Process for viral vector production

Abstract

Among other things, provided are compositions and methods for producing viral vectors. Provided herein are methods of producing a composition comprising viral vectors, the method comprising culturing producer cells that comprise one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors. In some embodiments, the viral vectors comprise a lipid bilayer, and the one or more nucleic acids comprise at least one nucleic acid that encodes one or more fusogens. In some embodiments, the one or more fusogens are present in the lipid bilayer. Methods herein are characterized by (i) a 10-fold increase in the number of active vector particles, (ii) a 10-fold reduction in host cell protein, and / or (iii) a 3000- fold reduction in host cell DNA, relative to an alternative process.

Description

PROCESS FOR VIRAL VECTOR PRODUCTIONRELATED APPLICATIONS

[0001] This present application claims priority to each of the following applications, the disclosure of each of which is hereby incorporated by reference in its entirety: United States Provisional Application No. 63 / 438243, filed January 10, 2023; United States Provisional Application No. 63 / 460258, filed April 18, 2023; and United States Provisional Application No. 63 / 466695, filed May 15, 2023.BACKGROUND

[0002] Replacement of deficient or damaged biologic components leading to disease with functional components has emerged as an effective approach for treating a number of diseases and disorders. Gene therapy and cell therapy are two such approaches. Gene therapy involves the delivery of a nucleic acid encoding a gene of interest into a cell, while cell therapy involves the delivery of a cell to a tissue or organism, both with the intention of treating a disease.

[0003] Beyond correcting genetic deficiencies, gene therapy and cell therapy can also endow a cell or organism with capabilities not present in the natural state. Adoptive cellular therapy using genetically engineered T cells is one of the most notable examples. Using an engineered gene, such as a chimeric antigen receptor (CAR) or cloned T-cell receptor (TCR), T cells can be endowed with the ability to recognize antigens that are not naturally recognized by their endogenous TCRs.SUMMARY

[0004] The present disclosure provides viral vectors, methods for producing viral vectors, and related technologies. Among other things, the present disclosure recognizes that current methods of producing viral vectors face challenges, including low yields of produced viral vectors, low potency of produced viral vectors, and undesirable levels of process related impurities (e.g., producer cell protein and DNA) in final products. The present disclosure alsorecognizes that viruses have a natural ability to enter into and deliver genetic material to cells. As such, viruses can be useful in both gene and cell therapies.

[0005] The present disclosure addresses these challenges by, inter alia, providing methods of making viral vectors that include expression of a fusogen, decreased producer cell aggregation during culture, nuclease treatment, or combinations thereof. The methods disclosed herein provide viral vectors having increased yield, increased titer and / or decreased process related impurities as compared to viral vectors produced with known methods. The present disclosure also provides compositions of viral vectors, e.g., gammaretroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors, made with the disclosed methods, as well as methods of using the same.

[0006] Accordingly, provided herein is a method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of viral vectors under conditions sufficient to produce viral vectors.

[0007] In some embodiments of any of the methods disclosed herein, one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid. In some embodiments, one or more nucleic acids further comprise a regulatory plasmid.

[0008] In some embodiments of any of the methods disclosed herein, viral vectors are chosen from: gammaretroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors. In some embodiments, viral vectors are lentiviral vectors. In some embodiments, viral vectors comprise a lipid bilayer.

[0009] In some embodiments of any of the methods disclosed herein, one or more nucleic acids comprise at least one nucleic acid that encodes one or more fusogens. In some embodiments, one or more fusogens are present in a lipid bilayer.

[0010] In some embodiments, an envelope plasmid encodes one or more fusogens.

[0011] In some embodiments of any of the methods disclosed herein, one or more fusogens comprise at least one fusogen that has a tropism for B cells, T cells, natural killer cells, islet cells, glial progenitor cells, neuronal cells, hematopoietic stem cells, cardiac cells, hepatocytes, stem cells, or induced pluripotent stem cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for B cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for T cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for islet cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for cardiac cells.

[0012] In some embodiments, one or more fusogens comprise at least one fusogen that has endogenous tropism.

[0013] In some embodiments, one or more fusogens comprise at least one fusogen that has engineered tropism.

[0014] In some embodiments, one or more fusogens comprise one or more viral fusogens. In some embodiments, one or more fusogens comprise at least one fusogen that is involved in attachment of a viral vector to a cell membrane.

[0015] In some embodiments, one or more fusogens comprise at least one fusogen that is involved in directing fusion of a lipid bilayer of a viral vector and a cell membrane.

[0016] In some embodiments, one or more fusogens comprise one or more paramyxovirus envelope proteins or biologically active portions thereof. In some embodiments, one or more paramyxovirus envelope proteins or biologically active portions thereof comprises a paramyxovirus glycoprotein G (“G protein”) or a biologically active portion thereof. In some embodiments, one or more paramyxovirus envelope proteins or biologically active portions thereof comprises a paramyxovirus fusion protein (“F protein”) or a biologically active portion thereof.

[0017] In some embodiments, one or more fusogens comprise one or more chimeric proteins. In some embodiments, one or more chimeric proteins comprise at least one chimeric protein that comprises a paramyxovirus envelope protein or biologically active portion thereof.

[0018] In some embodiments, one or more chimeric proteins comprise at least one chimeric protein that comprises an scFV. In some embodiments, one or more chimeric proteins comprise at least one chimeric protein that comprises (i) a paramyxovirus envelope protein or biologically active portion thereof and (ii) an scFV.

[0019] In some embodiments, an scFV targets an antigen present on the surface of a B cell, a T cell, a natural killer cell, an islet cell, a glial progenitor cell, a cardiac cell, a blood cell, a hepatocyte, a stem cell, or an induced pluripotent stem cell. In some embodiments, an scFV targets an antigen present on the surface of a B cell. In some embodiments, an scFV targets an antigen present on the surface of a T cell. In some embodiments, an scFV targets CD8. In some embodiments, an scFV targets CD4.

[0020] In some embodiments, an scFV targets an antigen present on the surface of an islet cell. In some embodiments, an islet cell is an alpha cell, a beta cell, or a delta cell. In some embodiments, an islet cell is an alpha cell. In some embodiments, an islet cell is a beta cell. In some embodiments, an islet cell is a delta cell.

[0021] In some embodiments of any of the methods disclosed herein, a transfer plasmid comprises a nucleotide sequence that encodes a nuclease. In some embodiments, a nuclease is a Cas, a TALEN, or a zinc-finger nuclease.

[0022] In some embodiments of any of the methods disclosed herein, a transfer plasmid comprises a nucleotide sequence that encodes a gRNA.

[0023] In some embodiments of any of the methods disclosed herein, a transfer plasmid comprises a nucleotide sequence that encodes an antibody or a biologically active portion thereof.

[0024] In some embodiments of any of the methods disclosed herein, a transfer plasmid comprises a nucleotide sequence that encodes a chimeric antigen receptor.

[0025] In some embodiments of any of the methods disclosed herein, a transfer plasmid comprises a nucleotide sequence that encodes an antigen.

[0026] In some embodiments of any of the methods disclosed herein, a transfer plasmid comprises a nucleotide sequence that encodes a therapeutic polypeptide. In some embodiments, a therapeutic polypeptide is useful for protein replacement therapy.

[0027] In some embodiments of any of the methods disclosed herein, a ratio of an envelope plasmid to a packaging plasmid within one or more nucleic acids is greater than 1:1, greater than 2:1, greater than 3:1, greater than 4:1, or greater than 5:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is greater than 1:1.

[0028] In some embodiments of any of the methods disclosed herein, a ratio of an envelope plasmid to a transfer plasmid within one or more nucleic acids is greater than 1:1, greater than 2:1, greater than 3:1, greater than 4:1, or greater than 5:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is greater than 1:1.

[0029] In some embodiments of any of the methods disclosed herein, a method comprises introducing at least one nucleic acid that encodes one or more fusogens into producer cells. In some embodiments, introducing at least one nucleic acid that encodes one or more fusogens into producer cells comprises transfecting at least one nucleic acid that encodes one or more fusogens into producer cells.

[0030] In some embodiments of any of the methods disclosed herein, a method comprises introducing one or more nucleic acids into producer cells. In some embodiments, introducing one or more nucleic acids into producer cells comprises transfecting one or more nucleic acids into producer cells.

[0031] In some embodiments of any of the methods disclosed herein, an amount of an envelope plasmid that is introduced into producer cells is greater than an amount of packaging plasmid that is introduced into producer cells.

[0032] In some embodiments of any of the methods disclosed herein, an amount of an envelope plasmid that is introduced into producer cells is greater than an amount of a transfer plasmid that is introduced into producer cells.

[0033] In some embodiments of any of the methods disclosed herein, a method further comprises treating producer cells with a nuclease. In some embodiments, producer cells arc treated with a nuclease at a time that is at least 30 minutes after at least one nucleic acid that encodes one or more fusogens are introduced into producer cells.

[0034] In some embodiments, producer cells are treated with a nuclease at a time that is no more than 6 hours after at least one nucleic acid that encodes one or more fusogens are introduced into producer cells.

[0035] In some embodiments, producer cells are cultured in a suspension culture.

[0036] In some embodiments, producer cells are cultured in serum free medium.

[0037] In some embodiments of any of the methods disclosed herein, a method further comprises agitating producer cells. In some embodiments, producer cells are cultured in medium that includes an anti-cell clumping agent. In some embodiments, producer cells are cultured in medium that includes a surfactant.

[0038] In some embodiments of any of the methods disclosed herein, a method further comprises enriching viral vectors from a producer cell culture.

[0039] In some embodiments of any of the methods disclosed herein, a method further comprises generating a crude drug substance from a producer cell culture. In some embodiments, a crude drug substance comprises viral vectors produced by producer cells or a subset thereof. In some embodiments, generating a crude drug substance from a producer cell culture comprisesenriching viral vectors from a producer cell culture. In some embodiments, generating a crude drug substance from a producer cell culture comprises removing producer cell DNA and / or producer cell protein from a producer cell culture. In some embodiments, a producer cell culture comprises producer cells and / or culture medium.

[0040] In some embodiments, a crude drug substance is characterized by: (i) an at least 3-fold increase in viral vector titer as compared to a crude drug substance produced by an alternative process; (ii) an at least 1.5-fold increase in infectivity as compared to a crude drug substance produced by an alternative process; (iii) an at least 1000-fold decrease in an amount of producer cell DNA as compared to a crude drug substance produced by an alternative process; (iv) an at least 15-fold decrease in an amount of producer cell protein as compared to a crude drug substance produced by an alternative process; (v) an at least 1.5-fold increase in transduction efficiency in a target cell exposed to a crude drug substance as compared to a crude drug substance produced by an alternative process; (vi) an at least 20-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process; (vii) an at least 3-fold increase in fusogen expression on viral vectors as compared to a crude drug substance produced by an alternative process; (viii) or a combination thereof.

[0041] In some embodiments of any of the methods disclosed herein, a method further comprises generating a drug substance from a crude drug substance. In some embodiments, a drug substance comprises viral vectors produced by producer cells or a subset thereof. In some embodiments, generating a drug substance from a crude drug substance comprises further enriching viral vectors from a crude drug substance. In some embodiments, generating a drug substance from a crude drug substance comprises further removing producer cell DNA and / or producer cell protein from a crude drug substance. In some embodiments, generating a drug substance from a crude drug substance comprises adding a pharmaceutically acceptable excipient.

[0042] In some embodiments, a drug substance is characterized by: (i) an at least 5-fold increase in viral vector titer as compared to a drug substance produced by an alternative process; (ii) an at least 5-fold increase in infectivity as compared to a drug substance produced by an alternative process; (iii) an at least 2000-fold decrease in an amount of producer cell DNA as compared to a drug substance produced by an alternative process; (iv) an at least 10-fold decrease in an amount of producer cell protein as compared to a drug substance produced by an alternative process; (v) an at least 2-fold increase in transduction efficiency in a target cell exposed to a drug substance as compared to a drug substance produced by an alternative process; (vi) an at least 50-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process; (vii) an at least 6-fold increase in fusogen expression on viral vectors as compared to a crude drug substance produced by an alternative process; (viii) or a combination thereof.

[0043] In some embodiments, an alternative process is a comparable process. In some embodiments, an alternative process comprises: (i) a ratio of an envelope plasmid to a packaging plasmid within one or more nucleic acids for production of viral vectors is equal to or less than 1:1; (ii) a ratio of an envelope plasmid to a transfer plasmid within one or more nucleic acids for production of viral vectors is equal to or less than 1:1; (iii) an amount of an envelope plasmid that is introduced into producer cells is equal to or less than an amount of an packaging plasmid that is introduced into producer cells; (iv) an amount of an envelope plasmid that is introduced into producer cells is equal to or less than an amount of a transfer plasmid that is introduced into producer cells; (v) producer cells arc treated with a nuclease at a time that is more than 6 hours after at least one nucleic acid that encodes one or more fusogens is introduced into producer cells; (vi) producer cells are not cultured in a suspension culture; (vii) cell aggregation of producer cells is not reduced; (viii) producer cells are not agitated during culture; (ix) culture medium for producer cells does not include an anti-clump agent or a surfactant; or (x) a combination thereof.

[0044] Also provided herein is a producer cell comprising one or more nucleic acids for the production of lentiviral vectors, wherein the one or more nucleic acids comprise a packaging plasmid and an envelope plasmid that encodes one or more fusogens.

[0045] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is greater than 1:1, greater than 2:1, greater than 3:1, greater than 4:1, or greater than 5:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is greater than 1:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is greater than 2:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is a ratio of said plasmids within a producer cell.

[0046] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is about 1.5:1, about 2:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, or about 15:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is about 1.5:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is about 2:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is a ratio of said plasmids within a producer cell.

[0047] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is between 1:1 to 2:1, between 1:1 to 4:1, between 1:1 to 5:1, between 1.5:1 to 6:1, between 1.5:1 to 7:1, between 1.5:1 to 8:1, between 1.5:1 to 9:1, between 1.5:1 to 10:1, between 1.5:1 to 11:1, between 1.5:1 to 12:1, between 1.5:1 to 13:1, between 1.5:1 to 14:1, or between 1.5:1 to 15:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is a ratio of said plasmids within a producer cell.

[0048] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is at most 15:1, at most 14:1, at most 13:1, at most 12:1, at most 11:1, at most 10:1, at most 9:1, or at most 8:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is a ratio of said plasmids within a producer cell.

[0049] In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is at least 1:1, at least 2:1, at least 3:1, at least 4:1, or at least 5:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is at least 1:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is at least 2:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is a ratio of said plasmids within a producer cell.

[0050] The disclosure further provides a producer cell comprising engineered lentiviral vectors, wherein a producer cell had been transfected with one or more nucleic acids for production of lentiviral vectors at a first time point and had been exposed to a nuclease at a second time point.

[0051] In some embodiments, a second time point is at least 30 minutes after and at most 6 hours after a first time point.

[0052] In some embodiments, a second time point is at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, or at least 5.5 hours after a first time point.

[0053] In some embodiments, a second time point is at most 6 hours, at most 5.5 hours, at most 5 hours, at most 4.5 hours, at most 4 hours, at most 3.5 hours, at most 3 hours, at most 2.5 hours, at most 2 hours, at most 1.5 hours, at most 1 hour or at most 30 minutes after a first time point.

[0054] In another aspect, this disclosure features method of producing a composition comprising viral vectors, comprising: (a) culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid; (b) treating the producer cell culture with a nuclease; and (c) harvesting the producer cell culture, wherein (b) is performed prior (c).

[0055] In another aspect, this disclosure features a method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid, wherein the ratio of the envelope plasmid to the packaging plasmid within the one or more nucleic acids is greater than 1:1.

[0056] In some embodiments, the ratio of the envelope plasmid to the packaging plasmid within the one or more nucleic acids is selected from a ratio of: 1 : 1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1:8, 1:9, and 1:10.

[0057] In some embodiments, the ratio of the packaging plasmid to the envelope plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0058] In another aspect, this disclosure features a method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid, wherein the ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is greater than 1 : 1

[0059] In some embodiments, the ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0060] In some embodiments, the ratio of the transfer plasmid to the envelope plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0061] In another aspect, this disclosure features a method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleicacids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid, wherein the ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is greater than 1:1.

[0062] In some embodiments, the ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0063] In another aspect, this disclosure features a method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, a first envelope plasmid, a second envelope plasmid, and a transfer plasmid, wherein the ratio of a first envelope plasmid to a second envelope plasmid within the one or more nucleic acids is greater than 1:1.

[0064] In some embodiments, the first envelope plasmid comprises a nucleic acid sequence encoding a first envelope protein and the second envelope plasmid comprises a nucleic acid sequence encoding a second envelope protein.

[0065] In some embodiments, the ratio of a first envelope plasmid to a second envelope plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0066] In some embodiments, the ratio of a second envelope plasmid to a first envelope plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0067] In some embodiments, the viral vectors are gammaretroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors.

[0068] In some embodiments, the viral vectors are lentiviral vectors.

[0069] In some embodiments, the one or more nucleic acids further comprise a regulatory plasmid.

[0070] In some embodiments, the viral vectors comprise a lipid bilayer.

[0071] In some embodiments, the one or more nucleic acids comprise at least one nucleic acid that encodes one or more fusogens.

[0072] In some embodiments, the one or more fusogens are present in the lipid bilayer.

[0073] In some embodiments, the one or more fusogens are present on the outside of the lipid bilayer.

[0074] In some embodiments, the envelope plasmid encodes the one or more fusogens.

[0075] In some embodiments, the one or more fusogens comprise at least one fusogen that has a tropism for B cells, T cells, natural killer cells, glial progenitor cells, neuronal cells, hematopoietic stem cells, cardiac cells, hepatocytes, stem cells, or induced pluripotent stem cells.

[0076] In some embodiments, the one or more fusogens comprise at least one fusogen that has a tropism for B cells. In some embodiments, the one or more fusogens comprise at least one fusogen that has a tropism for T cells. In some embodiments, the one or more fusogens comprise at least one fusogen that has a tropism for cardiac cells. In some embodiments, the one or more fusogens comprise at least one fusogen that has a tropism for liver cells. In some embodiments, the one or more fusogens comprise at least one fusogen that has a tropism for a neuron.

[0077] In some embodiments, the one or more fusogens comprise at least one fusogen that has natural tropism.

[0078] In some embodiments, the one or more fusogens comprise at least one fusogen that has engineered tropism.

[0079] In some embodiments, the one or more fusogens comprise one or more viral fusogens.

[0080] In some embodiments, the one or more fusogens comprise at least one fusogen that is involved in attachment of a viral vector to a cell membrane.

[0081] In some embodiments, the one or more fusogens comprise at least one fusogen that is involved in directing fusion of the lipid bilayer of a viral vector and a cell membrane.

[0082] In some embodiments, the one or more fusogens comprise one or more paramyxovirus envelope proteins or biologically active portions thereof.

[0083] In some embodiments, the one or more paramyxovirus envelope proteins or biologically active portions thereof is a variant paramyxovirus envelope attachment protein.

[0084] In some embodiments, the paramyxovirus envelope attachment protein is an envelope attachment protein from a Nipah virus, Hendra virus, or Measles virus, or is a variant or biologically active portion thereof.

[0085] In some embodiments, the paramyxovirus envelope attachment protein is a wildtype paramyxovirus G protein, H protein or HN protein or is a variant or biologically active portion of any of the foregoing.

[0086] In some embodiments, the paramyxovirus envelope attachment protein is a wildtype Nipah virus G (NiV-G) protein or is a variant or biologically active portion of a NiV-G.

[0087] In some embodiments, the paramyxovirus envelope attachment protein is a variant NiV-G that is a variant or a biologically active portion of a wild-type NiV-G.

[0088] In some embodiments, the variant paramyxovirus envelope attachment protein comprises one or more mutations that reduces native tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising the one or more mutations.

[0089] In some embodiments, the one or more paramyxovirus envelope proteins or biologically active portions thereof comprises a paramyxovirus fusion protein (“F protein”) or a biologically active portion thereof.

[0090] In some embodiments, the one or more paramyxovirus fusion (F) proteins is an F protein from a henipavirus or is a biologically active portion thereof or variant thereof.

[0091] In some embodiments, the henipavirus is a Hendra virus.

[0092] In some embodiments, the henipavirus is a Nipah virus.

[0093] In some embodiments, the paramyxovirus F protein is a wild-type NiV-F protein or a variant or a biologically active portion thereof.

[0094] In some embodiments, the paramyxovirus F protein is a variant NiV-F that is a variant or a biologically active portion of a wild-type NiV-F protein.

[0095] In some embodiments, the paramyxovirus F protein is an F0 precursor or is a proteolytically cleaved form thereof comprising Fl and F2 subunits.

[0096] In some embodiments, the proteolytically cleaved form is a cathepsin L cleavage product.

[0097] In some embodiments, the one or more fusogens comprise one or more chimeric proteins.

[0098] In some embodiments, the one or more chimeric proteins comprise at least one chimeric protein that comprises a paramyxovirus envelope protein or biologically active portion thereof,

[0099] In some embodiments, the paramyxovirus envelope protein is any one of paramyxovirus envelope proteins describe herein.

[0100] In some embodiments, the one or more chimeric proteins comprises a target binding domain.

[0101] In some embodiments, the targeting binding domain is an scFv.

[0102] In some embodiments, the one or more chimeric proteins comprise (i) a paramyxovirus envelope protein or biologically active portion thereof and (ii) an scFv.

[0103] In some embodiments, the scFv targets an antigen present on the surface of a B cell, a T cell, a natural killer cell, a glial progenitor cell, a cardiac cell, a blood cell, a hepatocyte, a stem cell, or an induced pluripotent stem cell.

[0104] In some embodiments, the scFv targets an antigen present on the surface of a B cell.

[0105] In some embodiments, the scFv targets an antigen present on the surface of a T cell.

[0106] In some embodiments, the scFv targets CD8.

[0107] In some embodiments, the scFv targets CD4.

[0108] In some embodiments, the transfer plasmid comprises a viral nucleic acid.

[0109] In some embodiments, the viral nucleic acid comprises one or more of (e.g., all of) the following nucleic acid sequences: 5’ LTR (c.g., comprising U5 and lacking a functionalU3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT) / central termination sequence (CTS) (e.g. DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g. WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).

[0110] In some embodiments, the transfer plasmid comprises a transgene.

[0111] In some embodiments, the transgene encodes a nuclease.

[0112] In some embodiments, the nuclease is a Cas, a TALEN, or a zinc-finger nuclease.

[0113] In some embodiments, the transfer plasmid comprises a nucleotide sequence that encodes a gRNA.

[0114] In some embodiments, the transgene encodes an antibody or a biologically active portion thereof.

[0115] In some embodiments, the transgene encodes a chimeric antigen receptor.

[0116] In some embodiments, the chimeric antigen receptor comprises an extracellular ligand-binding domain having specificity for CD 19, CD20, CD22, and / or BCMA, a hinge domain, a transmembrane domain, a co- stimulatory domain, and an intracellular signaling domain.

[0117] In some embodiments, the transgcnc encodes an antigen.

[0118] In some embodiments, the transgene encodes a therapeutic polypeptide.

[0119] In some embodiments, the therapeutic polypeptide is useful for protein replacement therapy.

[0120] In some embodiments, the culturing comprises introducing into the producer cells the one or more nucleic acids for the production of the viral vector.

[0121] In some embodiments, the one or more nucleic acid for the production of the viral vectors are stably integrated into the genome of one or more producer cells.

[0122] In some embodiments, introducing the one or more nucleic acids into the producer cells comprises transfecting the one or more nucleic acids into the producer cells.

[0123] In some embodiments, the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid, wherein the envelope plasmid comprises at least one nucleic acid that encodes one or more fusogens.

[0124] In some embodiments, the method further comprises treating the producer cells with a nuclease.

[0125] In some embodiments, the method comprises treating the producer cells with a nuclease at a time that is at least 30 minutes after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0126] In some embodiments, the method further comprising treating the producer cells with a nuclease at a time that is no more than 6 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0127] In some embodiments, the method further comprising treating the producer cells with a nuclease at a time that is no more than 4 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0128] In some embodiments, the method further comprising treating the producer cells with a nuclease at a time that is no more than 2 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0129] In some embodiments, the method further comprising treating the producer cells with a nuclease at a time that is no more than 1 hour after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0130] In some embodiments, the method further comprising treating the producer cells with a second nuclease treatment.

[0131] In some embodiments, the second nuclease treatment is performed concurrently with (c) harvesting the producer cells or the producer cell culture.

[0132] In some embodiments, the second nuclease treatment is performed after harvesting the producer cells or the producer cell culture.

[0133] In some embodiments, treating the producer cells with a nuclease treatment at a time that is at least 30 minutes and no more than 6 hours after one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producercells with a second nuclease treatment at a time that is about 48 hours after one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0134] In some embodiments, treating the producer cells with a nuclease treatment at a time that is no more than 6 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0135] In some embodiments, treating the producer cells with a nuclease treatment at a time that is no more than 4 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0136] In some embodiments, treating the producer cells with a nuclease treatment at a time that is no more than 2 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0137] In some embodiments, an amount of nuclease added to the producer cell culture medium is determined according to equations (eqn.) 1 and / or 2.

[0138] In some embodiments, an amount of nuclease added to the harvested producer cells is determined according to equations 3 and / or 4.

[0139] In some embodiments, the nuclease is an endonuclease capable of cleaving double-stranded and single- stranded nucleic acid.

[0140] In some embodiments, the nuclease is a salt activated nuclease.

[0141] In some embodiments, the salt activated nuclease is selected from: benzonase,RNase A, DNase I, micrococcal nuclease, and S7 nuclease.

[0142] In some embodiments, the nuclease is MSAN.

[0143] In some embodiments, the producer cells are treated with about 100,000 to500,000 units of MSAN.

[0144] In some embodiments, the producer cells are treated with about 1 Unit (U) of MSAN per 1 mL of cell culture medium to 100 U of MSAN per 1 mL of cell culture medium.

[0145] In some embodiments, the producer cells are treated with about 100U of MSAN per 1 mL of cell culture medium. In some embodiments, the producer cells are treated with about 50U of MSAN per 1 mL of cell culture medium. In some embodiments, the producer cells are treated with about 25U of MSAN per 1 mL of cell culture medium. In some embodiments, the producer cells are treated with about 10U of MSAN per 1 mL of cell culture medium. In some embodiments, the producer cells are treated with about 4U of MSAN per 1 mL of cell culture medium. In some embodiments, the producer cells are treated with about 3U of MSAN per 1 mL of cell culture medium. In some embodiments, the producer cells are treated with about 2U of MSAN per 1 mL of cell culture medium. In some embodiments, the producer cells are treated with about 1U of MSAN per 1 mL of cell culture medium.

[0146] In some embodiments, the producer cells are cultured in a suspension culture.

[0147] In some embodiments, the producer cells are cultured in serum free medium.

[0148] In some embodiments, the method further comprising agitating the producer cells.

[0149] In some embodiments, the producer cells are cultured in medium that includes an anti-cell clumping agent.

[0150] In some embodiments, the method comprising agitating the producer cells and culturing the cell producer cells in medium that includes an anti-clumping agent.

[0151] In some embodiments, the producer cells are cultured in medium that includes a surfactant.

[0152] In some embodiments, the producer cells are cultured in medium that includes a surfactant and an antidumping agent.

[0153] In some embodiments, the method comprises agitating the producer cells and culturing the cell producer cells in medium that includes a surfactant.

[0154] In some embodiments, the method comprises agitating the producer cells and culturing the cell producer cells in medium that includes a surfactant and an anti-clumping agent.

[0155] In some embodiments, the method further comprises a settling step, wherein after harvesting the producer cells are allowed to settle.

[0156] In some embodiments, settling is performed for about 4 hours to about 48 hours.

[0157] In some embodiments, settling is performed for about 4 hours to about 16 hours.

[0158] In some embodiments, the method further comprises enriching the viral vectors from the producer cell culture.

[0159] In some embodiments, the method further comprises generating a crude drug substance from the producer cell culture, wherein the crude drug substance comprises the viral vectors produced by the producer cells or a subset thereof.

[0160] In some embodiments, generating a crude drug substance from the producer cell culture comprises enriching the viral vectors from the producer cell culture.

[0161] In some embodiments, generating a crude drug substance from the producer cell culture comprises removing producer cell DNA and / or producer cell protein from the producer cell culture.

[0162] In some embodiments, the producer cell culture comprises the producer cells and / or culture medium.

[0163] In some embodiments, the crude drug substance is characterized by: (i) an at least 3-fold increase in viral vector titer as compared to a crude drug substance produced by an alternative process; (ii) an at least 1.5-fold increase in infectivity as compared to a crude drug substance produced by an alternative process; (iii) an at least 1000-fold decrease in the amount of producer cell DNA as compared to a crude drug substance produced by an alternative process; (iv) an at least 15-fold decrease in the amount of producer cell protein as compared to a crude drug substance produced by an alternative process; (v) an at least 1.5-fold increase in transduction efficiency in a target cell exposed to the crude drug substance as compared to a crude drug substance produced by an alternative process; or (vi) an at least 20-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process; (vii) an at least 3-fold increase in fusogen expression on the viral vectors as compared to a crude drag substance produced by an alternative process; (viii) or a combination thereof.

[0164] In some embodiments, the method further comprises generating a drug substance from the crude drug substance, wherein the drug substance comprises the viral vectors produced by the producer cells or a subset thereof.

[0165] In some embodiments, generating a drug substance from the crude drag substance comprises further enriching the viral vectors from the crude drug substance.

[0166] In some embodiments, generating a drug substance from the crude drag substance comprises further removing producer cell DNA and / or producer cell protein from the crude drug substance.

[0167] In some embodiments, generating a drug substance from the crude drag substance comprises adding a pharmaceutically acceptable excipient.

[0168] In some embodiments, the drag substance is characterized by: (i) an at least 5- fold increase in viral vector titer as compared to a drag substance produced by an alternative process; (ii) an at least 5-fold increase in infectivity as compared to a drag substance produced by an alternative process; (iii) an at least 2000-fold decrease in the amount of producer cellDNA as compared to a drug substance produced by an alternative process; (iv) an at least 10- fold decrease in the amount of producer cell protein as compared to a drug substance produced by an alternative process; (v) an at least 2-fold increase in transduction efficiency in a target cell exposed to the drug substance as compared to a drug substance produced by an alternative process; or (vi) an at least 50-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process; (vii) an at least 6-fold increase in fusogen expression on the viral vectors as compared to a crude drug substance produced by an alternative process; (viii) or a combination thereof.

[0169] In some embodiments, the alternative process is a comparable process wherein: (i) a ratio of an envelope plasmid to a packaging plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1; (ii) a ratio of an envelope plasmid to a transfer plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1; (iii) a ratio of a first envelope plasmid to a second envelope plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1; (iv) an amount of an envelope plasmid that is introduced into producer cells is equal to or less than an amount of an packaging plasmid that is introduced into the producer cells; (v) an amount of an envelope plasmid that is introduced into producer cells is equal to or less than the amount of a transfer plasmid that is introduced into the producer cells; (vi) an amount of a first envelope plasmid that is introduced into producer cells is equal to or less than the amount of a second envelope plasmid that is introduced into the producer cells; (vii) producer cells are treated with a nuclease at a time that is more than 6 hours after at least one nucleic acid that encodes one or more fusogens is introduced into the producer cells; (viii) producer cells are not cultured in a suspension culture; (ix) cell aggregation of producer cells is not reduced; (x) producer cells are not agitated during culture; (xi) culture medium for producer cells does not include an anti-clump agent or a surfactant; or (xii) a combination thereof.

[0170] In some embodiments, the first envelope plasmid encodes G protein.

[0171] In another aspect, this disclosure features a producer cell comprising one or more nucleic acids for the production of lenti viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid and an envelope plasmid that encodes one or more fusogens, and wherein the ratio of the envelope plasmid to the packaging plasmid within the producer cell is greater than 1:1.

[0172] In some embodiments, the ratio of the envelope plasmid to the packaging plasmid within the producer cell is greater than 2:1.

[0173] In another aspect, this disclosure features a producer cell comprising one or more nucleic acids for the production of lentiviral vectors, wherein the one or more nucleic acids comprise a first envelope plasmid and a second envelope plasmid, wherein the first envelope plasmid and second envelope plasmid each encode one or more fusogens, and wherein the ratio of the first envelope plasmid to the second envelope plasmid within the producer cell is greater than 1:1.

[0174] In some embodiments, the ratio of the first envelope plasmid to the second envelope plasmid within the producer cell is greater than 2:1.

[0175] In some embodiments, the first envelope plasmid encodes G protein.

[0176] In another aspect, this disclosure features a producer cell comprising engineered lentiviral vectors, wherein the producer cell had been transfected with one or more nucleic acids for the production of lentiviral vectors at a first time point and had been exposed to a nuclease at a second time point, wherein the second time point is at least 30 minutes after and at most 6 hours after the first time point.

[0177] In another aspect, this disclosure features a viral vector produced according to any of the methods described herein.

[0178] In another aspect, this disclosure features a viral vector produced using any of the producer cells described herein.BRIEF DESCRIPTION OF THE DRAWING

[0179] The Drawing included herein, which is composed of the following Figures, is for illustration purposes only and not for limitation.

[0180] FIG. 1 includes a table providing certain characteristics of a crude drug substance including lentivirus produced by an alternative process (“Baseline”) and a crude drug substance including lentivirus produced by an exemplary method described herein (“Improved Process”). The table also includes a column showing the fold increase observed the Baseline and Improved Process. As used in the table, “TU” means transducing unit, “VLP” means virus like particles, and “L BR” means liter of bioreactor.

[0181] FIG. 2 includes a bar graph depicting an exemplary fold increase in functional lentivirus titer. The “Baseline” bar shows the transducing units per milliliter in a drug substance including lentivirus produced by an alternative process. The “Improved” bar shows the transducing units per milliliter in a drug substance including lentivirus produced by an exemplary method described herein. As shown, the drug substance including lentivirus produced by an exemplary method described herein showed an almost 55x increase in TU / mL as compared to the drug substance including lentivirus produced by a prior method.

[0182] FIG. 3 includes a bar graph depicting an exemplary fold increase in yield in a crude drug substance including lentivirus and a drug substance including lentivirus. “Baseline” bars show the transducing units per liter of culture in a crude drug substance or a drug substance, both of which include lentivirus produced by an alternative process. “Improved” bars show the transducing units per liter of culture in a crude drug substance or a drug substance, both of which include lentivirus produced by an exemplary method described herein. As shown in the portion of the graph marked with “Crude,” the crude drug substance including lentivirus produced by an exemplary method described herein showed a 4.8x increase in TU / L of culture as compared to the crude drug substance including lentivirus produced by a prior method. As shown in theportion of the graph marked with “DS,” the drug substance including lentivirus produced by an exemplary method described herein showed a 6. lx increase in TU / L of culture as compared to the drug substance including lentivirus produced by a prior method.

[0183] FIG. 4 includes a bar graph depicting an exemplary increase in infectivity in a crude drag substance including lentivirus and a drug substance including lentivirus. Infectivity was reported as the number of viral particles required to deliver one transducing unit (VLP / TU). As such, a lower ratio indicates a more infectious preparation. “Baseline” bars show the VLP / TU of a crude drug substance or a drug substance, both of which include lentivirus produced by an alternative process. “Improved” bars show the VLP / TU of a crude drag substance or a drug substance, both of which include lentivirus produced by an exemplary method described herein. As shown in the portion of the graph marked with “Crude,” the crude drag substance including lentivirus produced by an exemplary method described herein showed an approximately 2x decrease in VLP / TU as compared to the crude drug substance including lentivirus produced by a prior method. As shown in the portion of the graph marked with “DS,” the drug substance including lentivirus produced by an exemplary method described herein showed an approximately 7x decrease in VLP / TU as compared to the drag substance including lentivirus produced by a prior method.

[0184] FIGs. 5A and 5B include bar graphs depicting an exemplary decrease in producer cell (PC) DNA and protein, respectively, measured in a crude drag substance including lentivirus and a drag substance including lentivirus. “Baseline” bars show the pg of producer cell DNA per transducing unit (pg DNA / TU) or pg of producer cell protein per transducing unit (pg PCP / TU) measured from a crude drug substance or a drug substance, both of which include lentivirus produced by an alternative process. “Improved” bars show the pg of producer cell DNA per transducing unit (pg DNA / TU) or pg of producer cell protein per transducing unit (pg PCP / TU) measured from a crude drug substance or a drug substance, both of which include lentivirus produced by an exemplary method described herein. As shown in the portion of FIG. 5A marked with “Crude,” the crude drag substance including lentivirus produced by anexemplary method described herein showed an approximately 1350x decrease in pg DNA / TU as compared to the crude drug substance including lentivirus produced by a prior method. As shown in the portion of FIG. 5A marked with “DS,” the drug substance including lentivirus produced by an exemplary method described herein showed an approximately 3000x decrease in pg DNA / TU as compared to the drug substance including lentivirus produced by a prior method. As shown in the portion of FIG. 5B marked with “Crude,” the crude drug substance including lentivirus produced by an exemplary method described herein showed an approximately 23x decrease in pg PCP / TU as compared to the crude drug substance including lentivirus produced by a prior method. As shown in the portion of FIG. 5B marked with “DS,” the drug substance including lentivirus produced by an exemplary method described herein showed an approximately 1 lx decrease in pg PCP / TU as compared to the drug substance including lentivirus produced by a prior method.

[0185] FIGs. 6A and 6B include schematics of exemplary lentiviral vectors having fusogens in their envelope bilayers. FIG. 6A includes a schemative of an exemplary lentiviral vector having paramyxovirus G protein and paramyxovirus F protein in its envelope bilayer. FIG. 6B includes a schemative of an exemplary lentiviral vector having a chimeric protein and paramyxovirus F protein in its envelope bilayer. The illustrated chimeric protein comprises a portion of paramyxovirus G protein and an scFV.

[0186] FIG. 7 includes a bar graph depicting an exemplary association between the amount of G protein envelope plasmid introduced into a producer cell relative to another envelope plasmid, and the amount of G protein that was on a lentiviral vector relative to capsid as produced by a method as described herein.

[0187] FIG. 8 includes a line graph depicting an exemplary increase in the intensity of G protein detected on an individual fusosome (e.g. lentiviral vectors comprising a fusogen in its envelope bilayer, produced by a method described herein) when an increased amount of G protein envelope plasmid was introduced into a producer cell relative to another envelopeplasmid. Line 1 represents the results obtained when equal ratios of G protein to another envelope plasmid were used. Line 2 and 3 represent replicate experiments where an increased ratio of G protein relative to another envelope plasmid was used. This line graph also depicts the frequency at which G protein was deteced on a fusosome. The grey dotted line represents the intensity of the negative control.

[0188] FIG. 9 includes a pie chart depicting an exemplary association between optimized G levels and the proportion of fusosomes that contain both G protein and F relative to just F protein.

[0189] FIG. 10 includes a bar graph depicting an exemplary association between the amount of G protein envelope plasmid introduced into a producer cell, relative to another envelope plasmid, and crude functional titer of a lentiviral vector produced by a method as described herein. Functional titer is depicted as transducing units per milliter of lentiviral culture of producer cells adapted for serum-free growth in suspension, as described herein.

[0190] FIG. 11 includes a line graph depicting an exemplary increase in the percentage of CD8 positive T cells that are positive for a CAR. CAR was delivered to T cell containing periphreal blood monuclear cells (PBMCs) by fusosomes either produced by a method described herein or produced by an alternative process. The amount of fusosomes per PBMC is depicted as international unit of fusosome per PBMC (IU / PBMC).

[0191] FIGs. 12A and 12B include bar graphs depicting an exemplary increase in the percentage of T cells expressing FMC63 CAR observed when PBMCS were exposed to increasing amounts of fusosomes produced either by a method described herein or an alternative process. FIG. 12A shows the percentage FMC63 CAR expression in CD8 positive T cells and FIG. 12B shows the percentage of FMC63 CAR expression in CD4 positive T cells. Controls were not exposed to fusosomes.

[0192] FIG. 13 depicts a bar graph showing an exemplary reduction in NALM6 tumor cells when exposed to increasing amounts of fusosomes produced by a method described hereinrelative to fusosomes produces by an alternative process. Controls were not exposed to fusosomes.CERTAIN DEFINITIONS

[0193] In general, terminology used herein is in accordance with its understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.

[0194] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0195] Antibody: As used herein, the term “antibody” refers to an immunoglobulin molecule, or fragment thereof, that binds specifically to an epitope (e.g., of an antigen). Naturally-occurring human antibodies typically include two identical heavy chains and two identical light chains, each of which includes a variable domain and a constant domain. The constant domain defines the antibody isotype (IgG, IgA, IgM, IgE, etc). Those skilled in the art aware that different animal species utilize different antibody structures in nature. For example, camelid antibodies are single chain antibodies. Those skilled in the art will further be aware that antibody variable domains are typically characterized by framework region (FR) sequences and complement-determining-region (CDR) sequences. Each variable domain typically includes three CDRs - CDR1, CDR2, and CDR3, which together contribute to specificity and / or affinity of epitope binding. In some embodiments, antibodies may be produced in or by organisms. In some embodiments, antibodies may be produced in or by cells in vitro (e.g., by hybridomas and / or by engineered cells). In some embodiments, antibody fragment(s) e.g., as may be produced by cleavage or recombinantly) may be generated and / or utilized in accordance with the present disclosure. Those skilled in the art are aware that a variety of technologies have beendeveloped to incorporate binding features (e.g., one or more CDRs and / or FR sequences, and in particular sets of 3 CDRs, optionally together with FR sequences) into new contexts. In some embodiments, CDR sequences may be maintained and other elements changed - e.g., as is done in humanization. Alternatively or additionally, in some embodiments, variable regions may be associated with alternative constant regions e.g., with constant regions from a different organism and / or that include one or more particular sequence features or elements desired, for example, to impart a particular attribute to the antibody agent. Those skilled in the art are further aware of a variety of technologies commonly utilized to associate binding features of two or more different antibodies with one another - e.g., in a single multispecific (most commonly bispecific) agent. Various known formats of antibody agents (i.e., agents that incorporate antibody binding sequences such as one or more CDRs, or a full set of CDRs) include, for instance: antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; KALBITOR®s, Zybodies®, etc.

[0196] Antigen -. The term “antigen”, as used herein, refers to an agent that elicits an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen- specific antibodies); in some embodiments, an elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen). In some embodiments, and antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, alipid, a polymer (in some embodiments other than a biologic polymer [e.g., other than a nucleic acid or amino acid polymer) etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those of ordinary skill in the ail will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen.

[0197] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0198] Crude Drug Substance: As used herein, the term “crude drug substance” is an intermediate composition generated in the production of a drug substance. In some embodiments, an active ingredient (e.g., viral vectors) in a crude drug substance have been enriched from other components (e.g., producer cell components, e.g., producer cell DNA and / or protein) that the active ingredient has been associated with during a production process. A crude drug substance often needs further processing to purify, isolate, or otherwise enrich the active ingredient prior to being classified as a drug substance or being incorporated into a drug product.

[0199] Drug Substance: As used herein, the term “drug substance” is an active ingredient (e.g., viral vectors) that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of a subject’s body, but does not include intermediates used in thesynthesis of such ingredient. A drug substance may need further processing to become a “drug product,” which is a finished dosage form (e.g., tablet or solution) to be administered to a subject. However, a drug substance does not require further processing to purify, isolate, or otherwise enrich the active ingredient prior to incorporation into a drug product.

[0200] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and / or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and is understoodby those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0201] Excipient: As used herein, refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.

[0202] “Improved ’'1“increased,” “decreased” or “reduced”: As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a method of interest may be “improved” relative to that obtained with a comparable reference method. Alternatively or additionally, in some embodiments, an assessed value achieved in a method of interest may be “improved” relative to that obtained in the same method under different conditions (e.g., prior to or after an event or step). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance.

[0203] Nucleic acid: As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleicacid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has a functional activity.

[0204] Plasmid', as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., transfection, e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

[0205] Polypeptide: As used herein refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only nonnatural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, apolypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide ofinterest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0206] Producer cell: As used herein, a “producer cell” is a cell capable of producing a viral vector when cultured under appropriate conditions. A number of cells are known to be capable of producing viral vectors, including for example, HEK293 cells, PER.C6 cells, VERO cells, HEK 293T cells, A549 cells, MRC5 cells, HeLa cells, Sf9 cells, and BHK-21 cells.

[0207] Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0208] Tropism: As used herein, the term “tropism” refers to the ability of a molecule (e.g., a fusogen) or viral vector to interact with molecules (e.g., receptors or antigens) associated with a cell (e.g., in its cell membrane). For example, if a fusogen has a tropism for T cells, the fusogen is able to interact with molecules on the surface of or in the cell membrane of a T cell. In some embodiments, this interaction allows for the fusion of the viral vector with the membrane of the cell and ultimately entry into the cell.

[0209] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differsfrom or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0210] The present disclosure provides viral vectors, methods for producing viral vectors, and related technologies. The present disclosure recognizes that viruses have a natural ability to enter into and deliver genetic material to cells. As such, viruses can be useful in both gene and cell therapies. The present disclosure therefore provides viral vectors including, e.g., gammaretroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. The present disclosure further provides methods for producing such viral vectors, e.g., by culturing producer cells.

[0211] Among other things, the present disclosure also provides the recognition that viral vectors, such as gammaretroviral vectors and lentiviral vectors comprise an envelope, which includes envelope proteins. Some of the envelope proteins are fusogens, which are proteins that mediate fusion of plasma membranes (e.g., a viral envelope with a cellular membrane).Increasing expression of fusogens, exploiting the endogenous tropism of fusogens or associated proteins, and / or modulating the tropism of fusogens can be useful for targeting certain cell types in a therapeutically acceptable manner. However, methods of efficiently producing viral vectors, including viral vectors expressing fusogens, has remained a challenge. For instance, current methods of producing such viral vectors face challenges, including low yields of produced viral vectors, low potency of produced viral vectors, and undesirable levels of process related impurities (e.g., producer cell protein and DNA) in final products. The present disclosure addresses these challenges by, inter alia, providing methods that include increased expression of a fusogen, decreased producer cell aggregation during culture, nuclease treatment at a time close to transfection, or a combination thereof.

[0212] The methods described herein can be useful for producing multiple viral vectors. In some embodiments, the methods described herein, particularly those comprising viral vectors comprising fusogens, are effective for increasing titer and / or infectivity of viral vectors. The methods provided herein are also useful in reducing unwanted impurities related to production of viral vectors in producer cells, e.g., producer cell DNA and proteins. The reduction of impurities can be especially beneficial when viral vectors are used in a drug substance (e.g., a pharmaceutical composition) for administration to a subject. Accordingly, the present disclosure further provides drug substances comprising viral vectors, e.g., produced by methods described herein. A non-limiting example of the viral vectors capble of being produced using the methods described herein include the viral vectors described in US 11,535,869, the entire contents of which is incorporated by reference herein.I. Lentivirus Systems

[0213] The present disclosure provides, in pail, lentiviral vector systems, and methods of making and using the same. The present disclosure further provides one or more nucleic acids for the production of a viral vector. In some embodiments, one or more nucleic acids comprise plasmids for the production of a viral vector. One or more nucleic acids (e.g., plasmids) for the production of a viral vector can include various components, which can be divided among the one or more nucleic acids (e.g., plasmids) in various ways, as known in the art. For example, for the production of lentiviral vectors, several lentiviral vector systems have been developed, including first-generation, second-generation, third-generation and more recently fourthgeneration vector systems. A brief description of those systems, including the nucleic acids (e.g., plasmids) is included below.A. First Generation

[0214] A first-generation lentiviral vector system typically includes three plasmids: a packaging plasmid, an envelope plasmid, and a transfer plasmid. A packaging plasmid of a first- generation lentivirus system usually encodes one or more accessory polypeptides (such as viralinfectivity factor (Vif), viral protein r (Vpr), viral protein u (Vpu), and negative factor (Nef)) and one or more polypeptides involved in viral production (such as group- specific antigen (Gag), polymerase (Pol), regulator of virion (Rev), and trans-activator of transcription (Tat) genes).

[0215] An envelope plasmid of a first-generation lentiviral vector system typically encodes an envelope protein (Env) which is usually the HIV-1 Env glycoprotein or the VSV-G glycoprotein.

[0216] A first-generation lentiviral vector system can also include a transfer plasmid which typically has a 5’ long terminal repeat (LTR), a Rev responsive element, a sequence for a promoter of interest, a sequence encoding a transgene of interest, and a 3’ LTR. The LTRs typically comprise a U3, an R, and a U5 region. A transgene as described herein can encode any gene product (e.g., RNA or polypeptide). Exemplary transgenes and encoded gene products are described herein.

[0217] In some embodiments, a lentiviral vector system disclosed herein is a first- generation lentiviral vector system. In some embodiments, a lentivirus system comprises one or more plasmids of a first-generation lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises one or more packaging plasmids, one or more envelope plasmids, and one or more transfer plasmids of a first-generation lentiviral vector system. In some embodiments, a lentivirus system comprises a packaging plasmid, an envelope plasmid and a transfer plasmid of a first-generation lentiviral vector system. In some embodiments, a lentivirus system comprises a packaging plasmid, two envelope plasmids and a transfer plasmid of a first-generation lentiviral vector system.

[0218] In some embodiments, when a lentiviral vector system disclosed herein is a first- generation lentiviral vector system, one or more envelope plasmids do not encode an Env polypeptide. In some embodiments, one or more envelope plasmids of a lentiviral vector system encode one or more fusogens or a biologically active portions thereof. In some embodiments, a fusogen comprises a glycoprotein, e.g., G protein or portion thereof, and / or F protein or portionsthereof. In some embodiments, one or more envelope plasmids encode paramyxovirus glycoprotein G (G protein) or a portion thereof and paramyxovirus fusion protein (F protein) or a portion thereof. In some embodiments, a fusogen comprises a chimeric protein. Various fusogens are contemplated as discussed further herein.B. Second Generation

[0219] A second-generation lentiviral vector system also generally includes three plasmids, similar to a first-generation lentiviral vector system. The three plasmids of a second- generation lentiviral vector system are: a packaging plasmid, an envelope plasmid and a transfer plasmid. One of the differences in a second-generation lentiviral vector system as compared to the earlier generation is that the packaging plasmid does not encode viral accessory polypeptides Vif, Vpr, Vpu, and Nef. In a second-generation lentiviral vector system, the packaging plasmid only encodes the Gag, Pol, Tat and Rev polypeptides.

[0220] In some embodiments, a lentiviral vector system disclosed herein is a second- generation lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises one or more plasmids of a second-generation lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises one or more packaging plasmids, one or more envelope plasmids, and one or more transfer plasmids of a second- generation lentiviral vector system. In some embodiments, a lentivirus system comprises a packaging plasmid, an envelope plasmid and a transfer plasmid of a second-generation lentiviral vector system. In some embodiments, a lentivirus system comprises a packaging plasmid, two envelope plasmids and a transfer plasmid of a second-generation lentiviral vector system.

[0221] In some embodiments, when a lentiviral vector system disclosed herein is a second-generation lentiviral vector system, one or more envelope plasmids do not encode an Env polypeptide. In some embodiments, one or more envelope plasmids of a lentiviral vector system encode one or more fusogens or a biologically active portions thereof. In some embodiments, a fusogen comprises a glycoprotein, e.g., G protein or portion thereof, and / or F protein or portionsthereof. In some embodiments, one or more envelope plasmids encode paramyxovirus glycoprotein G (G protein) or a portion thereof and paramyxovirus fusion protein (F protein) or a portion thereof. In some embodiments, a fusogen comprises a chimeric protein. Various fusogens are contemplated as discussed further herein.C. Third Generation

[0222] Third-generation lentiviral vector systems are newer and were developed to increase the safety of earlier generation vector systems. In the third-generation lentiviral vector system there are generally four plasmids: a packaging plasmid, an envelope plasmid, a regulatory plasmid, and a transfer plasmid. The envelope plasmid of third-generation lentiviral vector systems is relatively unchanged from envelope plasmids in prior generations in that it encodes an Env polypeptide. The packaging and transfer plasmids have several differences as described herein.

[0223] In a third-generation lentiviral vector system, the packaging plasmid only encodes the Gag and Pol polypeptides. A separate plasmid, the regulatory plasmid encodes the Rev polypeptide. The transfer plasmid also includes changes particularly to the LTRs. To enhance safety, the LTRs were modified in the U3 region. A transfer plasmid of a third-generation lentiviral vector system includes LTR regions comprising an R element, a U5 element, an RRE element, a posttranscriptional regulatory elements (PREs), and a self-inactivating (SIN) region.

[0224] In some embodiments, a lentiviral vector system disclosed herein is a third- generation lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises one or more plasmids of a third-generation lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises one or more packaging plasmids, one or more envelope plasmids, one or more regulatory plasmids, and one or more transfer plasmids of a third-generation lentiviral vector system. In some embodiments, a lentivirus system comprises a packaging plasmid, a regulatory plasmid, an envelope plasmid and a transfer plasmid of a third-generation lentiviral vector system. In some embodiments, a lentivirus systemcomprises a packaging plasmid, a regulatory plasmid, two envelope plasmids and a transfer plasmid of a third-generation lentiviral vector system.

[0225] In some embodiments, when a lentiviral vector system disclosed herein is a third- generation lentiviral vector system, one or more envelope plasmids do not encode an Env polypeptide. In some embodiments, one or more envelope plasmids of a lentiviral vector system encode one or more fusogens or a biologically active portions thereof. In some embodiments, a fusogen comprises a glycoprotein, e.g., G protein or portion thereof, and / or F protein or portions thereof. In some embodiments, one or more envelope plasmids encode paramyxovirus glycoprotein G (G protein) or a portion thereof and paramyxovirus fusion protein (F protein) or a portion thereof. In some embodiments, a fusogen comprises a chimeric protein. Various fusogens are contemplated as discussed further herein.D. Fourth Generation

[0226] Further iterations of lentiviral vector systems have been recently developed. For example, a fourth-generation lentiviral vector system having more than four plasmids has been reported to increases the number of recombination events required to generate replication- competent lentivirus (RCL). Such a fourth-generation lentiviral vector typically includes one or more plasmids whose expression is driven by a Tet-Off and / or Tat transactivator. A fourthgeneration lentiviral vector system can include five plasmids, wherein one or more plasmid(s) is / are pTre-gag-pro, LTRHIV-vpr-pol, pCMV-VSVG, pMV-tet-off, and pTre-tat-ires-rev.

[0227] In some embodiments, a lentiviral vector system disclosed herein is a fourthgeneration lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises one or more plasmids of a fourth-generation lentiviral vector system as disclosed herein.

[0228] In some embodiments, when a lentiviral vector system disclosed herein is a fourth-generation lentiviral vector system, one or more envelope plasmids do not encode an Env polypeptide. In some embodiments, one or more envelope plasmids of a lentiviral vector systemencode one or more fusogens or a biologically active portions thereof. In some embodiments, a fusogen comprises a glycoprotein, e.g., G protein or portion thereof, and / or F protein or portions thereof. In some embodiments, one or more envelope plasmids encode paramyxovirus glycoprotein G (G protein) or a portion thereof and paramyxovirus fusion protein (F protein) or a portion thereof. In some embodiments, a fusogen comprises a chimeric protein. Various fusogens are contemplated as discussed further herein.E. Certain Exemplary Plasmids

[0229] In some embodiments, a lentiviral vector system disclosed herein is a first- generation lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises a packaging plasmid, two envelope plasmids, and a transfer plasmid of a first- generation lentiviral vector system. In some embodiments, the envelope plasmids do not encode an Env polypeptide. In some embodiments, the envelope plasmids encode one or more fusogens or a biologically active portions thereof. In some embodiments, the one or more fusogens comprise one or more glycoproteins, e.g., G protein or a portion thereof, and / or F protein or a portion thereof. In some embodiments, a first envelope plasmid encodes a G protein or a biologically active portion thereof. In some embodiments, a second envelope plasmid encodes a F protein or a biologically active portion thereof. In some embodiments of such a lentiviral vector system, a transfer plasmid comprise a sequence encoding a transgene disclosed herein. In some embodiments, a transgene is or comprises a Chimeric Antigen Receptor, e.g., as disclosed herein.

[0230] In some embodiments, a lentiviral vector system disclosed herein is a second- generation lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises a packaging plasmid, two envelope plasmids, and a transfer plasmid of a second- generation lentiviral vector system. In some embodiments, the envelope plasmids do not encode an Env polypeptide. In some embodiments, the envelope plasmids encode one or more fusogens or a biologically active portions thereof. In some embodiments, the one or more fusogenscomprise one or more glycoproteins, e.g., G protein or a portion thereof, and / or F protein or a portion thereof. In some embodiments, a first envelope plasmid encodes a G protein or a biologically active portion thereof. In some embodiments, a second envelope plasmid encodes a F protein or a biologically active portion thereof. In some embodiments of such a lentiviral vector system, a transfer plasmid comprise a sequence encoding a transgene disclosed herein. In some embodiments, a transgene is or comprises a Chimeric Antigen Receptor, e.g., as disclosed herein.In some embodiments, a lentiviral vector system disclosed herein is a third-generation lentiviral vector system as disclosed herein. In some embodiments, a lentivirus system comprises a packaging plasmid, a regulatory plasmids, two envelope plasmids, and a transfer plasmid of a third-generation lentiviral vector system. In some embodiments, the envelope plasmids do not encode an Env polypeptide. In some embodiments, the envelope plasmids encode one or more fusogens or a biologically active portions thereof. In some embodiments, the one or more fusogens comprise one or more glycoproteins, e.g., G protein or a portion thereof, and / or F protein or a portion thereof. In some embodiments, a first envelope plasmid encodes a G protein or a biologically active portion thereof. In some embodiments, a second envelope plasmid encodes a F protein or a biologically active portion thereof. In some embodiments of such a lentiviral vector system, a transfer plasmid comprise a sequence encoding a transgene disclosed herein. In some embodiments, a transgene is or comprises a Chimeric Antigen Receptor, e.g., as disclosed herein.II. Transgenes

[0231] The present disclosure provides viral vectors and methods for producing viral vectors that can be used for numerous purposes. For example, as discussed above, in some embodiments, viral vectors as described herein can include a transgene. In some embodiments, a transfer plasmid, which includes one or more transgenes, is used in the production of a viral vector. Acceptable transgenes that can be included in such viral vectors can widely vary and canbe used for a large number of purposes. The only meaningful characteristic of such a transgene is that it be a nucleic acid that can be packaged and delivered by a viral vector.

[0232] In some embodiments, a transgene encodes a gene product. A gene product can be an RNA or a polypeptide.

[0233] In some embodiments, a transgene can encode an RNA. For example, a transgene can encode a gRNA, an siRNA, an shRNA, or miRNA.

[0234] In some embodiments, a transgene encodes a polypeptide.

[0235] In some embodiments, a transgene encodes a nuclease. In some embodiments, a nuclease is a Cas, a TALEN, or a zinc-finger nuclease. Viral vectors including transgenes encoding a nuclease can be useful for applications in which a viral vector is used to introduce genetic modifications into a cell. In such cases, a viral vector can enter into a cell and express the nuclease. In some embodiments, a viral vector may encode an RNA (e.g., a gRNA) and a polypeptide (e.g., a Cas) to impart targeted genetic modifications.

[0236] In some embodiments, a transgene encodes an antibody or portion thereof. Due to size limitations, in some embodiments, a transgene may encode an antibody having an alternative format that is smaller than a full canonical antibody (e.g., a Fab, a diabody, an scFv, a minibody, or nanobody). Viral vectors including transgenes encoding an antibody or portion thereof may be useful in applications involving targeted inhibition of molecules, e.g., molecules associated with specific cell types.

[0237] In some embodiments, a transgene encodes an antigen. Viral vectors including such transgenes can be helpful, e.g., in inducing desired immune responses.

[0238] In some embodiments, a transgene can encode a therapeutic polypeptide. In some embodiments, a transgene can encode a polypeptide used in protein replacement therapy. In some embodiments, a viral vector can include more than one transgene used in protein replacement therapy. For example, in some embodiments, a viral vector may include a firsttransgene encoding a nuclease that introduces a genetic modification knocking-out expression of an endogenous (e.g., dysfunctional) polypeptide and a second that delivers a (e.g., functional) replacement protein.

[0239] In some embodiments, a transgene can encode a chimeric antigen receptor (CAR). In some instances, a transgene encoding a CAR can be delivered to, e.g., a T-cell, for expression.

[0240] In some embodiments, the CAR binds to CD 19. In some embodiments, the CAR binds to CD22. In some embodiments, the CAR binds to CD20. In some embodiments, the CAR binds to BCMA. In some embodiments, the CAR binds to an EBV antigen. In some embodiments, the CAR binds to CD27. In some embodiments, the CAR binds to CD30. In some embodiments, the CAR binds to CD19 and CD20. In some embodiments, the CAR binds to CD 19 and CD22. In some embodiments, the CAR binds to CD 19 and CD27. In some embodiments, the CAR binds to EBNA1. In some embodiments, the CAR binds to EBNA3A. In some embodiments, the CAR binds to BRLF1. In some embodiments, the CAR binds to BALF4. In some embodiments, the CAR binds to EBNA3C. In some embodiments, the CAR binds to LMP1. In some embodiments, the CAR binds to LMP2. In some embodiments, the CAR binds to LMP2A. In some embodiments, the CAR binds to LMP2B. In some embodiments, the CAR binds to BZLF1 . In some embodiments, the CAR binds to BMLF1 . In some embodiments, the CAR binds to gp350. In some embodiments, the CAR binds to gH / gL. In some embodiments, the CAR binds to EBNA1 and LMP1. In some embodiments, the CAR binds to EBNA1 and LMP2A. In some embodiments, the CAR binds to EBNA1, LMP1 and LMP2A. In some embodiments, the CAR binds to LMP, BARF1 and EBNA1. In some embodiments, the CAR binds to CD 19 and an EBV antigen. In some embodiments, the CAR binds to CD20 and an EBV antigen. In some embodiments, the CAR binds to CD22 and an EBV antigen. In some embodiments, the CAR is selected from the group consisting of a first generation CAR, a second generation CAR, a third generation CAR, and a fourth generation CAR. In some embodiments, the CAR includes a single binding domain that binds to a single target antigen. In some embodiments, the CAR includes a single binding domain that binds to more than one targetantigen, e.g., 2, 3, or more target antigens. In some embodiments, the CAR includes two binding domains such that each binding domain binds to a different target antigens. In some embodiments, the CAR includes two binding domains such that each binding domain binds to the same target antigen. Detailed descriptions of exemplary CARs including CD19-specific, CD20-specific and CD19 / CD20-bispecific CARs can be found in WO2012 / 079000, WO2016 / 149578 and W02020 / 014482, the disclosures including the sequence listings and figures are incorporated herein by reference in their entirety. In some embodiments, the CAR includes two binding domains such that each binding domain binds to the same target antigen. Detailed descriptions of exemplary CARs including CD19-specific, CD22-specific and CD19 / CD22-bispecific CARs can be found in W02012 / 079000, WO2016 / 149578 and W02020 / 014482, the disclosures including the sequence listings and figures are incorporated herein by reference in their entirety. Detailed descriptions of exemplary CARs, TCRs or scFvs including CD27-specific, CD30- specific, EBNAl-specific, EBNA3C-specific, LMP1- specific, LMP2-specific, LMP2A-specific, gp35O-specific CARs, gH / gL- specific CARs can be found in EP 2 520 589, US 9403914, US 11180566, US 2021 / 0009706, EP 2 558 498 Bl, WO 2021 / 222929, US 2021 / 10206863, WO 2015 / 199617A1, WO 2012 / 109659A1, US 7786269B2, WO 2021 / 211455A1, US 2016 / 0199479, WO2019 / 201995A1, and US 11116835, the disclosures including the sequence listings and figures are incorporated herein by reference in their entireties. In some embodiments, the CAR includes two binding domains such that each binding domain binds to the same target antigen.

[0241] In some embodiments, the CD 19 specific CAR includes an anti-CD19 singlechain antibody fragment (scFv), a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co- stimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, the CD20 specific CAR includes an anti-CD20 scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co- stimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, the CD19 / CD20-bispecific CAR includes an anti-CD19 scFv, an anti-CD20 scFv, a transmembrane domain such as one derivedfrom human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, the CD22 specific CAR includes an anti-CD22 scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) costimulatory signaling domain, and a Cf)3^ signaling domain. In some embodiments, the CD19 / CD22-bispecific CAR includes an anti-CD19 scFv, an anti-CD22 scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3(^ signaling domain. In some embodiments, the EBNA1 specific CAR includes an anti-EBNAl scFv, a transmembrane domain such as one derived from human CD8a, a 4- IBB (CD137) co-stimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, the EBNA3A CAR includes an anti-EBNA3A scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3(^ signaling domain. In some embodiments, the EBNA3C CAR includes an anti-EBNA3C scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co- stimulatory signaling domain, and a CD32 signaling domain. In some embodiments, the LMP1 specific CAR includes an anti-LMPl scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3(^ signaling domain. In some embodiments, the LMP2 specific CAR includes an anti-LMP2 scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, the LMP2A CAR includes an anti-LMP2A scFv, a transmembrane domain such as one derived from human CD8a, a 4- IBB (CD137) co-stimulatory signaling domain, and a CD3(^ signaling domain. In some embodiments, the BZLF1 CAR includes an anti-BZLFl scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3(^ signaling domain. In some embodiments, the BMLF1 CAR includes an anti-BMLFl scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co- stimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, the gp35O CAR includes an anti-gp35O scFv, a transmembrane domain such as one derived from humanCD8a, a 4-1BB (CD137) co- stimulatory signaling domain, and a CD3^ signaling domain. In some embodiments, the gH / gL specific CAR includes an anti-gH / gL scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co- stimulatory signaling domain, and a CD3(^ signaling domain.

[0242] In some embodiments, the CAR comprises a commercial CAR construct canned by a T cell. Non-limiting examples of commercial CAR-T cell based therapies include brexucabtagene autoleucel (TECARTUS®), axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®), Descartes-08 and Descartes- 11 from Cartesian Therapeutics, CTL119 from Novartis, P-BMCA-101 from Poseida Therapeutics, PBCAR19B and PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics, and CYAD-211 from Clyad Oncology.

[0243] In some embodiments, a hypoimmunogenic cell described herein comprises a polynucleotide encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, a hypoimmunogenic cell described herein comprises a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the polynucleotide is or comprises a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and at least one signaling domain (e.g., one, two or three signaling domains). In some embodiments, the CAR comprises a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains. In some embodiments, the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, the antigen binding domain is or comprises an antibody, an antibody fragment, an scFv or a Fab.

[0244] In certain embodiments, the CAR may comprise a hinge domain, also referred to as a spacer. The terms “hinge” and “spacer” may be used interchangeably in the present disclosure. Non-limiting examples of hinge domains include CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table 1 below.Table 1. Exemplary sequences of hinge domains

[0245] In certain embodiments, the transmembrane domain of the CAR may comprise a transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154, or a functional variant thereof, including the human versions of each of these sequences. In other embodiments, the transmembrane domain may comprise a transmembrane region of CD8a, CD8P, 4-1BB / CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40 / CD134, CD3^, CD3s, CD3y, CD36, TCRa, TCRp, TCR^ CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or a functional variantthereof, including the human versions of each of these sequences. Table 2 provides the amino acid sequences of a few exemplary transmembrane domains.Table 2. Exemplary sequences of transmembrane domains

[0246] In certain embodiments, the intracellular signaling domain and / or intracellular costimulatory domain of the CAR may comprise one or more signaling domains selected from B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, PDCD6, 4- 1BB / TNFSF9 / CD137, 4-1BB Ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 Ligand / TNFSF7, CD30 / TNFRSF8, CD30 Ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 Ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR Ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lympho toxin- alpha / TNFp, OX40 / TNFRSF4, 0X40 Ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1 A / TNFSF15, TNFa, TNF RII / TNFRSF1B, 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, SLAM / CD150, CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thyl, CD96, CD160, CD200, CD300a / LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4 / CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7 / LPAM-l, LAG- 3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1 / CLEC7 A, DPPIV / CD26, EphB6, TIM-l / KIM- 1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), NKG2C, CD3 an immunoreceptor tyrosine-based activation motif (IT AM), CD27, CD28, 4- IBB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and afunctional variant thereof including the human versions of each of these sequences. In some embodiments, the intracellular signaling domain and / or intracellular costimulatory domain comprises one or more signaling domains selected from a CD3^ domain, an IT AM, a CD28 domain, 4- IBB domain, or a functional variant thereof. Table 3 provides the amino acid sequences of a few exemplary intracellular costimulatory and / or signaling domains. In certain embodiments, the CD3c signaling domain of SEQ ID NO: 12 may have a mutation, e.g., a glutamine (Q) to lysine (K) mutation, at amino acid position 14 (see SEQ ID NO: 13).Table 3Exemplary sequences of intracellular costimulatory and / or signaling domains

[0247] In some embodiments, a CD 19 specific CAR is comprises an FMC63 anti-CD19 variable domain (FMC63 CAR). In some embodiments, a CD19 specific CAR (e.g., a CAR19) comprises an FMC63 anti-CD19 scFv. In some embodiments, a cell that carries an FMC63 CAR targets a cell (e.g., B cells) that expresses CD19.

[0248] In some embodiments, the extracellular binding domain of the CD 19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16-17): 1157- 1165 (1997) and PCT Application Publication No.WO2018 / 213337, the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequences of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 4 below. In some embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14, 15, or 20, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14, 15, or 20. In some embodiments, the CD19-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 16-18 and 21-12. In some embodiments, the CD19- specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 16-18. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 21- 23. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD 19 CAR comprises or consists of the one or more CDRs as described herein.

[0249] In some embodiments, the linker linking the VH and the VL portions of the scFv is a Whitlow linker having an amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the Whitlow linker may be replaced by a different linker, for example, a 3xG4S linker having an amino acid sequence set forth in SEQ ID NO:25, which gives rise to a differentFMC63-derived scFv having an amino acid sequence set forth in SEQ ID NO:24. In certain of these embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:24 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:24.Table 4. Exemplary sequences of anti-CD19 scFv and components

[0250] In some embodiments, the extracellular binding domain of the CD 19 CAR is derived from an antibody specific to CD 19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094- 4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381(1989)). In any of these embodiments, the extracellular binding domain of the CD 19 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0251]

[0656] In some embodiments, the hinge domain of the CD 19 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:1 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:1. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain.In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:2. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:6.

[0252] In some embodiments, the transmembrane domain of the CD 19 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:8.

[0253] In some embodiments, the intracellular costimulatory domain of the CD 19 CAR comprises a 4- IBB costimulatory domain. 4- IBB, also known as CD137, transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. In some embodiments, the 4- IBB costimulatory domain is human. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain. CD28 is another co-stimulatory molecule on T cells. In some embodiments, the CD28 costimulatory domain is human. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:11 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain as described.

[0254] In some embodiments, the intracellular signaling domain of the CD 19 CAR comprises a CD3 zeta (Q signaling domain. CD3(^ associates with T cell receptors (TCRs) to produce a signal and contains immunoreceptor tyrosine-based activation motifs (IT AMs). The CD3^ signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In some embodiments, the CD3^ signaling domain is human. In some embodiments, the CD32 signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 12.

[0255] In some embodiments, a transgene can encode a chimeric B-cell autoantibody receptor (BAR). A BAR recognizes and binds to certain antibody-expressing B cells. In some embodiments, a BAR comprises an antigen. An antigen of a BAR can be bound by neutralizing antibodies. The neutralizing antibodies may be undesirable because they can block or inhibit an effect or function of antigen to which they bind. For example, hemophilia patients can receive therapeutic factor VIII (FVIII) as part of their treatment. However, a patient’s body may develop an immune response against the FVIII, including the production of anti-FVIII antibodies from B cells. When the patient produces anti-FVIII antibodies that bind to FVIII, FVIII is not able to perform its therapeutic functions. Accordingly, it may be beneficial to remove the anti-FVIII antibodies and / or the B-cells producing those antibodies from the patient. A BAR, which includes an FVIII antigen, can be used for this purpose.

[0256] In some embodiments, a BAR comprises a transmembrane domain. In some embodiments, a BAR comprises a signaling domain. In some embodiments, a BAR comprises one or more signaling domains.

[0257] In some embodiments, a BAR comprises an antigen, a transmembrane domain, and a signaling domain. In some embodiments, a BAR comprises an antigen, a transmembrane domain, and one or more signaling domains.

[0258] A BAR can be expressed by, e.g., a hypoimmunogenic T-cell. BAR T-cells can recognize and can bind target select antibodies and / or the B cells producing those antibodies. Once a BAR T-cell binds a target antibody, the BAR T-cell can destroy the antibodies and / or the B cells producing those antibodies. In some embodiments, a BAR T-cell is a BAR T-cell (Treg), e.g., a regulatory T-cell (Treg) comprising a BAR.

[0259] A BAR can be expressed by, e.g., a hypoimmunogenic NK-cell. BAR NK-cells can recognize and can bind target select antibodies and / or the B cells producing those antibodies.Once a BAR NK-cell binds a target antibody, the BAR NK-cell can destroy the antibodies and / or the B cells producing those antibodies.

[0260] In some embodiments, a transgene can encode a chimeric autoantibody receptor (CAAR). In some embodiments, a CAAR comprises an antigen, e.g., an autoantigen that can be bound by autoantibodies. In some embodiments, a CAAR comprises a transmembrane domain. In some embodiments, a CAAR comprises a signaling domain. In some embodiments, a CAAR comprises one or more signaling domains. In some embodiments, a CAAR comprises an antigen, a transmembrane domain, and a signaling domain. In some embodiments, a CAAR comprises an antigen, a transmembrane domain, and one or more signaling domains.

[0261] A CAAR can be expressed by, e.g., a hypoimmunogenic T-cell. CAAR T-cells can recognize and can bind target autoantibodies expressed on autoreactive cells via an antigen of a CAAR. Once a CAAR T-cell binds a target autoantibody expressed on an autoreactive cell, the CAAR T-cell can destroy the autoreactive cell.

[0262] A CAAR can be expressed by, e.g., a hypoimmunogenic NK-cell. CAAR NK- cells can recognize and can bind target autoantibodies expressed on autoreactive cells via an antigen of a CAAR. Once a CAAR NK-cell binds a target autoantibody expressed on an autoreactive cell, the CAAR NK-cell can destroy the autoreactive cell.

[0263] In some embodiments, a transgene can encode a tolerogenic factor. In some embodiments, a tolerogenic factor is CD47, DUX4, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF, and Serpinb9, A20 / TNFAIP3, CD39, CR1, HLA-F, IL15-RF, or MANF. In some embodiments, a tolerogenic factor is CD47.

[0264] In some embodiments, a viral vector includes one or more transgenes. In some embodiments, a viral vector includes one, two, three, four, five or more transgenes.

[0265] In some embodiments, one or more nucleic acids for the production of the viral vector can include one or more transgenes. In some embodiments, one or more nucleic acids for the production of the viral vector include one, two, three, four, five or more transgenes.

[0266] In some embodiments, one or more nucleic acids for the production of the viral vector include a transfer plasmid. In some embodiments, a transfer plasmid can include one or more transgenes. In some embodiments, a transfer plasmid includes one, two, three, four, five or more transgenes.

[0267] In some embodiments, a transgene can encode a chimeric antigen receptor (CAR). In some instances, a transgene encoding a CAR can be delivered to, e.g., a T-cell, for expression.IIL Fusogens

[0268] Disclosed herein are methods of producing viral vectors, e.g., lentiviral vectors, comprising one or more fusogens. Also disclosed are one or more nucleic acids for the production of viral vectors (e.g., lentiviral vectors) that comprise one or more nucleic acid sequences encoding a fusogen. In some embodiments, one or more nucleic acids for the production of viral vectors (e.g., lentiviral vectors) comprise one or more envelope plasmids. In some embodiments, one or more envelope plasmids encode one or more fusogens as described herein. In some embodiments, two or more envelope plasmids encode two or more fusogens as described herein.

[0269] In some embodiments, the viral vector (e.g., the lentiviral vector) is pseudotyped with a retargeted fusion, wherein the retargeted fusogen includes a fusogen protein fused to an antibody, antigen binding domain, or target binding domain (the fusogen (e.g., retargeted fusogen) fused to the antibody or antigen binding domain is also referred to herein as a chimeric protein). In such cases, the antibody or antigen binding domain impacts tropism. Non-limiting examples of viral vectors (e.g., lentiviruses) comprising re-targeted fusogens include are as described in US11 ,535,869, the entire contents of which is incorporated by reference herein.

[0270] In some embodiments, one or more fusogens are present in a lipid bilayer of a viral vector described herein (e.g., produced by a method provided herein).

[0271] In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for B cells, T cells, natural killer cells, islet cells, glial progenitor cells, neuronal cells, hematopoietic stem cells, cardiac cells, hepatocytes, stem cells, or induced pluripotent stem cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for B cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for T cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for islet cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a tropism for cardiac cells. In some embodiments, one or more fusogens comprise at least one fusogen that has a natural tropism (e.g., a fusogen with a wild type or naturally occurring amino acid sequence). In some embodiments, one or more fusogens comprise at least one fusogen that has engineered tropism. In some embodiments, one or more fusogens comprise one or more viral fusogens.

[0272] In some embodiments, one or more fusogens comprise at least one fusogen that binds a protein. In some embodiments, one or more fusogens comprise at least one fusogen that binds a cellular membrane protein. In some embodiments, one or more fusogens comprise at least one fusogen that binds a cellular membrane protein involved in viral attachment and / or fusion. In some embodiments, one or more fusogens comprise at least one fusogen that is involved in directing fusion of the lipid bilayer of a viral vector and a cell membrane.

[0273] In some embodiments, one or more fusogens comprise one or more paramyxovirus envelope proteins or biologically active portions thereof. In some embodiments, one or more fusogens comprise one or more modified paramyxovirus envelope proteins or biologically active portions thereof. In some embodiments, one or more paramyxovirus envelope proteins or biologically active portions thereof comprises a paramyxovirus glycoprotein (“G protein”) or a portion thereof. In some embodiments, one or more paramyxovirus envelopeproteins or biologically active portions thereof comprises a paramyxovirus fusion protein (“F protein”) or a portion thereof.

[0274] In some embodiments, one or more fusogens comprise one or more chimeric proteins. In some embodiments, one or more chimeric proteins comprise at least one chimeric protein that comprises a paramyxovirus envelope protein or biologically active portion thereof. In some embodiments, one or more chimeric proteins comprise at least one chimeric protein that comprises a target binding protein. In some embodiments, a target is an antigen, a receptor, or a ligand. In some embodiments, a target is present on or expressed by a target cell (e.g., a B cell, a T cell, a natural killer cell, an islet cell, a glial progenitor cell, a cardiac cell, a blood cell, a hepatocyte, a stem cell, or an induced pluripotent stem cell). In some embodiments, a target is present on or expressed by an immune cell. In some embodiments, a target binding protein is a receptor (e.g., a receptor that binds a target ligand). In some embodiments, a target binding protein is a ligand (e.g., a ligand that is bound by a target receptor). In some embodiments, a target binding protein is an antibody or fragment thereof (e.g., an antibody or fragment thereof that binds a target antigen). In some embodiments, a target binding protein is an scFv. In some embodiments, one or more chimeric proteins comprise at least one chimeric protein that comprises an scFv. In some embodiments, one or more chimeric proteins comprise at least one chimeric protein that comprises (i) a paramyxovirus envelope protein or biologically active portion thereof and (ii) an scFv (see, e.g., FIG. 6B). In some embodiments, an scFv targets an antigen present on the surface of a B cell, a T cell, a natural killer cell, an islet cell, a glial progenitor cell, a cardiac cell, a blood cell, a hepatocyte, a stem cell, or an induced pluripotent stem cell.

[0275] In some embodiments, an scFv targets an antigen present on the surface of a B cell. In some embodiments, an scFv targets an antigen present on the surface of a T cell. In some embodiments, an scFv targets CD8. Non-limiting examples of an antibody or target binding domain that targets CD8 include those described in US 11,535,869, the entire contents of which is incorporated by reference herein. In some embodiments, an scFV targets CD4. Non-limiting examples of an antibody or target binding domain that targets CD4 include those described in PCT publication WO2023193003 A2, the entire contents of which is incorporated by reference herein. In some embodiments, an scFv targets an antigen present on the surface of an islet cell. In some embodiments, an islet cell is an alpha cell, a beta cell, or a delta cell. In some embodiments, an islet cell is a beta cell.

[0276] Methods for measuring an amount of fusogen expressed by a producer cell and / or present on a lentiviral vector can be carried out using one or more methods known in the art. For example, in some embodiments, an amount of fusogen on a particle (e.g., lentiviral vector, fusosome, virus like particle, virus, etc.) is measured using per particle analysis. In some embodiments, fusogen expression is measured using an enzyme-linked immunosorbent assay (ELISA). In some embodiments, an ELISA uses Meso Scale Discovery technology (MSD). In some embodiments, fusogen expression is measured using Western blot. In some embodiments, fusogen expression can be measure by a technique that utilizes fluorescence imaging and single particle interferometry to provide complete characterization of viruses and exosomes. Such an approach can provide biophysical and proteomic information at the single-particle level. In some embodiments, a technique that combines fluorescence imaging and single particle interferometry can be “ExoView.”IV. Methods of Producing

[0277] Disclosed herein are methods of producing viral vectors, e.g., lentiviral vectors. Typically, a producer cell line is transfected with one or more plasmids (e.g., as disclosed herein), and cultured under conditions sufficient to transfer and / or express one or more polypeptides encoded by said plasmids in the producer cell line.

[0278] In some embodiments, a method of producing viral vectors (e.g., a lentiviral vector) includes treating the producer cell culture with a nuclease. In one embodiment, a method of producing a composition comprising viral vectors, includes (a) culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditionssufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid; (b) treating the producer cell culture with a nuclease; and (c) harvesting the producer cell culture; wherein (b) is performed prior (c). In one embodiment, a method of producing a composition comprising viral vectors, includes (a) culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid; (b) treating the producer cell culture with a nuclease; and (c) harvesting the producer cell culture; and (d) treating the harvested producer cell culture with a nuclease, wherein (b) is performed prior (c) and (d) is performed after or concurrently with (c).

[0279] In some embodiments, a method of producing a composition comprising viral vectors includes culturing producer cells comprising one or more nucleic acids for the production of the viral vector, wherein the one or more nucleic acids comprise a packaging plasmid, one or more envelope plasmids, and a transfer plasmid, wherein the ratio of the envelope plasmid to packaging plasmid within the one or more nucleic acids is greater than 1:1.

[0280] In some embodiments, a method of producing a composition comprising viral vectors includes culturing producer cells comprising one or more nucleic acids for the production of the viral vector, wherein the one or more nucleic acids comprise a packaging plasmid, one or more envelope plasmids, and a transfer plasmid, wherein the ratio of the envelope plasmid to transfer plasmid within the one or more nucleic acids is greater than 1:1.

[0281] In another embodiment, a method of producing a composition comprising viral vectors includes culturing producer cells comprising one or more nucleic acids for the production of the viral vector, wherein the one or more nucleic acids comprise a packaging plasmid, one or more envelope plasmids, and a transfer plasmid, wherein the ratio of the packaging plasmid to envelope plasmid within the one or more nucleic acids is greater than 1:1.

[0282] In another embodiment, a method of producing a composition comprising viral vectors includes culturing producer cells comprising one or more nucleic acids for the production of the viral vector, wherein the one or more nucleic acids comprise a packaging plasmid, a first envelope plasmid, a second envelope plasmid, and a transfer plasmid, wherein present the ratio of a first envelope plasmid to a second envelope plasmid within the one or more nucleic acids is greater than 1:1.

[0283] In some embodiments, a method of producing viral vectors (e.g., a lentiviral vector) includes introducing into the producer cells one or more nucleic acids for the production of the viral vector (e.g., a packaging plasmid, one or more envelope plasmids, and a transfer plasmid), where the one or more nucleic acids arc introduced at different ratios.

[0284] In one embodiment, a method of producing a composition comprising viral vectors includes introducing into the producer cells one or more nucleic acids for the production of the viral vector, wherein the one or more nucleic acids comprise a packaging plasmid, one or more envelope plasmids, and a transfer plasmid, and wherein the ratio of the envelope plasmid to the packaging plasmid introduced into the producer cells is greater than 1:1.

[0285] In one embodiment, a method of producing a composition comprising viral vectors includes introducing into the producer cells one or more nucleic acids for the production of the viral vector, wherein the one or more nucleic acids comprise a packaging plasmid, one or more envelope plasmids, and a transfer plasmid, and wherein the ratio of the envelope plasmid to the transfer plasmid introduced into the producer cells is greater than 1:1.

[0286] In another embodiment, a method of producing a composition comprising viral vectors includes introducing into the producer cells one or more nucleic acids for the production of the viral vector, wherein the one or more nucleic acids comprise a packaging plasmid, one or more envelope plasmids, and a transfer plasmid, and wherein the ratio of the packaging plasmid to the envelope plasmid introduced into the producer cells is greater than 1:1.

[0287] In another embodiment, a method of producing a composition comprising viral vectors includes introducing into the producer cells one or more nucleic acids for the production of the viral vector, wherein the one or more nucleic acids comprise a packaging plasmid, a first envelope plasmid and a second envelope plasmid, and a transfer plasmid, wherein the ratio of a first envelope plasmid to a second envelope plasmid introduced into the producer cells is greater than 1:1.

[0288] A. Transfection and Ratios

[0289] Methods for producing a composition comprising a viral vector include introducing into a producer cell (e.g., transfecting a producer cells) one or more nucleic acids (e.g., plasmids) described herein. Nucleic acids (e.g., plasmids) that can be used for transfecting a produce cell include: one or more packaging plasmids, one or more envelope plasmids, one or more regulatory plasmids, and one or more transfer plasmids as described herein.

[0290] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is about 1:1.

[0291] In some embodiments, a relative amount of an envelope plasmid is increased, e.g., to increase the amount of an encoded fusogen present in a produced lentiviral vector. In some embodiments, the fusogen is an attachment protein. In some embodiments, a fusogen is a paramyxovirus G protein or a portion thereof. In some embodiments, a fusogen is a chimeric protein that comprises a paramyxovirus G protein or a biologically active portion thereof and an scFV. A fusogen can also comprise other fusogens as described herein.

[0292] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is greater than 1:1, greater than 2:1, greater than 3:1, greater than 4:1, greater than 5 : 1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is greater than 1:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is at least 2:1. In someembodiments, a ratio of an envelope plasmid to a packaging plasmid is a ratio of said plasmids within a producer cell.

[0293] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is about 1.5:1, about 2:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, or about 15:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is about 1.5 : 1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is about 2:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is a ratio of said plasmids within a producer cell.

[0294] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is between 1:1 to 2:1, between 1:1 to 4:1, between 1:1 to 5 : 1 , between 1.5:1 to 6:1, between 1.5:1 to 7:1, between 1.5:1 to 8:1, between 1.5:1 to 9:1, between 1.5:1 to 10:1, between 1.5:1 to 11:1, between 1.5:1 to 12:1, between 1.5:1 to 13:1, between 1.5:1 to 14:1, or between 1.5:1 to 15:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is a ratio of said plasmids within a producer cell.

[0295] In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is at most 15:1, at most 14:1, at most 13:1, at most 12:1, at most 11:1, at most 10:1, at most 9:1, or at most 8:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is a ratio of said plasmids within a producer cell.

[0296] In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is at least 1:1, at least 2:1, at least 3:1, at least 4:1, or at least 5:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is at least 1:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is at least 2:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is a ratio of said plasmids within a producer cell.

[0297] In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is about 1 :1 , about 2: 1 , about 4: 1 , about 5:1 , about 6:1 , about 7:1 , about 8:1 , about 9: 1 , about 10:1 , about 11:1, about 12:1, about 13:1, about 14:1, or about 15:1. In some embodiments, a ratio of anenvelope plasmid to a transfer plasmid is about 1:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is about 2: 1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is a ratio of said plasmids within a producer cell.

[0298] In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is between 1:1 to 2: 1 , between 1:1 to 4: 1 , between 1:1 to 5: 1 , between 1 : 1 to 6: 1 , between 1 : 1 to 7:1, between 1:1 to 8:1, between 1:1 to 9:1, between 1:1 to 10:1, between 1:1 to 11:1, between 1:1 to 12:1, between 1:1 to 13:1, between 1:1 to 14:1, or between 1:1 to 15:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is a ratio of said plasmids within a producer cell.

[0299] In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is at most 15:1, at most 14:1, at most 13:1, at most 12:1, at most 11:1, at most 10:1, at most 9:1, or at most 8:1. In some embodiments, a ratio of an envelope plasmid to a transfer plasmid is a ratio of said plasmids within a producer cell.

[0300] In some embodiments, two envelope plasmids are introduced to a producer cell. In some embodiments, a first and a second envelope plasmid each encoding a different fusogen (e.g., a first fusogen and a second fusogen, respectively) are introduced to a producer cell. In some embodiments, a ratio of a first envelope plasmid to a second envelope plasmid is at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, or at least 15:1. In some embodiments, a ratio of an envelope plasmid to a packaging plasmid is greater than 1:1. In some embodiments, a ratio of a first envelope plasmid to a second envelope plasmid is a ratio of said plasmids within a producer cell. In some embodiments, a first envelope plasmid encodes G protein and a second envelope plasmid encodes F protein.

[0301] In some embodiments, a ratio of a first envelope plasmid to a second envelope plasmid is between 1:1 to 2:1, between 1:1 to 4:1, between 1:1 to 6:1, between 1:1 to 8:1, between 1:1 to 10:1, between 1:1 to 12:1, or between 1:1 to 15:1. In some embodiments, a ratio of a first envelope plasmid to a second envelope plasmid is a ratio of said plasmids within aproducer cell. In some embodiments, a first envelope plasmid encodes G protein and a second envelope plasmid encodes F protein.

[0302] In some embodiments, a ratio of a first envelope plasmid to a second envelope plasmid is at most 24:1, at most 22:1, at most 20:1, at most 18:1, or at most 16:1. In some embodiments, a ratio of a first envelope plasmid to a second envelope plasmid is a ratio of said plasmids within a producer cell. In some embodiments, a first envelope plasmid encodes G protein and a second envelope plasmid encodes F protein.

[0303] In some embodiments, a ratio of a first envelope plasmid to a second envelope plasmid is 1:1, is 2:1, is 4:1, is 6:1, is 8:1, is 10:1, or is 12:1. In some embodiments, a first envelope plasmid encodes G protein and a second envelope plasmid encodes F protein.

[0304] In some embodiments, a ratio of first envelope plasmid to second envelope plasmid is optimized for increased expression of G protein on a lentiviral vector. In some embodiments the lentiviral vector is a fusosome. In some embodiments, a ratio of first envelope plasmid to second envelope plasmid is optimized to increase expression of G protein by a producer cell. In some embodiments, a ratio of first envelope plasmid to second envelope plasmid is optimized so a greater proportion of fusosomes comprise both G protein and F protein (e.g., complete fusosome). In some embodiments, a ratio of one fusogen relative to another fusogen on a lentiviral vector (e.g., fusosome) as described herein, confers tropism of the lentiviral vector to one or more cell(s) (e.g., B cells, T cells, natural killer cells, islet cells, glial progenitor cells, neuronal cells, hematopoietic stem cells, cardiac cells, hepatocytes, stem cells, or induced pluripotent stem cells). In some embodiments, a lentiviral vector (e.g., fusosome) with tropism to one more or cell(s) carries one or more CAR(s). In some embodiments, an optimized ratio of G protein relative to F protein produces a lentiviral vector (e.g., fusosome) that targets CD8 positive T cells. In some embodiments, a lentiviral vector (e.g., fusosome) that targets CD8 positive T cells carries a nucleic acid sequence encoding a FMC63 CAR.

[0305] In some embodiments, a lentiviral vector encodes a FMC63 CAR (e.g., fusosome), which targets CD8 positive T cells. CD8 positive T cells can express a FMC63 CAR, which allows the CD8 positive T cells to target CD19 expressing cells (e.g., B cell) via an affinity of the FMC63 CAR with CD 19.

[0306] In some embodiments, introduction of nucleic acids into producer cells occurs by transfection. In some embodiments, transfection can be performed using lipofectamine. In some embodiments, a 2: 1 ratio of lipofectamine to DNA is used for transfection. In some embodiments, transfection can be performed with electroporation. In some embodiments, transfection can be performed with nucleofection.

[0307] In some embodiments, one or more of the one or more nucleic acids for the production of the viral vectors are stably integrated (e.g., stable integration results in stable expression of the encoded viral proteins) into the genome of one or more producer cells. In some embodiments, one or more of the one or more nucleic acids for the production of the viral vectors are stably integrated (e.g., stable integration results in stable expression of the encoded viral proteins) into the genome of one or more producer cells prior to culturing the producer cells under conditions sufficient to produce the viral vectors. In some embodiments, stable integration is achieved via random (i.e., insertion into a random genomic locus of the host cell) integration of the one or more nucleic acids into the genome of a producer cell. In some embodiments, stable integration is achieved via targeted integration (i.e., insertion into a specific genomic locus of the host cell) into the genome of a producer cell. As known to a person skilled in the art, viral vectors, including, for example, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors, are commonly used to deliver genetic material into producer cells and randomly insert the gene(s) encoding viral proteins into the host cell genome to facilitate stable expression and replication of the viral genes.

[0308] A number of gene editing methods can be used to insert a polynucleotide (e.g., gene encoding viral proteins for production of a viral vector) into a specific genomic locus ofchoice. Gene editing is a type of genetic engineering in which a nucleotide sequence is inserted, deleted, modified, or replaced in the genome of a living organism. In some embodiments, the gene editing technologies are systems involving nucleases, integrases, transposases, and / or recombinases. In some embodiments, the gene editing technology mediates single-strand breaks (SSB). In some embodiments, the gene editing technology mediates double-strand breaks (DSB), including in connection with non-homologous end-joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the gene editing technologies are DNA-based editing or prime-editing. In some embodiments, the gene editing technology is Programmable Addition via Site-specific Targeting Elements (PASTE). In some embodiments, the gene editing technology is TnpB polypeptides. Many gene editing techniques generally utilize the innate mechanism for cells to repair double-strand breaks (DSBs) in DNA.

[0309] Eukaryotic cells repair DSBs by two primary repair pathways: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). HDR typically occurs during late S phase or G2 phase, when a sister chromatid is available to serve as a repair template. NHEJ is more common and can occur during any phase of the cell cycle, but it is more error prone. In gene editing, NHEJ is generally used to produce insertion / deletion mutations (indels), which can produce targeted loss of function in a target gene by shifting the open reading frame (ORF) and producing alterations in the coding region or an associated regulatory region. HDR, on the other hand, is a preferred pathway for producing targeted knock-ins, knockouts, or insertions of specific mutations in the presence of a repair template with homologous sequences. Several methods are known to a skilled artisan to improve HDR efficiency, including, for example, chemical modulation (e.g., treating cells with inhibitors of key enzymes in the NHEJ pathway); timed delivery of the gene editing system at S and G2 phases of the cell cycle; cell cycle arrest at S and G2 phases; and introduction of repair templates with homology sequences. The methods provided herein may utilize HDR-mediated repair, NHEJ-mediated repair, or a combination thereof.

[0310] In some embodiments, the methods provided herein for HDR-mediated insertion utilize a site-directed nuclease, including, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, transposases, and clustered regularly interspaced short palindromic repeat (CRISPR) / Cas systems.B. Producer Cell Culture

[0311] Also disclosed herein are methods for introducing one or more nucleic acids into producer cells for the production of viral vectors. In some embodiments, a producer cell is a cell capable of producing a viral vector when cultured under appropriate conditions. In some embodiments, a producer cell is cultured at 37°C. In some embodiments, a producer cell is cultured at least at 37°C. In some embodiments, a producer cell is cultured between 35°C and 45°C. In some embodiments, a producer cell is cultured while exposed to, among other gases, 5% carbon dioxide. In some embodiments, a producer cell is cultured at a temperature that is permissive to viral vector production by a producer cell. In some embodiments, a producer cell is cultured with one or more gas(es) that is / are permissive to viral vector production by a producer cell.

[0312] In some embodiments, a producer cell is any one of a number of cells known to be capable of producing viral vectors. In some embodiments, a producer cell comprises one or more HEK293 cells, PER.C6 cells, VERO cells, HEK 293T cells, A549 cells, MRC5 cells, HeLa cells, Sf9 cells, and BHK-21 cells.

[0313] In some embodiments, a method provided herein comprises one or more steps to reduce producer cell aggregation.

[0314] In some embodiments, a producer cell is cultured in a suspension culture. In some embodiments, a producer cell is cultured in serum free medium. In some embodiments, a producer cell is agitated, e.g., mixed, rotated, shook, stirred, during culturing. In some embodiments, a producer cell is exposed to increased agitation relative to as compared to a process that does not include agitation.

[0315] In some embodiments, a producer cell (e.g., HEK 293 or HEK 293T cells) is adapted to serum-free growth. In some embodiments, a producer cell is adapted to suspension culture. In some embodiments, adapted (e.g., to serum-free media and / or suspension culture) producer cell clones are selected based on production of a higher crude functional titer of lentiviral vector. In some embodiments, adapted producer cell clones are selected based on morphology. In some embodiments, adapted producer cell clones are selected based on higher productivity of a fusosome. In some embodiments, adapted producer cell clones are selected based on higher productivity of a fusosome targeting one or more cell(s). In some embodiments, adapted producer cell clones are selected based on higher productivity of a CD8-targeted fusosome. In some embodiments, adapted producer cell clones are selected based on fusosomes that carry a CAR transgene. In some embodiments, adapted producer cell clones are selected based on fusosomes that carry a CD19-directed CAR transgene. In some embodiments, adapted producer cell clones are selected based on one or more of the following; increase in crude functional lentiviral vector titer, morphology, increase in productivity of a specific fusosomes, increase in productivity of CD8-targeting fusosomes, increase in productivity of fusosomes that carry a CAR transgene, increase in productivity of fusosomes that carry a CD19-directed CAR transgene. In some embodiments, adapted producer cell clones are selected based on feature(s) (e.g., functional titer, morphology, productivity, transgene expression) exhibited relative to each other, to a control cell line that has not been adapted to serum-free growth in a suspension culture, or both.

[0316] In some embodiments, a producer cell is cultured in medium (cell culture medium or producer cell culture medium) that includes an anti-cell clumping agent. Non-limiting examples of anti-cell clumping agents include: albumin, heparin, and chelating agents.

[0317] In some embodiments, a producer cell is cultured in medium (cell culture medium or producer cell culture medium) that includes a surfactant. Non-limiting examples of surfactants that can be added to producer cell culture medium include: pluronic F-68, Tween 20 (polysorbate 20), Tween 80 (polysorbate 80), and polyethylene glycol (PEG).

[0318] In some embodiments, a producer cell is cultured at a temperature that reduces producer cell aggregation relative to a lower or a higher temperature.

[0319] In some embodiments, a producer cell is cultured in a vessel that reduces producer cell aggregation. In some embodiments, a producer cell is cultured at a density, e.g., concentration of producer cells, that reduces producer cell aggregation.C. Nuclease Treatment

[0320] Methods provided herein comprises treating producer cells (or producer cell culture) with a nuclease. In some embodiments, the method of producing a composition comprising viral vectors, includes (a) culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid; (b) treating the producer cell culture with a nuclease; and (c) harvesting the producer cell culture; wherein (b) is performed prior (c).

[0321] In some embodiments, the methods provided herein include treating the producer cells (or producer cell culture) with a nuclease at a time that is at least 30 minutes, at least 60 minutes, at least 1 .5 hours, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours, after the one or more nucleic acids used for the production of the viral vector are introduced into the producer cells. In some embodiments, the methods provided herein include treating the producer cells (or producer cell culture) with a nuclease at a time that is at least 30 minutes after the one or more nucleic acids used for the production of the viral vector are introduced into the producer cells.

[0322] In some embodiments, a nuclease is or comprises a DNase. In some embodiments, a DNase is or comprises an exonuclease. Is some embodiments, a DNase is or comprises an endonuclease. In some embodiments, a DNase is both an endonuclease and an exonuclease.

[0323] In some embodiments, a nuclease is a salt activated nuclease. Non-limiting examples of salt activated nucleases include: benzonase, RNase A, DNase I, micrococcal nuclease, and S7 nuclease.

[0324] In some embodiments, a salt activated nuclease is derived from yeast (e.g., Pichia pastoris). In some embodiments, a nuclease is MS AN. In some embodiments, the producer cells (or producer cell culture) are treated with about 50,000 to 100,000, 100,000 to 500,000 units of nuclease, 200,000 to 400,000 units of nuclease. In some embodiments, the producer cells are treated with about 50,000 to 100,000, 100,000, 200,000, 300,000, 400,000, 500,000 units of nuclease. In some embodiments, the producer cells are treated with 400,000 units of nuclease.

[0325] In some embodiments, the producer cells (or the producer cell culture) are treated with about 1 Unit (U) of MSAN per 1 mL of cell culture medium to about 100 U of MSAN per 1 mL of cell culture medium, wherein the producer cells are treated with about 100U of MSAN per 1 mL of cell culture medium.

[0326] In some embodiments, the producer cells (or the producer cell culture) are treated with about 50U of MSAN per 1 mL of producer cell culture medium. In some embodiments, the producer cells (or the producer cell culture) are treated with about 25U of MSAN per 1 mL of producer cell culture medium. In some embodiments, the producer cells (or the producer cell culture) are treated with about 10U of MSAN per 1 mL of producer cell culture medium. In some embodiments, the producer cells (or the producer cell culture) are treated with about 4U of MSAN per 1 mL of producer cell culture medium. In some embodiments, the producer cells (or the producer cell culture) are treated with about 3U of MSAN per 1 mL of producer cell culture medium. In some embodiments, the producer cells (or the producer cell culture) are treated with about 2U of MSAN per 1 mL of producer cell culture medium. In some embodiments, the producer cells (or the producer cell culture) are treated with about 1U of MSAN per 1 mL of producer cell culture medium.

[0327] In some embodiments, a method provided herein comprises introducing one or more nucleic acids for the production of the viral vector into producer cells. In some embodiments, a method provided herein comprises treating producer cells with a nuclease. In some embodiments, treating producer cells with a nuclease occurs after one or more nucleic acids for the production of the viral vector are introduced into producer cells.

[0328] In some embodiments, treating producer cells with a nuclease occurs at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, or at least 5.5 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells.

[0329] In some embodiments, treating producer cells with a nuclease occurs at a time that is no more than 10 hours, no more than 9 hours, no more than 8 hour, no more than 7 hours, no more than 6 hours, no more than 5.5 hours, no more than 5 hours, no more than 4.5 hours, no more than 4 hours, no more than 3.5 hours, no more than 3 hours, no more than 2.5 hours, no more than 2 hours, no more than 1.5 hours, no more than 1 hour, and no more than 30 minutes after one or more nucleic acids for the production of the viral vector are introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs at a time that is no more than 6 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs at a time that is no more than 4 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs at a time that is no more than 2 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells.

[0330] In some embodiments, treating producer cells with a nuclease occurs at most 6 hours, at most 5.5 hours, at most 5 hours, at most 4.5 hours, at most 4 hours, at most 3.5 hours, at most 3 hours, at most 2.5 hours, at most 2 hours, at most 1.5 hours, at most 1 hour or at most 30minutes after one or more nucleic acids for the production of the viral vector are introduced into producer cells.

[0331] In some embodiments, treating producer cells with a nuclease occurs between 30 minutes and 6 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs between 30 minutes and 5 hours after one or more nucleic acids for the production of the viral vector arc introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs between 30 minutes and 5 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs between 30 minutes and 4 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs between 30 minutes and 3 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs between 90 minutes and 6 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells. In some embodiments, treating producer cells with a nuclease occurs between 60 minutes and 6 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells.

[0332] In some embodiments, treating producer cells with a nuclease occurs 1, 2, 3, 4, 5, or 6 hours after one or more nucleic acids for the production of the viral vector are introduced into producer cells.

[0333] In some embodiments, the methods provided herein include a first nuclease treatment that occurs a first time period and a second nuclease treatment that occurs at a second time period.

[0334] In some embodiments, a method of producing a composition comprising viral vectors, includes (a) culturing producer cells comprising one or more nucleic acids for theproduction of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid; (b) treating the producer cell culture with a nuclease (i.e., first nuclease treatment); and (c) harvesting the producer cell culture; and (d) treating the harvested producer cell culture with a nuclease (i.e., second nuclease treatment), wherein (b) is performed prior (c) and (d) is performed after or concurrently with (c).

[0335] In some embodiments, the method includes treating the producer cells with a nuclease treatment at a time that is at least 30 minutes and no more than 6 hours after one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0336] In some embodiments, the method includes treating the producer cells with a nuclease treatment at a time that is no more than 6 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0337] In some embodiments, the method includes treating the producer cells with a nuclease treatment at a time that is no more than 4 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0338] In some embodiments, the method includes treating the producer cells with a nuclease treatment at a time that is no more than 2 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producercells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

[0339] In some embodiments, the amount of nuclease added to the producer cell culture medium is determined according to equations (eqn.) 1 and / or 2. In some embodiments, the amount of nuclease added to the producer cell culture medium for the first nuclease treatment is determined according to equations (eqn.) 1 and / or 2.

[0340] Equation 1:

[0341] Equation 2:Vol. of Media (mL) = 2000 mL - (1000 mL 1.0 M Mg [Cl] 2 +Vol. of MSAN HQ) ; (Eqn. 2)

[0342] In some embodiments, the amount of nuclease added to the harvested producer cells is determined according to equations 3 and / or 4. In some embodiments, the amount of nuclease added to the producer cell culture medium for the second nuclease treatment is determined according to equations 3 and / or 4.

[0343] Equation 3:

[0344] Equation 4:

[0345] Vol. of Media (mL) = 410 mL - (Vol. of MSAN HQ) ; (Eqn.4)

[0346] In some embodiments that include a first nuclease treatment and a first nuclease treatment, the nuclease used in the first nuclease treatment and the nuclease used in the second nuclease treatment are the same. In some embodiments that include a first nuclease treatmentand a first nuclease treatment, the nuclease used in the first nuclease treatment and the nuclease used in the second nuclease treatment are different.

[0347] In some embodiments, the method further comprises agitating the producer cells.

[0348] In some embodiments, the producer cells are cultured in medium that includes an anti-cell clumping agent.

[0349] In some embodiments, the method includes agitating the producer cells and culturing the cell producer cells in medium that includes an anti-clumping agent.

[0350] In some embodiments, the producer cells are cultured in medium that includes a surfactant.

[0351] In some embodiments, the producer cells are cultured in medium that includes a surfactant and an antidumping agent.

[0352] In some embodiments, the method includes agitating the producer cells and culturing the cell producer cells in medium that includes a surfactant.

[0353] In some embodiments, the method includes agitating the producer cells and culturing the cell producer cells in medium that includes a surfactant and an anti-clumping agent.

[0354] In some embodiments, the method includes further comprising a settling step, wherein after harvesting the producer cells (or the producer cell culture) are allowed to settle.

[0355] In some embodiments, the settling is performed for about 4 hours to about 48 hours (e.g., about 4 hours to about 44 hours, about 4 hours to about 40 hours, about 4 hours to about 36 hours, about 4 hours to about 32 hours, about 4 hours to about 28 hours, about 4 hours to about 24 hours, about 4 hours to about 20 hours, about 4 hours to about 16 hours, about 4 hours to about 12 hours, about 4 hours to about 8 hours). In some embodiments, the settling is performed for about 4 hours to about 16 hours. In some embodiments, the settling is performed for about 4 hours. In some embodiments, the settling is performed for about 5 hours. In some embodiments, the settling is performed for about 6 hours. In some embodiments, the settling isperformed for about 8 hours. In some embodiments, the settling is performed for about 10 hours. In some embodiments, the settling is performed for about 12 hours. In some embodiments, the settling is performed for about 14 hours. In some embodiments, the settling is performed for about 16 hours. In some embodiments, the settling is performed for about 20 hours. In some embodiments, the settling is performed for about 24 hours. In some embodiments, the settling is performed for about 30 hours. In some embodiments, the settling is performed for about 36 hours. In some embodiments, the settling is performed for about 42 hours. In some embodiments, the settling is performed for about 48 hours.

[0356] In some embodiments, the method further includes enriching the viral vectors from the producer cell culture.

[0357] In some embodiments, a producer cell culture is passed (e.g., filtered, clarified) through a chromatography resin. In some embodiments, a chromatography resin or membrane is one or more of the following: an affinity resin, and ion exchange resin, a hydrophobic interaction resin, a size exclusion resin, a reversed phase resin, a Capto type resin, a Capto MMC, a protein A resin, a Capto Core resin, a Capto S resin, a his-tag resin, a Mustang type membrane, a Mustang S membrane, a Mustang Q membrane, a glutathione S-transferase resin, a Sepharose type resin, a SP Sepharose Fast Flow resin, a Q Sepharose Fast Flow resin, a DEAE Sepharose resin, or a ANX Sepharose 4 Fast Flow resin. In some embodiments, a producer cell culture comprises a lentiviral vector.V. Drug Substance

[0358] Methods provided herein are useful for the production of viral vectors. In some embodiments, such viral vectors will be utilized in a drug substance. A “drug substance” is an active ingredient (e.g., viral vectors) that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of a subject’s body, but does not include intermediates used in the synthesis of such ingredient. A drug substance may need further processing to become a “drugproduct,” which is a finished dosage form (e.g., tablet or solution) to be administered to a subject. However, a drug substance does not require further processing to purify, isolate, or otherwise enrich the active ingredient prior to incorporation into a drug product.

[0359] Viral vectors produced by methods provided herein can have improved properties. For example, in some embodiments, viral vectors produced by methods provided herein have increased viral vectors titers, increased biologically active viral vector titers, increased infectivity, and decreased impurities (e.g., producer cell DNA and / or proteins).

[0360] In some embodiments, viral vectors are obtained from a producer cell culture. In some embodiments, a method can comprise enriching viral vectors from a producer cell culture. In some embodiments, a method comprises generating a crude drug substance from a producer cell culture, where the crude drug substance comprises viral vectors produced by the producer cells or a subset thereof. In some embodiments, generating a crude drug substance from a producer cell culture comprises enriching viral vectors from the producer cell culture. In some generating a crude drug substance from a producer cell culture comprises removing producer cell DNA and / or producer cell protein from the producer cell culture (e.g., producer cells and / or culture medium).

[0361] In some embodiments, a crude drug substance is characterized by an at least 3- fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold increase in viral vector titer as compared to a crude drug substance produced by an alternative process.

[0362] In some embodiments, a crude drug substance is characterized by an at least 1.5- fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, or at least 2.5-fold increase in infectivity as compared to a crude drug substance produced by an alternative process.

[0363] In some embodiments, a crude drug substance is characterized by an at least 500- fold, at least 750-fold, at least 1000-fold, at least 1100-fold, at least 1200-fold, at least 1300-fold, or at least 1400-fold decrease in the amount of producer cell DNA as compared to a crude drug substance produced by an alternative process.

[0364] Producer cell DNA can be measured using various techniques known to a skilled artisan. In some embodiments, producer cell DNA is measured using quantitative PCR (qPCR). In some embodiments, producer cell DNA is measure using probe based PCR. In some embodiments, producer cell DNA is measured using a spectrophotometer.

[0365] In some embodiments, a crude drug substance is characterized by an at least 15- fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold decrease in the amount of producer cell protein as compared to a crude drug substance produced by an alternative process.

[0366] In some embodiments, a crude drug substance is characterized by an at least 1.5- fold, at least 2-fold, at least 2.5-fold, or at least 3.0-fold increase in transduction efficiency in a target cell exposed to the crude drug substance as compared to a crude drug substance produced by an alternative process.

[0367] In some embodiments, a crude drug substance is characterized by an at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50- fold, at least 55-fold, or at least 60-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process.

[0368] In some embodiments, a crude drug substance is characterized by an at least 2- fold, at least 3-fold, or at least 4-fold increase in fusogen expression on the viral vectors as compared to a crude drug substance produced by an alternative process.

[0369] In some embodiments, a method provided herein can comprise generating a drug substance. In some embodiments, a method provided herein can comprise generating a drug substance from the crude drug substance. In some embodiments, a drug substance comprises the viral vectors produced by the producer cells or a subset thereof. In some embodiments, generating a drug substance from a crude drug substance comprises further enriching the viral vectors from the crude drug substance. In some embodiments, generating a drug substance from a crude drug substance comprises further removing producer cell DNA and / or producer cellprotein from the crude drug substance. In some embodiments, generating a drug substance from the crude drug substance comprises adding a pharmaceutically acceptable excipient.

[0370] In some embodiments, a drug substance is characterized by an at least 5-fold, at least 5.5-fold, at least 6-fold, or at least 6.5-fold increase in viral vector titer as compared to a drug substance produced by an alternative process.

[0371] In some embodiments, a drug substance is characterized by an at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, or at least?.5-fold increase in infectivity as compared to a drug substance produced by an alternative process.

[0372] In some embodiments, a drug substance is characterized by an at least 2000-fold, at least 2250-fold, at least 2500-fold, at least 2750-fold, at least 3000-fold, or at least 3250-fold decrease in the amount of producer cell DNA as compared to a drug substance produced by an alternative process.

[0373] In some embodiments, a drug substance is characterized by an at least 7.5-fold, at least 10-fold, at least 12.5-fold, or at least 15-fold decrease in the amount of producer cell protein as compared to a drug substance produced by an alternative process.

[0374] In some embodiments, a drug substance is characterized by an at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, or at least 4.5-fold increase in transduction efficiency in a target cell exposed to the drug substance as compared to a drug substance produced by an alternative process.

[0375] In some embodiments, a drug substance is characterized by an at least 45-fold, at least 50-fold, at least 55-fold, or at least 60-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process.

[0376] In some embodiments, a drug substance is characterized by an at least 5-fold, at least 6-fold, at least 6.5-fold, at least 7.5-fold, or at least 10-fold increase in fusogen expression on the viral vectors as compared to a crude drug substance produced by an alternative process.

[0377] In some embodiments, an alternative process is a comparable process that differs in one or more steps and / or conditions. In some embodiments, wherein the alternative process is a comparable process in which a ratio of an envelope plasmid to a packaging plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1. In some embodiments, wherein the alternative process is a comparable process in which a ratio of an envelope plasmid to a packaging plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1. In some embodiments, wherein the alternative process is a comparable process in which a ratio of an envelope plasmid to a transfer plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1. In some embodiments, wherein the alternative process is a comparable process in which an amount of an envelope plasmid that is introduced into producer cells is equal to or less than an amount of an packaging plasmid that is introduced into the producer cells. In some embodiments, wherein the alternative process is a comparable process in which an amount of an envelope plasmid that is introduced into producer cells is equal to or less than the amount of a transfer plasmid that is introduced into the producer cells. In some embodiments, wherein the alternative process is a comparable process in which producer cells are treated with a nuclease at a time that is more than 6 hours after at least one nucleic acid that encodes one or more fusogens is introduced into the producer cells. In some embodiments, wherein the alternative process is a comparable process in which producer cells are not cultured in a suspension culture. In some embodiments, wherein the alternative process is a comparable process in which cell aggregation of producer cells is not reduced. In some embodiments, wherein the alternative process is a comparable process in which producer cells are not agitated during culture. In some embodiments, wherein the alternative process is a comparable process in which culture medium for producer cells does not include an anti-clump agent or a surfactant.

[0378] In some embodiments, a drug substance or drug product as described herein can include a pharmaceutically acceptable carrier or excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspensionaids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, glycerol, sugars such as mannitol, sucrose, or others, dextrose, fatty acid esters, etc., as well as combinations thereof.

[0379] A drug substance or drug product as described herein can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and / or aromatic substances and the like), which do not deleteriously react with the active compounds or interfere with their activity. In certain embodiments, a water-soluble carrier suitable for intravenous administration is used. In some embodiments, a pharmaceutical composition can be sterile.

[0380] A suitable a drug substance or drug product as described herein, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0381] A drug product formulated from a drug substance described herein can be formulated in accordance with the routine procedures as a drug substance, e.g., adapted for administration to human beings. The formulation of a drug substance should suit the mode of administration. For example, in some embodiments, a drug substance for intravenous administration is typically a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active agent. Where a drug substance is to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water, saline or dextrose / water. Where a drug substance is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0382] Although the descriptions of drug substances provided herein are principally directed to drug substances that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts or cells in vitro or ex vivo. Modification of drug substances suitable for administration to humans in order to render the compositions suitable for administration to various animals or cells in vitro or ex vivo is well understood, and the ordinarily skilled practitioner, e.g., a veterinary pharmacologist, can design and / or perform such modification with merely ordinary, if any, experimentation.

[0383] Formulations of drug substances described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a diluent or another excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0384] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.

[0385] In some embodiments, a crude drug substance is characterized by an at least 1.5- fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, or at least a 4-fold increase in yield as compared to a crude drug substance produced by an alternative process.

[0386] In some embodiments, a crude drug substance is characterized by a 4.8-fold increase in yield as compared to a crude drug substance produced by an alternative process.

[0387] In some embodiments, a drug substance is characterized by an at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least a 5-fold, or at least a 5.5-fold, increase in yield as compared to a drug substance produced by an alternative process.

[0388] In some embodiments, a drug substance is characterized by a 6.1-fold increase in yield as compared to a drug substance produced by an alternative process.CERTAIN NUMBERED EMBODIMENTS

[0389] Embodiment A:

[0390] Embodiment Al. A method of producing a lentiviral vector, said method comprising culturing producer cells comprising one or more nucleic acids for the production of the lentiviral vector under conditions for producing the lentiviral vector by the producer cells, wherein the method is characterized by:(i) a 10-fold increase in the number of active vector particles,(ii) a 10-fold reduction in producer cell protein, and / or(iii) a 3000- fold reduction in producer cell DNA, relative to an alternative process.

[0391] Embodiment A2. The method of embodiment Al, wherein the method is characterized by a 50-fold increase in functional titer relative to an alternative process.

[0392] Embodiment A3. The method of embodiment Al or A2, wherein one of the one or more nucleic acids further encodes one or more fusogens.

[0393] Embodiment A4. The method of embodiment A3, wherein the nucleic acid encoding the one or more fusogens is introduced into the producer cell, optionally by transfection of the one or more nucleic acids.

[0394] Embodiment A5. The method of any of embodiments Al -A4, wherein the lentiviral vector produced by the method comprises a lipid bilaycr and the one or more fusogens are embedded in the lipid bilayer.

[0395] Embodiment A6. The method of any of embodiments A1-A5, wherein the one or more fusogens are a paramyxovirus envelope protein or a biologically active portion thereof.

[0396] Embodiment A7. The method of any of embodiments A3-A6, wherein the method is characterized by a 6-fold increase in fusogen expression relative to an alternative process.

[0397] Embodiment A8. The method of any of embodiments A1-A7, wherein the method is characterized by one or more steps to reduce producer cell aggregation, optionally wherein the method is characterized by culturing producer cells with an agent to reduce cell aggregation (e.g., anti-clump reagent or surfactant) and / or wherein the method is characterized by culturing producer cells with increased agitation relative to an alternative process.

[0398] Embodiment A9. The method of any of embodiments A1-A8, wherein the method is characterized by one or more steps of nuclease treatment, optionally wherein the culturing of producer cells and / or transfection of the one or more nucleic acids comprises nuclease treatment for 30 minutes to 6 hours, or any value between the foregoing.

[0399] Embodiment A10. The method of embodiment A9, wherein the method is characterized by a 3-5-fold increase in lentiviral vector production.

[0400] Embodiment B:

[0401] Embodiment Bl. A method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors.

[0402] Embodiment B2. The method of embodiment Bl, wherein the viral vector are gammaretroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors.

[0403] Embodiment B3. The method of embodiment Bl or B2, wherein the viral vectors are lentiviral vectors.

[0404] Embodiment B4. The method of embodiment B3, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid.

[0405] Embodiment B5. The method of embodiment B4, wherein the one or more nucleic acids further comprise a regulatory plasmid.

[0406] Embodiment B6. The method of any one of embodiments B3-B5, wherein the viral vectors comprise a lipid bilayer.

[0407] Embodiment B7. The method of any one of embodiments B3-B6, wherein the one or more nucleic acids comprise at least one nucleic acid that encodes one or more fusogens.

[0408] Embodiment B8. The method of embodiment B6 or B7, wherein the one or more fusogens are present in the lipid bilayer.

[0409] Embodiment B9. The method of any one of embodiments B4-B8, wherein the envelope plasmid encodes the one or more fusogens.

[0410] Embodiment B 10. The method of any one of embodiments B 1-B9, wherein the one or more fusogens comprise at least one fusogen that has a tropism for B cells, T cells, natural killer cells, islet cells, glial progenitor cells, neuronal cells, hematopoietic stem cells, cardiac cells, hepatocytes, stem cells, or induced pluripotent stem cells.

[0411] Embodiment B 11. The method of embodiment B10, wherein the one or more fusogens comprise at least one fusogen that has a tropism for B cells.

[0412] Embodiment B 12. The method of embodiment B10, wherein the one or more fusogens comprise at least one fusogen that has a tropism for T cells.

[0413] Embodiment B 13. The method of embodiment B10, wherein the one or more fusogens comprise at least one fusogen that has a tropism for islet cells

[0414] Embodiment B 14. The method of embodiment BIO, wherein the one or more fusogens comprise at least one fusogen that has a tropism for cardiac cells.

[0415] Embodiment B 15. The method of any one of embodiments B 1-B 14, wherein the one or more fusogens comprise at least one fusogen that has endogenous tropism.

[0416] Embodiment B 16. The method of any one of embodiments B 1-B 15, wherein the one or more fusogens comprise at least one fusogen that has engineered tropism.

[0417] Embodiment B 17. The method of any one of embodiments B 1 -B 16, wherein the one or more fusogens comprise one or more viral fusogens.

[0418] Embodiment B 18. The method of any one of embodiments B 1-B 17, wherein the one or more fusogens comprise at least one fusogen that is involved in attachment of a viral vector to a cell membrane.

[0419] Embodiment B19. The method of any one of embodiments B1-B18, wherein the one or more fusogens comprise at least one fusogen that is involved in directing fusion of the lipid bilayer of a viral vector and a cell membrane.

[0420] Embodiment B20. The method of any one of embodiments B 1 -B 19, wherein the one or more fusogens comprise one or more paramyxovirus envelope proteins or biologically active portions thereof.

[0421] Embodiment B21. The method of embodiment B20, wherein the one or more paramyxovirus envelope proteins or biologically active portions thereof comprises a paramyxovirus glycoprotein (“G protein”) or a biologically active portion thereof.

[0422] Embodiment B22. The method of embodiment B20 or B21, wherein the one or more paramyxovirus envelope proteins or biologically active portions thereof comprises a paramyxovirus fusion protein (“F protein”) or a biologically active portion thereof.

[0423] Embodiment B23. The method of any one of embodiments B1-B22, wherein the one or more fusogens comprise one or more chimeric proteins.

[0424] Embodiment B24. The method of embodiment B23, wherein the one or more chimeric proteins comprise at least one chimeric protein that comprises a paramyxovirus envelope protein or biologically active portion thereof.

[0425] Embodiment B25. The method of embodiment B23 or B24, wherein the one or more chimeric proteins comprise at least one chimeric protein that comprises an scFV.

[0426] Embodiment B26. The method of any one of embodiments B23-B25, wherein the one or more chimeric proteins comprise at least one chimeric protein that comprises (i) a paramyxovirus envelope protein or biologically active portion thereof and (ii) an scFV.

[0427] Embodiment B27. The method of embodiment B25 or B26, wherein the scFV targets an antigen present on the surface of a B cell, a T cell, a natural killer cell, an islet cell, a glial progenitor cell, a cardiac cell, a blood cell, a hepatocyte, a stem cell, or an induced pluripotent stem cell.

[0428] Embodiment B28. The method of embodiment B27, wherein the scFV targets an antigen present on the surface of a B cell.

[0429] Embodiment B29. The method of embodiment B27, wherein the scFV targets an antigen present on the surface of a T cell.

[0430] Embodiment B30. The method of embodiment B27 or B29, wherein the scFV targets CD8.

[0431] Embodiment B31. The method of embodiment B27 or B29, wherein the scFV targets CD4.

[0432] Embodiment B32. The method of embodiment B27, wherein the scFV targets an antigen present on the surface of an islet cell.

[0433] Embodiment B33. The method of embodiment B32, wherein the islet cell is an alpha cell, a beta cell, or a delta cell.

[0434] Embodiment B34. The method of embodiment B32, wherein the islet cell is a beta cell.

[0435] Embodiment B35. The method of any one of embodiments B4-B34, wherein the transfer plasmid comprises a nucleotide sequence that encodes a nuclease.

[0436] Embodiment B36. The method of embodiment B35, wherein the nuclease is a Cas, a TALEN, or a zinc-finger nuclease.

[0437] Embodiment B37. The method of embodiment B35, wherein the transfer plasmid comprises a nucleotide sequence that encodes a gRNA.

[0438] Embodiment B38. The method of embodiment B35, wherein the transfer plasmid comprises a nucleotide sequence that encodes an antibody or a biologically active portion thereof.

[0439] Embodiment B39. The method of embodiment B35, wherein the transfer plasmid comprises a nucleotide sequence that encodes a chimeric antigen receptor.

[0440] Embodiment B40. The method of embodiment B35, wherein the transfer plasmid comprises a nucleotide sequence that encodes an antigen.

[0441] Embodiment B41. The method of embodiment B35, wherein the transfer plasmid comprises a nucleotide sequence that encodes a therapeutic polypeptide.

[0442] Embodiment B42. The method of embodiment B41, wherein the therapeutic polypeptide is useful for protein replacement therapy.

[0443] Embodiment B43. The method of any one of embodiments B4-B42, wherein the ratio of the envelope plasmid to the packaging plasmid within the one or more nucleic acids is greater than 1:1.

[0444] Embodiment B44. The method of any one of embodiments B4-B43, wherein the ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is greater than 1:1.

[0445] Embodiment B45. The method of any one of embodiments B4-B42, wherein the ratio of a first envelope plasmid to a second envelope plasmid within the one or more nucleic acids is greater than 1:1.

[0446] Embodiment B46. The method of embodiment B45, wherein a first envelope plasmid encodes glycoprotein G (“G protein”).

[0447] Embodiment B47. The method of any one of embodiments B 1-B44, wherein the method comprises introducing the at least one nucleic acid that encodes one or more fusogens into the producer cells.

[0448] Embodiment B48. The method of embodiment B47, wherein introducing the at least one nucleic acid that encodes one or more fusogens into the producer cells comprises transfecting the at least one nucleic acid that encodes one or more fusogens into the producer cells.

[0449] Embodiment B49. The method of any one of embodiments B 1-B48, wherein the method comprises introducing the one or more nucleic acids into the producer cells.

[0450] Embodiment B50. The method of embodiment B49, wherein introducing the one or more nucleic acids into the producer cells comprises transfecting the one or more nucleic acids into the producer cells.

[0451] Embodiment B51. The method of embodiment B49 or B50, wherein the amount of the envelope plasmid that is introduced into the producer cells is greater than the amount of the packaging plasmid that is introduced into the producer cells.

[0452] Embodiment B52. The method of any one embodiments B49-B51, wherein the amount of the envelope plasmid that is introduced into the producer cells is greater than the amount of the transfer plasmid that is introduced into the producer cells.

[0453] Embodiment B53. The method of any one of embodiments B 1-B52, further comprising treating the producer cells with a nuclease.

[0454] Embodiment B54. The method of embodiment B47-B53, further comprising treating the producer cells with a nuclease at a time that is at least 30 minutes after the at least one nucleic acid that encodes one or more fusogens arc introduced into the producer cells.

[0455] Embodiment B55. The method of embodiment B47-B54, further comprising treating the producer cells with a nuclease at a time that is no more than 6 hours after the at least one nucleic acid that encodes one or more fusogens are introduced into the producer cells.

[0456] Embodiment B56. The method of any one of embodiments B47-B54, further comprising treating the producer cells with a nuclease at a time that is no more than 4 hours after the at least one nucleic acid that encodes one or more fusogens are introduced into the producer cells.

[0457] Embodiment B57. The method of any one of embodiments B53-B56, wherein the nuclease is a salt activated nuclease.

[0458] Embodiment B58. The method of any one of embodiments B53-B56, wherein the nuclease is MSAN.

[0459] Embodiment B59. The method of embodiment B58, wherein the producer cells are treated with about 100,000 to 500,000 units of MSAN.

[0460] Embodiment B60. The method of any one of embodiments B 1-B59, wherein the producer cells are cultured in a suspension culture.

[0461] Embodiment B61. The method of any one of embodiments B1-B60, wherein the producer cells are cultured in serum free medium.

[0462] Embodiment B62. The method of any one of embodiments B 1-B61, further comprising agitating the producer cells.

[0463] Embodiment B63. The method of any one of embodiments B1-B62, wherein the producer cells are cultured in medium that includes an anti-cell clumping agent.

[0464] Embodiment B64. The method of any one of embodiments B 1-B63, wherein the producer cells are cultured in medium that includes a surfactant.

[0465] Embodiment B65. The method of any one of embodiments B 1 -B64, further comprising enriching the viral vectors from the producer cell culture.

[0466] Embodiment B66. The method of any one of embodiments B 1-B65, further comprising generating a crude drug substance from the producer cell culture, wherein the crude drug substance comprises the viral vectors produced by the producer cells or a subset thereof.

[0467] Embodiment B67. The method of embodiment B66, wherein generating a crude drug substance from the producer cell culture comprises enriching the viral vectors from the producer cell culture.

[0468] Embodiment B68. The method of embodiment B66 or B67, wherein generating a crude drug substance from the producer cell culture comprises removing producer cell DNA and / or producer cell protein from the producer cell culture.

[0469] Embodiment B69. The method of any one of embodiments B65-B68, wherein the producer cell culture comprises the producer cells and / or culture medium.

[0470] Embodiment B70. The method of any one of embodiments B66-B69, wherein the crude drug substance is characterized by: (i) an at least 3-fold increase in viral vector titer as compared to a crude drug substance produced by an alternative process; (ii) an at least 1.5-fold increase in infectivity as compared to a crude drug substance produced by an alternative process; (iii) an at least 1000-fold decrease in the amount of producer cell DNA as compared to a crude drug substance produced by an alternative process; (iv) an at least 15-fold decrease in theamount of producer cell protein as compared to a crude drug substance produced by an alternative process; (v) an at least 1.5-fold increase in transduction efficiency in a target cell exposed to the crude drug substance as compared to a crude drug substance produced by an alternative process; or (vi) an at least 20-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process; (vii) an at least 3-fold increase in fusogen expression on the viral vectors as compared to a crude drug substance produced by an alternative process; (viii) or a combination thereof.

[0471] Embodiment B71 . The method of any one of embodiments B66-B70, further comprising generating a drug substance from the crude drug substance, wherein the drug substance comprises the viral vectors produced by the producer cells or a subset thereof.

[0472] Embodiment B72. The method of embodiment B71, wherein generating a drug substance from the crude drug substance comprises further enriching the viral vectors from the crude drug substance.

[0473] Embodiment B73. The method of embodiment B71 or B72, wherein generating a drug substance from the crude drug substance comprises further removing producer cell DNA and / or producer cell protein from the crude drug substance.

[0474] Embodiment B74. The method of any one of embodiments B71-B73, wherein generating a drug substance from the crude drug substance comprises adding a pharmaceutically acceptable excipient.

[0475] Embodiment B75. The method of any one of embodiments B71-B74, wherein the drug substance is characterized by: (i) an at least 5-fold increase in viral vector titer as compared to a drug substance produced by an alternative process; (ii) an at least 5-fold increase in infectivity as compared to a drug substance produced by an alternative process; (iii) an at least 2000-fold decrease in the amount of producer cell DNA as compared to a drug substance produced by an alternative process; (iv) an at least 10-fold decrease in the amount of producer cell protein as compared to a drug substance produced by an alternative process; (v) an at least 2-fold increase in transduction efficiency in a target cell exposed to the drug substance as compared to a drug substance produced by an alternative process; or (vi) an at least 50-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process; (vii) an at least 6-fold increase in fusogen expression on the viral vectors as compared to a crude drug substance produced by an alternative process; (viii) or a combination thereof.

[0476] Embodiment B76. The method of any one of embodiments B70-B75, wherein the alternative process is a comparable process wherein: (i) a ratio of an envelope plasmid to a packaging plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1; (ii) a ratio of an envelope plasmid to a transfer plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1; (iii) a ratio of a first envelope plasmid to a second envelope plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1; (iv) an amount of an envelope plasmid that is introduced into producer cells is equal to or less than an amount of an packaging plasmid that is introduced into the producer cells; (v) an amount of an envelope plasmid that is introduced into producer cells is equal to or less than the amount of a transfer plasmid that is introduced into the producer cells; (vi) an amount of a first envelope plasmid that is introduced into producer cells is equal to or less than the amount of a second envelope plasmid that is introduced into the producer cells; (vii) producer cells are treated with a nuclease at a time that is more than 6 hours after at least one nucleic acid that encodes one or more fusogens is introduced into the producer cells; (viii) producer cells are not cultured in a suspension culture; (ix) cell aggregation of producer cells is not reduced; (x) producer cells are not agitated during culture; (xi) culture medium for producer cells does not include an anti-clump agent or a surfactant; or (xii) a combination thereof.

[0477] Embodiment B77. The method of embodiment B76, wherein the first envelope plasmid encodes G protein.

[0478] Embodiment B78. A producer cell comprising one or more nucleic acids for the production of lentiviral vectors, wherein the one or more nucleic acids comprise a packaging plasmid and an envelope plasmid that encodes one or more fusogens, and wherein the ratio of the envelope plasmid to the packaging plasmid within the producer cell is greater than 1:1.

[0479] Embodiment B79. The producer cell of embodiment B78, wherein the ratio of the envelope plasmid to the packaging plasmid within the producer cell is greater than 2:1.

[0480] Embodiment B80. A producer cell comprising one or more nucleic acids for the production of lentiviral vectors, wherein the one or more nucleic acids comprise a first envelope plasmid and a second envelope plasmid, wherein the first envelope plasmid and second envelope plasmid each encode one or more fusogens, and wherein the ratio of the first envelope plasmid to the second envelope plasmid within the producer cell is greater than 1:1.

[0481] Embodiment B81. The producer cell of embodiment B80, wherein the ratio of the first envelope plasmid to the second envelope plasmid within the producer cell is greater than 2:1.

[0482] Embodiment B82. The producer cell of any one of embodiments B78-B81, wherein the first envelope plasmid encodes G protein.

[0483] Embodiment B83. A producer cell comprising engineered lentiviral vectors, wherein the producer cell had been transfected with one or more nucleic acids for the production of lentiviral vectors at a first time point and had been exposed to a nuclease at a second time point, wherein the second time point is at least 30 minutes after and at most 6 hours after the first time point.EXAMPLESExample 1: Production of Lentiviral Vectors

[0484] This Example describes an exemplary for the production of lentiviral vectors according to a method as provided herein.

[0485] HEK 293T producer cells are plated in the presence of serum-containing culture medium. HEK 293T producer cells are subsequently adapted for serum-free growth in a suspension culture. Adapted HEK 293T producer cells were sub-cloned and selected based on growth and functional titer production in suspension and serum free media, relative to each other and to producer cells that were not adapted to serum-free suspension growth. Selected clones were further screened and selected based on their morphology. Adapted HEK 293T are grown in a suspension culture and allowed to expand in a flask prior to transfection. Transfection is carried out using a third-generation lentiviral vector system comprising a packaging plasmid encoding gag and pol, a regulatory plasmid encoding rev, 2 envelope plasmids encoding Henipavirus (e.g., nipah virus) G protein and F respectively, and a transfer plasmid carrying a CD 19 CAR transgene. The ratio of G protein envelope plasmid relative to F protein envelope plasmid is increased for (i) optimal amounts of G protein on a lentiviral vector and (ii) for increased crude functional titer. Analysis of G protein content on a lentiviral vector (e.g., fusosome) was carried out via an ELISA (Meso Scale Discovery technology) (FIG. 7), Western blot analysis, and per particle analysis (ExoView TWO platform) (FIG. 8). These results demonstrate that an increase in G protein envelope plasmid produces a greater amount of G protein on a lentiviral vector. Per particle analysis further demonstrated that a greater amount of G protein on a lentiviral vector leads to a greater proportion of transduction competent lentiviral vector with both G protein and F on its surface (e.g., complete lentiviral vector) (FIG. 9).

[0486] The relationship between an increase in G protein plasmid and functional titer of a lentiviral vector was measured using flow cytometry. Crude lentiviral vector supernatants, produced by a method as described herein, were used to transduce cells that express CD8. GFP expression was analyzed by flow cytometry 4 days post-transduction, and the serial dilution point that corresponded to 5-15% GFP-positive cells was used to calculate the lentiviral vector functional titer (FIG. 10). These results indicate that there is an optimal range of increase in the amount of G protein envelope plasmid relative F protein envelope plasmid that leads to increased lentiviral functional titer.

[0487] Plasmids are complexed with polyethylenimine (PEI) to create a plasmid DNA / PEI complex that is transfected into HEK293 producer cells. Transfected producer cells are placed in a bioreactor where the cells are exposed to physical (e.g., agitation, mixing, stirring) and / or chemical (e.g., anti-clump agent, surfactant) conditions to prevent cell aggregation. 4 hours post-transfection, producer cells are exposed to a salt activated nuclease (e.g., MSAN). A volume of MSAN is prepared according to the formula:

[0488] Once prepared, M-SAN is added to a volume of media determined by the formula:Vol. of Media (mL) = 2000 mL - (1000 mL 1.0 M Mg [Cl] _2 + Vol. of MSAN HQ) ; (Eqn. 2)

[0489] Approximately two days (e.g., 44-48 hours) after transfection, producer cells are harvested and again exposed to a volume of MSAN prepared in media per the following equations for M-SAN preparation:Vol. of Media mL) = 410 mL - (Vol. of MSAN HQ) ; (Eqn.4)

[0490] Following nuclease treatment, harvested samples are allowed to settle for 4-16 hours before clarification by passage of the sample through a .45 micron glass microfiber filter. After clarification, the clarified samples are passed through a chromatography membrane and / orresin two times. Samples are then passed through a 0.2 micron filter resulting in an enrichment of lentiviral vectors and removal of producer cell impurities, including producer cell DNA and protein.Example 2: Production of Lentiviral Vectors that Express a Fusogen and encode a Transgene and Target CD8 positive T cells for Transduction.

[0491] This Example describes an exemplary method for the production of lentiviral vectors that produce CD8-targeted fusosomes (e.g., lentiviral vectors comprising a fusogen in its envelope bilayer) as described herein.

[0492] Lentiviral vectors were produced as described in Example 1. CD8-targeted fusogen were generated through targeted mutation to ablate binding to its native receptor. This “blinded” fusogen was subsequently engineered to display a novel scFv that is specific for human CD8a. Viral vector encoding either FMC63 or human CD19 CARs were pseudotyped with CD8-specific fusogen to produce CD8-targeted fusosomes. Methods of producing fusogen pseudotyped CARs are described in the art, for example in US 11,535,869, the entire contents of which is incorporated by reference herein. For example, a CAR-T cell is produced with an FMC63 CAR, a CAR with an FMC63 binder to direct the CAR T to CD 19, an scFv that binds human CD8-alpha, and a fusogen. Fusosome titer was quantified by ddPCR directly for physical titer.

[0493] Transduction of T cells was achieved by exposing resting peripheral blood mononuclear cells (PBMCs) to CD8 targeted fusosomes that encode a CAR, produced by a method as described herein, for 2 hours. PBMCS were then washed and cultured for 8 days in the presence of T cell activating IL-2 and anti CD3 / CD28 beads before CAR expression, measured as percent of CD8 positive T cells that are positive for CAR, was assessed by flow cytometry (FIG. 11). These results indicate that CD8-targeted fusosomes that encode a CAR produced by a method as described herein transduced PBMCs better than CD8-targeted fusosomes that encode a CAR produced by an alternative process.

[0494] Fusosomes, produced by a method as described herein, were assayed for their ability to specifically transduce CD8 positive T cells. Specificity of transduction was measured via vector copy number (VCN) and flow cytometry analysis. Flow cytometry was conducted by measuring the percentage of either CD8 positive or CD4 positive T cells that are positive for an FMC3 CAR, at various multiplicities of infection (MOI) and relative to fusosomes produced by an alternative process (FIG. 12A-B). Vector copy number analysis showed fusosomes produced by a method as described herein resulted in lower off-target (C8 negative cells) transduction and over 3 times higher on-target transduction (CD8 positive T cells) relative to fusosomes produced by an alternative process.

[0495] Negative controls consisted of a tumor and PBMC sample, a tumor only sample, a PBMC only sample, and saline.

[0496] Fusosomes, produced by a method as described herein, were assayed for their ability to clear tumors. To measure tumor clearance, NALM6 tumor cells expressing firefly luciferase were intravenously injected into immunodeficient NSG mice at 5xl05tumor cells / mouse on day -4. On day -1, PMCs were injected into the same mice, followed 24 hours later by CD8-targeted fusosomes encoding a CD19 CAR (e.g., CD-8 T cell targeting fusosome), produced by a method as described herein. Tumor growth was then monitored by bioluminescence imaging. Results are displayed as area under curve of normalized total flux (photons / sec) through day 20. Results compare tumor clearance by CD8-targeted fusosomes that encode a CD8 / CD19 CAR, produced by a method as described herein, to fusosomes produced by an alternative process (FIG. 13). Negative controls consist of a tumor and PBMC sample, a tumor only sample, a PBMC only sample, and saline.Example 3; Characterization of Exemplary Lenti viral Vectors

[0497] This Example describes exemplary assays performed to characterize selected qualities of lentiviral vectors produced by methods described herein.

[0498] Lentiviral vectors comprising G protein and F protein in their lipid bilayer were produced as described in Example 1. A crude drug substance was generated and assayed. Following further lentiviral vector enrichment steps, a drug substance was also generated and assayed. A table summarizing the various results as compared to lentiviral vectors generated by an alternative process is shown in Fig. 1.

[0499] As shown in Fig. 2, lentiviral vectors produced by a method as described herein showed an approximately 55-fold increase in the number of transducing units per milliliter as compared to lentiviral vectors produced by an alternative process. This data demonstrated that methods described herein produced drug substances having an increased functional lentiviral vector titer.

[0500] As shown in Fig. 3 (left), a crude drug substance comprising lentiviral vectors produced by a method as described herein showed an approximately 4.8-fold increase in the number of transducing units per liter as compared to lentiviral vectors produced by an alternative process. Further, in Fig. 3 (right) a drug substance comprising lentiviral vectors produced by a method as described herein showed an approximately 6.1 -fold increase in the number of transducing units per liter as compared to lentiviral vectors produced by an alternative process. This data demonstrated that methods described herein produced crude drug substances and drug substances having an overall increased lentiviral vector titer.

[0501] As shown in Fig. 4 (left), a crude drug substance comprising lentiviral vectors produced by a method as described herein showed an approximately 2-fold increase in the number of viral like particles per transducing unit as compared to lentiviral vectors produced by an alternative process. Additionally, in Fig. 4 (right), a drug substance comprising lentiviral vectors produced by a method as described herein showed an approximately 7-fold increase in the number of viral like particles per transducing unit as compared to lentiviral vectors produced by an alternative process. This data demonstrated that methods described herein produced crude drug substances and drug substances having lentiviral vectors with increased infectivity.

[0502] As shown in Fig. 5A, a crude drug substance comprising lentiviral vectors produced by a method as described herein showed an approximately 1350-fold decrease in the pg of producer cell DNA / transducing unit of lentiviral vector as compared to lentiviral vectors produced by an alternative process. Additionally, in Fig. 5A, a drug substance comprising lentiviral vectors produced by a method as described herein showed an approximately 3000-fold decrease in the pg of producer cell DNA / transducing unit of lentiviral vector as compared to lentiviral vectors produced by an alternative process. Producer cell DNA was measured using quantitative PCR. This data demonstrated that methods described herein produced crude drug substances and drug substances having less impurities (e.g., producer cell DNA).

[0503] As shown in Fig. 5B, a crude drug substance comprising lentiviral vectors produced by a method as described herein showed an approximately 23-fold decrease in the pg of producer cell protein / transducing unit of lentiviral vector as compared to lentiviral vectors produced by an alternative process. Moreover, in Fig. 5B, a drug substance comprising lentiviral vectors produced by a method as described herein showed an approximately 11-fold decrease in the pg of producer cell protein / transducing unit of lentiviral vector as compared to lentiviral vectors produced by an alternative process. This data demonstrated that methods described herein produced crude drug substances and drug substances having less impurities (e.g., producer cell produced).EQUIVALENTS

[0504] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

PCT / US24 / 11000 10 January 2024 (10.01.2024) _WHAT IS CLAIMED IS:

1. A method of producing a composition comprising viral vectors, comprising(a) culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid;(b) treating the producer cell culture with a nuclease; and(c) harvesting the producer cell culture, wherein (b) is performed prior (c).

2. A method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid, wherein the ratio of the envelope plasmid to the packaging plasmid within the one or more nucleic acids is greater than 1:1.

3. The method of claim 2, wherein the ratio of the envelope plasmid to the packaging plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

4. The method of claim 2, wherein the ratio of the packaging plasmid to the envelope plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

5. A method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid, wherein thePCT / US24 / 11000 10 January 2024 (10.01.2024) _ ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is greater than 1 :

16. The method of claim 5, wherein the ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

7. The method of claim 5, wherein the ratio of the transfer plasmid to the envelope plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

8. A method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid, wherein the ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is greater than 1:1.

9. The method of claim 8, wherein the ratio of the envelope plasmid to the transfer plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

10. A method of producing a composition comprising viral vectors, comprising culturing producer cells comprising one or more nucleic acids for the production of the viral vector under conditions sufficient to produce the viral vectors, wherein the one or more nucleic acids comprise a packaging plasmid, a first envelope plasmid, a second envelope plasmid, and a transfer plasmid, wherein the ratio of a first envelope plasmid to a second envelope plasmid within the one or more nucleic acids is greater than 1:1.PCT / US24 / 11000 10 January 2024 (10.01.2024) _11. The method of claim 10, wherein the first envelope plasmid comprises a nucleic acid sequence encoding a first envelope protein and the second envelope plasmid comprises a nucleic acid sequence encoding a second envelope protein.

12. The method of 10 or 11, wherein the ratio of a first envelope plasmid to a second envelope plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

13. The method of 10 or 11, wherein the ratio of a second envelope plasmid to a first envelope plasmid within the one or more nucleic acids is selected from a ratio of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

14. The method of any one of claims 1-13, wherein the viral vectors are gammaretroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors.

15. The method of any one of claims 1-14, wherein the viral vectors are lentiviral vectors.

16. The method of any one of claims 1-15, wherein the one or more nucleic acids further comprise a regulatory plasmid.

17. The method of any one of claims 1-16, wherein the viral vectors comprise a lipid bilayer.

18. The method of any one of claims 1-17, wherein the one or more nucleic acids comprise at least one nucleic acid that encodes one or more fusogens.

19. The method of claim 18, wherein the one or more fusogens are present in the lipid bilayer.PCT / US24 / 11000 10 January 2024 (10.01.2024) _20. The method of claim 18 or 19, wherein the one or more fusogens are present on the outside of the lipid bilayer.

21. The method of any one of claims 1-20, wherein the envelope plasmid encodes the one or more fusogens.

22. The method of any one of claims 18-21, wherein the one or more fusogens comprise at least one fusogen that has a tropism for B cells, T cells, natural killer cells, glial progenitor cells, neuronal cells, hematopoietic stem cells, cardiac cells, hepatocytes, stem cells, or induced pluripotent stem cells.

23. The method of claim 22, wherein the one or more fusogens comprise at least one fusogen that has a tropism for B cells.

24. The method of claim 22, wherein the one or more fusogens comprise at least one fusogen that has a tropism for T cells.

25. The method of claim 22, wherein the one or more fusogens comprise at least one fusogen that has a tropism for cardiac cells.

26. The method of claim 22, wherein the one or more fusogens comprise at least one fusogen that has a tropism for liver cells.

27. The method of claim 22, wherein the one or more fusogens comprise at least one fusogen that has a tropism for a neuron.

28. The method of any one of claims 1-27, wherein the one or more fusogens comprise at least one fusogen that has natural tropism.

29. The method of any one of claims 1-28, wherein the one or more fusogens comprise at least one fusogen that has engineered tropism.PCT / US24 / 11000 10 January 2024 (10.01.2024) _30. The method of any one of claims 1-29, wherein the one or more fusogens comprise one or more viral fusogens.

31. The method of any one of claims 1-30, wherein the one or more fusogens comprise at least one fusogen that is involved in attachment of a viral vector to a cell membrane.

32. The method of any one of claims 1-31, wherein the one or more fusogens comprise at least one fusogen that is involved in directing fusion of the lipid bilayer of a viral vector and a cell membrane.

33. The method of any one of claims 1-32, wherein the one or more fusogens comprise one or more paramyxovirus envelope proteins or biologically active portions thereof.

34. The method of claim 33, wherein the one or more paramyxovirus envelope proteins or biologically active portions thereof is a variant paramyxovirus envelope attachment protein.

35. The method of claim 33 or 34, wherein the paramyxovirus envelope attachment protein is an envelope attachment protein from a Nipah virus, Hendra virus, or Measles virus, or is a variant or biologically active portion thereof.

36. The method of claim 33, wherein the paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein or HN protein or is a variant or biologically active portion of any of the foregoing.

37. The method of claim 36, wherein the paramyxovirus envelope attachment protein is a wild- type Nipah virus G (NiV-G) protein or is a variant or biologically active portion of a NiV-G.

38. The lipid particle of claim 36, wherein the paramyxovirus envelope attachment protein is a variant NiV-G that is a variant or a biologicallv active portion of a wild-type NiV-G.PCT / US24 / 11000 10 January 2024 (10.01.2024) _39. The method of claim 36, wherein the variant paramyxovirus envelope attachment protein comprises one or more mutations that reduces native tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising the one or more mutations.

40. The method of claim 33 or 34, wherein the one or more paramyxovirus envelope proteins or biologically active portions thereof comprises a paramyxovirus fusion protein (“F protein”) or a biologically active portion thereof.

41. The method of claim 40, wherein the one or more paramyxovirus fusion (F) proteins is an F protein from a henipavirus or is a biologically active portion thereof or variant thereof.

42. The method of claim 40 or 41, wherein the henipavirus is a Hendra vims.

43. The method of claim 40 or 41, wherein the henipavirus is a Nipah vims.

44. The method of any one of claims 40-43, wherein the paramyxovirus F protein is a wild-type NiV-F protein or a variant or a biologically active portion thereof.

45. The method of any one of claims 40-43, wherein the paramyxovirus F protein is a variant NiV-F that is a variant or a biologically active portion of a wild-type NiV-F protein.

46. The method of any one of claims 40-45, wherein the paramyxovirus F protein is an F0 precursor or is a proteolytically cleaved form thereof comprising Fl and F2 subunits.

47. The method of any one of claims 40-46, wherein the proteolytically cleaved form is a cathepsin L cleavage product.

48. The method of any one of claims 1-47, wherein the one or more fusogens comprise one or more chimeric proteins.PCT / US24 / 11000 10 January 2024 (10.01.2024) _49. The method of claim 48, wherein the one or more chimeric proteins comprise at least one chimeric protein that comprises a paramyxovirus envelope protein or biologically active portion thereof,50. The method of claim 49, wherein the paramyxovirus envelope protein is any one of paramyxovirus envelope protein of claims 36-39.

51. The method of claim 49, wherein the one or more chimeric proteins comprises a target binding domain.

52. The method of claim 51, wherein the targeting binding domain is an scFv.

53. The method of any one of claims 48-52, wherein the one or more chimeric proteins comprise (i) a paramyxovirus envelope protein or biologically active portion thereof and (ii) an scFv.

54. The method of claim 52 or 53, wherein the scFv targets an antigen present on the surface of a B cell, a T cell, a natural killer cell, a glial progenitor cell, a cardiac cell, a blood cell, a hepatocyte, a stem cell, or an induced pluripotent stem cell.

55. The method of claim 54, wherein the scFv targets an antigen present on the surface of a B cell.

56. The method of claim 54, wherein the scFV targets an antigen present on the surface of a T cell.

57. The method of claim 54 or 56, wherein the scFv targets CD8.

58. The method of claim 54 or 56, wherein the scFv targets CD4.PCT / US24 / 11000 10 January 2024 (10.01.2024) _59. The method of any one of claims 1-58, wherein the transfer plasmid comprises a viral nucleic acid.

60. The method of claim 59, wherein the viral nucleic acid comprises one or more of (e.g., all of) the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT)Zcentral termination sequence (CTS) (e.g. DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g. WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).

61. The method of any one of claims 1-60, wherein the transfer plasmid comprises a transgene.

62. The method of claim 61, wherein the transgene encodes a nuclease.

63. The method of claim 62, wherein the nuclease is a Cas, a TALEN, or a zinc-finger nuclease.

64. The method of any one of claims 1-63, wherein the transfer plasmid comprises a nucleotide sequence that encodes a gRNA.

65. The method of claim 61, wherein the transgene encodes an antibody or a biologically active portion thereof.

66. The method of claim 61, wherein the transgene encodes a chimeric antigen receptor.

67. The method of claim 66, wherein the chimeric antigen receptor comprises an extracellular ligand-binding domain having specificity for CD 19, CD20, CD22, and / or BCMA, a hinge domain, a transmembrane domain, a co- stimulatory domain, and an intracellular signaling domain.PCT / US24 / 11000 10 January 2024 (10.01.2024) _68. The method of claim 61, wherein the transgene encodes an antigen.

69. The method of claim 61, wherein the transgene encodes a therapeutic polypeptide.

70. The method of claim 69, wherein the therapeutic polypeptide is useful for protein replacement therapy.

71. The method of any one of claims 1-70, wherein the culturing comprises introducing into the producer cells the one or more nucleic acids for the production of the viral vector.

72. The method of any one of claims 1-70, wherein the one or more nucleic acid for the production of the viral vectors are stably integrated into the genome of one or more producer cells.

73. The method of claim 71, wherein introducing the one or more nucleic acids into the producer cells comprises transfecting the one or more nucleic acids into the producer cells.

74. The method of claim 71 or 73, wherein the one or more nucleic acids comprise a packaging plasmid, an envelope plasmid, and a transfer plasmid, wherein the envelope plasmid comprises at least one nucleic acid that encodes one or more fusogens.

75. The method of any one of claims 2-74, further comprising treating the producer cells with a nuclease.

76. The method of any one of claim 1-75, comprising treating the producer cells with a nuclease at a time that is at least 30 minutes after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.PCT / US24 / 11000 10 January 2024 (10.01.2024) _77. The method of any one of claims 1-76, further comprising treating the producer cells with a nuclease at a time that is no more than 6 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

78. The method of any one of claims 1-76, further comprising treating the producer cells with a nuclease at a time that is no more than 4 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

79. The method of any one of claims 1-76, further comprising treating the producer cells with a nuclease at a time that is no more than 2 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

80. The method of any one of claims 1-76, further comprising treating the producer cells with a nuclease at a time that is no more than 1 hour after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

81. The method of any one of claims 1-81, further comprising treating the producer cells with a second nuclease treatment.

82. The method of claim 81, wherein the second nuclease treatment is performed concurrently with (c) harvesting the producer cells or the producer cell culture.

83. The method of claim 81, wherein the second nuclease treatment is performed after harvesting the producer cells or the producer cell culture.

84. The method of claim 81, wherein treating the producer cells with a nuclease treatment at a time that is at least 30 minutes and no more than 6 hours after one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after one or more nucleic acids for the production of the viral vector are introduced into the producer cells.PCT / US24 / 11000 10 January 2024 (10.01.2024) _85. The method of claim 81, wherein treating the producer cells with a nuclease treatment at a time that is no more than 6 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

86. The method of claim 81, wherein treating the producer cells with a nuclease treatment at a time that is no more than 4 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

87. The method of claim 81, wherein treating the producer cells with a nuclease treatment at a time that is no more than 2 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells, and treating the producer cells with a second nuclease treatment at a time that is about 48 hours after the one or more nucleic acids for the production of the viral vector are introduced into the producer cells.

88. The method of any one of claims 1-87, wherein an amount of nuclease added to the producer cell culture medium is determined according to equations (eqn.) 1 and / or 2.

89. The method of any one of claims 81-87, wherein an amount of nuclease added to the harvested producer cells is determined according to equations 3 and / or 4.

90. The method of any one of claims 1-89, wherein the nuclease is an endonuclease capable of cleaving double- stranded and single- stranded nucleic acid.

91. The method of any one of claims 1-90, wherein the nuclease is a salt activated nuclease.PCT / US24 / 11000 10 January 2024 (10.01.2024) _92. The method of claim 91, wherein the salt activated nuclease is selected from: benzonase, RNase A, DNase I, micrococcal nuclease, and S7 nuclease.

93. The method of any one of claims 1-91, wherein the nuclease is MS AN.

94. The method of claim 93, wherein the producer cells are treated with about 100,000 to 500,000 units of MSAN.

95. The method of claim 94, wherein the producer cells are treated with about 1 Unit (U) of MSAN per 1 mL of cell culture medium to 100 U of MSAN per 1 mL of cell culture medium.

96. The method of claim 95, wherein the producer cells are treated with about 100U of MSAN per 1 mL of cell culture medium.

97. The method of claim 95, wherein the producer cells are treated with about 50U of MSAN per 1 mL of cell culture medium.

98. The method of claim 95, wherein the producer cells are treated with about 25U of MSAN per 1 mL of cell culture medium.

99. The method of claim 95, wherein the producer cells are treated with about 10U of MSAN per 1 mL of cell culture medium.

100. The method of claim 95, wherein the producer cells are treated with about 4U of MSAN per 1 mL of cell culture medium.

101. The method of claim 95, wherein the producer cells are treated with about 3U of MSAN per 1 mL of cell culture medium.PCT / US24 / 11000 10 January 2024 (10.01.2024) _102. The method of claim 95, wherein the producer cells are treated with about 2U of MSAN per 1 mL of cell culture medium.

103. The method of claim 95, wherein the producer cells are treated with about 1U of MSAN per 1 mL of cell culture medium.

104. The method of any one of claims 1-103, wherein the producer cells are cultured in a suspension culture.

105. The method of any one of claims 1-104, wherein the producer cells are cultured in serum free medium.

106. The method of any one of claims 1-105, further comprising agitating the producer cells.

107. The method of any one of claims 1-106, wherein the producer cells are cultured in medium that includes an anti-cell clumping agent.

108. The method of any one of claims 1-107, wherein the method comprising agitating the producer cells and culturing the cell producer cells in medium that includes an anti-clumping agent.

109. The method of any one of claims 1-108, wherein the producer cells are cultured in medium that includes a surfactant.

110. The method of any one of claims 1-109, wherein the producer cells are cultured in medium that includes a surfactant and an antidumping agent.

111. The method of any one of claims 1-110, wherein the method comprises agitating the producer cells and culturing the cell producer cells in medium that includes a surfactant.PCT / US24 / 11000 10 January 2024 (10.01.2024) _112. The method of any one of claims 1-111, wherein the method comprises agitating the producer cells and culturing the cell producer cells in medium that includes a surfactant and an anti-clumping agent.

113. The method of any one of claims 1-112, further comprising a settling step, wherein after harvesting the producer cells are allowed to settle.

114. The method of claim 113, wherein the settling is performed for about 4 hours to about 48 hours.

115. The method of claim 114, wherein the settling is performed for about 4 hours to about 16 hours.

116. The method of any one of claims 1-115, further comprising enriching the viral vectors from the producer cell culture.

117. The method of any one of claims 1-116, further comprising generating a crude drug substance from the producer cell culture, wherein the crude drug substance comprises the viral vectors produced by the producer cells or a subset thereof.

118. The method of claim 117, wherein generating a crude drug substance from the producer cell culture comprises enriching the viral vectors from the producer cell culture.

119. The method of claim 117 or 118, wherein generating a crude drug substance from the producer cell culture comprises removing producer cell DNA and / or producer cell protein from the producer cell culture.

120. The method of any one of claims 116-119, wherein the producer cell culture comprises the producer cells and / or culture medium.PCT / US24 / 11000 10 January 2024 (10.01.2024) _121. The method of any one of claims 117-120, wherein the crude drug substance is characterized by:(i) an at least 3-fold increase in viral vector titer as compared to a crude drug substance produced by an alternative process;(ii) an at least 1.5-fold increase in infectivity as compared to a crude drug substance produced by an alternative process;(iii) an at least 1000-fold decrease in the amount of producer cell DNA as compared to a crude drug substance produced by an alternative process;(iv) an at least 15-fold decrease in the amount of producer cell protein as compared to a crude drug substance produced by an alternative process;(v) an at least 1.5-fold increase in transduction efficiency in a target cell exposed to the crude drug substance as compared to a crude drug substance produced by an alternative process; or(vi) an at least 20-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process;(vii) an at least 3-fold increase in fusogen expression on the viral vectors as compared to a crude drug substance produced by an alternative process;(viii) or a combination thereof.

122. The method of any one of claims 117-121, further comprising generating a drug substance from the crude drug substance, wherein the drug substance comprises the viral vectors produced by the producer cells or a subset thereof.PCT / US24 / 11000 10 January 2024 (10.01.2024) _123. The method of claim 122, wherein generating a drug substance from the crude drug substance comprises further enriching the viral vectors from the crude drug substance.

124. The method of claim 122 or 123, wherein generating a drug substance from the crude drug substance comprises further removing producer cell DNA and / or producer cell protein from the crude drug substance.

125. The method of any one of claims 122-124, wherein generating a drug substance from the crude drug substance comprises adding a pharmaceutically acceptable excipient.

126. The method of any one of claims 122-125, wherein the drug substance is characterized by:(i) an at least 5-fold increase in viral vector titer as compared to a drug substance produced by an alternative process;(ii) an at least 5-fold increase in infectivity as compared to a drug substance produced by an alternative process;(iii) an at least 2000-fold decrease in the amount of producer cell DNA as compared to a drug substance produced by an alternative process;(iv) an at least 10-fold decrease in the amount of producer cell protein as compared to a drug substance produced by an alternative process;(v) an at least 2-fold increase in transduction efficiency in a target cell exposed to the drug substance as compared to a drug substance produced by an alternative process; or(vi) an at least 50-fold increase in functional viral vector titer as compared to a crude drug substance produced by an alternative process;PCT / US24 / 11000 10 January 2024 (10.01.2024) _(vii) an at least 6-fold increase in fusogen expression on the viral vectors as compared to a crude drug substance produced by an alternative process;(viii) or a combination thereof.

127. The method of any one of claims 121-126, wherein the alternative process is a comparable process wherein:(i) a ratio of an envelope plasmid to a packaging plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1;(ii) a ratio of an envelope plasmid to a transfer plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1;(iii) a ratio of a first envelope plasmid to a second envelope plasmid within one or more nucleic acids for the production of viral vectors is equal to or less than 1:1;(iv) an amount of an envelope plasmid that is introduced into producer cells is equal to or less than an amount of an packaging plasmid that is introduced into the producer cells;(v) an amount of an envelope plasmid that is introduced into producer cells is equal to or less than the amount of a transfer plasmid that is introduced into the producer cells;(vi) an amount of a first envelope plasmid that is introduced into producer cells is equal to or less than the amount of a second envelope plasmid that is introduced into the producer cells;(vii) producer cells are treated with a nuclease at a time that is more than 6 hours after at least one nucleic acid that encodes one or more fusogens is introduced into the producer cells;(viii) producer cells are not cultured in a suspension culture;PCT / US24 / 11000 10 January 2024 (10.01.2024)(ix) cell aggregation of producer cells is not reduced;(x) producer cells are not agitated during culture;(xi) culture medium for producer cells does not include an anti-clump agent or a surfactant; or(xii) a combination thereof.

128. The method of claim 127, wherein the first envelope plasmid encodes G protein.

129. A producer cell comprising one or more nucleic acids for the production of lentiviral vectors, wherein the one or more nucleic acids comprise a packaging plasmid and an envelope plasmid that encodes one or more fusogens, and wherein the ratio of the envelope plasmid to the packaging plasmid within the producer cell is greater than 1:1.

130. The producer cell of claim 129, wherein the ratio of the envelope plasmid to the packaging plasmid within the producer cell is greater than 2: 1.

131. A producer cell comprising one or more nucleic acids for the production of lentiviral vectors, wherein the one or more nucleic acids comprise a first envelope plasmid and a second envelope plasmid, wherein the first envelope plasmid and second envelope plasmid each encode one or more fusogens, and wherein the ratio of the first envelope plasmid to the second envelope plasmid within the producer cell is greater than 1:1.

132. The producer cell of claim 131, wherein the ratio of the first envelope plasmid to the second envelope plasmid within the producer cell is greater than 2:1.

133. The producer cell of any one of claims 129-132, wherein the first envelope plasmid encodes G protein.PCT / US24 / 11000 10 January 2024 (10.01.2024)134. A producer cell comprising engineered lentiviral vectors, wherein the producer cell had been transfected with one or more nucleic acids for the production of lentiviral vectors at a first time point and had been exposed to a nuclease at a second time point, wherein the second time point is at least 30 minutes after and at most 6 hours after the first time point.

135. A viral vector produced according to the methods of any one of claims 1-128.

136. A viral vector produced using the producer cells of any one of claims 129-134.

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