5, and 6, but also include individual values and sub-ranges within the indicated range, such as 1 to 3.5, 4 to 4.9, 3, 3.1, 3.14, 3.16, and 5, as well as 3.14 to 3.16. The same applies to ranges including averages, such as "between 100 and 200." Unless otherwise stated, a disclosed range includes its endpoints.

[0065] Without being bound by any particular theory, standard viral purification techniques tend to result in loss of viral product during filtration. This is particularly true when the viral product is concentrated prior to sterile filtration, which is standard practice. Passing a pre-rinse protein solution through the sterile filter prior to filtering the viral product results in increased viral yield, which can be due to the protein solution preventing the viral product from being trapped in the filter due to charge interactions. In addition, it was also found that performing sterile filtration prior to concentrating the viral product increases product yield.

[0066] Purification of viral vectors and / or production of concentrates comprising viral vectors Method of sterilizing a solution

[0067] In some embodiments, a method of purifying a viral vector or producing a concentrated sterile solution comprising a viral vector is provided. In some embodiments, the method comprises clarifying a solution comprising cell culture media and a viral vector; filtering the clarified solution comprising the viral vector through a first chromatography filter to produce a filtered clarified solution comprising the viral vector; passing the filtered clarified solution comprising the viral vector through a protein-coated sterile membrane to produce a sterile solution comprising the viral vector; and concentrating the sterile solution comprising the viral vector to produce a concentrated sterile solution comprising the viral vector.

[0068] In some embodiments, the method comprises clarifying a solution comprising cell culture media and a viral vector; filtering the clarified solution comprising the viral vector through a first chromatography filter to produce a filtered clarified solution comprising the viral vector; concentrating the filtered clarified solution comprising the viral vector to produce a concentrated solution comprising the viral vector; and passing the concentrated solution comprising the viral vector through a protein-coated sterile membrane to produce a concentrated sterile solution comprising the viral vector.

[0069] In some embodiments, the method further comprises the step of mixing the concentrated sterile solution comprising the viral vector with one or more sterile, non-pyrogenic buffers and / or excipients to produce a sterile composition comprising the viral vector. In some embodiments, the concentrated sterile solution comprising the viral vector is mixed with one non-pyrogenic buffer and / or excipient. In some embodiments, the concentrated sterile solution comprising the viral vector is mixed with more than one non-pyrogenic buffer and / or excipient. In some embodiments, the method of purifying a viral vector comprises the steps of clarifying a solution comprising cell culture media and a viral vector, filtering the clarified solution comprising the viral vector through a first chromatography filter to produce a filtered clarified solution comprising the viral vector, passing the filtered clarified solution comprising the viral vector through a protein-coated sterile membrane to produce a sterile solution comprising the viral vector, concentrating the sterile solution comprising the viral vector to produce a concentrated sterile solution comprising the viral vector, and mixing the concentrated sterile solution comprising the viral vector with one or more sterile, non-pyrogenic buffers and / or excipients to produce a sterile composition comprising the viral vector. In some embodiments, the concentrated sterile solution comprising the viral vector is mixed with one non-pyrogenic buffer and / or excipient. In some embodiments, the concentrated sterile solution comprising the viral vector is mixed with more than one non-pyrogenic buffer and / or excipient.

[0070] In some embodiments, the method further comprises the step of collecting media from a cell culture producing a viral vector prior to clarifying the solution. In some embodiments, the media collected from the cell culture producing a viral vector comprises the viral vector.

[0071] In some embodiments, filtering the clarified solution through a first chromatography filter comprises filtering the clarified solution via a 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.

[0072] In some embodiments, the method further comprises the step of filtering the filtered clarified solution comprising the viral vector through a second chromatography filter to produce a twice-filtered clarified solution comprising the viral vector. In some embodiments, the second filtration step is performed prior to passing the filtered clarified solution through the protein-coated sterile membrane. In some embodiments, the twice-filtered clarified solution comprising the viral vector is passed through the protein-coated sterile membrane.

[0073] In some embodiments, filtering the first filtered solution through a second chromatography filter comprises filtering the first filtered solution via a polishing chromatography. In some embodiments, the polishing chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multi-modal 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 multi-modal chromatography.

[0074] In some embodiments, the protein-coated sterile membrane comprises a sterile membrane pre-rinsed with a protein solution. In some embodiments, the sterile membrane is pre-rinsed with a protein solution to produce the protein-coated sterile membrane. In some embodiments, the method further comprises the step of pre-rinsing the sterile membrane with a protein solution to produce the protein-coated sterile membrane. In some embodiments, the method further comprises the step of coating the sterile membrane with a protein solution to produce the protein-coated sterile membrane.

[0075] In some embodiments, the method comprises passing the filtered, clarified solution comprising the viral vector through a protein-coated sterile membrane to produce a sterilized solution comprising the viral vector, wherein the protein-coated sterile membrane has a pore size of about 0.01 pm to about 0.45 pm. In some embodiments, the pore size of the protein-coated sterile membrane is about 0.01 pm, about 0.10 pm, about 0.11 pm, about 0.12 pm, about 0.13 pm, about 0.14 pm, about 0.15 pm, about 0.16 pm, about 0.17 pm, about 0.18 pm, about 0.19 pm, about 0.2 pm, about 0.21 pm, about 0.22 pm, about 0.23 pm, about 0.24 pm, about 0.25 pm, about 0.26 pm, about 0.27 pm, about 0.28 pm, about 0.29 pm, about 0.30 pm, about 0.31 pm, about 0.32 pm, about 0.33 pm, about 0.34 pm, about 0.35 pm, about 0.36 pm, about 0.37 pm, about about 0.38 pm, about 0.39 pm, about 0.40 pm, about 0.41 pm, about 0.42 pm, about 0.43 pm, about 0.44 pm, or about 0.45 pm, or any value or range therein. In some embodiments, the protein-coated sterile membrane has a pore size of 0.01 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.10 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.11 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.12 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.13 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.14 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.15 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.16 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.17 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.18 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.19 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.20 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.21 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.22 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.23 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.24 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.25 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.26 pm. In some embodiments, the protein-coated sterile membrane has a pore size of 0.27 pm.In some embodiments, the protein-coated sterile film has a pore size of 0.28 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.29 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.30 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.31 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.32 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.33 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.34 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.35 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.36 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.37 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.38 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.39 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.40 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.41 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.42 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.43 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.44 pm. In some embodiments, the protein-coated sterile film has a pore size of 0.45 pm.

[0076] In some embodiments, the protein-coated sterile membrane does not significantly retain viral vectors, such as when a solution comprising viral vectors is passed through the membrane. In some embodiments, the recovery of viral vectors from a solution comprising viral vectors 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 a solution comprising viral vectors is at least about 60%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 61%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 62%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 63%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 64%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 65%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 66%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 67%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 68%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 69%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 70%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 71%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 72%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 73%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 74%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 75%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 76%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 77%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 78%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 79%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 80%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 81%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 82%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 83%. In some embodiments, the recovery of a solution comprising viral vectors is at least about 84%.In some embodiments, the recovery of the solution comprising the viral vector is at least about 85%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 86%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 87%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 88%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 89%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 90%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 91%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 92%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 93%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 94%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 95%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 96%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 97%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 98%. In some embodiments, the recovery of the solution comprising the viral vector is at least about 99%. In some embodiments, the recovery of the solution comprising the viral vector is about 100%.

[0077] In some embodiments, the protein solution comprises one or more components selected from the group consisting of a protein, 2-amino-2-(hydroxymethyl)propane-l,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-l-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-l,3-diol (TRIS). In some embodiments, the protein solution comprises histidine, phosphate. In some embodiments, the protein solution comprises HEPES ((4-(2-hydroxyethyl)-l-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 the group consisting of 2-amino-2-(hydroxymethyl)propane-l,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), sodium chloride, or a combination thereof. In some embodiments, the protein solution comprises a protein and 2-amino-2-(hydroxymethyl)propane-l,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)-l-piperazineethanesulfonic acid). In some embodiments, the protein solution comprises a protein and sodium chloride.

[0078] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v of a protein or any value or range therein. In some embodiments, the protein solution comprises about 0.2% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 0.3% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 0.4% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 0.5% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 0.6% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 0.7% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 0.8% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 0.9% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 1% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 1.2% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 1.4% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 1.6% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 1.8% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 2% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 2.2% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 2.4% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 2.6% to about 3% w / v of a protein. In some embodiments, the protein solution comprises about 2.8% to about 3% w / v of a protein.

[0079] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 2.8% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 2.6% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 2.4% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 2.2% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 2% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 1.8% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 1.6% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 1.4% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 1.2% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 1.0% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 0.9% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 0.8% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 0.7% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 0.6% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 0.5% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 0.4% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 0.3% w / v of protein. In some embodiments, the protein solution comprises about 0.1% to about 0.2% w / v of protein.

[0080] 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 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 of protein. In some embodiments, the protein solution comprises about 0.4% w / v of protein. In some embodiments, the protein solution comprises about 0.5% w / v of protein. In some embodiments, the protein solution comprises about 0.6% w / v of protein. In some embodiments, the protein solution comprises about 0.7% w / v of protein. In some embodiments, the protein solution comprises about 0.8% w / v of protein. In some embodiments, the protein solution comprises about 0.9% w / v of protein. In some embodiments, the protein solution comprises about 1.0% w / v of protein. In some embodiments, the protein solution comprises about 1.2% w / v of protein. In some embodiments, the protein solution comprises about 1.4% w / v of protein. In some embodiments, the protein solution comprises about 1.6% w / v of protein. In some embodiments, the protein solution comprises about 1.8% w / v of protein. In some embodiments, the protein solution comprises about 2.0% w / v of protein. In some embodiments, the protein solution comprises about 2.2% w / v of protein. In some embodiments, the protein solution comprises about 2.4% w / v of protein. In some embodiments, the protein solution comprises about 2.6% w / v of protein. In some embodiments, the protein solution comprises about 2.8% w / v of protein. In some embodiments, the protein solution comprises about 3.0% w / v of protein.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] In some embodiments, the protein solution comprises about 5 mM to about 50 mM 2-amino-2-(hydroxymethyl)propane-l,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.

[0085] In some embodiments, the protein solution comprises about 5 mM to about 50 mM 2-amino-2-(hydroxymethyl)propane-l,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.

[0086] 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-l,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.

[0087] 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.

[0088] 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. In some embodiments, the protein solution comprises about 5 mM to about 10 mM histidine buffer.

[0089] 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.

[0090] In some embodiments, the pH of the protein solution is about 5.5 to about 8.5 or any value or range therein. In some embodiments, the pH of the protein solution is about 6.0 to about 8.5. In some embodiments, the pH of the protein solution is about 6.5 to about 8.5. In some embodiments, the pH of the protein solution is about 7.0 to about 8.5. In some embodiments, the pH of the protein solution is about 7.5 to about 8.5. In some embodiments, the pH of the protein solution is about 8.0 to about 8.5. In some embodiments, the pH of the protein solution is about 6.0 to about 8.0.

[0091] In some embodiments, the pH of the protein solution is about 5.5 to about 8.5. In some embodiments, the pH of the protein solution is about 5.5 to about 8.0. In some embodiments, the pH of the protein solution is about 5.5 to about 7.5. In some embodiments, the pH of the protein solution is about 5.5 to about 7.0. In some embodiments, the pH of the protein solution is about 5.5 to about 6.5. In some embodiments, the pH of the protein solution is about 5.5 to about 6.0.

[0092] In some embodiments, the pH of the protein solution is 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 pH of the protein solution is about 5.5. In some embodiments, the pH of the protein solution is about 6.0. In some embodiments, the pH of the protein solution is about 6.5. In some embodiments, the pH of the protein solution is about 6.6. In some embodiments, the pH of the protein solution is about 6.7. In some embodiments, the pH of the protein solution is about 6.8. In some embodiments, the pH of the protein solution is about 6.9. In some embodiments, the pH of the protein solution is about 7.0. In some embodiments, the pH of the protein solution is about 7.1. In some embodiments, the pH of the protein solution is about 7.2. In some embodiments, the pH of the protein solution is about 7.3. In some embodiments, the pH of the protein solution is about 7.4. In some embodiments, the pH of the protein solution is about 7.5. In some embodiments, the pH of the protein solution is about 7.6. In some embodiments, the pH of the protein solution is about 7.7. In some embodiments, the pH of the protein solution is about 7.8. In some embodiments, the pH of the protein solution is about 7.9. In some embodiments, the pH of the protein solution is about 8.0. In some embodiments, the pH of the protein solution is about 8.5.

[0093] 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-l,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-l,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-l,3-diol (TRIS), and about 100 mM to about 400 mM sodium chloride.

[0094] 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-l,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-l,3-diol (TRIS), 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 20 mM 2-amino-2-(hydroxymethyl)propane-l,3-diol (TRIS), 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-l,3-diol (TRIS), about 100 mM to about 400 mM sodium chloride, and has a pH of about 6.0 to about 8.0.

[0095] 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.

[0096] In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v of a protein, about 5 mM to about 100 mM of a histidine buffer, and about 100 mM to about 400 mM of sodium chloride. In some embodiments, the protein solution comprises about 0.1% w / v to about 3.0% w / v of a protein, about 5 mM to about 100 mM of a histidine buffer, about 100 mM to about 400 mM of 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 of a protein, about 5 mM to about 100 mM of a histidine buffer, about 100 mM to about 400 mM of 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 of a protein, about 5 mM to about 100 mM of a histidine buffer, about 100 mM to about 400 mM of sodium chloride, and has a pH of about 6.0 to about 8.0.

[0097] 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.

[0098] In some embodiments, the protein solution comprises a protein selected from, but not limited to, myosin, serum albumin, bovine serum albumin, human serum albumin, immunoglobulin, immunoglobulin fragment, fibronectin, vitronectin, or a combination thereof. In some embodiments, the protein solution comprises 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 of making human serum albumin are known in the art, and any such methods are within the scope of the present application. In some embodiments, the human serum albumin is produced from a bacterial source (e.g., bacterial cells). In some embodiments, the human serum albumin is produced from an insect source (e.g., insect cells). In some embodiments, the human serum albumin is produced from a mammalian source (e.g., mammalian cells). In some embodiments, the human serum albumin is produced from a plant source. Likewise, recombinant human serum albumin can be recombinantly produced from any source. Methods of making recombinant human serum albumin are known in the art, and any such methods are within the scope of the present application. In some embodiments, the human serum albumin is recombinantly produced from a bacterial source (e.g., bacterial cells). In some embodiments, the recombinant human serum albumin is recombinantly produced from an insect source (e.g., insect cells). In some embodiments, the recombinant human serum albumin is recombinantly produced from a mammalian source (e.g., mammalian cells). In some embodiments, the recombinant human serum albumin is recombinantly produced from a plant source.

[0099] 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. In some embodiments, the protein solution comprises about 2.8% to about 3% w / v human serum albumin.

[0100] 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, the protein solution comprises about 0.1% to about 0.3% w / v human serum albumin. In some embodiments, the protein solution comprises about 0.1% to about 0.2% w / v human serum albumin.

[0101] 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 human serum albumin or any value therein. In some embodiments, the protein solution comprises about 0.1% w / v 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.

[0102] 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.

[0103] 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.

[0104] 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 recombinant human serum albumin or any value therein. In some embodiments, the protein solution comprises about 0.1% w / v 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.

[0105] 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.

[0106] In some embodiments, the protein solution comprises about 0.1% to about 3% w / v non-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 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.

[0107] 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.

[0108] 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,

[0109] 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,

[0110] about 2.6% w / v, about 2.8% w / v, or about 3.0% w / v non-recombinant human serum albumin or any value therein. In some embodiments, the protein solution comprises about 0.1% w / v 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.

[0111] 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 an FDA approved USP grade non-recombinant human serum albumin. FDA approved USP grade non-recombinant human serum albumin can be obtained via any appropriate commercial supplier such as, but not limited to, Nova, Octapharma, Grifols Bio Supplies, and the like.

[0112] In some embodiments, the method of purifying a viral vector further comprises utilizing a sterile membrane coated with a positively charged protein prior to coating with the protein. In some embodiments, the membrane is a polyether sulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane. In some embodiments, the membrane is a polyether sulfone (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 method of purifying a viral vector or producing a concentrated sterile solution comprising a viral vector comprises digesting DNA in a solution comprising cell culture media and a viral vector prior to clarifying the solution. In some embodiments, the method of purifying a viral vector comprises a step of digesting DNA in a solution comprising a viral vector after filtering a clarified solution. In some embodiments, the method of purifying a viral vector comprises a first DNA digestion step comprising digesting DNA in a solution comprising cell culture media and a viral vector prior to clarifying the solution; and a second DNA digestion step comprising digesting DNA in a solution comprising a viral vector after filtering a clarified solution.

[0114] In some embodiments, the method comprises mixing the concentrated sterilized solution comprising a viral vector with one or more sterile, pyrogen-free buffers and / or excipients in a sterile environment to produce a sterile pharmaceutical composition comprising a viral vector. In some embodiments, the method comprises mixing the concentrated sterilized solution comprising a viral vector with one or more sterile, pyrogen-free buffers and / or excipients to produce a sterile composition comprising a viral vector. In some embodiments, the sterile, pyrogen-free buffers and / or excipients comprise, for example, without limitation, TRIS, HEPES, histidine buffer, phosphate buffer, sucrose, trehalose, polyethylene glycol, or any combination thereof.

[0115] In some embodiments, the method comprises digesting DNA in a solution comprising viral vectors to produce a first digested solution comprising viral vectors; clarifying the digested solution comprising viral vectors; filtering the clarified, digested solution comprising viral vectors through a first chromatography filter to produce a filtered, clarified, digested solution comprising viral vectors; digesting DNA in the filtered, clarified, digested solution comprising viral vectors to produce a twice digested solution comprising viral vectors; filtering the twice digested solution comprising viral vectors through a second chromatography filter to produce a twice filtered, clarified, twice digested solution comprising viral vectors; concentrating the twice filtered, clarified, twice digested solution comprising viral vectors to produce a concentrated, twice filtered, clarified, twice digested solution comprising viral vectors; and passing the concentrated, twice filtered, clarified, twice digested solution comprising viral vectors through a protein-coated sterile membrane to produce a sterilized solution comprising viral vectors.

[0116] In some embodiments, the method comprises digesting DNA in a solution comprising viral vectors to produce a first digested solution comprising viral vectors; clarifying the digested solution comprising viral vectors; filtering the clarified, digested solution comprising viral vectors through a first chromatography filter to produce a filtered, clarified, digested solution comprising viral vectors; digesting DNA in the filtered, clarified, digested solution comprising viral vectors to produce a twice digested solution comprising viral vectors; filtering the twice digested solution comprising viral vectors through a second chromatography filter to produce a twice filtered, clarified, twice digested solution comprising viral vectors; concentrating the twice filtered, clarified, twice digested solution comprising viral vectors to produce a concentrated, twice filtered, clarified, twice digested solution comprising viral vectors; and passing the concentrated, twice filtered, clarified, twice digested solution comprising viral vectors through a protein-coated sterile membrane to produce a sterilized solution comprising viral vectors.

[0117] In some embodiments, DNA digestion is performed using an endonuclease. In some embodiments, the endonuclease is a DNase. In some embodiments, the endonuclease has both DNase and RNase activity. In some embodiments, the endonuclease is selected from the group comprising, 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.

[0118] In some embodiments, filtering the clarified, digested solution through a first chromatography filter comprises filtering the clarified, digested solution via a 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.

[0119] In some embodiments, the second chromatography filter is a filter as defined herein. In some embodiments, the second chromatography step is a membrane as provided herein, a membrane filter unit as provided herein, or a stationary phase as provided herein. In some embodiments, the stationary phase is a membrane filter unit. In some embodiments, the stationary phase is a resin.

[0120] In some embodiments, the second chromatography filter is a resin.

[0121] In some embodiments, filtering the filtered, clarified, twice-digested solution through a second chromatography filter comprises filtering the filtered, clarified, twice-digested solution via a 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.

[0122] In some embodiments, the method of purifying a viral vector further comprises the step of collecting media from a cell culture producing the viral vector. In some embodiments, the method of purifying a viral vector further comprises the step of collecting media from a cell culture producing the viral vector prior to digesting DNA. In some embodiments, the method of purifying a viral vector further comprises the step of collecting media from a cell culture producing the viral vector prior to clarifying the digested solution.

[0123] In some embodiments, the protein-coated sterile membrane comprises a sterile membrane pre-rinsed with a protein solution. In some embodiments, the sterile membrane is pre-rinsed with a protein solution to produce the protein-coated sterile membrane. In some embodiments, the method further comprises the step of pre-rinsing the sterile membrane with a protein solution to produce the protein-coated sterile membrane. In some embodiments, the method further comprises the step of coating the sterile membrane with a protein solution to produce the protein-coated sterile membrane.

[0124] In some embodiments, the membrane is a charged sterile membrane. In some embodiments, the protein-coated sterile membrane is charged prior to coating with the protein solution. In some embodiments, the membrane is positively charged. In some embodiments, the protein-coated sterile membrane is positively charged prior to coating with the protein solution. In some embodiments, the membrane is a polyether sulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane. In some embodiments, the membrane is a polyether sulfone (PES) membrane. In some embodiments, the membrane is a nylon membrane. In some embodiments, the membrane is a polyvinylidene fluoride (PVDF) membrane.

[0125] In some embodiments, the protein-coated sterile membrane has a pore size as provided herein. In some embodiments, the protein-coated sterile membrane has a pore size of about 0.01 pm to about 0.45 pm or any value or range therein as provided herein. In some embodiments, the protein-coated sterile membrane has a pore size of 0.01 pm, 0.10 pm, 0.11 pm, 0.12 pm, 0.13 pm, 0.14 pm, 0.15 pm, 0.16 pm, 0.17 pm, 0.18 pm, 0.19 pm, 0.2 pm, 0.21 pm, 0.22 pm, 0.23 pm, 0.24 pm, 0.25 pm, 0.26 pm, 0.27 pm, 0.28 pm, 0.29 pm, 0.30 pm, 0.31 pm, 0.32 pm, 0.33 pm, 0.34 pm, 0.35 pm, 0.36 pm, 0.37 pm, 0.38 pm, 0.39 pm, 0.40 pm, 0.41 pm, 0.42 pm, 0.43 pm, 0.44 pm, or 0.45 pm as provided herein.

[0126] In some embodiments, the protein-coated sterile membrane does not significantly retain viral vectors. In some embodiments, the recovery of the solution comprising viral vectors is as provided herein. In some embodiments, the recovery of the solution comprising viral vectors 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 provided herein.

[0127] In some embodiments, the method of purifying viral vectors further comprises freezing the concentrated sterile solution comprising viral vectors. In some embodiments, the freezing is performed by controlled rate freezing.

[0128] In some embodiments, the method for purifying the viral vector further includes diluting the concentrated sterile solution containing the viral vector prior to one or more steps.

[0129] In some embodiments, the concentration of the viral vector is approximately 1 × 10⁻⁶ before the solution is passed through a sterile filter. 3 To approximately 1×10 7 Transduction units (TU) / mL. In some embodiments, the concentration of the viral vector is approximately 1 × 10⁻⁶ before the solution is passed through a sterile filter. 4 To approximately 1×10 6 Transduction units (TU) / mL. In some embodiments, the concentration of the viral vector is 1 × 10⁻⁶ prior to filtration of the solution. 6 Up to 1×10 10 One viral particle (vp) / mL. In some embodiments, the concentration of the viral vector is 1 × 10⁻⁶ prior to filtration of the solution. 7 Up to 1×10 9 Viral particles (vp) / mL.

[0130] In some embodiments, the methods provided herein can be used to purify any viral vector. In some embodiments, the viral vector is adenovirus, adeno-associated virus, lentivirus, or retrovirus. In some embodiments, the viral vector is adenovirus. In some embodiments, the viral vector is adeno-associated virus. In some embodiments, the viral vector is lentivirus. In some embodiments, the viral vector is 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.

[0131] Exemplary methods

[0132] Figure 1 Non-limiting exemplary methods for purifying viral vectors and / or producing concentrated sterile solutions containing viral vectors, as provided herein, are shown. It should be understood that... Figure 1 The methods shown are merely illustrative and are not intended to limit in any way.

[0133] like Figure 1 As shown, step 110 includes thawing a vial of cells. The cells can be any suitable cell line capable of producing a viral vector (such as the viral vectors 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.

[0134] The cells are passaged (step 115) to allow the cells to recover to an appropriate growth rate, and then transfected (step 120) with one or more nucleic acid molecules encoding one or more viral vectors. In some embodiments, the one or more nucleic acid molecules encode a viral vector as provided herein. In some embodiments, the one or more nucleic acid molecules further encode additional elements for viral production, such as but not limited to Gag-Pol and Rev helper elements. The skilled artisan will readily recognize that additional elements for viral production will vary depending on the viral vector being produced, and thus any such elements are within the scope of the present disclosure.

[0135] At step 125, the transfection is allowed to proceed for a predetermined amount of time. In various embodiments, the transfection proceeds for between 1 hour and 120 hours. In various embodiments, the transfection proceeds for between 2 hours and 90 hours, between 3 hours and 80 hours, between 5 hours and 70 hours, between 10 hours and 60 hours, between 20 hours and 55 hours, or between 30 hours and 50 hours. In various embodiments, the transfection proceeds for between 40 and 50 hours, such as 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, the transfection proceeds for 48 hours. Following transfection, the cell supernatant is collected. The cell supernatant is then treated with an endonuclease to digest DNA in the supernatant (step 130). In some embodiments, the DNA digestion is performed using an endonuclease as provided herein.

[0136] Following DNA digestion, the digested cell media is clarified (step 135). In some embodiments, the clarification step comprises passing the digested cell media through a filter. In some embodiments, the clarification step comprises passing the digested cell media through a series of filters. In some embodiments, the clarification step comprises passing the digested cell media through a first filter, and then passing the digested cell media through a second filter, wherein the first filter has a larger pore size than the second filter.

[0137] The clarified solution is then filtered using capture chromatography (step 140). Further details of capture chromatography are provided herein. Following chromatographic filtration, the filtered solution is subjected to a second round of DNA digestion (step 145). In various embodiments, the DNA digestion is performed using an endonuclease. Further details of exemplary endonucleases are provided herein. In some embodiments, the endonuclease used for step 145 is the same as the endonuclease used in step 130.

[0138] The twice-digested solution is then filtered using a polishing chromatography (step 150). Further details of polishing chromatography are provided herein. Following polishing filtration, the solution is sterile filtered (step 155). In some embodiments, the sterile filtration step includes a first step of pre-rinsing the sterile filter membrane with a protein solution prior to passing the digested solution through the sterile filter. Further details of an exemplary protein solution are provided herein. In some embodiments, the protein in the protein solution is human serum albumin (HSA), such as recombinant HSA or non-recombinant HSA. Further details of exemplary proteins in the protein solution are provided herein.

[0139] The sterile filtered solution is then concentrated, and the product formulation is buffer exchanged (step 160). In some embodiments, the concentration step is performed via ultracentrifugation. In some embodiments, the concentration step is performed via dialysis. In some embodiments, the concentration step is performed via centrifugation. In some embodiments, the concentration step is performed via a chromatography method. In some embodiments, the concentration step is performed via tangential flow filtration. In some embodiments, the tangential flow filtration is two-stage tangential flow filtration. Once the solution is appropriately concentrated, a buffer exchange is performed to provide a final concentrated viral particle formulation. Following the buffer exchange, the product can be subjected to additional concentration steps via the methods provided herein. Once the final product is prepared, the formulation is subjected to controlled rate freezing (step 165) for long-term storage.

[0140] Viral vectors

[0141] In some embodiments, the pseudotyped viral vector comprises a VSV-G polypeptide.

[0142] In some embodiments, a VSV-G protein comprising a mutation at position 198 as compared to SEQ ID NO: 1 or at position 182 as compared to SEQ ID NO: 2 is provided. SEQ ID NO: 1 is the full-length protein of VSV-G and SEQ ID NO: 2 is the ectodomain. The 16-mer signal peptide of MKCLLYLAFLFIGVNC (SEQ ID NO: 65) as shown at the N-terminus of SEQ ID NO: 1 is cleaved, leaving the protein of SEQ ID NO: 2. Thus, although mutations can be mentioned in the context of SEQ ID NO: 2, it is understood that the mutations are also made in the context of SEQ ID NO: 1 (which contains the leader sequence), and thus will be position numbering 16 more than the positions recited 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 1182D mutation as compared to SEQ ID NO: 2. In some embodiments, the mutation is an 1182E mutation as compared to SEQ ID NO: 2.

[0143] In some embodiments, a VSV-G protein comprising a mutation at position 198 as compared to SEQ ID NO: 1 or at position 182 as compared to SEQ ID NO: 2 is provided. SEQ ID NO: 1 is the full-length protein of VSV-G and SEQ ID NO: 2 is the ectodomain. The 16-mer signal peptide of MKCLLYLAFLFIGVNC (SEQ ID NO: 65) as shown at the N-terminus of SEQ ID NO: 1 is cleaved, leaving the protein of SEQ ID NO: 2. Thus, although mutations can be mentioned in the context of SEQ ID NO: 2, it is understood that the mutations are also made in the context of SEQ ID NO: 1 (which contains the leader sequence), and thus will be position numbering 16 more than the positions recited 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 1182D mutation as compared to SEQ ID NO: 2. In some embodiments, the mutation is an 1182E mutation as compared to SEQ ID NO: 2.

[0144] In some embodiments, a VSV-G protein is provided that comprises a mutation at position 198 as compared to SEQ ID NO: 12 or at position 182 as compared to SEQ ID NO: 13. SEQ ID NO: 12 is a full-length protein of VSV-G and SEQ ID NO: 13 is the ectodomain. The 16-mer signal peptide of MLRLFLFCFLALGAHS (SEQ ID NO: 67) as shown at the N-terminus of SEQ ID NO: 12 is cleaved, leaving the protein of SEQ ID NO: 13. Thus, although mutations can be mentioned in the context of SEQ ID NO: 13, it is understood that the mutations are also made in the context of SEQ ID NO: 12 (which contains the leader sequence), and thus will be position numbering that is 16 greater than the positions recited 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 as compared to SEQ ID NO: 13. In some embodiments, the mutation is an A182E mutation as compared to SEQ ID NO: 13.

[0145] In some embodiments, a VSV-G protein is provided that comprises a mutation at position 203 as compared to SEQ ID NO: 14 or at position 182 as compared to SEQ ID NO: 15. SEQ ID NO: 14 is a full-length protein of VSV-G and SEQ ID NO: 15 is the ectodomain. The 21-mer signal peptide of MKMKMV IAGLILCIGILPAIG (SEQ ID NO: 68) as shown at the N-terminus of SEQ ID NO: 14 is cleaved, leaving the protein of SEQ ID NO: 15. Thus, although mutations can be mentioned in the context of SEQ ID NO: 15, it is understood that the mutations are also made in the context of SEQ ID NO: 14 (which contains the leader sequence), and thus will be position numbering that is 21 greater than the positions recited 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 as compared to SEQ ID NO: 15. In some embodiments, the mutation is a V182E mutation as compared to SEQ ID NO: 15.

[0146] In some embodiments, a VSV-G protein comprising a mutation at position 199 as compared to SEQ ID NO: 16 or at position 182 as compared to SEQ ID NO: 17 is provided. SEQ ID NO: 16 is a full-length protein of a VSV-G protein, and SEQ ID NO: 17 is an ectodomain. The 17-mer signal peptide of MTPAFILC MLLAGSSWA (SEQ ID NO: 69) as shown at the N-terminus of SEQ ID NO: 16 is cleaved, leaving the protein of SEQ ID NO: 17. Thus, although mutations can be mentioned in the context of SEQ ID NO: 17, it is understood that the mutations are also made in the context of SEQ ID NO: 16, which contains a leader sequence, and thus will be position numbering 17 greater than the positions recited 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 as compared to SEQ ID NO: 17. In some embodiments, the mutation is a V182E mutation as compared to SEQ ID NO: 17.

[0147] In some embodiments, a VSV-G protein comprising a mutation at position 199 as compared to SEQ ID NO: 18 or at position 182 as compared to SEQ ID NO: 19 is provided. SEQ ID NO: 18 is a full-length protein of a VSV-G protein, and SEQ ID NO: 19 is an ectodomain. The 17-mer signal peptide of MNFLLLTFIVLPLCSHA (SEQ ID NO: 70) as shown at the N-terminus of SEQ ID NO: 18 is cleaved, leaving the protein of SEQ ID NO: 19. Thus, although mutations can be mentioned in the context of SEQ ID NO: 19, it is understood that the mutations are also made in the context of SEQ ID NO: 18, which contains a leader sequence, and thus will be position numbering 17 greater than the positions recited 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 as compared to SEQ ID NO: 19. In some embodiments, the mutation is a V182E mutation as compared to SEQ ID NO: 19.

[0148] In some embodiments, a VSV-G protein is provided that comprises a mutation at position 199 as compared to SEQ ID NO:20 or at position 182 as compared to SEQ ID NO:21. SEQ ID NO:20 is a full-length protein of VSV-G and SEQ ID NO:21 is the ectodomain. The 17-mer signal peptide of MLVLYLL LSLLALGAQC (SEQ ID NO:71) as shown at the N-terminus of SEQ ID NO:20 is cleaved, leaving the protein of SEQ ID NO:21. Thus, although mutations can be mentioned in the context of SEQ ID NO:21, it is understood that the mutations are also in the context of SEQ ID NO:20, which contains the leader sequence, and thus will be position numbering 17 more than the positions recited 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 an 1182D mutation as compared to SEQ ID NO:21. In some embodiments, the mutation is an 1182E mutation as compared to SEQ ID NO:21.

[0149] As used herein, when a polypeptide is referred to as having a mutation as compared to a reference sequence, this comparison is based on an alignment such as using BlastP or ClustalW or ClutalOmega alignment software using default parameters. For example, position 182 can be found in SEQ ID NO:2 and is also compared to other strains as shown in Table 1. Figure 3 Figure 3 ​A clustal alignment of wild-type sequences of the ectodomain of the VSV-G protein of various strains is shown. The bolded and underlined residues are the residues of the various strains that align with position 182 of SEQ ID NO: 2. SEQ ID NO: 2 refers to the ectodomain of the VSV-G protein of the Indiana strain. SEQ ID NO: 11 refers to the ectodomain of the VSV-G protein of the New Jersey strain. SEQ ID NO: 13 refers to the ectodomain of the VSV-G protein of the Marraba strain. SEQ ID NO: 15 refers to the ectodomain of the VSV-G protein of the Carajas strain. SEQ ID NO: 17 refers to the ectodomain of the VSV-G protein of the Alagoa strain. SEQ ID NO: 19 refers to the ectodomain of the VSV-G protein of the Cocal strain. SEQ ID NO: 21 refers to the ectodomain of the VSV-G protein of the Morreton strain. Thus, as compared to SEQ ID NO: 2, the residue that aligns with residue 182 can also be mutated as provided herein.

[0150] In some embodiments, the mutation at position 182 as compared to SEQ ID NO: 2 is not alanine. In some embodiments, the mutation at position 182 as compared to SEQ ID NO: 2 is not valine.

[0151] In some embodiments, the mutation at position 182 as compared to SEQ ID NO: 2 is I182S, I182H, I182T, I182Q, or I182N. In some embodiments, the mutation at position 182 as compared to SEQ ID NO: 11 is T182S, T182H, T182Q, or T182N. In some embodiments, the mutation at position 182 as compared to SEQ ID NO: 13 is A182S, A182H, A182T, A182Q, or A182N. In some embodiments, the mutation at position 182 as compared to SEQ ID NO: 15 is V182S, V182H, V182T, V182Q, or V182N. In some embodiments, the mutation at position 182 as compared to SEQ ID NO: 17 is V182S, V182H, V182T, V182Q, or V182N. In some embodiments, the mutation at position 182 as compared to SEQ ID NO: 19 is V182S, V182H, V182T, V182Q, or V182N. In some embodiments, the mutation at position 182 as compared 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.

[0152] While the mutations can be described with reference to SEQ ID NO: 1 or SEQ ID NO: 2, which is the VSV-G protein from the Indiana strain, the mutations can also be used in VSV-G proteins of other strains. For example, the mutations can be made in the VSV-G of the New Jersey strain, the Maraba strain, the Carajas strain, the Alagoas strain, the Coetal strain, or the Moreton strain. In some embodiments, the respective sequences are as provided herein. Examples of these can be found, for example, in U.S. Patent Application Publication No. 20200216502, which is hereby incorporated by reference. For example, the wild-type full length or ectodomain of the VSV-G of the New Jersey strain is SEQ ID NO: 10 and SEQ ID NO: 11, respectively, the wild-type full length or ectodomain of the VSV-G of the Maraba strain is SEQ ID NO: 12 and SEQ ID NO: 13, respectively, the wild-type full length or ectodomain of the VSV-G of the Carajas strain is SEQ ID NO: 14 and SEQ ID NO: 15, respectively, the wild-type full length or ectodomain of the VSV-G of the Alagoas strain is SEQ ID NO: 16 and SEQ ID NO: 17, respectively, the wild-type full length or ectodomain of the VSV-G of the Coetal strain is SEQ ID NO: 18 and SEQ ID NO: 19, respectively, or the wild-type full length or ectodomain of the VSV-G of the Moreton strain is SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0153] The VSV-G protein comprising a mutation at position 182 as 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 hereby incorporated by reference in its entirety. For example, the VSV-G protein can comprise a mutation at a position corresponding to position 8, 47, 209, and / or 354 of SEQ ID NO: 2.

[0154] In some embodiments, the substitution at position 8 is any amino acid substitution other than Y that is different from the amino acid indicated at that position in the sequence SEQ ID NO: 2. In some embodiments, the substitution at position 209 is any amino acid substitution other than H that is different from the amino acid indicated at that position in the sequence SEQ ID NO: 2. In some embodiments, the substitution at position 47 is any amino acid substitution other than K or R that is different from the amino acid indicated at that position in the sequence SEQ ID NO: 2. In some embodiments, the substitution at position 354 is any amino acid substitution other than K or R that is different from the amino acid indicated at that position in the sequence SEQ ID NO: 2.

[0155] In some embodiments, the substitution is at position 47 or at position 354, or at both positions 47 and 354, with A, G, F, or Q. In some embodiments, the substitution is A or Q.

[0156] In some embodiments, the substitution at position 8 is alanine, i.e., H8A.

[0157] In some embodiments, the substitution at position 47 is Q or N, i.e., K47Q or K47N.

[0158] In some embodiments, the protein comprises a mutation (substitution) at position 10. In some embodiments, the substitution / mutation is Q10A, Q10R, or Q10K.

[0159] In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 2 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 2 (or SEQ ID NO: 1 if a full-length protein is used). 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.

[0160] In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 11 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 11 (or SEQ ID NO: 10 if a full-length protein is used). 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.

[0161] In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 13 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 13 (or SEQ ID NO: 12 if using a full-length protein). 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.

[0162] In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 15 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 15 (or SEQ ID NO: 14 if using a full-length protein). 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.

[0163] In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 17 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 17 (or SEQ ID NO: 16 if using a full-length protein). 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.

[0164] In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 19 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 19 (or SEQ ID NO: 18 if using a full-length protein) as compared to SEQ ID NO: 19. 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.

[0165] In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 21 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 21 (or SEQ ID NO: 20 if using a full-length protein) as compared to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a I182D or I182E mutation. In some embodiments, the VSV-G protein comprises a I182S, I182H, I182T, I182Q, or I182N mutation.

[0166] viral particles

[0167] Mutant VSV-G proteins can be used, for example, to pseudotype viruses, such as, but not limited to, lentiviruses. Accordingly, in some embodiments, viral particles comprising a mutant VSV-G protein as provided herein are provided. In some embodiments, the viral particles comprise a VSV-G protein comprising a mutation at position 198 as compared to SEQ ID NO: 1. In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 2 comprises a mutation at position 182 and has at least 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% identity as compared to SEQ ID NO: 2 (or SEQ ID NO: 1 if using a full-length protein). In some embodiments, the polypeptide comprises an I182D or I182E mutation as compared to SEQ ID NO: 2. In some embodiments, the VSV-G protein comprises an I182S, I182H, I182T, I182Q, or I182N mutation.

[0168] In some embodiments, the viral particles comprise a VSV-G protein comprising a mutation at position 198 as compared to SEQ ID NO: 10. In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO: 11 comprises a mutation at position 182 and has at least 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% identity as compared to SEQ ID NO: 11 (or SEQ ID NO: 10 if using a full-length protein). In some embodiments, the polypeptide comprises a T182D or T182E mutation as compared to SEQ ID NO: 11. In some embodiments, the VSV-G protein comprises a T182S, T182H, T182Q, or T182N mutation.

[0169] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 198 as compared to SEQ ID NO: 12. In some embodiments, the protein comprising a mutation at position 182 as compared to SEQ ID NO: 13 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 13 (or SEQ ID NO: 12 if a full-length protein is used). In some embodiments, the polypeptide comprises an A182D or A182E mutation as compared to SEQ ID NO: 13. In some embodiments, the VSV-G protein comprises an A182S, A182H, A182T, A182Q, or A182N mutation.

[0170] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 203 as compared to SEQ ID NO: 14. In some embodiments, the protein comprising a mutation at position 182 as compared to SEQ ID NO: 15 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 15 (or SEQ ID NO: 14 if a full-length protein is used). In some embodiments, the polypeptide comprises a V182D or V182E mutation as compared to SEQ ID NO: 15. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0171] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 199 as compared to SEQ ID NO: 16. In some embodiments, the protein comprising a mutation at position 182 as compared to SEQ ID NO: 17 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 17 (or SEQ ID NO: 16 if a full-length protein is used). In some embodiments, the polypeptide comprises a V182D or V182E mutation as compared to SEQ ID NO: 17. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0172] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 199 as compared to SEQ ID NO: 18. In some embodiments, the protein comprising a mutation at position 182 as compared to SEQ ID NO: 19 comprises a mutation at position 182 and has at least 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% identity to SEQ ID NO: 19 (or SEQ ID NO: 18 if a full-length protein is used). In some embodiments, the polypeptide comprises a V182D or V182E mutation as compared to SEQ ID NO: 19. In some embodiments, the VSV-G protein comprises a V182S, V182H, V182T, V182Q, or V182N mutation.

[0173] In some embodiments, the viral particle comprises a VSV-G protein comprising a mutation at position 199 as compared to SEQ ID NO:20. In some embodiments, a protein comprising a mutation at position 182 as compared to SEQ ID NO:21 comprises a mutation at position 182 and has at least 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% identity as compared to SEQ ID NO:21 (or SEQ ID NO:20, if a full-length protein is used). In some embodiments, the polypeptide comprises an I182D or I182E mutation as compared to SEQ ID NO:21. In some embodiments, the VSV-G protein comprises an I182S, I182H, I182T, I182Q, or I182N mutation.

[0174] 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 corresponding to a T214N mutation as compared to SEQ ID NO:2. In some embodiments, the VSV-G protein comprises a mutation corresponding to a T352A mutation as compared to SEQ ID NO:2. In some embodiments, the VSV-G protein comprises a T214N and a T352A mutation as compared to SEQ ID NO:2. These mutations can be combined with any other mutations as provided herein. In some embodiments, the T214N and / or T352A mutation is combined with an I182E or I182D mutation. In some embodiments, the VSV-G protein comprises the amino acid sequence of SEQ ID NO:22 and SEQ ID NO:23, which combine I182D or I182E with T214N and T352A mutations, respectively. Sequences with leader sequences, which are removed during protein processing, are also shown below.

[0175] VSV-G protein I196D, T230N, and T368A mutations (with leader sequence and adjusted numbering)

[0176] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLDSMDITFFSEDGELSSLGKEGTGFRSNYFAYENGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSR MVGMISGTTAERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 24)

[0177] VSV-G protein I182D, T214N, and T352A mutations (without leader sequence)

[0178] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLDSMDITFFSEDGELSSLGKEGTGFRSNYFAYENGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTAERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 22)

[0179] VSV-G protein with I196E, T230N, and T368A mutations (with leader sequence and adjusted numbering)

[0180] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLESMDITFFSEDGELSSLGKEGTGFRSNYFAYENGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSR MVGMISGTTAERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 25)

[0181] VSV-G protein with I182E, T214N, and T352A mutations (without leader sequence)

[0182] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLESMDITFFSEDGELSSLGKEGTGFRSNYFAYENGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTAERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 23)

[0183] In some embodiments, a VSV-G protein comprising a mutation at position 182 as compared to SEQ ID NO: 2 further comprises a mutation at a position corresponding to position 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 corresponding to a T38A mutation as compared to SEQ ID NO: 2. In some embodiments, the VSV-G protein comprises a mutation corresponding to a T320A mutation as compared to SEQ ID NO: 2. In some embodiments, the VSV-G protein comprises a T38A and a T320A mutation as compared to SEQ ID NO: 2. These mutations can be combined with any other mutations as provided herein.

[0184] In some embodiments, the VSV-G protein of other strains as described herein further can comprise one or more mutations corresponding to any other mutations as compared to SEQ ID NO: 2 and as provided herein. For example, the VSV-G protein of other strains as described herein also can comprise mutations corresponding to T38A, T214N, T320A, and / or T352A in SEQ ID NO: 2. In some embodiments, the VSV-G protein of other strains as described herein also can comprise mutations corresponding to T214N and / or T352A in SEQ ID NO: 2 and as set forth in SEQ ID NO: 22 and SEQ ID NO: 23.

[0185] In some embodiments, the composition comprises mutations as described in Hwang et al., Gene Ther. 2013 Aug;20(8):807-15. (Epub 2013 Jan 31), which is hereby incorporated by reference in its entirety. For example, the mutations can be at positions 230, 368, 66, and / or 162 corresponding to SEQ ID NO: 1. When the leader sequence is removed, the positions will be 16 positions less as compared to SEQ ID NO: 2. In some embodiments, the mutations at those positions are, for example, T230N, T368A, K66T, S162T, or any combination thereof. In some embodiments, the VSV-G protein comprises T230N and T368A mutations. In some embodiments, the VSV-G polypeptide comprises K66T, S162T, T230N, and T368A. These positions are those corresponding to positions 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, a 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 hereby incorporated by reference in its entirety. For example, the VSV-G protein further can comprise mutations at positions corresponding to positions 8, 47, 209, and / or 354 of SEQ ID NO: 2.

[0186] In some embodiments, the substitution at position 8 is any amino acid substitution other than Y from the amino acid indicated at that position in sequence SEQ ID NO: 2. In some embodiments, the substitution at position 209 is any amino acid substitution other than H from the amino acid indicated at that position in sequence SEQ ID NO: 2. In some embodiments, the substitution at position 47 is any amino acid substitution other than K or R from the amino acid indicated at that position in sequence SEQ ID NO: 2. In some embodiments, the substitution at position 354 is any amino acid substitution other than K or R from the amino acid indicated at that position in sequence SEQ ID NO: 2. In some embodiments, the substitution is at position 47 or at position 354, or at both positions 47 and 354, with A, G, F, or Q. 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.

[0187] In addition, in some embodiments, instead of a VSV-G protein or a mutant thereof, the virus can be pseudotyped with other viral structural proteins. Suitable examples of alternative viral structural proteins can be found at least in WO2023 / 064884, WO2023 / 114698, WO2023 / 114884, and WO2023 / 154858, each incorporated by reference in its entirety.

[0188] Targeting moiety

[0189] In some embodiments, the viral particle comprises a targeting moiety. The targeting moiety can be used to target the viral particle comprising the mutant VSV-G protein to a cell expressing a target to which the targeting moiety binds. In some embodiments, the targeting moiety is an antibody, an scFv antibody, an antigen binding domain, an ankyrin repeat sequence (e.g., DARPIN), a VHH domain antibody, a nanobody, a single domain antibody, a FN3 domain, or any combination thereof. The targeting moiety can be attached to the viral surface via an IgG Fc stalk. 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 attached (fused or linked) to the envelope glycoprotein G or H of a virus of the family Paramyxoviridae, such as a morbillivirus (such as a measles virus) or a henipavirus (such as a Nipah virus, a Cedar virus, or a Hendra virus). In some embodiments, the targeting moiety can be attached (fused or linked) to the glycoprotein of a virus of the family Rhabdoviridae, such as a vesicular stomatitis New Jersey virus, a vesicular stomatitis Indiana virus, a vesicular stomatitis Alagoas virus, a vesicular stomatitis Maraba virus, a vesicular stomatitis Caracas virus, a parainfluenza virus, a Spodoptera frugiperda rhabdovirus isolate Sf G, a Drosophila obscura sigma virus 10A, a Wuhan insect virus 7, a Perch virus, or a Spring viremia of carp virus. In some embodiments, the VSV protein is a mutant protein, such as those provided herein. In some embodiments, the targeting moiety is attached to the glycoprotein of a virus of the family Filoviridae (such as an Ebola virus) or the glycoprotein of a virus of the family Arenaviridae (such as a Machupo virus).

[0190] 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.

[0191] In some embodiments, the targeting moiety binds CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCRa, TCRp, TCRy, TCRd, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells; a glycosylated CD43 epitope expressed on non-hematopoietic cancers; A kinase anchor protein 4 (AKAP-4); adrenergic receptor beta 3 (ADRB3); AFP; anaplastic lymphoma kinase (ALK); androgen receptor; cell surface receptor 2 for angiopoietin (Tie 2); autoantibodies against desmoglein 1 (Dsgl); autoantibodies against desmoglein 3 (Dsg3); B7H3 (CD 276); 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 (BORIS or Brother of the Regulator of Imprinted Site); CCR4; CD5; CD19; CD20; CD22; CD24; CD30; CD32 (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; X-chromosomal open reading frame 61 (CXORF61); Claudin 6 (CLDN6); Claudin 18.2 (CLD18A2 or CLDN18A.2); CMV pp65; C-MYC epitope tag; Cripto; CS1 (also known as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24); CSF2R A (GM-CSFR-a); C-type lectin domain family 12 member A (CLEC12A); C-type lectin-like molecule 1 (CLL-1 or CLECL1); Cyclin Bl; Cytochrome P450 IB 1 (CYP1B 1); DLL3; EBV-EBNA3c; EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); Mutated elongation factor 2 (ELF2M); Ephrin B2; Ephrin type-A receptor 2 (EphA2); Epidermal growth factor receptor (EGF R);Epidermal growth factor receptor variant III (EGFRviii); Epithelial cell adhesion molecule (EpCAM); ERG; ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); Fc fragment of IgA receptor (FCAR or CD89); Fc receptor like 5 (FCRL5); Fibroblast 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 (FSH R); Fos-related antigen 1; Fucosyl-GM1; 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)bD Glcp(l-l)Cer); Ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l-4)bDGlcp(l-l)Cer); GD3; GFRa4; Glycoprotein 100 (gplOO); Glypican-3 (GPC3); Gonadotropin receptor (CGHR or GR); GpA33; GpNMB; GPRC 5D; Guanylate cyclase C (GCC); Mutated heat shock protein 70-2 (mut hsp70-2); Hepatitis A virus cellular receptor 1 (HAVCR1); Hexose portion of globoH ceramide (GloboH); High molecular weight-melanoma-associated antigen (HMWMAA); HIV1 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; HTLVl-Tax; Human papilloma virus E6 (HPV E6); Human papilloma virus E7 (HPV E7); Human telomerase reverse transcriptase (hTERT); IgE; IL13Ra2; ILl lRa; Immunoglobulin lambda-like polypeptide 1 (IGLL1); Influenza A hemagglutinin (HA); Insulin-like growth factor 1 receptor (I GF-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; Lew s Ag; Livl; Locus K 9 (LY6K);Low conductance chloride channel; lymphocyte antigen 6 complex; lymphocyte antigen 75 (LY75); lymphocyte-specific protein tyrosine kinase (LCK); breast cancer differentiation antigen (NY-BR-1); melanoma antigen recognized by T cells 1 (Melan A 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 apoptosis inhibitory protein (ML-IAP); mesothelin; MPL; cell surface associated mucin 1 (MUC1); N-acetylglucosaminyltransferase V (NA17); nectin-4; neural cell adhesion molecule (NCAM); NKG2D; NYBR1; O-acetyl-GD2 ganglioside (OAcGD2); olfactory receptor 51E2 (OR51E2); oncogenic 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; previtellogenin binding protein sp32 (OY-TES1); prostase; prostate cancer tumor antigen 1 (PCT A-l 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 (Prosome Macropain) subunit beta type 9 (LMP2); PTK7; Ras G12V; Ras homolog family member C (RhoC); rat sarcoma (Ras) mutant; receptor for advanced glycation end products (RAG E-1); receptor tyrosine kinase-like orphan receptor 1 (ROR1); receptor tyrosine-protein kinase E RBB2 or Her-22 / neu; renal ubiquitous protein 1 (RU1); renal ubiquitous protein 2 (RU2); sarcoma translocation breakpoints; serine 2 (TMPRSS2) ETS fusion gene; sialyl Lewis adhesion molecule (sLe); SLAMF4; SLAMF6; Slea (CA19.9 or sialyl Lewis antigen); sperm protein 17 (SPA17); 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 variable reading frame protein (TAR P); 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 antibody; thyroid stimulating hormone receptor (TSHR); Tim 1- / 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 protein (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 CD 8.

[0192] 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 as, but not limited to, a T cell, a B cell; an NK cell, a dendritic cell, a neutrophil, a macrophage, a cancer cell; or, for example, a CD3+T cell; a CD4+T cell; a CD7+T cell, a CD8+T cell; a CD19+B cell; a CD19+cancer cell; a CD20+B cell; a CD20+cancer cell; a CD30+lung epithelial cell; a CD34+hematopoietic stem cell; a CD105+endothelial cell; a CD105+hematopoietic stem cell; a CD117+hematopoietic stem cell; a CD133+cancer cell; an EpCAM+cancer cell; a GluA2+neuron; a GluA4+neuron; a hematopoietic stem cell; a hepatocyte; a Her2 / Neu+cancer cell; a NKG2D+natural killer cell; a SLC1A3+astrocyte; a SLC7A10+adipocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a CD7+T cell and / or a CD8+T cell.

[0193] In some embodiments, the targeting moiety (polypeptide) can bind to CD7.

[0194] 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):

[0195] MAGPPRLLLLPLLLALARGLPGALAAQEVQQSPHCTTVPVGASVNITCSTSGGLRGIYLRQLGPQPQDIIYYEDGVVPTTDRRFRGRIDFSGSQDNLTITMHRLQLSDTGTYTCQAITEVNVYGSGTLVLVTEE QSQGWHRCSDAPPRASALPAPPTGSALPDPQTASALPDPPAASALPAALAVISFLLGLGLGVACVLARTQIKKLCSWRDKNSAACVVYEDMSHSRCNTLSSPNQYQ (SEQ ID NO: 29)

[0196] In some embodiments, the CD7 antibody comprises an Fc region. The Fc region can be linked to the heavy chain or light chain of the antibody. The Fc region can be directly fused to the heavy chain or light chain of the antibody, or can be indirectly fused to the heavy chain or light chain of the antibody via, for example, a peptide linker provided herein. In some embodiments, the Fc region is an IgG Fc. In some embodiments, the IgG is selected from IgGl, IgG2, IgG3, or IgG4. In some embodiments, the IgG Fc is an IgGl Fc. In some embodiments, the antibody comprises an Fc constant region of SEQ ID NO: 26 as shown below:

[0197] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26)

[0198] In some embodiments, the IgG fc is an IgG2 Fc. In some embodiments, the antibody comprises an Fc constant region of SEQ ID NO: 27 as shown below:

[0199] STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27)

[0200] In some embodiments, the IgG fc is an IgG4 Fc. In some embodiments, the antibody comprises an Fc constant region of SEQ ID NO: 28 as shown below:

[0201] STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 28)

[0202] In some embodiments, the IgG Fc is a variant of the IgGl Fc protein (SEQ ID NO: 26). In some embodiments, the variant IgGl Fc protein 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 can be present or absent, and the mutations can be combined in any combination. In some embodiments, the variant IgGl Fc protein comprises mutations corresponding to L234A and L235A of SEQ ID NO: 26. In some embodiments, the variant IgGl Fc protein comprises a mutation corresponding to N297A of SEQ ID NO: 26. In some embodiments, the variant IgGl Fc protein comprises a mutation corresponding to P329G of SEQ ID NO: 26. In some embodiments, the variant IgGl Fc protein comprises mutations corresponding to L234A, L235A, N297A, and P329G of SEQ ID NO: 83. In some embodiments, the variant IgGl Fc protein comprises a mutation corresponding to I253A of SEQ ID NO: 26. In some embodiments, the variant IgGl Fc protein comprises a mutation corresponding to H310A of SEQ ID NO: 26. In some embodiments, the variant IgGl Fc protein comprises a mutation corresponding to H435A of SEQ ID NO: 26. In some embodiments, the variant IgGl Fc protein comprises mutations corresponding to I253A, H310A, and H435A of SEQ ID NO: 26.

[0203] In some embodiments, the IgG Fc is a variant of an IgG2 Fc protein (SEQ ID NO: 27). In some embodiments, the variant IgG2 Fc protein comprises one or more mutations selected from the group consisting of N297A, P329G, I253A, H310A, and H435A, those positions corresponding to SEQ ID NO: 27. Any of the mutations N297A, P329G, I253A, H310A, and H435A of SEQ ID NO: 27 can be present or absent, and the mutations can be combined in any combination. In some embodiments, the variant IgG2 Fc protein comprises a mutation corresponding to N297A of SEQ ID NO: 27. In some embodiments, the variant IgG2 Fc protein comprises a mutation corresponding to P329G of SEQ ID NO: 27. In some embodiments, the variant IgG2 Fc protein comprises mutations corresponding to N297A and P329G of SEQ ID NO: 27. In some embodiments, the variant IgG2 Fc protein comprises a mutation corresponding to I253A of SEQ ID NO: 27. In some embodiments, the variant IgG2 Fc protein comprises a mutation corresponding to H310A of SEQ ID NO: 27. In some embodiments, the variant IgG2 Fc protein comprises a mutation corresponding to H435A of SEQ ID NO: 27. In some embodiments, the variant IgG2 Fc protein comprises mutations corresponding to I253A, H310A, and H435A of SEQ ID NO: 27.

[0204] In some embodiments, the IgG Fc protein is a variant of an IgG4 Fc protein (SEQ ID NO: 28). In some embodiments, the variant IgG4 Fc protein comprises one or more mutations selected from the group consisting of S228P, L235E, N297A, P329G, I253A, H310A, and H435A, those positions corresponding to SEQ ID NO: 28. Any of the mutations S228P, L235E, N297A, P329G, I253A, H310A, and H435A of SEQ ID NO: 28 can be present or absent, and the mutations can be combined in any combination. In some embodiments, the variant IgG4 Fc protein comprises a mutation corresponding to S228P of SEQ ID NO: 28. In some embodiments, the variant IgG4 Fc protein comprises a mutation corresponding to L235E of SEQ ID NO: 28. In some embodiments, the variant IgG4 Fc protein comprises a mutation corresponding to N297A of SEQ ID NO: 28. In some embodiments, the variant IgG4 Fc protein comprises a mutation corresponding to P329G of SEQ ID NO: 28. In some embodiments, the variant IgG4 Fc protein comprises mutations corresponding to S228P, L235E, N297A, and P329G of SEQ ID NO: 28. In some embodiments, the variant IgG4 Fc protein comprises a mutation corresponding to I253A of SEQ ID NO: 28. In some embodiments, the variant IgG4 Fc protein comprises a mutation corresponding to H310A of SEQ ID NO: 28. In some embodiments, the variant IgG4 Fc protein comprises a mutation corresponding to H435A of SEQ ID NO: 28. In some embodiments, the variant IgG4 Fc protein comprises mutations corresponding to I253A, H310A, and H435A of SEQ ID NO: 28.

[0205] In some embodiments, the Fc region comprises a variant Fc polypeptide. In some embodiments, the variant Fc polypeptide is a variant Fc polypeptide as provided in PCT Publication No. WO2024026284, which is hereby incorporated by reference in its entirety. In some embodiments, the variant Fc polypeptide 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:

[0206] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK(SEQ ID NO:82)

[0207] In some implementations, the variant Fc polypeptide contains the amino acid sequence of SEQ ID NO:82.

[0208] In some implementations, the targeting portion binds to CD7 and includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, and VL as described in Tables 1 and 2 below:

[0209] Table 1 - CD7 Target Part CD7AB1 CDR Sequence

[0210]

[0211]

[0212] Table 2 - CD7 Targeting Region: CD7AB1 Variable Region

[0213]

[0214] The VH and VL sequences can be of any form, including but not limited to scFv forms in which the VH and VL regions are linked by peptide linkers. Examples of peptide linkers that can be used to link the various peptides presented herein include, but are not limited to: (GGGGS) n (SEQ ID NO: 64), where each n is independently 1-4. 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 region is not linked to the peptide linker. In some embodiments, the polypeptide comprises SEQ ID NO: 42 and SEQ ID NO: 43. In some embodiments, the targeting portion comprises a peptide of formula V. L -ZV Ha connecting peptide represented by the formula Z-V, wherein Z is a peptide linker. In some embodiments, the targeting moiety comprises a heavy chain variable region as set forth in SEQ ID NO: 42 linked to a light chain variable region as set forth in SEQ ID NO: 43 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the targeting moiety comprises a heavy chain variable region as set forth in SEQ ID NO: 42 linked to a light chain variable region as set forth in SEQ ID NO: 43 via a peptide linker. H V L The targeting moiety of V

[0215] DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNDSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPTTFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKLSCKASGYPFTSYWIHWVKQRPGRGLEWLGRIDPNSGDTKYNEKFKNKATLTVDKSSTTAYMQLSSLTSEDSAVYYCARSPYYSNDNSMDYWGQGTSVTVSS (SEQ ID NO: 44)

[0216] In some embodiments, the targeting moiety comprises a heavy chain variable region as set forth in SEQ ID NO: 42 linked to a light chain variable region as set forth in SEQ ID NO: 43 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the targeting moiety comprises a heavy chain variable region as set forth in SEQ ID NO: 42 linked to a light chain variable region as set forth in SEQ ID NO: 43 via a peptide linker. H -Z-V L a connecting peptide represented by the formula Z-V, wherein Z is a peptide linker. In some embodiments, the targeting moiety comprises a heavy chain variable region as set forth in SEQ ID NO: 42 linked to a light chain variable region as set forth in SEQ ID NO: 43 via the linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the targeting moiety comprises a heavy chain variable region as set forth in SEQ ID NO: 42 linked to a light chain variable region as set forth in SEQ ID NO: 43 via a peptide linker. L V H The targeting moiety of V

[0217] QVQLQQPGAELVKPGASVKLSCKASGYPFTSYWIHWVKQRPGRGLEWLGRIDPNSGDTKYNEKFKNKATLTVDKSSTTAYMQLSSLTSEDSAVYYCARSPYYSNDNSMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNDSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPTTFGGGTKLEIKR (SEQ ID NO: 45)

[0218] In some embodiments, the targeting moiety (polypeptide) can bind to CD8.

[0219] 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 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):

[0220] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGCYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 46)

[0221] The sequence of human CD8-beta (UniProtKB Q8TD28) is as follows (SEQ ID NO: 47):

[0222] MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQLYK (SEQ ID NO: 47)

[0223] In some embodiments, the CD8 antibody comprises an Fc region. The Fc region can be linked to a heavy chain or a light chain of the antibody. The Fc region can be directly fused to a heavy chain or a light chain of the antibody, or can be indirectly fused to a heavy chain or a light chain of the antibody via, for example, a peptide linker provided herein. In some embodiments, the Fc region is an IgG Fc as provided herein. In some embodiments, the IgG is selected from IgGl, IgG2, IgG3, or IgG4. In some embodiments, the IgG fc is an IgGl Fc as provided herein. In some embodiments, the antibody comprises the Fc constant region of SEQ ID NO: 26. In some embodiments, the IgG fc is an IgG2 Fc as provided herein. In some embodiments, the antibody comprises the Fc constant region of SEQ ID NO: 27. In some embodiments, the IgG fc is an IgG4 Fc as provided herein. In some embodiments, the antibody comprises the Fc constant region of SEQ ID NO: 28.

[0224] In some embodiments, the targeting moiety binds to CD8 and comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, a LCDR3, a VH, and a VL as set forth in Tables 3 and 4 below:

[0225] Table 3 - CD8 targeting moiety CD8AB1 CDR sequences

[0226]

[0227] Table 4 - CD8 targeting moiety CD8AB1 variable regions

[0228]

[0229] The VH and VL sequences can be in any format, including but not limited to a scFv format in which the VH and VL regions are connected by a peptide linker. Examples of peptide linkers that can be used to connect the various peptides provided herein include, but are not limited to: (GGGGS) n (SEQ ID NO: 64), wherein each n is independently 1-4. 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 region is not connected to a peptide linker. In some embodiments, the polypeptide comprises SEQ ID NO: 60 and SEQ ID NO: 61.

[0230] In some embodiments, the targeting moiety comprises a connecting peptide represented by Formula V L -Z-V H wherein Z is a peptide linker. In some embodiments, the targeting moiety comprises a heavy chain variable region as set forth in SEQ ID NO: 60 connected to a light chain variable region as set forth in SEQ ID NO: 61 via a linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the targeting moiety comprising V H connected to V L has a sequence as set forth below:

[0231] NIVLTQSPASLAVSLGQRATISCRASESVDGFGNSFMNWYQQKPGQSPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPYTFGGGTKLEIKRGGGGSGGGGSGGGGSG GGGSEVQLQQSGPELVKPGASVKISCKASRYTFTDYNLHWVKLSHEKSLEWIGFIYPYNGGTGYNQKFKNKAKLTVDYSSSTAYMELRSLTSVDAAVYYCARDHRYNEGVSFDYWGQGTTLTVSS(SEQ ID NO:62)

[0232] In some implementations, the targeting portion includes formula V H -ZV L The term represents a linker peptide, where Z is a peptide linker. In some embodiments, the targeting portion comprises a heavy chain variable region, as shown in SEQ ID NO:60, linked to a light chain variable region, as shown in SEQ ID NO:61, via a linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:72). In some embodiments, it comprises a heavy chain variable region, as shown in SEQ ID NO:60, linked to a light chain variable region, as shown in SEQ ID NO:61, via a peptide linker. L Connected V H The target portion has the following sequence:

[0233] EVQLQQSGPELVKPGASVKISCKASRYTFTDYNLHWVKLSHEKSLEWIGFIYPYNGGTGYNQKFKNKAKLTVDYSSSTAYMELRSLTSVDAAVYYCARDHRYNEGVSFDYWGQGTTLTVSSGGGGSGG GGSGGGGSGGGGSNIVLTQSPASLAVSLGQRATISCRASESVDGFGNSFMNWYQQKPGQSPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPYTFGGGTKLEIKR(SEQ ID NO:63)

[0234] In some embodiments, the targeting portion comprises an amino acid sequence having at least 70% identity with SEQ ID NO:44, or being substantially similar to SEQ ID NO:44, or being the active fragment of SEQ ID NO:44. In some embodiments, the targeting portion 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 with SEQ ID NO:44. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO:44.

[0235] In some embodiments, the targeting portion comprises an amino acid sequence having at least 70% identity with SEQ ID NO:45, or being substantially similar to SEQ ID NO:45, or the active fragment of SEQ ID NO:45. In some embodiments, the targeting portion 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 with SEQ ID NO:45. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO:45.

[0236] In some embodiments, the targeting portion comprises an amino acid sequence having at least 70% identity with SEQ ID NO:62, or being substantially similar to SEQ ID NO:62, or the active fragment of SEQ ID NO:62. In some embodiments, the targeting portion 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 with SEQ ID NO:62. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO:62.

[0237] In some embodiments, the targeting portion comprises an amino acid sequence having at least 70% identity with SEQ ID NO:63, or being substantially similar to SEQ ID NO:63, or being the active fragment of SEQ ID NO:63. In some embodiments, the targeting portion 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 with SEQ ID NO:63. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO:63.

[0238] In some embodiments, the targeting portion as provided herein is attached to the surface of the virus via a stem portion S1. In some embodiments, the targeting portion is represented by the formula T-S1, where T is the targeting portion as provided herein and S1 is the stem portion. In some embodiments, the stem portion S1 is as provided in PCT Publication WO2024026284 (which is hereby incorporated herein by reference in its entirety). In some embodiments, the stem portion S1 comprises a variant Fc protein as provided herein and is given by the formula L1-Fc-L2-X1, where L1 is a linker or is absent; Fc is a variant Fc protein; L2 is a linker or is absent; and X1 is a polypeptide containing a transmembrane domain. Thus, in some embodiments, the formula representing the targeting portion can also be written as T-L1-Fc-L2-X1. In some embodiments, the stem portion S1 does not contain a variant Fc region and is given by the formula L3-X1, where L3 is a flexible polypeptide linker and X1 is a polypeptide containing a transmembrane domain. Therefore, in some embodiments, the formula representing the targeting portion can also be written as T-L3-X1. In some embodiments, the polypeptide X1 containing a transmembrane domain comprises a peptide with the formula ECD-T M -ICD polypeptides, wherein ECD is an extracellular domain of a cell surface protein or a fragment thereof, or is absent, T M It is the transmembrane domain of a transmembrane protein, and ICD is either the intracellular domain of a protein, or a protein that facilitates the incorporation of the target portion into the viral particle's envelope, or it may not exist. Therefore, the formula representing the target portion connected to the stem portion can also be written as T-L1-Fc-L2-ECD-T M -ICD or T-L3-ECD-T M -ICD. L1, L2, L3, Fc, ECD, T M An exemplary identity of the ICD can be found in PCT Publication WO2024026284 (which is hereby incorporated herein by reference in its entirety).

[0239] In some implementations, the target portion includes T-L1-Fc-L2-ECD-T M -ICD. In some embodiments, T comprises an amino acid sequence having at least 90% identity with 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 with SEQ ID NO:82. In some embodiments, L2 is absent. In some embodiments, ECD comprises an amino acid sequence having at least 90% identity with SEQ ID NO:83: KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(SEQ ID NO:83)

[0240] NO:83)

[0241] In some implementations, T M It contains an amino acid sequence that is at least 90% identical to SEQ ID NO:84:

[0242] FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:84)

[0243] In some embodiments, the ICD comprises an amino acid sequence containing an env-incorporated motif, wherein the env-incorporated motif comprises the amino acid sequence of SEQ ID NO:85:

[0244] NRVRQGYS (SEQ ID NO:85)

[0245] In some implementations, the target portion includes T-L1-Fc-L2-ECD-T M -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, 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 MThe ICD contains the amino acid sequence of SEQ ID NO:84. In some embodiments, the ICD contains an amino acid sequence with an env-incorporated motif, wherein the env-incorporated motif contains the amino acid sequence of SEQ ID NO:85.

[0246] In some implementations, the target portion includes T-L1-Fc-L2-ECD-T M -ICD. 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 The ICD contains the amino acid sequence of SEQ ID NO:84. In some embodiments, the ICD contains an amino acid sequence with an env-incorporated motif, wherein the env-incorporated motif contains the amino acid sequence of SEQ ID NO:85.

[0247] In some implementations, the target portion includes T-L1-Fc-L2-ECD-T M -ICD. In 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 The ICD contains the amino acid sequence of SEQ ID NO:84. In some embodiments, the ICD contains an amino acid sequence with an env-incorporated motif, wherein the env-incorporated motif contains the amino acid sequence of SEQ ID NO:85.

[0248] In some implementations, the target portion includes T-L3-ECD-T M -ICD. In some embodiments, T comprises an amino acid sequence having at least 90% identity with 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, wherein n is 1, 2, or 4. In some embodiments, ECD comprises an amino acid sequence having at least 90% identity with SEQ ID NO:86.

[0249] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT

[0250] RGLDFACD (SEQ ID NO:86)

[0251] In some implementations, T M It contains an amino acid sequence that is at least 90% identical to SEQ ID NO:87:

[0252] IYIWAPLAGTCGVLLLSLVITLYCNNHRN(SEQ ID NO:87)

[0253] In some embodiments, the ICD comprises an amino acid sequence containing an env-incorporated motif, wherein the env-incorporated motif comprises the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:88:

[0254] GGTETSQVAPA (SEQ ID NO:88).

[0255] In some implementations, the target portion includes T-L3-ECD-T M -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 The ICD contains the amino acid sequence of SEQ ID NO:87. In some embodiments, the ICD contains an amino acid sequence with an env-incorporated motif, wherein the env-incorporated motif contains the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:88.

[0256] In some implementations, the target portion includes T-L3-ECD-T M -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 MThe ICD contains the amino acid sequence of SEQ ID NO:87. In some embodiments, the ICD contains an amino acid sequence with an env-incorporated motif, wherein the env-incorporated motif contains the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:88.

[0257] In some implementations, the target portion includes T-L3-ECD-T M -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 M The ICD contains the amino acid sequence of SEQ ID NO:87. In some embodiments, the ICD contains an amino acid sequence with an env-incorporated motif, wherein the env-incorporated motif contains the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:88.

[0258] In some embodiments, the targeting portion of the inclusion 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 with SEQ ID NO:89.

[0259] METDTLLLWVLLLWVPGSTGDSAQVQLQQPGAELVKPGASVKLSCKASGYPFTSYWIHWVKQRPGRGLEWLGRIDPNSGDTKYNEKFKNKATLTVDKSSSTTAYMQLSSLTS EDSAVYYCARSPYYSNDNSMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNDSPRLLIKYASESISGI PSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPTTFGGGTKLEIKRASGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSL SLSPGKKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNRVRQGYS(SEQ ID NO:89)

[0260] In some embodiments, the targeting portion of the included T-S1 comprises an amino acid sequence having 90% identity with SEQ ID NO:89. In some embodiments, the targeting portion of the included T-S1 comprises an amino acid sequence having 95% identity with SEQ ID NO:89. In some embodiments, the targeting portion of the included T-S1 comprises an amino acid sequence having 98% identity with SEQ ID NO:89. In some embodiments, the targeting portion of the included T-S1 comprises the amino acid sequence of SEQ ID NO:89.

[0261] In some embodiments, viral particles comprising the mutant VSV-G protein as provided herein and the targeting portion as provided herein further comprise a nucleic acid molecule encoding a target heterologous molecule or "cargo". For example, a target heterologous molecule means any product that can be encoded by a nucleic acid molecule. As a non-limiting example, "cargo" or "target heterologous molecule" can refer to siRNA, shRNA, peptide, polypeptide, protein, viral payload, viral genome, or combinations thereof. In some embodiments, the polypeptide is a chimeric antigen receptor ("CAR").

[0262] As used herein, a "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 consists of a single-chain variable fragment (scFv) derived from the variable heavy and light chain regions of a fusion mouse or humanized monoclonal antibody. Alternatively, scFvs derived from Fabs (rather than antibody-derived, e.g., obtained from a Fab library) can be used. In various embodiments, the scFv is 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 the cell. For example, the antigen-binding domain of a CAR can be an antibody, an scFv antibody, an antigen-binding domain, an ankyrin repeat sequence (e.g., DARPIN), a VHH domain antibody, a nanobody, a single-domain antibody, an FN3 domain, or any combination thereof. In some embodiments, CARs include those that provide only CD3ζ signaling upon antigen binding. In some embodiments, CARs include those that provide co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ). In some embodiments, CARs include those that provide multiple co-stimulations (e.g., CD28 and CD137) and activation (CD3ζ). In various embodiments, CARs are selected to have high affinity or affinity for the antigen. In some embodiments, the CAR also contains a 4-1BB domain. These are merely illustrative in nature and do not limit embodiments of the invention, and any chimeric antigen receptor can be co-delivered with the viral particles and vectors provided herein. These are non-limiting examples of CARs, and any CAR construct can be encoded by a nucleic acid molecule.

[0263] In some implementations, the antigen-binding domain of the CAR contains V H Structural domain, V L Domain, or V H and V L Structural domain. In some implementations, the V HThe domain contains 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 with SEQ ID NO:73, or any value or range between the two.

[0264] EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAP GKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSL RAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS(SEQ ID NO:73)

[0265] In some implementations, the V H The domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:73. In some embodiments, the V H The domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:73. In some embodiments, the V H The domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:73. In some embodiments, the V H The domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:73. In some embodiments, the V H The domain contains an amino acid sequence having the sequence of SEQ ID NO:73.

[0266] In some implementations, the V L The domain contains 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 with SEQ ID NO:74, or any value or range between the two.

[0267] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQR SNWPITFGQGTRLEIK(SEQ ID NO:74)

[0268] In some implementations, the V LThe domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:74. In some embodiments, the V L The domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:74. In some embodiments, the V L The domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:74. In some embodiments, the V L The domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:74. In some embodiments, the V L The domain contains an amino acid sequence having the sequence of SEQ ID NO:74.

[0269] In some implementations, the antigen-binding domain of the CAR contains V H Domain and V L Structural domain. In some implementations, the V H and V L The domains are not linked via linker peptides. In some embodiments, the V... H and V L The domains are linked by adaptor peptides, such as those provided herein, including but not limited to: (GGGGS) n (SEQ ID NO:64), where each n is independently 1-5. 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.

[0270] In some implementations, V is included H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:73. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:74. L In some implementations, V is included. H Domain and V LThe antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:73. H The structural domain, and contains a V that is at least 90% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:73. H The structural domain, and contains a V that is at least 95% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:73. H The structural domain, and contains a V that is at least 98% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:73. H The structural domain, and contains a V that is at least 99% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:73. HThe structural domain, and contains a V with the sequence having SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 90% identical to that of SEQ ID NO:73. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 95% identical to that of SEQ ID NO:73. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 98% identical to that of SEQ ID NO:73. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 99% identical to that of SEQ ID NO:73. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V amino acid sequence having SEQ ID NO:73. HThe structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 90% identical to that of SEQ ID NO:73. H The structural domain, and contains a V that is at least 90% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 95% identical to that of SEQ ID NO:73. H The structural domain, and contains a V that is at least 90% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 90% identical to that of SEQ ID NO:73. H The structural domain, and contains a V that is at least 95% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 95% identical to that of SEQ ID NO:73. H The structural domain, and contains a V that is at least 95% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 98% identical to that of SEQ ID NO:73. H The structural domain, and contains a V that is at least 98% identical to SEQ ID NO:74. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 99% identical to that of SEQ ID NO:73. H The structural domain, and contains a V that is at least 99% identical to SEQ ID NO:74. L In some implementations, V is included.H Domain and V L The antigen-binding domain of the CAR contains a V amino acid sequence having SEQ ID NO:73. H The domain contains a V with the amino acid sequence having SEQ ID NO:74. L .

[0271] In some implementations, the antigen-binding domain of the CAR includes the formula V H -ZV L V H Z is the heavy chain variable region containing the amino acid sequence of SEQ ID NO:73, Z is the linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:77), and V L It is a light chain variable region containing the amino acid sequence of SEQ ID NO:74. In some embodiments, it includes formula V. H -ZV L The antigen-binding domain of the CAR has the following amino acid sequence:

[0272] EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGG GGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK(SEQ ID NO:75)

[0273] 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 with 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 with 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 with 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 with 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 with 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.

[0274] In some implementations, the antigen-binding domain of the CAR includes the formula V L -ZV H V L Z is the light chain variable region containing the amino acid sequence of SEQ ID NO:74, Z is the linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:77), and V H It is a light chain variable region containing the amino acid sequence of SEQ ID NO:73. In some embodiments, it includes formula V. L -ZV H The antigen-binding domain of the CAR has the following amino acid sequence:

[0275] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSGGGGSGGGGSEV QLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS(SEQ ID NO:76)

[0276] 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 with the sequence of SEQ ID NO:76. In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO:76. In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:76. In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% identity with the sequence of SEQ ID NO:76. In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 99% identity with the sequence of SEQ ID NO:76. In some embodiments, the antigen-binding domain of the CAR comprises the amino acid sequence of SEQ ID NO:76.

[0277] In some implementations, the antigen-binding domain of the CAR contains V H Structural domain, V L Domain, or V H and V L Structural domain. In some implementations, the V H The domain contains 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 with SEQ ID NO:78, or any value or range between the two.

[0278] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSS PKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQ QWSFNPPTFGGGTKLEIKGSTS(SEQ ID NO:78)

[0279] In some implementations, the V H The domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:78. In some embodiments, the V H The domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:78. In some embodiments, the V HThe domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:78. In some embodiments, the V H The domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:78. In some embodiments, the V H The domain contains an amino acid sequence having the sequence of SEQ ID NO:78.

[0280] In some implementations, the V L The domain contains 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 with SEQ ID NO:79, or any value or range between the two.

[0281] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSS LTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS(SEQ ID NO:79)

[0282] In some implementations, the V L The domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:79. In some embodiments, the V L The domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:79. In some embodiments, the V L The domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:79. In some embodiments, the V L The domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:79. In some embodiments, the V L The domain contains an amino acid sequence having the sequence of SEQ ID NO:79.

[0283] In some implementations, V is included H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:78. HThe structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:78. H The structural domain, and contains a V that is at least 90% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:78. H The structural domain, and contains a V that is at least 95% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:78. H The structural domain, and contains a V that is at least 98% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:78. H The structural domain, and contains a V that is at least 99% identical to SEQ ID NO:79. L In some implementations, V is included. HDomain and V L The antigen-binding domain of the CAR contains a V domain that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:78. H The structural domain, and contains a V with the sequence having SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 90% identical to that of SEQ ID NO:78. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 95% identical to that of SEQ ID NO:78. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 98% identical to that of SEQ ID NO:78. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 99% identical to that of SEQ ID NO:78. HThe structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V amino acid sequence having SEQ ID NO:78. H The structural domain, and includes a V that has at least 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 90% identical to that of SEQ ID NO:78. H The structural domain, and contains a V that is at least 90% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 95% identical to that of SEQ ID NO:78. H The structural domain, and contains a V that is at least 90% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 90% identical to that of SEQ ID NO:78. H The structural domain, and contains a V that is at least 95% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 95% identical to that of SEQ ID NO:78. H The structural domain, and contains a V that is at least 95% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 98% identical to that of SEQ ID NO:78.H The structural domain, and contains a V that is at least 98% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V domain that is at least 99% identical to that of SEQ ID NO:78. H The structural domain, and contains a V that is at least 99% identical to SEQ ID NO:79. L In some implementations, V is included. H Domain and V L The antigen-binding domain of the CAR contains a V amino acid sequence having SEQ ID NO:78. H The domain contains a V with an amino acid sequence having SEQ ID NO:79. L .

[0284] In some implementations, the antigen-binding domain of the CAR includes the formula V H -ZV L V H Z is the heavy chain variable region containing the amino acid sequence of SEQ ID NO:78, Z is the linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:77), and V L It is a light chain variable region containing the amino acid sequence of SEQ ID NO:79. In some embodiments, it includes formula V. H -ZV L The antigen-binding domain of the CAR has the following amino acid sequence:

[0285] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGGSGGGGSGGGGSS EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS(SEQ ID NO:80)

[0286] 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 with 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 with 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 with 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 with 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 with 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.

[0287] In some implementations, the antigen-binding domain of the CAR includes the formula V L -ZV H V L Z is the light chain variable region containing the amino acid sequence of SEQ ID NO:79, Z is the linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:77), and V H It is a light chain variable region containing the amino acid sequence of SEQ ID NO:78. In some embodiments, it includes formula V. L -ZV H The antigen-binding domain of the CAR has the following amino acid sequence:

[0288] SEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGG GSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTS(SEQ ID NO:81)

[0289] 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 with the sequence of SEQ ID NO:81. In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO:81. In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:81. In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% identity with the sequence of SEQ ID NO:81. In some embodiments, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 99% identity with the sequence of SEQ ID NO:81. In some embodiments, the antigen-binding domain of the CAR comprises the amino acid sequence of SEQ ID NO:81.

[0290] In some embodiments, the antigen-binding domain of the CAR comprises rituximab, ocrelizumab, obinutuzumab, ofa tumumab, ibritumomab tiuxetan, tositumob, or ublituximab. In some embodiments, the antigen-binding domain comprises rituximab. In some embodiments, the antigen-binding domain comprises ofa tumumab. In some embodiments, the CAR also comprises a 4-1BB domain. These are merely illustrative in nature and do not limit the embodiments of the invention, and any chimeric antigen receptor can be co-delivered with the viral particles and vectors provided herein. These are non-limiting examples of CARs, and any CAR construct can be encoded by a nucleic acid molecule.

[0291] In some embodiments, 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 with the sequence of SEQ ID NO:90.

[0292] MALPPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNY YYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPI TFGQGTRLEIKSGLDFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:90)

[0293] Alternatively, it may be substantially similar to SEQ ID NO:90, or it may be the active fragment of SEQ ID NO:90. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO:90. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:90. In some embodiments, the CAR comprises an amino acid sequence having at least 98% identity with 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, co-stimulatory domain, signal transduction domain) are as provided in PCT Publication WO2024026284 (which is hereby incorporated herein by reference in its entirety).

[0294] In some embodiments, the pseudotyped viral particles further comprise a heterologous nucleic acid molecule encoding the target cargo. The nucleic acid molecule can be used to regulate the expression of a target gene. In some embodiments, the cargo can be used to regulate cell activity or express proteins transported to the surface of target cells. Therefore, in some embodiments, the nucleic acid can comprise siRNA or shRNA. The nucleic acid can also encode the target cargo. Therefore, in some embodiments, the target cargo can comprise a polypeptide or a portion thereof, a protein or a portion thereof, a chimeric antigen receptor or a portion thereof, or a tumor antigen or a portion thereof. In some embodiments, the target cargo is an antibody produced by the virus, which can then be secreted by virus-infected cells. The term "protein" can refer to any polypeptide carrying a natural function in the cellular environment. Therefore, in some embodiments, the protein encoded by the target nucleic acid cargo can include enzymes, nuclear receptors, transport proteins, ribosomal proteins, membrane-bound proteins, cytoplasmic proteins, G protein-coupled receptors, voltage-gated ion channels, secretory proteins, mitochondrial proteins, cytokines, chimeric antigen receptors, tumor antigens, or portions or chimeric substances thereof.

[0295] Unbound by any particular theory, viral particles containing a mutant VSV-G protein with a targeting motif, as provided herein, can be used to express a target heterologous molecule in target cells. Thus, for example, CARs can be expressed in T cells targeted by viral particles pseudotyped with VSV-G protein as provided herein. When T cells are the intended target, the viral particles may contain a targeting motif that binds to a target on the surface of the T cells, such as, 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.

[0296] In some implementations, the pseudotyped viral particles are recombinant lentiviruses. In some implementations, the recombinant pseudotyped viral particles are capable of replication. In some implementations, the recombinant pseudotyped viral particles are not capable of replication.

[0297] Exemplary virus particles:

[0298] In some embodiments, a viral particle is provided comprising a heterologous viral glycoprotein and a targeting portion, 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 wherein the targeting portion comprises an amino acid sequence of SEQ ID NO:89. In some embodiments, the viral particle further comprises a nucleic acid molecule encoding a target heterologous molecule. In some embodiments, the target heterologous molecule is as provided herein. In some embodiments, the target heterologous molecule is a CAR as provided herein. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity with SEQ ID NO:90, at least 90% identity with SEQ ID NO:90, at least 95% identity with SEQ ID NO:90, at least 99% identity with SEQ ID NO:90, or at least 100% identity with SEQ ID NO:90.

[0299] In some embodiments, a viral particle is provided comprising a heterologous viral glycoprotein and a targeting portion, wherein the heterologous viral glycoprotein comprises an amino acid sequence selected from SEQ ID NO:22 or SEQ ID NO:23, and wherein the targeting portion comprises an amino acid sequence of SEQ ID NO:89. In some embodiments, the viral particle further comprises a nucleic acid molecule encoding a target heterologous molecule. In some embodiments, the target heterologous molecule is as provided herein. In some embodiments, the target heterologous molecule is a CAR as provided herein. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity with SEQ ID NO:90, at least 90% identity with SEQ ID NO:90, at least 95% identity with SEQ ID NO:90, at least 99% identity with SEQ ID NO:90, or at least 100% identity with SEQ ID NO:90.

[0300] In some embodiments, a viral particle is provided comprising a heterologous viral glycoprotein and a targeting portion, wherein the heterologous viral glycoprotein comprises the amino acid sequence of SEQ ID NO:22, and wherein the targeting portion comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the viral particle further comprises a nucleic acid molecule encoding a target heterologous molecule. In some embodiments, the target heterologous molecule is as provided herein. In some embodiments, the target heterologous molecule is a CAR as provided herein. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity with SEQ ID NO:90, at least 90% identity with SEQ ID NO:90, at least 95% identity with SEQ ID NO:90, at least 99% identity with SEQ ID NO:90, or at least 100% identity with SEQ ID NO:90.

[0301] In some embodiments, a viral particle is provided comprising a heterologous viral glycoprotein and a targeting portion, wherein the heterologous viral glycoprotein comprises the amino acid sequence of SEQ ID NO:23, and wherein the targeting portion comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the viral particle further comprises a nucleic acid molecule encoding a target heterologous molecule. In some embodiments, the target heterologous molecule is as provided herein. In some embodiments, the target heterologous molecule is a CAR as provided herein. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity with SEQ ID NO:90, at least 90% identity with SEQ ID NO:90, at least 95% identity with SEQ ID NO:90, at least 99% identity with SEQ ID NO:90, or at least 100% identity with SEQ ID NO:90.

[0302] List of implementation plans

[0303] The following examples are illustrative of the compounds, compositions, and methods described herein, and are not intended to limit the scope thereof. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.

[0304] 1. A method for producing a concentrated sterile solution containing a viral vector, the method comprising the following steps:

[0305] Clarify the solution containing the cell culture medium and the viral vector;

[0306] The clarified solution containing the viral vector is filtered through a first chromatographic filter to produce a filtered clarified solution containing the viral vector.

[0307] The filtered, clarified solution containing the viral vector is passed through a protein-coated sterile membrane to produce a sterile solution containing the viral vector; and

[0308] The sterile solution containing the viral vector is concentrated to produce a concentrated sterile solution containing the viral vector.

[0309] 2. The method of embodiment 1, wherein the method further comprises mixing the concentrated sterile solution containing the viral vector with one or more sterile pyrogen-free buffers and / or excipients to produce a sterile pharmaceutical composition containing the viral vector.

[0310] 3. The method of embodiment 1, wherein the method further comprises collecting cell culture medium from the cell culture that produced the viral vector before clarifying the solution.

[0311] 4. The method of any one of embodiments 1-3, the method further comprising filtering the filtered clarified solution containing the viral vector through a second chromatographic filter before passing the filtered clarified solution through the protein-coated sterile membrane to produce a twice-filtered clarified solution containing the viral vector.

[0312] 5. The method of embodiment 1, wherein the protein-coated sterile membrane comprises a membrane pre-rinsed with a protein solution.

[0313] 6. The method of embodiment 1, wherein the method further comprises coating a sterile membrane with a protein solution to produce a protein-coated sterile membrane.

[0314] 7. The method of embodiment 5 or 6, wherein the protein solution comprises one or more components selected from the group consisting of: protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), sodium chloride, or combinations thereof.

[0315] 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), about 100 mM to about 400 mM sodium chloride and a pH of about 6.0 to about 8.0.

[0316] 9. The method of embodiment 7, wherein the protein comprises human serum albumin, myosin, bovine serum albumin, immunoglobulin, immunoglobulin fragments, fibronectin, hylocinin, or any combination thereof.

[0317] 10. The method of embodiment 9, wherein the human serum albumin is recombinant human serum albumin, such as that produced by plants.

[0318] 11. The method of embodiment 9, wherein the human serum albumin is produced by a plant.

[0319] 12. The method of embodiment 9, wherein the human serum albumin is non-recombinant human serum albumin.

[0320] 13. The method of embodiment 12, wherein the non-recombinant human serum albumin is USP grade non-recombinant human serum albumin.

[0321] 14. The method of embodiment 6, wherein the protein-coated sterile membrane is positively charged prior to being coated with the protein.

[0322] 15. The method of embodiment 6, wherein the membrane is a polyethersulfone (PES) membrane, a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane.

[0323] 16. The method of any one of embodiments 1-15, wherein the clarified solution is filtered through the first chromatographic filter by capture chromatography.

[0324] 17. The method of embodiment 16, wherein the capture chromatography is ion exchange chromatography, affinity chromatography, hydrophobic chromatography, size exclusion chromatography, or multimode chromatography.

[0325] 18. The method of embodiment 4, wherein the filtered solution is filtered through a second chromatographic filter by fine purification chromatography.

[0326] 19. The method of embodiment 18, wherein the fine purification chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimode chromatography.

[0327] 20. The method of any one of embodiments 1-19, the method further comprising digesting the DNA in the solution containing the cell culture medium and the viral vector prior to clarifying the solution.

[0328] 21. The method of any one of embodiments 1-20, the method further comprising digesting the DNA in the solution containing the viral vector after filtering the clarified solution.

[0329] 22. The method of any one of embodiments 1-21, wherein the concentration step comprises concentrating the viral vector by tangential flow filtration.

[0330] 23. The method of embodiment 22, wherein the tangential flow filtering comprises two-stage tangential flow filtering.

[0331] 24. The method as described in embodiment 22 or 23, wherein the tangential flow filter utilizes 1.0 mm ID fiber and a 750 kDa pore size.

[0332] 25. The method of any one of embodiments 1-24, wherein a concentrated sterile solution containing the viral vector is mixed with one or more sterile pyrogen-free buffers and / or excipients in a sterile environment to produce a sterile pharmaceutical composition containing the viral vector.

[0333] 26. The method of embodiment 25, wherein the mixing with one or more sterile pyrogen-free buffers and / or excipients includes a buffer exchange step.

[0334] 27. The method as described in embodiment 1, wherein the method further comprises:

[0335] Before clarifying the solution:

[0336] i) Obtain a solution containing cell culture medium and the viral vector; and

[0337] ii) Digest the DNA in a solution containing cell culture medium and the viral vector to produce a first digested solution containing the viral vector;

[0338] Clarifying the solution includes clarifying the digested solution containing the viral vector to produce a clarified, digested solution containing the viral vector;

[0339] After filtering the clarified solution and before passing the filtered clarified solution through a protein-coated sterile membrane:

[0340] i) Digesting the DNA in a filtered, clarified, digested solution containing the viral vector to produce 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 chromatographic filter to produce a twice-filtered, clarified, twice-digested solution containing the viral vector.

[0341] The protein-coated sterile membrane described herein comprises a membrane pre-rinsed with a protein solution.

[0342] The protein solution contains about 0.1% w / v to 2.0% w / v of protein, about 10 mM to about 30 mM of 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), about 100 mM to about 400 mM of sodium chloride, and a pH of about 6.0 to about 8.0.

[0343] 28. A method for producing a concentrated sterile solution containing a viral vector, the method comprising the following steps:

[0344] The DNA in the solution containing the viral vector is digested to produce a first digested solution containing the viral vector;

[0345] Clarify the digested solution containing the viral vector to produce a clear, digested solution containing the viral vector;

[0346] The clarified, digested solution containing the viral vector is filtered through a first chromatographic filter to produce a filtered, clarified, digested solution containing the viral vector.

[0347] The DNA in a filtered, clarified, digested solution containing the viral vector is digested to produce a filtered, clarified, twice-digested solution containing the viral vector; the filtered, clarified, twice-digested solution containing the viral vector is filtered through a second chromatographic filter to produce a twice-filtered, clarified, twice-digested solution containing the viral vector.

[0348] The twice-filtered, clarified, twice-digested solution containing the viral vector is passed through a protein-coated sterile membrane to produce a sterile solution containing the viral vector; and

[0349] The sterile solution containing the viral vector is concentrated to produce a concentrated sterile solution containing the viral vector.

[0350] 29. The method of embodiment 28, wherein the digestion of DNA is performed using a nuclease having DNase activity, RNase activity, or a combination thereof, such as DNase I, DENARASE, Cryonase, etc.

[0351] 30. The method of embodiment 28, wherein the second chromatographic filter is a resin.

[0352] 31. The method of embodiment 28, the method further comprising mixing the concentrated sterile solution containing the viral vector with one or more sterile pyrogen-free buffers and / or excipients to produce a sterile pharmaceutical composition containing the viral vector.

[0353] 32. The method of embodiment 28, wherein the method further comprises collecting culture medium from the cell culture that produces the viral vector.

[0354] 33. The method of embodiment 28, wherein the method further comprises collecting culture medium from the cell culture that produced the viral vector prior to digesting DNA.

[0355] 34. The method of embodiment 28, the method further comprising collecting culture medium from the cell culture that produced the viral vector before clarifying the digested solution.

[0356] 35. The method of embodiment 28, wherein the protein-coated sterile membrane comprises a membrane pre-rinsed with a protein solution.

[0357] 36. The method of embodiment 35, the method further comprising coating a sterile membrane with a protein solution to produce a protein-coated sterile membrane.

[0358] 37. The method of embodiment 36, wherein the protein solution comprises one or more of the following: protein, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), histidine, phosphate, HEPES ((4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), sodium chloride, or combinations thereof.

[0359] 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), about 100 mM to about 400 mM sodium chloride and a pH of about 6.0 to about 8.0.

[0360] 39. The method of any one of embodiments 37-38, wherein the protein is human serum albumin.

[0361] 40. The method of embodiment 39, wherein the human serum albumin is recombinant human serum albumin.

[0362] 41. The method of embodiment 39, wherein the human serum albumin is produced by a plant.

[0363] 42. The method of embodiment 39, wherein the human serum albumin is non-recombinant human serum albumin.

[0364] 43. The method of embodiment 42, wherein the non-recombinant human serum albumin is USP grade non-recombinant human serum albumin.

[0365] 44. The method of any one of embodiments 35-43, wherein the protein-coated sterile membrane is positively charged prior to being coated with protein.

[0366] 45. The method of any one of embodiments 35-44, wherein the membrane is a polyethersulfone (PES), a nylon membrane, or a polyvinylidene fluoride (PVDF) membrane.

[0367] 46. ​​The method of any one of embodiments 28-45, wherein 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, Approximately 0.25 μm, approximately 0.26 μm, approximately 0.27 μm, approximately 0.28 μm, approximately 0.29 μm, approximately 0.30 μm, approximately 0.31 μm, approximately 0.32 μm, approximately 0.33 μm, approximately 0.34 μm, approximately 0.35 μm, approximately 0.36 μm, approximately 0.37 μm, approximately 0.38 μm, approximately 0.39 μm, approximately 0.40 μm, approximately 0.41 μm, approximately 0.42 μm, approximately 0.43 μm, approximately 0.44 μm, or approximately 0.45 μm.

[0368] 47. The method of any one of embodiments 28-46, wherein the protein-coated sterile membrane does not significantly retain the viral vector.

[0369] 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%, or 99% or about 100%.

[0370] 49. The method of any one of embodiments 28-48, wherein the clarified solution is filtered through the first chromatographic filter by capture chromatography.

[0371] 50. The method of embodiment 49, wherein the capture chromatography is ion exchange chromatography, affinity chromatography, hydrophobic chromatography, size exclusion chromatography, or multimode chromatography.

[0372] 51. The method of embodiment 28, wherein the first filtered solution is filtered through a second filter by fine purification chromatography.

[0373] 52. The method of embodiment 51, wherein the fine purification chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimode chromatography.

[0374] 53. The method of any one of embodiments 28-52, wherein the concentration step comprises concentrating the viral vector by tangential flow filtration.

[0375] 54. The method of embodiment 53, wherein the tangential flow filtering comprises two-stage tangential flow filtering.

[0376] 55. The method of embodiment 53 or embodiment 54, wherein the tangential flow filter utilizes 1.0 mm ID fiber and a 750 kDa pore size.

[0377] 56. The method of any one of embodiments 28-55, wherein a concentrated sterile solution containing the viral vector is mixed with an excipient in a sterile environment to produce a sterile pharmaceutical composition containing the viral vector.

[0378] 57. The method of embodiment 56, wherein the mixing with the excipient includes a buffer exchange step.

[0379] 58. The method of any one of embodiments 1-57, the method further comprising freezing the concentrated sterile solution containing the viral vector or the sterile pharmaceutical composition containing the viral vector.

[0380] 59. The method of embodiment 58, wherein the freezing is performed by controlled-rate freezing.

[0381] 60. The method of any one of embodiments 1-59, wherein the viral vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector.

[0382] 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 described.

[0383] 62. The method as described in any one of embodiments 1-61, wherein the concentration of the viral vector is about 1 × 10⁻⁶ before the solution is passed through the sterile filter. 4 To approximately 1×10 6 Transduction units (TU) / mL.

[0384] 63. The method as described in any one of embodiments 1-62, wherein the concentration of the viral vector is 1 × 10⁻⁶ before the solution is passed through the sterile filter. 7 Up to 1×10 9 Viral particles (vp) / mL.

[0385] 64. The method of any one of the foregoing embodiments, wherein the viral vector comprises a VSV-G polypeptide.

[0386] 65. The method as described in any of the preceding embodiments, wherein the viral vector, such as a lentiviral vector, is pseudotyped with a VSV-G polypeptide containing a mutation corresponding to the mutation at position 182 of SEQ ID NO:2.

[0387] 66. The method of embodiment 64 or 65, wherein the VSV-G polypeptide comprises the amino acid sequence of SEQ ID NO:2 having a mutation at position 182 and having at least 95% identity with SEQ ID NO:2.

[0388] 67. The method as described in embodiments 64-66, wherein the VSV-G polypeptide contains an I182E or I182D mutation, as compared to SEQ ID NO:2.

[0389] 68. The method of any one of embodiments 64-67, wherein the VSV-G polypeptide comprises the amino acid sequence of SEQ ID NO:1 having a mutation at position 198 and having at least 70% identity with SEQ ID NO:2.

[0390] 69. The method of any one of embodiments 64-68, wherein the VSV-G polypeptide comprises a mutation corresponding to I182D or I182E compared to the sequence of SEQ ID NO:2.

[0391] 70. The method of any one of embodiments 64-69, wherein the VSV-G polypeptide comprises an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:4.

[0392] 71. The method of any one of embodiments 64-70, wherein the VSV-G polypeptide comprises the amino acid sequence of SEQ ID NO:4.

[0393] 72. The method of any one of embodiments 64-69, wherein the VSV-G polypeptide comprises an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:5.

[0394] 73. The method of any one of embodiments 64-72, wherein the VSV-G polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0395] 74. The method of any one of embodiments 64-73, wherein the VSV-G polypeptide further comprises a mutation corresponding to positions 8, 10, 47, 209 and / or 354 in the VSV-G protein compared to SEQ ID NO:2.

[0396] 75. The method of any one of embodiments 64-74, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 8 in SEQ ID NO:2, wherein the mutation is any amino acid other than Y that is different from the amino acid indicated at that position in SEQ ID NO:2.

[0397] 76. The method of any one of embodiments 64-75, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 209 in SEQ ID NO:2, wherein the mutation is any amino acid other than H that is different from the amino acid indicated at that position in SEQ ID NO:2.

[0398] 77. The method of any one of embodiments 64-76, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 47 in SEQ ID NO:2, wherein the mutation is any amino acid other than K or R that is different from the amino acid indicated at that position in SEQ ID NO:2.

[0399] 78. The method of any one of embodiments 64-77, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 354 in SEQ ID NO:2, wherein the mutation is any amino acid other than K or R that is different from the amino acid indicated at that position in SEQ ID NO:2.

[0400] 79. The method of any one of embodiments 64-78, wherein the VSV-G polypeptide further comprises a mutation corresponding to position 10 in SEQ ID NO:2, wherein the mutation is any amino acid other than Q or N that is different from the amino acid indicated at that position in SEQ ID NO:2.

[0401] 80. The method of any one of embodiments 64-79, wherein the VSV-G polypeptide further comprises a substitution at position 47 or at position 354 or at both positions 47 and 354, wherein each position is independently substituted by A, G, F, Q or N.

[0402] 81. The method of any one of embodiments 64-80, wherein the VSV-G polypeptide comprises a substitution at position 8, wherein the substitution is H8A, H8I, H8V, H8L, etc.

[0403] 82. The method of any one of embodiments 64-81, wherein the VSV-G polypeptide comprises a substitution at position 47, wherein the substitution is K47Q or K47N.

[0404] 83. The method of any one of embodiments 64-82, wherein the VSV-G polypeptide comprises a substitution H8A and / or a K47Q mutation.

[0405] 84. The method of any one of embodiments 94-83, wherein the VSV-G polypeptide comprises Q10A, Q10R or Q10K substitution.

[0406] 85. The method of any one of embodiments 64-84, wherein the VSV-G polypeptide further comprises a mutation corresponding to the mutation at position 214 and / or 352 of SEQ ID NO:2.

[0407] 86. The method of embodiment 85, wherein the VSV-G polypeptide comprises a T214N and / or T352A mutation.

[0408] 87. The method of any one of embodiments 64-86, wherein the viral particles comprise a VSV-G polypeptide, the VSV-G polypeptide comprising a substitution at at least one of position I182 and T214 and T352 of SEQ ID NO:2.

[0409] 88. The method of embodiment 87, wherein the VSV-G polypeptide comprises substitutions at positions I182, T214 and T352 of SEQ ID NO:2.

[0410] 89. The method as described in embodiment 87 or 88, wherein the substitution at position 182 is I182D or I182E, the substitution at position 214 is T214N, and the substitution at position 352 is T352A.

[0411] 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.

[0412] 91. The method of any one of embodiments 87-89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO:23.

[0413] 92. The method of any one of embodiments 87-89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO:22.

[0414] 93. The method of any one of embodiments 87-89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO:24.

[0415] 94. The method of any one of embodiments 87-89, wherein the VSV-G polypeptide comprises the sequence of SEQ ID NO:25.

[0416] 95. The method of any one of embodiments 1-94, wherein the viral vector comprises a targeting portion.

[0417] 96. The method of embodiment 95, wherein the targeting portion is bound to CD7.

[0418] 97. The method of embodiment 96, wherein the targeting portion comprises a polypeptide comprising: (i) a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO:30; the heavy chain CDR2 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, or a variant thereof; and (ii) a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, 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; or a variant thereof.

[0419] 98. The method of embodiment 96 or 97, wherein the targeting portion comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:42 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:43.

[0420] 99. The method of any one of embodiments 96-98, wherein the targeting portion comprises an antigen-binding domain comprising the amino acid sequence of SEQ ID NO:44 or SEQ ID NO:45.

[0421] 100. The method as described in embodiment 95, wherein the targeting portion is bound to CD8.

[0422] 101. The method of embodiment 100, wherein the targeting portion comprises a polypeptide comprising: (i) a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO:48; the heavy chain CDR2 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, or a variant thereof; and (ii) a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, 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; or a variant thereof.

[0423] 102. The method as described in embodiment 100 or 101, wherein the targeting portion comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:60 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:61.

[0424] 103. The method of any one of embodiments 100-102, wherein the targeting portion comprises an antigen-binding domain, the antigen-binding domain comprising the amino acid sequence of SEQ ID NO:62 or SEQ ID NO:63.

[0425] 104. The method of any one of embodiments 95-103, wherein the targeting portion comprises T-S1, wherein T is a target-binding domain and S1 is a stem portion, wherein the targeting portion is attached to the surface of the viral vector via the stem portion S1.

[0426] 105. The method of embodiment 104, wherein the stem portion S1 comprises a variant Fc protein.

[0427] 106. The method of embodiment 105, wherein the stem portion S1 comprises the formula L1-Fc-L2-X1, wherein:

[0428] L1 is a connector or does not exist;

[0429] Fc is a variant of the Fc protein;

[0430] L2 is a connector or does not exist; and

[0431] X1 is a polypeptide containing a transmembrane domain.

[0432] The target portion having formula T-S1 has formula T-L1-Fc-L2-X1.

[0433] 107. The method as described in embodiment 106, wherein X1 comprises having the formula ECD-T M -ICD polypeptide, wherein:

[0434] ECD is the extracellular domain or a fragment thereof of a cell surface protein, or it may not exist.

[0435] T M It is a transmembrane domain of a transmembrane protein; and

[0436] ICD is an intracellular domain or a protein that helps incorporate the target portion into the envelope of the viral particle, or it may be absent.

[0437] The targeting portion having the formula T-L1-Fc-L2-X1 has the formula T-L1-Fc-L2-ECD-T M -ICD.

[0438] 108. The method as described in implementation scheme 107, wherein:

[0439] T contains the amino acid sequence of SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:62 or SEQ ID NO:63;

[0440] L1 contains the amino acid sequence of SEQ ID NO:72;

[0441] Fc contains the amino acid sequence of SEQ ID NO:82;

[0442] L2 does not exist;

[0443] ECD contains the amino acid sequence of SEQ ID NO:83;

[0444] T M The amino acid sequence containing SEQ ID NO:84; and

[0445] The ICD contains an amino acid sequence with an env-incorporated motif, wherein the env-incorporated motif contains the amino acid sequence of SEQ ID NO:85.

[0446] 109. The method of embodiment 104, wherein the stem portion S1 does not contain the variant Fc protein.

[0447] 110. The method of embodiment 109, wherein the stem portion S1 comprises formula L3-X1, wherein:

[0448] L3 is a flexible peptide linker, and

[0449] X1 is a polypeptide containing a transmembrane domain.

[0450] The target portion having formula T-S1 has formula T-L3-X1.

[0451] 111. The method as described in embodiment 110, wherein X1 comprises having the formula ECD-T M -ICD polypeptide, wherein:

[0452] ECD is the extracellular domain or a fragment thereof of a cell surface protein, or it may not exist.

[0453] T M It is a transmembrane domain of a transmembrane protein; and

[0454] ICD is an intracellular domain or a protein that helps incorporate the target portion into the envelope of the viral particle, or it may be absent.

[0455] The targeting portion having formula T-L3-X1 has formula T-L3-ECD-T M -ICD.

[0456] 112. The method as described in implementation scheme 111, wherein:

[0457] T contains the amino acid sequence of SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:62 or SEQ ID NO:63;

[0458] L3 contains the amino acid sequence of SEQ ID NO:64, where n is 1, 2 or 4;

[0459] ECD contains the amino acid sequence of SEQ ID NO:86;

[0460] T M The amino acid sequence containing SEQ ID NO:87; and

[0461] The ICD contains an amino acid sequence with an env-incorporated motif, wherein the env-incorporated motif contains an amino acid sequence of SEQ ID NO:85 or SEQ ID NO:88.

[0462] 113. The method of embodiment 104, wherein the targeting portion of the included T-S1 comprises an amino acid sequence having at least 90% identity with SEQ ID NO:89.

[0463] 114. The method of embodiment 104, wherein the targeting portion of the included T-S1 comprises an amino acid sequence having at least 95% identity with SEQ ID NO:89.

[0464] 115. The method of embodiment 104, wherein the targeting portion of the included T-S1 comprises the amino acid sequence of SEQ ID NO:89.

[0465] 116. The method of any one of embodiments 1-115, wherein the viral vector further comprises a nucleic acid molecule encoding a target heterologous molecule.

[0466] 117. The method of embodiment 116, wherein the target heterologous molecule is siRNA, shRNA, non-coding RNA (e.g., guide RNA of the CRISPR system), peptide, polypeptide, protein, viral payload, viral genome, or a combination thereof.

[0467] 118. The method as described in embodiment 116 or 117, wherein the target heterologous molecule is a chimeric antigen receptor (“CAR”).

[0468] 119. The method of embodiment 118, wherein the CAR comprises an antigen-binding domain, the antigen-binding domain comprising an antibody or a fragment thereof.

[0469] 120. The method of embodiment 119, wherein the antibody or fragment thereof is an antibody, scFv antibody, antigen-binding domain, an ankyrin repeat sequence, VHH domain antibody, nanobody, single-domain antibody or FN3 antibody.

[0470] 121. The method as described in embodiment 119 or 120, wherein the antigen-binding domain of the CAR binds to CD20.

[0471] 122. The method of embodiment 121, wherein the antigen-binding domain binding to CD20 comprises a polypeptide containing a light chain and a heavy chain, the light chain and the heavy chain comprising: a heavy chain variable region of the heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO:73; and a light chain variable region of the light chain having at least 90% identity with the amino acid sequence of SEQ ID NO:74.

[0472] 123. The method as described in embodiment 121 or 122, wherein the antigen-binding domain that binds to CD20 comprises the amino acid sequence of SEQ ID NO:75 or SEQ ID NO:76.

[0473] 124. The method of embodiment 121, wherein the antigen-binding domain binding to CD20 comprises a polypeptide containing a light chain and a heavy chain, said light chain and heavy chain comprising: a heavy chain variable region of the heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO:78; and a light chain variable region of the light chain having at least 90% identity with the amino acid sequence of SEQ ID NO:79.

[0474] 125. The method as described in embodiment 123 or 124, wherein the antigen-binding domain that binds to CD20 comprises the amino acid sequence of SEQ ID NO:80 or SEQ ID NO:81.

[0475] 126. The method of any one of embodiments 118-121, wherein the CAR comprises an amino acid sequence having at least 90% identity with SEQ ID NO:90.

[0476] 127. The method of any one of embodiments 118-121, wherein the CAR comprises an amino acid sequence having at least 95% identity with SEQ ID NO:90.

[0477] 128. The method of any one of embodiments 118-121, wherein the CAR comprises the amino acid sequence of SEQ ID NO:90.

[0478] Example

[0479] Various embodiments are described in further detail with reference to the following examples. Unless otherwise stated, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the embodiments should in no way be construed as limited to the following embodiments, but should be construed as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0480] Example 1: Method for manufacturing viral vector.

[0481] Viral vector generation: Viral vectors were generated in 293T cells. During transfection, a five-plasmid system (pModified-VSVg, pGag-Pol, pRev, pBinder, and LV plasmids expressing GFP or CAR20) was used to generate self-inactivated lentiviruses (LVs) pseudotyped with VSV-G protein mutations corresponding to I182E, T214N, and T352A, expressing GFP or other target genes (such as chimeric antigen receptors (CAR20, chimeric antigen receptors with an antigen-binding domain that binds to CD20) under the EF1A promoter. 300 ng / cm 2 The plasmids were used in T175 and CellSTACK TMTransfection was performed in cell culture dishes using PEIpro-mediated transfection at a ratio of 1 mcg:1 mcL DNA:polyvinylimine (PEI). The cell culture medium used during transfection was DMEM supplemented with 10% or 3% fetal bovine serum (FBS).

[0482] DNase digestion: 48 hours after transfection, the cell culture was spiked with 2 mM MgCl2 and 150 units of DNase / mL for 2 hours. The cell culture was incubated at 37°C in an incubator. After incubation, the cell culture was cooled to room temperature.

[0483] Clarification: at 250L / m 2 The flux use of h(LMH) has PP3 1.2 microns in series A PP3 0.45-micron peristaltic pump clarifies digested cell cultures. Load 80-200 L of digested cell cultures / m³ into the clarification filter. 2 Each clarifier filter area. Pre-condition the filter with water, and finally use DMEM + 3% FBS (10L / m²). 2 Then use DMEM (5L / m 2 After filtration, the filter is rinsed with DMEM with the minimum filter pore volume and emptied for product collection.

[0484] Ion exchange chromatography: Q-ion exchange membranes were used for LV capture and purification steps after clarification. The membranes were conditioned to 25 membrane volumes (MV) using 20 mM Tris, 100 mM NaCl, pH 7.5 (with or without 4% sucrose) at a flow rate of 5 mV / min. After conditioning, the clarified cell cultures were used... The Avant 25 or Avant 150 chromatography system was pumped through at a flow rate of 5 mV / min. Q membrane. 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 loading and weakly bound molecules, and then eluted with 20 mM Tris 1000 mM NaCl pH 7.5 (with or without 4% sucrose) to recover LV. The eluted LV was collected at 1 mV immediately after the start of the elution step until the UV signal at 280 nm flattened. Immediately after elution, the LV eluent was diluted with 20 mM Tris pH 7.5 (with or without 4% sucrose) at a 1:2 or 1:6 ratio to stabilize LV functionality. A 1:2 dilution ratio resulted in a higher conductivity (approximately 50 mS / cm), while a 1:6 dilution ratio resulted in a conductivity of approximately 12.5 mS / cm.

[0485] Post-chromatographic DNase: The diluted chromatographic product was DNase digested using the same type of DNase as the first post-harvest digestion. The diluted chromatographic product was spiked with 2 mM MgCl2 and 100 units of DNase / mL and stored at 4°C for 16–24 hours. After incubation, the product was warmed to room temperature.

[0486] Fine purification chromatography: Pack Capto Core 700 resin to a bed height of 10-20 cm. After packing, condition the Capto Core 700 column with 10 column volumes (CV) of 20 mM Tris, 100 mM NaCl, pH 7.5 (with or without 4% sucrose). The Q-chromatographic product was loaded onto a CaptoCore 700 column at a column loading rate of 10–20 mL / mL resin volume. LV flowed through the CaptoCore 700, and after loading, the column was washed to recover any remaining LV flow-through. Product collection was based on UV absorbance at 280 nm, starting when UV280 began to rise and ending when UV280 decreased after washing.

[0487] Aseptic filtration: Data on the use of two types of aseptic filters in the production of the positively charged aseptic filters described in this disclosure: 2XLG has a 0.8 μm / 0.2 μm bilayer film and Pall. It has a 0.8-micron / 0.2-micron double membrane. Rinse the sterile filter with water, and then rinse with 1% w / v water without pre-rinsing. Pre-rinse (20 mL rinse / cm) 2 Operating at the filter area, then at 50-200 L / m² 2 The Capto Core 700 product was introduced at a loading ratio and a flow rate of 250 LMH. After loading the feed, the sterile filter was flushed with 20 mM Tris, 100 mM NaCl, and pH 7.5 to recover the retained volume in the filter and tubing.

[0488] Final Concentration, Buffer Exchange, and Controlled-Rate Freezing: For the final concentration step and buffer exchange, a two-stage tangential flow filtration (TFF) was used to allow for a high concentration factor. For both stages, modified PES (mPES) hollow fiber modules with 1.0 mm ID fibers and a 750 kDa pore size were used, except that the first-stage filter area was approximately 20 times larger than the second-stage filter area. The hollow fibers were rinsed with water and 20 mM Tris, 100 mM NaCl (pH 7.5) before use. In the first stage, the aseptically filtered product was concentrated to approximately 20×. The 20× concentrated product was then further concentrated to approximately 15× using the second-stage TFF, at which point the product was buffer-exchanged for 6–8 perfiltration volumes (DV) with 20 mM Tris, 100 mM NaCl, and 4% sucrose (pH 7.5). After the buffer exchange, the product was further concentrated by 2×, and human serum albumin was added to a final concentration of 0.5% v / v. The product was then frozen at -80°C.

[0489] Example 2: Viral vector recovery rate with and without pre-rinsing charged sterile filters.

[0490] In the initial development of the aseptic filtration procedure, the filter was passed through three types of aseptic filters (Pall) in a 20 mM Tris 100 mM NaCl pH 7.5 buffer system. 0.8 / 0.2 micrometers 2XLG 0.8 / 0.2 microns and Millipore Multimedia Unconcentrated purified lentiviral vectors (approximately 2 × 10⁻⁶ micrometers) at 1.2 / 0.5 / 0.22 micrometers 8 The recovery rate (vp / mL) was less than 50%. The sterile filters tested were made of modified polyethersulfone (PES) or polyvinylidene fluoride (PVDF), which are reported to carry a positive charge at approximately pH 6 to approximately 8 (Basic Aspects of Membrane Science and Engineering, N. Hilal, D. Johnson, in Comprehensive Membrane Science and Engineering, 2010).

[0491] For two feed conductivity values ​​(12.5 mS / cm and 41.0 mS / cm), carrier recovery was compared between un-pre-rinsed and pre-rinsed PES sterile filters with 1% recombinant human albumin. At lower conductivity values, pre-rinsing with 1% recombinant human albumin solution before sterile filtration resulted in twice the recovery compared to un-pre-rinsed PES filters. For higher conductivity feeds, pre-rinsing the sterile filter resulted in increased marginal yield compared to un-pre-rinsed feeds. These results are presented in... Figure 2 middle.

[0492] Conversely, when using Sartobran P sterile filters, pre-rinsing with recombinant human albumin solution resulted in a modest increase in virus recovery. Figure 3 Sartobran P is made of cellulose acetate material, which has been reported to carry a negative charge (Li, Nancy et al., “Synthesis and Characterization of a High Flux Nanocellulose-Cellulose Acetate Nanocomposite Membrane.” Membranes, Vol. 9, 670. June 6, 2019, doi:10.3390 / membranes9060070). This suggests that the loss of viral vectors during sterile filtration is primarily due to adsorption onto the filter via charge, which can be avoided by coating with proteins such as recombinant human serum albumin.

[0493] Example 3: Initial pre-coating of sterile filters.

[0494] The effect of protein pre-coating of sterile filters to reduce carrier loss was investigated. Prior to lentivirus filtration, water and 20 mM Tris + 100 M NaCl at pH 7.5 were used, or water was followed by 20 mM Tris + 100 mM NaCl + 1%. Viral vector recovery of unconcentrated purified viral vectors was evaluated using a sterile filter rinsed at pH 7.5 at conductivity levels of 12.5 mS / cm and 30 mS / cm. The sterile filter used in this study was a Pall filter. 0.8 / 0.2 micrometers, composed of modified polyethersulfone (PES). The protein used for pre-coating is recombinant human serum albumin, such as... (Invitria). Results are shown in Figure 4 In. Figure 4Experimental filtration results are represented as Exb(+), and control filtration results without recombinant human serum albumin washing are represented as Exb(-). As demonstrated by three orthogonal analysis methods, pre-coating the sterile filter with recombinant human serum albumin solution increased the recovery rate of lentiviral vectors passing through the sterile filter by up to 2-fold. Figure 4 Therefore, this indicates that the loss of viral vectors during sterile filtration is mainly due to adsorption onto the filter, which can be avoided by coating with proteins such as recombinant human serum albumin.

[0495] Example 4: Pre-coating of a negatively charged sterile filter.

[0496] The effects of protein pre-coating on negatively charged sterile filters were also investigated. Protein pre-coating was applied to... P sterile filter, which is made of cellulose acetate material, reportedly carrying a negative charge (Synthesis and Transaction of a High Flux Nanocellulose-Cellulose Acetate Nanocomposite Membrane; Nancy Li, Jackie Zheng, Pejman Hadi, Mengyi ng Yang, Xiangyu Huang, Hongyang Ma, Harold W. Walker and Beja min S. Hsiao; Membranes Vol. 9 (6), 70, (2019)). The observed improvement was smaller compared to the improvement observed with positively charged sterile filters ( Figure 5 The control experiment without protein pre-coating yielded approximately 80% vector recovery, while the experiment with protein pre-coating showed approximately 70% yield. This result indicates that the lentiviral vector does not tend to be lost due to charge interactions with the Sartobran P filter (see [link to study]). Figure 5Lentiviral vectors themselves carry a negative charge 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, it is likely that viral vector recovery will not be affected when using a negatively charged filter during pre-rinsing of the filter, since the lentiviral vector carries the same charge as the filter.

[0497] Example 5: Effect of pre-coating sterile filters with HSA and recombinant HSA.

[0498] Pre-coating sterile filters with human serum albumin (HSA) to reduce the impact of carrier loss, and recombinant human serum albumin. Comparison. Under the first condition ( Figure 6 (The leftmost data) Before lentivirus filtration, use water and 20mM Tris + 100mM NaCl at pH 7.5, or use water followed by 20mM Tris + 100mM NaCl + 1%.

[0499] The viral vector recovery rate of the unconcentrated purified viral vector was evaluated using a sterile filter rinsed at pH 7.5 at 12.5 mS / cm. Under the second condition ( Figure 6 (Intermediate data) Before lentivirus filtration, the viral vector recovery rate of the unconcentrated purified viral vector was evaluated at 12.5 mS / cm using a sterile filter rinsed with water and 20 mM Tris and 100 mM NaCl at pH 7.5, or with water followed by rinsing with 20 mM Tris and 100 mM NaCl + 1% HSA at pH 7.5. Under the third condition ( Figure 6(Data on the far right) Before lentivirus filtration, the viral vector recovery rate of the unconcentrated purified viral vector was evaluated at 12.5 mS / cm using a sterile filter rinsed with water and 20 mM Tris-100 M NaCl pH 7.5, or with water followed by rinsing with 20 mM Tris-100 M NaCl + 0.1% HSA pH 7.5. The sterile filter used in this study was... 2XLG 0.8 / 0.2 microns, composed of modified polyethersulfone (PES). When determined by genome copy number or viral particle count via p24, use 1% or 0.1% HSA, or... Solution pre-coating of sterile filters resulted in comparable lentiviral vector recoveries. This indicates that both HSA (e.g., non-recombinant HSA) and recombinant HSA can be used as pre-coated protein solutions for sterile filters.

[0500] Example 6: The mutation at position 182 of VSV-G eliminated the LDL-R interaction but preserved the fusion properties, such as... Figures 7A-8B As shown.

[0501] 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). Previous studies have shown that individual mutations of H8A and K47Q partially “blind” VSV-G, reducing its binding to LDL-R (the natural cellular receptor for VSV) (PMID:29531262, DOI:10.1038 / s41467-018-03432-4). In this experiment, a single binder molecule consisting of a CD7-targeting scFv (clone MT701) fused to an IgG “stem” carrying the CD28 transmembrane domain was used.

[0502] 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 TherapySystems) 48 hours before transduction.

[0503] Lentiviral particle generation. Recombinant lentiviral particles co-expressing VSV-G glycoprotein and binding molecule were generated by transfecting HEK293T cells with plasmids using Lipofectamine 3000 (ThermoFisher Scientific). A total of five plasmids were transfected: (1) a plasmid expressing VSV-G glycoprotein, (2) a plasmid expressing the binding protein, (3) a plasmid expressing the lentiviral transfer genome encoding eGFP, (4) a plasmid expressing gag-pol, and (5) a plasmid expressing rev. The transfected cell supernatant was harvested after 48 hours. The virus in the cell supernatant was concentrated by centrifugation with a sucrose pad and resuspended in PBS. Lentiviral particle titers were determined using the Lenti-X p24 Rapid Tit Kit (Takara Bio, San Jose, CA).

[0504] Lentiviral transduction assay. Concentrated lentivirus was subjected to a series of 10-fold dilutions (in cell culture medium) 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 viability staining agent and anti-CD7 antibody to detect CD7-positive cells (PeCy7 mouse anti-human CD7, clone CD7-6B7, BD Biosciences). eGFP expression was measured to calculate transduction efficiency.

[0505] Novel structure-guided design of blinding 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 sites in VSV-G with side chains facing the binding interface on LDL-R. Figure 7A and Figure 7B Residue Q10 (SEQ ID NO:2) appears to form several interactions with residues in both CR2 and CR3. In CR3, this includes interactions with positively charged arginine residues. Therefore, three substitutions were tested: Q10A to reduce side-chain interactions that could potentially stabilize LDL-R binding, and Q10R and Q10K to generate electrostatic repulsion.

[0506] Residue I182 (SEQ ID NO:2) also appears to contact several residues in both CR2 and CR3. Three substitutions were tested: I182A to reduce side-chain interactions that could stabilize LDL-R binding, and I182D and I182E to generate electrostatic repulsion against the primary binding interface on LDL-R.

[0507] Adding a negative charge to the binding interface eliminated native tropism without altering fusion. Titration of viral supernatant from the CD7+ T cell line SupT1 validated the structural prediction of residue I182. In the absence of any compensating binding molecule, WT VSV-G achieved a titer of 3.0e8, while both I182D and I182E were approximately three orders of magnitude lower. Figure 8A The substitution at residue I182 preserves fusion, as the titer is restored to 1e8 in the presence of a binding agent that redirects the virion to CD7.

[0508] Data such as Figure 8A and Figure 8B As shown, it demonstrates that adding a negative charge to the bonding interface eliminates the natural tropism without altering the fusion property. Figure 8A (Top) shows the titration of the VSV-G construct on SupT1 cells. For the p24 antigen, the percentage of SupT1 cells expressing GFP at each viral input level is plotted. Dashed / hollow circles indicate individual VSV-G constructs, and solid / closed circles indicate the same construct with trans-expressed CD7 targeting molecules. Figure 8B The functional titer for each construct, calculated according to titration in A, is shown and expressed as transduction units / mL concentrated viral supernatant (TU / mL).

[0509] Therefore, these embodiments demonstrate that mutation site 182 is sufficient to eliminate LDL-R interactions, but retains fusion properties when combined with the targeting portion of the target bound to the target cell.

[0510] Therefore, the embodiments provided herein demonstrate that virus recovery can be significantly increased when a sterile filter (such as a positively charged sterile filter) is pre-coated with a protein (such as recombinant human serum albumin). This effect appears to be even greater for positively charged filters, which is surprising and unpredictable. Furthermore, the method provided herein results in increased recovery even after the step of passing the solution through a sterile filter, and even while producing a sterile composition. This contrasts with other methods where the final step is sterile filtration. Therefore, the methods and embodiments of the present invention result in increased recovery without sacrificing the sterility of the product, which is important for pharmaceutical compositions. These methods can be used to administer higher doses of smaller volumes of viral vector-containing pharmaceutical compositions, which is advantageous for patients treated with viral vector-based therapies.

[0511] This specification contains extensive references to patents, patent applications, and publications. Each of these is hereby incorporated by reference for all purposes.

[0512] This specification also references various sequences, such as those provided herein and below. Comparisons of the extracellular domains of different VSV-G proteins from different strains are shown below. Figure 9 As shown.

[0513] VSV-G Indiana full length WT:

[0514] MKCLLYLAFLFIGVNC KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQ FINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAG LPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:1)

[0515] VSV-G Indiana ectodomain WT:

[0516] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:2)

[0517] VSV-G Indiana ectodomain I182A:

[0518] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLASMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWD DWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:3)

[0519] VSV-G Indiana ectodomain I182D:

[0520] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLDSMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:4)

[0521] VSV-G Indiana ectodomain I182E:

[0522] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLESMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQ VFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:5)

[0523] VSV-G Indiana ectodomain H8A+K47Q:

[0524] KFTIVFPANQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:6)

[0525] VSV-G Indiana ectodomain Q10A:

[0526] KFTIVFPHNAKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIAS FFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:7)

[0527] VSV-G Indiana ectodomain Q10R:

[0528] KFTIVFPHNRKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:8)

[0529] VSV-G Indiana ectodomain Q10K:

[0530] KFTIVFPHNKKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:9)

[0531] VSV-G New Jersey full length WT:

[0532] MLSYLIFALVVSPILGKIEIVFPQHTTGDWKRVPHEYNYCPTSADKNSHGTQTGIPVELTMPKGLTTHQVDGFMCHSALWMTTCDFRWYGPKYITHSIHNEEPTDYQCLEAIKAYKDGVSFNPGFPPQSCGYGTVTDAEAHIVTVTPHSVKVDEYTGEWIDPHFIGGRCKGQICETVHNSTKWFTSSDGESVCSQLFTLVGGTFFSDSEEITSMGLPETGIRSNYFPYVSTEGICKMPFCRKPGYKLKNDLWFQITDPDLDKTVRDLPHIKDCDLSSSIVTPGEHATDISLISDVERILDYALCQNTWSKIEAGEPITPVDLSYLGPKNPGAGPVFTIINGSLHYFMSKYLRVELESPVIPRMEGKVAGTRIVRQLWDQWFPFGEVEIGPNGVLKTKQGYKFPLHIIGTGEVDNDIKMERIVKHWEHPHIEAAQTFLKKDDTEEVLYYGDTGVSKNPVELVEGWFSGWRSSIMGVLAVIIGFVILIFLIRLIGVLSSLFRQKRRPIYKSDVEMAHFR(SEQ IDNO:10)

[0533] VSV-G New Jersey ectodomain WT:

[0534] KIEIVFPQHTTGDWKRVPHEYNYCPTSADKNSHGTQTGIPVELTMPKGLTTHQVDGFMCHSALWMTTCDFRWYGPKYITHSIHNEEPTDYQCLEAIKAYKDGVSFNPGFPPQSCGYGTVTDAEAHIVTVTPHSVKVDEYTGEWIDPHFIGGRCKGQICETVHNSTKWFTSSDGESVCSQLFTLVGGTFFSDSEEITSMGLPETGIRSNYFPYVSTEGICKMPFCRKPGYKLKNDLWFQITDPDLDKTVRDLPHIKDCDLSSSIVTPGEHATDISLISDVERILDYALCQNTWSKIEAGEPITPVDLSYLGPKNPGAGPVFTIINGSLHYFMSKYLRVELESPVIPRMEGKVAGTRIVRQLWDQWFPFGEVEIGPNGVLKTKQGYKFPLHIIGTGEVDNDIKMERIVKHWEHPHIEAAQTFLKKDDTEEVLYYGDTGVSKNPVELVEGWFSGWRSSIMGVLAVIIGFVILIFLIRLIGVLSSLFRQKRRPIYKSDVEMAHFR(SEQ ID NO:11)

[0535] VSV-G Maraba full length WT:

[0536] MLRLFLFCFLALGAHSKFTIVFPHHQKGNWKNVPSTYHYCPSSSDQNWHNDLTGVSLHVKIPKSHKAIQADGWMCHAAKWVTTCDFRWYGPKYITHSIHSMSPTLEQCKTSIEQTKQGVWINPGFPPQSCGYATVTDAEVVVVQATPHHVLVDEYTGEWIDSQLVGGKCSKEVCQTVHNSTVWHADYKITGLCESNLASVDITFFSEDGQKTSLGKPNTGFRSNHFAYESGEKACRMQYCTQWGIRLPSGVWFELVDKDLFQAAKLPECPRGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAKLPVSPVDLSYLAPKNPGSGPAFTIINGTLKYFETRYIRVDISNPIIPHMVGTMSGTTTERELWNDWYPYEDVEIGPNGVLKTPTGFKFPLYMIGHGMLDSDLHKSSQAQVFEHPHAKDAASQLPDDETLFFGDTGLSKNPVELVEGWFSSWKSTLASFFLIIGLGVALIFIIRIIVAIRYKYKGRKTQKIYNDVEMSRLGNK(SEQ ID NO:12)

[0537] VSV-G Maraba ectodomain WT:

[0538] KFTIVFPHHQKGNWKNVPSTYHYCPSSSDQNWHNDLTGVSLHVKIPKSHKAIQADGWMCHAAKWVTTCDFRWYGPKYITHSIHSMSPTLEQCKTSIEQTKQGVWINPGFPPQSCGYATVTDAEVVVVQATPHHVLVDEYTGEWIDSQLVGGKCSKEVCQTVHNSTVWHADYKITGLCESNLASVDITFFSEDGQKTSLGKPNTGFRSNHFAYESGEKACRMQYCTQWGIRLPSGVWFELVDKDLFQAAKLPECPRGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAKLPVSPVDLSYLAPKNPGSGPAFTIINGTLKYFETRYIRVDISNPIIPHMVGTMSGTTTERELWNDWYPYEDVEIGPNGVLKTPTGFKFPLYMIGHGMLDSDLHKSSQAQVFEHPHAKDAASQLPDDETLFFGDTGLSKNPVELVEGWFSSWKSTLASFFLIIGLGVALIFIIRIIVAIRYKYKGRKTQKIYNDVEMSRLGNK(SEQ ID NO:13)

[0539] VSV-G Caracas full length WT:

[0540] MKMKMVIAGLILCIGILPAIGKITISFPQSLKGDWRPVPKGYNYCPTSADKNLHGDLIDIGLRLRAPKSFKGISADGWMCHAARWITTCDFRWYGPKYITHSIHSFRPSNDQCKEAIRLTNEGNWINPGFPPQSCGYASVTDSESVVVTVTKHQVLVDEYSGSWIDSQFPGGSCTSPICDTVHNSTLWHADHTLDSICDQEFVAMDAVLFTESGKFEEFGKPNSGIRSNYFPYESLKDVCQMDFCKRKGFKLPSGVWFEIEDAEKSHKAQVELKIKRCPHGAVISAPNQNAADINLIMDVERILDYSLCQATWSKIQNKEALTPIDISYLGPKNPGPGPAFTIINGTLHYFNTRYIRVDIAGPVTKEITGFVSGTSTSRVLWDQWFPYGENSIGPNGLLKTASGYKYPLFMVGTGVLDADIHKLGEATVIEHPHAKEAQKVVDDSEVIFFGDTGVSKNPVEVVEGWFSGWRSSLMSIFGIILLIVCLVLIVRILIALKYCCVRHKKRTIYKEDLEMGRIPRRA(SEQ ID NO:14)

[0541] VSV-G Caracas ectodomain WT:

[0542] KITISFPQSLKGDWRPVPKGYNYCPTSADKNLHGDLIDIGLRLRAPKSFKGISADGWMCHAARWITTCDFRWYGPKYITHSIHSFRPSNDQCKEAIRLTNEGNWINPGFPPQSCGYASVTDSESVVVTVTKHQVLVDEYSGSWIDSQFPGGSCTSPICDTVHNSTLWHADHTLDSICDQEFVAMDAVLFTESGKFEEFGKPNSGIRSNYFPYESLKDVCQMDFCKRKGFKLPSGVWFEIEDAEKSHKAQVELKIKRCPHGAVISAPNQNAADINLIMDVERILDYSLCQATWSKIQNKEALTPIDISYLGPKNPGPGPAFTIINGTLHYFNTRYIRVDIAGPVTKEITGFVSGTSTSRVLWDQWFPYGENSIGPNGLLKTASGYKYPLFMVGTGVLDADIHKLGEATVIEHPHAKEAQKVVDDSEVIFFGDTGVSKNPVEVVEGWFSGWRSSLMSIFGIILLIVCLVLIVRILIALKYCCVRHKKRTIYKEDLEMGRIPRRA(SEQ ID NO:15)

[0543] VSV-G Alagoas full length WT:

[0544] MTPAFILCMLLAGSSWAKFTIVFPQSQKGDWKDVPPNYRYC PSSADQNWHGDLLGVNIRAKMPKVHKAIKADGWMCHAAKWVTTCDYRWYGPQYITHSIHSFIPTKAQCEESIKQTKEGVWINPGFPPKNCGYASVSDAESIIVQATAHSVMIDEYSGDWLDSQFPTGRCTGSTCETIHNSTLWYADYQVTGLCDSALVSTEVTFYSEDGLMTSIGRQNTGYRSNYFPYEKGAAACRMKYCTHEGIRLPSGVWFEMVDKELLESVQMPECPAGLTISAPTQTSVDVSLILDVERMLDYSLCQETWSKVHSGLPISPVDLGYIAPKNPGAGPAFTIVNGTLKYFDTRYLRIDIEGPVLKKMTGKVSGTPTKRELWTEWFPYDDVEIGPNGVLKTPEGYKFPLYMIGHGLLDSDLQKTSQAEVFHHPQIAEAVQKLPDDETLFFGDTGISKNPVEVIEGWFSNWRSSVMAIVFAILLLVITVLMVRLCVAFRHFCCQKRHKIYNDLEMNQLRR(SEQ ID NO:16)

[0545] VSV-G Alagoas ectodomain WT:

[0546] KFTIVFPQSQKGDWKDVPPNYRYCPSSADQNWHGDLLGVNIRAKMPKVHKAIKADGWMCHAAKWVTTCDYRWYGPQYITHSIHSFIPTKAQCEESIKQTKEGVWINPGFPPKNCGYASVSDAESIIVQATAHSVMIDEYSGDWLDSQFPTGRCTGSTCETIHNSTLWYADYQVTGLCDSALVSTEVTFYSEDGLMTSIGRQNTGYRSNYFPYEKGAAACRMKYCTHEGIRLPSGVWFEMVDKELLESVQMPECPAGLTISAPTQTSVDVSLILDVERMLDYSLCQETWSKVHSGLPISPVDLGYIAPKNPGAGPAFTIVNGTLKYFDTRYLRIDIEGPVLKKMTGKVSGTPTKRELWTEWFPYDDVEIGPNGVLKTPEGYKFPLYMIGHGLLDSDLQKTSQAEVFHHPQIAEAVQKLPDDETLFFGDTGISKNPVEVIEGWFSNWRSSVMAIVFAILLLVITVLMVRLCVAFRHFCCQKRHKIYNDLEMNQLRR(SEQ ID NO:17)

[0547] VSV-G CoCAL full length WT:

[0548] MNFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTC DFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK(SEQ ID NO:18)

[0549] VSV-G CoCAL ectodomain WT:

[0550] KFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK(SEQ ID NO:19)

[0551] VSV-G Moreton full length WT:

[0552] MLVLYLLLSLLALGAQCKFTIVFPHNQKGNWKNVPANYQYCPSSSDLNWHNGLIGTSLQVKMPKSHKAIQADGWMCHAAKWVTTCDFRWYGPKYVTHSIKSMIPTVDQCKESIAQTKQGTWLNPGFPPQ SCGYASVTDAEAVIVKATPHQVLVDEYTGEWVDSQFPTGKCNKDICPTVHNSTTWHSDYKVTGLCDANLISMDITFFSEDGKLTSLGKEGTGFRSNYFAYENGDKACRMQYCKHWGVRLPSGVWFEMADKDIYNDAKFPDCPEGSSIAAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAHLPISPVDLSYLSPKNPGTGPAFTIINGTLKYFETRYIRVDIAGPIIPQMRGVISGTTTERELWTDWYPYEDVEIGPNGVLKTATGYKFPLYMIGHGMLDSDLHISSKAQVFEHPHIQDAASQLPDDETLFFGDTGLSKNPIELVEGWFSGWKSTIASFFFIIGLVIGLYLVLRIGIALCIKCRVQEKRPKIYTDVEMNRLDR(SEQ ID NO:20)

[0553] VSV-G Moreton ectodomain WT:

[0554] KFTIVFPHNQKGNWKNVPANYQYCPSSSDLNWHNGLIGTSLQVKMPKSHKAIQADGWMCHAAKWVTTCDFRWYGPKYVTHSIKSMIPTVDQCKESIAQTKQGTWLNPGFPPQSCGYASVTDAEAVIVKATPHQVLVDEYTGEWVDSQFPTGKCNKDICPTVHNSTTWHSDYKVTGLCDANLISMDITFFSEDGKLTSLGKEGTGFRSNYFAYENGDKACRMQYCKHWGVRLPSGVWFEMADKDIYNDAKFPDCPEGSSIAAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAHLPISPVDLSYLSPKNPGTGPAFTIINGTLKYFETRYIRVDIAGPIIPQMRGVISGTTTERELWTDWYPYEDVEIGPNGVLKTATGYKFPLYMIGHGMLDSDLHISSKAQVFEHPHIQDAASQLPDDETLFFGDTGLSKNPIELVEGWFSGWKSTIASFFFIIGLVIGLYLVLRIGIALCIKCRVQEKRPKIYTDVEMNRLDR(SEQ ID NO:21)

Claims

1. A method for producing a concentrated sterile solution containing a viral vector, the method comprising the following steps: Clarify the solution containing the cell culture medium and the viral vector; The clarified solution containing the viral vector is filtered through a first chromatographic filter to produce a filtered clarified solution containing the viral vector. The filtered, clarified solution containing the viral vector is passed through a protein-coated sterile membrane to produce a sterile solution containing the viral vector. as well as The sterile solution containing the viral vector is concentrated to produce a concentrated sterile solution containing the viral vector.

2. 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 excipients to produce a sterile pharmaceutical composition containing the viral vector.

3. The method of claim 1, further comprising collecting the cell culture medium from the cell culture that produced the viral vector before clarifying the solution.

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

5. The method of claim 1, wherein the protein-coated sterile membrane comprises a membrane pre-rinsed with a protein solution.

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

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

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), about 100 mM to about 400 mM sodium chloride, and a pH of about 6.0 to about 8.

0.

9. The method of claim 7, wherein the protein comprises human serum albumin, myosin, bovine serum albumin, immunoglobulin, immunoglobulin fragments, fibronectin, hylocinin, or any combination thereof.

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

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

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

13. The method of claim 6, wherein the protein-coated sterile membrane is positively charged prior to being coated with the protein.

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

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

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

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

18. The method of claim 17, wherein the fine purification chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimode chromatography.

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

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

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

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

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

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

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

26. The method of claim 1, further comprising: Before clarifying the solution: i) Obtain a solution containing cell culture medium and the viral vector; and ii) Digest the DNA in a solution containing cell culture medium and the viral vector to produce a first digested solution containing the viral vector; Clarifying the solution includes clarifying the first digested solution containing the viral vector to produce 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: i) Digest the DNA in a filtered, clarified, digested solution containing the viral vector to produce 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 chromatographic filter to produce a twice-filtered, clarified, twice-digested solution containing the viral vector; The protein-coated sterile membrane described herein comprises a membrane pre-rinsed with a protein solution. The protein solution contains about 0.1% w / v to 2.0% w / v of protein, about 10 mM to about 30 mM of 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), about 100 mM to about 400 mM of sodium chloride, and a pH of about 6.0 to about 8.

0.

27. A method for producing a concentrated sterile solution containing a viral vector, the method comprising the following steps: The DNA in the solution containing the viral vector is digested to produce a first digested solution containing the viral vector; Clarify the digested solution containing the viral vector to produce a clear, digested solution containing the viral vector; The clarified, digested solution containing the viral vector is filtered through a first chromatographic filter to produce a filtered, clarified, digested solution containing the viral vector. The DNA in a filtered, clarified, digested solution containing the viral vector is digested to produce a filtered, clarified, twice-digested solution containing the viral vector; the filtered, clarified, twice-digested solution containing the viral vector is filtered through a second chromatographic filter to produce a twice-filtered, clarified, twice-digested solution containing the viral vector. The twice-filtered, clarified, twice-digested solution containing the viral vector is passed through a protein-coated sterile membrane to produce a sterile solution containing the viral vector. as well as The sterile solution containing the viral vector is concentrated to produce a concentrated sterile solution containing the viral vector.

28. The method of claim 27, wherein the digestion of DNA is performed using a nuclease having DNase activity, RNase activity, or a combination thereof.

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

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

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

32. The method of claim 27, further comprising collecting culture medium from the cell culture that produced the viral vector before clarifying the digested solution.

33. The method of claim 27, wherein the protein-coated sterile membrane comprises a membrane pre-rinsed with a protein solution.

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

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

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), about 100 mM to about 400 mM sodium chloride, and a pH of about 6.0 to about 8.

0.

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

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

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

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

41. The method of any one of claims 27-40, wherein the protein-coated sterile membrane is positively charged prior to being coated with the protein.

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

43. The method of any one of claims 27-42, wherein 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, Approximately 0.25 μm, approximately 0.26 μm, approximately 0.27 μm, approximately 0.28 μm, approximately 0.29 μm, approximately 0.30 μm, approximately 0.31 μm, approximately 0.32 μm, approximately 0.33 μm, approximately 0.34 μm, approximately 0.35 μm, approximately 0.36 μm, approximately 0.37 μm, approximately 0.38 μm, approximately 0.39 μm, approximately 0.40 μm, approximately 0.41 μm, approximately 0.42 μm, approximately 0.43 μm, approximately 0.44 μm, or approximately 0.45 μm.

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

45. The method of claim 44, 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%, or 99% or about 100%.

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

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

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

49. The method of claim 48, wherein the fine purification chromatography is ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or multimode chromatography.

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

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

52. The method of claims 50 and 51, wherein the tangential flow filter utilizes 1.0 mm ID fiber and a 750 kDa pore size.

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

54. The method of claim 53, wherein the mixing with the excipient includes a buffer exchange step.

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

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

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

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

59. The method of any one of claims 1-58, wherein the concentration of the viral vector is about 1 × 10⁻⁶ before the solution is passed through the sterile filter. 4 To approximately 1×10 6 Transduction units (TU) / mL.

60. The method of any one of claims 1-59, wherein the concentration of the viral vector is 1 × 10⁻⁶ before the solution is passed through the sterile filter. 7 Up to 1×10 9 Viral 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. 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. The method of any one of claims 2-26 or 28-60, wherein the viral vector, such as a lentiviral vector, is pseudotyped with a VSV-G polypeptide containing a mutation corresponding to the mutation at position 182 of SEQ ID NO:2.

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