WO2023081831A2

WO2023081831A2 - Methods and compositions for immune tolerance to aav antigens and transgene products in gene therapy

Info

Publication number: WO2023081831A2
Application number: PCT/US2022/079310
Authority: WO
Inventors: Bernhard J. Hering; Thomas Henley; Sabarinathan RAMACHANDRAN
Original assignee: Diabetes-Free, Inc.; Regents Of The University Of Minnesota
Priority date: 2021-11-05
Filing date: 2022-11-04
Publication date: 2023-05-11
Also published as: WO2023081831A3

Abstract

Provided herein are methods and compositions for tolerizing a recipient to a viral gene therapy vector and/or a transgene product by reducing or eliminating undesired immune reactions to the gene therapy vector and/or transgene product in the recipient and/or inducing, activating and/or amplifying desired immune regulatory responses in the recipient. Such methods can also induce T cell exhaustion. Specific methods and compositions provided herein include compositions that comprise a leukocyte, a chemical crosslinking agent and a viral antigen or an antigenic fragment or variant thereof, wherein the viral antigen or antigenic fragment or variant thereof and/or the transgene product is conjugated or cross-linked to the leukocyte.

Description

METHODS AND COMPOSITIONS FOR IMMUNE TOLERANCE TO AAV ANTIGENS AND TRANSGENE PRODUCTS IN GENE THERAPY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 63/276,530 filed November 5, 2021 and U.S. Provisional Application No. 63/295,977, filed January 3, 2022, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE DISCLOSURE

[0002] Existing gene therapy technologies, including those employing adeno-associated viruses (AAV), have shown significant promise for treatment of genetic diseases. However, the use of AAV gene therapy vectors often elicit an immune response by the recipient. The safety, efficacy, and durability of the therapy are often limited by these immune responses. Therefore, there is a great unmet need for therapies that tolerize a subject to a gene therapy vector and/or a transgene product and prevent unwanted immune reactions.

SUMMARY OF THE DISCLOSURE

[0003] Provided herein are methods and compositions that reduce, alleviate, or eliminate undesired immune responses to a gene therapy in a subject. In some embodiments, the gene therapy is an adeno-associated virus (AAV) gene therapy. In some embodiments, the method comprises administering to a subject a tolerizing regimen, wherein the totalizing regimen comprises: a tolerizing composition comprising: a population of leukocytes, a crosslinking agent, and a viral antigen from the gene therapy viral vector or antigenic fragment or variant thereof, wherein the viral antigen or antigenic fragment or variant thereof is conjugated or cross-linked to the population of leukocytes. In some embodiments, the totalizing regimen tolerizes the subject to a transgene product (e.g., a protein or an RNA) encoded by the gene therapy vector. In some embodiments, the tolerizing composition is crosslinked with the transgene product. The tolerizing composition can be administered to a subject as part of a totalizing regimen that also includes one or more agents that induce transient immunosuppression in the subject, e.g., CD40/CD40L inhibitors, mTOR inhibitors, pro-inflammatory cytokine inhibitors, etc. In some embodiments, provided herein are totalizing regimens designed to reduce or eliminate both innate and adaptive immune responses from the subject to the gene therapy, thereby resulting in a reduction or elimination of adverse immune reaction and associated symptoms, for instance, hepatotoxicity, in the subject as compared to the gene therapy in the absence of a tolerizing regimen.

[0004] Provided herein are compositions, wherein the compositions comprise: a leukocyte; a crosslinking agent; and a viral antigen, an antigenic fragment or a variant thereof. In some embodiments, the viral antigen, antigenic fragment or variant thereof is conjugated or cross-linked to the leukocyte. In some embodiments, the viral antigen, the antigenic fragment or the variant thereof is selected from the group consisting of: a capsid protein or an antigenic fragment of the capsid protein; an envelope protein or an antigenic fragment of the envelope protein; or a viral capsid protein. In some embodiments, the viral antigen, antigenic fragment, or variant thereof is from a recombinant viral vector. In some embodiments, the recombinant viral vector is a viral vector for use in a gene therapy. In some embodiments, the recombinant viral vector is selected from the group consisting of: a recombinant herpes simplex virus (HSV) vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picomavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector. In some embodiments, the viral antigen comprises an empty capsid or a nucleocapsid.

[0005] Further provided herein is a viral vector comprising a transgene (e.g., a transgene used in a gene therapy). In some embodiments, a composition provided herein further comprises a transgene product or fragment thereof crosslinked in conjugation with the leukocyte and viral antigen or antigenic fragment or variant thereof in the presence of the crosslinking agent. In some embodiments, the transgene product comprises a nucleic acid, a protein, or a fragment of a protein. In some embodiments, the crosslinking agent comprises a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof. The diimidoester can comprise cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof. In some embodiments, the crosslinking agent comprises a carbodiimide that comprises l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (ECDI); N,N'-diisopropylcarbodiimide (DIC); N,N'- dicyclohexylcarbodiimide (DCC); or a combination thereof.

[0006] Provided herein are compositions, wherein the compositions comprise: a population of leukocytes that are rendered apoptotic by crosslinking with a crosslinking agent provided herein; a viral antigen, an antigenic fragment, or a variant thereof; and optionally, a protein or fragment thereof. In some embodiments, the protein is a transgene product that is encoded by a viral vector. In some embodiments, the transgene product is for use in a gene therapy. In some embodiments, the transgene product is a transgene product listed in Table 2. In some embodiments, the population of leukocytes comprise a pre-apoptotic leukocyte, an apoptotic leukocyte, a late apoptotic leukocyte, or. In some embodiments, the population of leukocytes comprise mammalian leukocytes. In some embodiments, the population of leukocytes comprise human leukocytes. In some embodiments, the population of leukocytes comprise a cadaveric leukocyte (e.g., derived from a subject that is deceased or not living). In some embodiments, the cadaveric leukocytes comprises a leukocyte from a non-heart beating donor or a brain dead donor. In some embodiments, the population of leukocytes comprise a stem cell-derived leukocyte. In some embodiments, the population of leukocytes are derived from a living donor. In some embodiments, the living donor is a subject in need of a gene therapy or a subject that is a recipient of a gene therapy comprising a viral vector provided herein.

[0007] Further provided herein are compositions for use in tolerizing a living donor to a viral antigen. In some embodiments, the use comprises administering to a living donor a composition comprising crosslinked leukocytes as provided herein. In some embodiments, the leukocytes for use in the compositions provided herein are obtained by ex vivo differentiation of a stem cell, pluripotent cell, or induced pluripotent stem cell. In some embodiments, the leukocyte is isolated from a spleen, peripheral blood, a lymph node, a secondary lymphoid organ e.g., tonsils, mucous membrane layers, etc.), a tissue, or bone marrow. In some embodiments, the leukocytes for use in the compositions provided herein are isolated from a living donor or a cadaveric donor that is MHC class II matched to the subject that is the recipient of the compositions.

[0008] In some embodiments, leukocytes provided herein comprise an ex vivo expanded leukocyte. In some embodiments, the leukocytes provided herein comprise a B-lymphocyte. In some embodiments, the leukocytes are fixed with a crosslinking agent for a pre-determined amount of time. In some embodiments, the pre-determined amount of time is at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes or 240 minutes. In some embodiments, the pre-determined amount of time is at least about 10 minutes up to 6 hours. The compositions provided herein are used to tolerize a recipient. In some embodiments, the leukocytes provided herein comprise an MHC class II molecule that is matched with that of the recipient, wherein the use is administering the composition to the recipient. In some embodiments, the leukocytes comprise an MHC class II molecule or one or more peptides derived from the MHC class II molecule, wherein the MHC class II molecule or the one or more peptides derived from the MHC class II molecule is conjugated with the leukocyte. In some embodiments, the composition can be used in tolerizing a recipient, wherein the MHC class II molecule is matched with that of the recipient. In some embodiments, said use comprises administering the composition to the recipient. In some embodiments, the MHC class II molecule comprises HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, the MHC class II molecule HLA-DP comprises a HLA-DPA (a chain), or a HLA-DPB (P chain). In some embodiments, the MHC class II molecule HLA-DQ comprise HLA-DQA, or HLA-DQB. In some embodiments, the MHC class II molecule HLA-DR comprises HLA-DRA, or HLA-DRB. In some embodiments, the MHC class II molecule HLA-DRB comprises HLA-DR1, HLA-DR2, HLA- DR3, HLA-DR4, HLA-DR5 or a combination thereof. In some embodiments, the MHC class II molecule is encoded by HLA-DRBl*01, HLA-DRBl*03, HLA-DRB1*O4, HLA-DRB1*O7 HLA-DRB1*11, HLA-DRB1*15, or HLA-DRB 1*16 allele of the recipient. In some embodiments, the one or more peptides derived from the MHC class II molecule comprise a sequence from a hypervariable region or constant region of the MHC class II molecule. In some embodiments, the one or more peptides derived from the MHC class II molecule comprises at least 10 to 30 amino acid residues in length. In some embodiments, the one or more peptides derived from the MHC class II molecule are synthesized or recombinant. In some embodiments, the one or more peptides derived from the HLA class II molecule can exhibit a high affinity for binding to the peptide binding groove of a recipient’s MHC class II molecule binding groove.

[0009] Further provided herein are tolerizing compositions, wherein the tolerizing compositions comprise: a plurality of leukocytes conjugated by use of a crosslinking agent to a viral antigen, an antigen fragment thereof, or variant thereof from a viral vector designed for use in gene therapy. In some embodiments, the viral vector is an adenovirus associated virus (AAV). In some embodiments, the viral antigen, the antigen fragment or can comprise an adenovirus associated virus (AAV) antigen. In some embodiments, the AAV antigen is from a recombinant adenovirus associated virus (AAV) vector that has an AAV serotype selected from the group consisting of: AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i, AAV-DJ or any combination thereof. In some embodiments, the capsid protein comprises an AAV VP1, VP2, or VP3 capsid protein. In some embodiments, the VP1, VP2 or VP3 capsid protein comprise an amino acid sequence that is at least 60% identical to the corresponding capsid protein of an AAV serotype selected from the group consisting of: AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ. In some embodiments, the AAV is an AAV9. In some embodiments, the recombinant viral vector can comprise a recombinant herpes simplex virus vector which comprises a recombinant herpes simplex virus 1 (HSV1) vector, or a recombinant herpes simplex virus 2 (HSV2) vector, a recombinant retrovirus vector which comprises a recombinant Moloney murine sarcoma virus (MMSV) vector, or a recombinant murine stem cell virus (MSCV) vector, a recombinant lentivirus vector which comprises a recombinant human immunodeficiency virus 1 (HIV-1) vector or a recombinant human immunodeficiency virus 2 (HIV-2) vector, a recombinant alphavirus vector which comprises a recombinant Semliki forest virus (SFV) vector, Sindbis virus (SIN) vector, a recombinant Venezuelan equine encephalitis virus (VEE) vector, or a recombinant alphavirus Ml, a recombinant flavivirus vector which comprises a recombinant Kunjin virus vector, a recombinant West Nile virus vector, or a recombinant Dengue virus vector, a recombinant rhabdovirus vector which comprises a recombinant Rabies virus vector, or a recombinant vesicular stomatitis virus vector, a recombinant measles virus vector which comprises a recombinant MV Edmonston strain (MV-Edm) vector, a recombinant poxvirus vector which comprises a recombinant vaccinia virus (VV) vector, or a recombinant picornavirus vector which comprises a recombinant Coxsackievirus vector.

[0010] Further provided herein are compositions, wherein the compositions comprise: (a) a nanoparticle; (b) an MHC class II molecule or one or more peptides derived from the MHC class II molecule; and (c) a viral antigen, wherein the viral antigen is selected from the group consisting of a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a capsid, wherein (b) and (c) are encapsulated in, conjugated to, or crosslinked to the nanoparticle. In some embodiments, the viral antigen is derived from a recombinant viral vector. In some embodiments, the compositions further comprise a transgene product or fragment thereof. In some embodiments, the recombinant viral vector is selected from the group consisting of a recombinant herpes simplex virus (HSV) vector, recombinant alphavirus vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picornavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector. The capsid can comprise an empty capsid or a nucleocapsid. In some embodiments, the nanoparticle comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises one or more cationic lipids. In some embodiments, the recombinant viral vector comprises a transgene. In some embodiments, the lipid nanoparticle comprises one or more noncationic lipids. In some embodiments, the lipid nanoparticle comprises one or more PEG modified lipids. In some embodiments, the lipid nanoparticle comprises C12- 200, DLin-KC2-DMA, CHOL, DMGPEG2K, DOPE, and DMG-PEG-2000. In some embodiments, the lipid nanoparticle comprises a cleavable lipid. In some embodiments, the nanoparticle can comprise a polymer nanoparticle. In some embodiments, the polymer nanoparticle comprises a polymer that is biodegradable. In some embodiments, the nanoparticle can comprise a solid-lipid nanoparticle. In some embodiments, the nanoparticle comprises a micelle. In some embodiments, the micelle comprises a polymer, which can be an amphiphilic polymer. In some embodiments, the micelle comprises a water soluble micelle. In some embodiments, the micelle can coat a solid core. In some embodiments, the core comprises a traceable inorganic material selected from the group consisting of: iron oxide, CdSe/CdS/ZnS, silver and gold. In some embodiments, the diameter of the core is about 5 nanometers up to 30 nm. In some embodiments, the nanoparticle is negatively charged. In some embodiments, the nanoparticle comprises a zeta potential from about -100 mV to about 0 mV or about -60 mV to about -40 mV. In some embodiments, the nanoparticle surface comprises a functionalized surface group which can comprise hydroxyl group, amine group, a thiol group, an alcohol group, or a carboxylic acid group. The polymer comprises a synthetic polymer selected from group consisting of poly(maleic anhydride-alt-l-octa-decene), poly(maleic anhydride-alt-1 -tetradecene), and polyisoprene-block poly-ethylene-oxide block copolymer, polylactide-polyglycolide copolymers, polyacrylates, polycaprolactones, poly(D,L -lactide), polycyanoacrylate and poly(lactic-co- glycolic acid) (PLGA) or poly(lactic acid), and poly(ethyl methacrylate) (PEMA). The polymer comprises PLGA modified with PEMA as a surfactant. The polymer comprises a natural polymer selected from a group consisting of albumin, gelatin, alginate, collagen, chitosan, and dextran. In some embodiments, the nanoparticle is formulated for targeting to a splenic marginal zone antigen presenting cell or a non-splenic marginal zone macrophage, dendritic cell, or antigen presenting cell in vitro or in vivo. In some embodiments, the nanoparticle comprises a diameter in the range of about 10-1000 nm, 20-900 nm, or 500 nm. In some embodiments, the nanoparticle is coated with polyethylene glycol. In some embodiments, the composition is for use in tolerizing a recipient, wherein the MHC class II molecule is matched with that of said recipient. In some embodiments, said tolerizing comprises administering said composition to said recipient. The MHC class II molecule comprises HLA-DP, HLA-DQ, or HLA-DR. HLA-DP comprises HLA- DPA (a chain), or HLA-DPB (P chain). HLA-DQ can comprise HLA-DQ A, or HLA-DQB. HLA- DR can comprise HLA-DRA, or HLA-DRB. HLA-DR can comprise HLA-DRB, and wherein said HLA-DRB is selected from HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, or HLA-DR5. In some embodiments, the composition is for use in tolerizing a recipient, wherein the MHC class II molecule is encoded by HLA-DRB 1*03 or HLA-DRB 1*04 allele of said recipient, and wherein said tolerizing comprises administering the composition to said recipient. In some embodiments, the one or more peptides derived from the MHC class II molecule comprises a sequence from a hypervariable region of the MHC class II molecule. In some embodiments, the one or more peptides derived from the MHC class II molecule is at least about 10 up to 30 amino acid residues in length. In some embodiments, the one or more peptides derived from the MHC class II molecule are synthetic or recombinant. In some embodiments, the one or more peptides derived from the MHC class II molecule are synthesized or recombinant. In some embodiments, the one or more peptides derived from the HLA class II molecule can exhibit a high affinity for binding to the peptide binding groove of a recipient’s MHC class II molecule binding groove. In some embodiments, the recombinant viral vector comprises the recombinant adenovirus associated virus (AAV) vector. In some embodiments, the recombinant adenovirus associated virus (AAV) vector comprises an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, - 8, -9, -10, -11, -rh74, -rhlO, AAV-2i, chimera or combinations thereof. In some embodiments, the capsid protein comprises a VP1, VP2, or VP3 capsid protein. In some embodiments, the VP1 capsid protein comprises an amino acid sequence that is at least 60% identical to that of an AAV serotype selected from the group consisting of: AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. In some embodiments, the VP2 capsid protein comprises an amino acid sequence that is at least 60% identical to that of the AAV serotype selected from the group consisting of: AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. In some embodiments, the VP3 capsid protein comprises an amino acid sequence that is at least 60% identical to that of the AAV serotype selected from the group consisting of: AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. In some embodiments, the empty capsid or the nucleocapsid comprises that of an AAV serotype selected from the group consisting of: AAV-1, - 2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. In some embodiments, the recombinant adenovirus vector comprises an adenovirus serotype 5 (Ad5) vector. In some embodiments, the recombinant herpes simplex virus vector comprises a recombinant herpes simplex virus 1 (HSV1) vector, or a recombinant herpes simplex virus 2 (HSV2) vector. In some embodiments, the recombinant retrovirus vector comprises a recombinant Moloney murine sarcoma virus (MMSV) vector, or a recombinant murine stem cell virus (MSCV) vector. In some embodiments, the recombinant lentivirus vector comprises a recombinant human immunodeficiency virus 1 (HIV-1) vector or a recombinant human immunodeficiency virus 2 (HIV-2) vector. In some embodiments, the recombinant alphavirus vector comprises a recombinant Semliki forest virus (SFV) vector, Sindbis virus (SIN) vector, a recombinant Venezuelan equine encephalitis virus (VEE) vector, or a recombinant alphavirus Ml. In some embodiments, the recombinant flavivirus vector comprises a recombinant Kunjin virus vector, a recombinant West Nile virus vector, or a recombinant Dengue virus vector. In some embodiments, the recombinant rhabdovirus vector comprises a recombinant Rabies virus vector, or a recombinant vesicular stomatitis virus vector. In some embodiments, the recombinant measles virus vector can comprise a recombinant MV Edmonston strain (MV-Edm) vector. The recombinant poxvirus vector comprises a recombinant vaccinia virus (VV) vector. The recombinant picornavirus vector comprises is a recombinant Coxsackievirus vector. The recombinant adenovirus vector comprises an AAV chimera. In some embodiments, the recombinant adenovirus vector comprises the AAV chimera AAV-DJ. In some embodiments, the nanoparticle comprises a peptide tag, detecting agent, a therapeutic agent, a one or more immunomodulatory agents or a combination thereof encapsulated in, or conjugated with the nanoparticle. In some embodiments, the composition comprises an immunomodulatory agent, wherein said immunomodulatory agent is an anti-CD40 agent, anti-CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor, IFNy, an NFKB inhibitor, vitamin D3, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, alpha 1 antitrypsin inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), or any combination thereof. In some embodiments, the one or more immunomodulatory agents blocks CD40:CD40-L costimulation. In some embodiments, the NF-kB inhibitor comprises curcumin, triptolide, Bay- 117085, or a combination thereof. In some embodiments, the anti-CD40 agent comprises CD40 siRNA. In some embodiments, the anti-CD40 agent comprises a CD40 binding peptide inhibitor, anti-CD40 monoclonal antibody, a Fab’ anti-CD40 monoclonal antibody fragment, FcR- engineered, Fc silent anti-CD40 monoclonal domain antibody. In some embodiments, the anti CD40-L agent comprises an anti-CD40-L monoclonal antibody, a Fab’ anti-CD40-L monoclonal antibody fragment CDP7657, a FcR-engineered, Fc silent anti-CD40-L monoclonal domain antibody, a Fab’ anti-CD40-L antibody, anti-CD40 siRNA, CD40-L-binding fusion protein, CD40 binding peptides or an Fc-engineered anti-CD40-L antibody.

[0011] In some embodiments, a tolerogenic composition comprises: (a) the composition comprising a leukocyte, a crosslinking agent, and a viral antigen or antigenic fragment or variant thereof, wherein the viral antigen or antigenic fragment or variant thereof is conjugated or crosslinked to the leukocyte and/or (b) the composition comprising (i) a nanoparticle, (ii) an MHC class II molecule, or one or more peptides derived from the MHC class II molecule, and (iii) a viral antigen, wherein the viral antigen is selected from the group consisting of a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a capsid, wherein (ii) and (iii) are encapsulated in, conjugated, or crosslinked to the nanoparticle, wherein the tolerogenic composition is capable of modulating an immune response to the recombinant viral vector in a recipient that is administered said composition. The leukocyte can express a MHC class II molecule that is matched with that of the recipient. In some embodiments, the leukocyte comprises an MHC molecule or one or more peptides derived from the MHC molecule that is matched with that of the recipient. In some embodiments, the leukocyte comprises the MHC molecule in combination with one or more peptides derived from the MHC molecule that is matched with that of the recipient. In some embodiments, the tolerogenic composition comprises a leukocyte derived from the recipient. In some embodiments, the nanoparticle comprises a MHC class II molecule that is matched with that of the recipient. In some embodiments, the MHC class II molecule is from a donor with an MHC class II or an MHC class I molecule. In some embodiments, the MHC Class II or the MHC class I molecule includes a peptide sequence that binds to the peptide binding groove of the recipient MHC class II molecule. In some embodiments, a transgene provided herein can be crosslinked to the surface of the nanoparticle. In some embodiments, the recombinant viral vector comprises a transgene. In some embodiments, the transgene encodes a nucleic acid or a polypeptide. In some embodiments, modulating immune responses comprises inhibiting an immune response to the viral antigen. In some embodiments, inhibiting an immune response comprises one or more of: inhibiting a B- cell response, inhibiting a T cell response, inhibiting B-cell activation, inhibiting T-cell proliferation, inhibiting T cell migration, inhibiting B-cell proliferation, inhibiting B-cell migration, inhibiting macrophage activation, inhibiting production of one or more cytokines, inhibiting production of antibodies specific for the viral antigen or a combination thereof.

[0012] Provided herein are methods of modulating an immune response. In some embodiments, modulating an immune response comprises in vivo generation, expansion and/or activation of Treg cells, CD4+ Tregs, CD8+ Tregs, CD4+ Tri cells, CD8+ Natural Suppressor cells, Breg cells, BIO cells, myeloid derived suppressor cells or other immune regulatory subsets in the recipient. In some embodiments, modulating an immune response comprises contraction or exhaustion of CD4+ and/or CD8+ T cells specific to said viral antigen in said recipient as compared to corresponding amounts of said CD4+ and/or CD8+ T cells absent administration of the tolerogenic composition. In some embodiments, modulating an immune response comprises modulating the retention of T cells to a secondary lymphoid organ or tissue. In some embodiments, modulating an immune response comprises modulating the retention of B cells to a secondary lymphoid organ or tissue. In some embodiments, modulating an immune response comprises modulating T cell activation, or T cell exhaustion.

[0013] Provided herein is a method for inducing tolerance to a recombinant viral vector comprising a transgene in a subject, the method comprising: administering to a subject the tolerogenic composition provided herein, wherein the tolerogenic composition comprises a leukocyte crosslinked to a recombinant viral vector, a viral antigen, fragment, or variant thereof in an amount effective to induce tolerance to the recombinant viral vector comprising a transgene. In some embodiments, the method further comprises administering to said recipient an immunomodulatory agent to induce transient immunosuppression in the subject, wherein said immunomodulatory agent comprises an anti-CD40 agent, anti-CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor for instance compstatin or a derivative thereof, IFNy, an NFKB inhibitor, vitamin D3, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, an alpha 1 anti-trypsin inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), or any combination thereof.

[0014] In some embodiments, the administration of a composition provided herein is local or systemic. In some embodiments, the administration is intravenous administration, intracardiac injection, ocular injection, intravitreal injection, otic administration (e.g., drops to the ear), muscular injection, or via a drug delivery device.

[0015] Further provided herein is a method of tolerizing a recipient of an AAV vector to said AAV vector, the method comprising: administering to said recipient a tolerizing regimen that comprises a population of leukocytes from said recipient or a population of leukocytes differentiated in vitro from stem cells extracted from said recipient, wherein the population of leukocytes have been contacted with a crosslinking agent, and an AAV viral antigen or an antigenic fragment or variant thereof, wherein the AAV viral antigen or antigenic fragment or variant thereof shares a serotype with said AAV vector, and wherein said AAV viral antigen is conjugated or cross-linked with the leukocyte. In some embodiments, the method comprises administering to the recipient an agent that blocks the binding of CD40 and CD40L. In some embodiments, the method comprises administering to the recipient an mTOR inhibitor and/or an inhibitor of at least one pro- inflammatory cytokine. The inhibitor of at least one pro-inflammatory cytokine can comprise a TNF-alpha inhibitor and/or an IL-6 inhibitor. In some embodiments, the tolerogenic regimen is administered intravenously or via local injection on days -7 and +1 relative to a first administration of said AAV vector. In some embodiments, the transgene encodes an autoantigen.

[0016] In some embodiments, the administering is performed prior to, simultaneously and/or subsequent to administering the recombinant viral vector to the recipient. Administering the composition can inhibit a B- cell response, a T cell response, macrophage activation, cytokine production, or a combination thereof in said recipient, thereby inducing tolerance. The B cell response can comprise at least one of B- cell activation, B-cell proliferation, and production of neutralizing antibodies specific for the viral antigen. The T cell response can comprise at least one of T cell activation, T cell proliferation, generation of memory T cells, and generation of T cell effector function involving cytokines or cytolytic mechanisms. The administering can induce in vivo generation, expansion and/or activation of Treg cells, CD4+ Tregs, CD8+ Tregs, CD4+ Tri cells, CD8+ Natural Suppressor cells, Breg cells, B10 cells, myeloid derived suppressor cells or other immune regulatory subsets in the recipient, thereby inducing tolerance. Further provided herein is a method of modulating an immune response in a subject to a transduced cell, wherein the method comprises: administering to the subject the tolerogenic composition provided herein in an amount effective to modulate the immune response to the transduced cell in the subject. In some embodiments, the transduced cell is generated by contacting a cell with the recombinant viral vector. Further provided herein is a method for sustained expression of a transgene in a subject, wherein the method comprise: administering to the subject the tolerogenic composition provided herein prior to, simultaneously, and/or subsequent to administering a recombinant viral vector comprising the transgene to the subject. In some embodiments, the administering of a composition provided herein induces contraction of T cell clones with specificity for viral antigens. In some embodiments, the administering of a composition provided herein induces exhaustion of T cells with specificity for viral antigens, including memory T cells and de novo primed T cells.

[0017] Further provided herein is a composition for tolerizing a subject to a viral gene therapy vector and an associated transgene that encodes for a transgene product. In some embodiments, the composition comprises an autologous leukocyte crosslinked to (i) a component of the viral gene therapy vector, and (ii) the transgene product or a fragment or derivative thereof. In some embodiments, the viral gene therapy vector comprises an AAV. In some embodiments, the transgene product comprises a nucleic acid. In some embodiments, the nucleic acid comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In some embodiments, the transgene product comprises a protein or polypeptide fragment or derivative thereof. In some embodiments, the AAV comprises a capsid. In some embodiments, the capsid is empty. In some embodiments, the AAV comprises at least one serotype. In some embodiments, the AAV is an AAV9. In some embodiments, the transgene product or fragment thereof comprises microdystrophin, RPE65, Human FVIII, Cas9, or a similar transgene product associated with a respective gene therapy (e.g., a therapy for muscular dystrophy).

[0018] Provided herein are methods of producing a tolerizing composition, wherein the methods comprise: contacting a leukocyte with a crosslinking agent; and a viral antigen, an antigenic fragment, or variant thereof, wherein the viral antigen, the antigenic fragment, or variant thereof is conjugated or cross-linked to the leukocyte via the crosslinking agent, thereby producing a tolerizing composition.

[0019] Provided herein are tolerizing compositions produced by the methods provided herein.

[0020] Provided herein are methods of producing a gene therapy tolerization composition, wherein the methods comprise: contacting a population of leukocytes for at least about 10 minutes up to 6 hours with: (a) a crosslinking agent; (b) a viral antigen, an antigenic fragment, or variant thereof, wherein the viral antigen, an antigenic fragment, or variant thereof is conjugated or cross-linked to a leukocyte; and (c) a bioactive agent, wherein the bioactive agent is conjugated or cross-linked to the leukocyte, thereby producing a gene therapy tolerization composition. [0021] Provided herein are gene therapy tolerization compositions made by the methods provided herein. Further provided herein are gene therapy compositions that comprise the gene therapy tolerization composition provided herein; and a viral vector. Further provided herein are gene therapy compositions, wherein the viral vector is an adenovirus associated viral (AAV) vector, a recombinant AAV (rAAV), a lentiviral vector, a retroviral vector, or an alphaviral vector. Further provided herein are gene therapy compositions, wherein the gene therapy compositions, further comprise a transgene.

[0022] Provided herein are methods of treating a disease or condition in a subject, wherein the methods comprise: (a) contacting a population of leukocytes with a crosslinking agent; and a plurality of viral antigens, antigenic fragments, or variants thereof, wherein at least one viral antigen, antigenic fragment, or variant thereof is conjugated or cross-linked to a leukocyte within the population of leukocytes via the crosslinking agent, thereby producing a tolerizing composition; (b) administering to a subject the tolerizing composition; and (c) administering a gene therapy composition to the subject, thereby treating the disease or the condition.

[0023] Provided herein are methods of tolerizing a population of immune cells to a gene therapy composition, wherein the methods comprise: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: (a) viral vector capsid, a fragment, or a variant thereof; and (b) a protein or a fragment thereof, wherein (a) and (b) are conjugated or crosslinked to the modified leukocyte via a crosslinking agent, thereby tolerizing the population of immune cells to the gene therapy composition. Further provided herein are methods, wherein the method further comprises contacting the population of immune cells with a gene therapy composition. Further provided herein are methods, wherein the gene therapy composition comprises a viral vector; and a transgene encoding the protein in (b). Further provided herein are methods, wherein the gene therapy composition comprises a viral vector comprising the viral vector capsid, the fragment, or the variant thereof in (a). Further provided herein are methods, wherein the contacting is in vitro, in vivo, or ex vivo.

[0024] Provided herein are methods of tolerizing a population of immune cells to an adeno- associated virus (AAV) vector and a spinal motor neuron 1 protein, wherein the methods comprise: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: (a) an AAV capsid, a fragment, or a variant thereof; and (b) a spinal motor neuron 1 (SMN1) protein or a fragment thereof, wherein (a) and (b) are conjugated or crosslinked to the modified leukocyte via a crosslinking agent, thereby tolerizing the population of immune cells to the AAV vector and the SMN1 protein. Further provided herein are methods, wherein the contacting is in vitro, in vivo, or ex vivo. [0025] Provided herein are methods of tolerizing a subject to a gene therapy composition, wherein the methods comprise: (a) administering to a subject a population of modified leukocytes, wherein the population of modified leukocytes comprise: (i) a viral vector capsid, a fragment, or variant thereof; and (ii) a protein or a fragment thereof, wherein (i) and (ii) are conjugated or crosslinked to a modified leukocyte via a crosslinking agent, (b) administering to a subject a gene therapy composition, wherein the gene therapy composition comprises a viral vector and a transgene encoding the protein crosslinked or conjugated to the modified leukocyte, wherein the administering of population of modified leukocytes tolerizes the subject to the gene therapy composition.

[0026] Provided herein are methods of increasing Type 1 regulatory T (Tri) cell proliferation, wherein the methods comprise: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: (a) a viral antigen, antigenic fragment, or variant thereof; or (b) a viral vector, wherein (a) or (b) is conjugated or crosslinked to the modified leukocyte, thereby increasing Tri cell proliferation within the population of immune cells relative to a comparable population of immune cells that have not been contacted with the modified leukocyte.

[0027] Provided herein are methods of increasing the level of CD33 and the level of PD-L1 in a monocyte, wherein the methods comprise: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: (a) a viral antigen, antigenic fragment, or variant thereof; or (b) a viral vector, wherein (a) or (b) is conjugated or crosslinked to the modified leukocyte, and wherein the population of immune cells comprise a population of monocytes, thereby increasing the level of CD33 and the level of PD-L1 in a monocyte relative to a comparable population of immune cells that have not been contacted with the modified leukocyte.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [0029] FIG. 1 provides an exemplary treatment regimen comprising administering to a recipient a tolerizing composition of adeno-associated virus (AAV) Capsid Antigen-and Transgene Product-Conjugated Apoptotic Autologous Leukocytes provided herein to tolerize the subject to an AAV gene therapy vector and transgene product and reduce an adverse immune reaction to said gene therapy vector and transgene product as compared to the adverse immune reaction absent administration of the tolerizing regimen. In the embodiment illustrated in FIG. 1, B cells derived from the recipient (or differentiated in vitro from stem cells derived from the recipient) are expanded ex vivo, and conjugated to an AAV capsid protein and the transgene product by use of a crosslinking agent, (e.g. ECDI). The composition of cross-linked cells is administered intravenously or via local injection to the subject on days -7 and +1, in relation to a first administration on day 0 of an AAV gene therapy vector of the same serotype as the AAV capsid protein conjugated to the B cells. In addition to the tolerizing composition of cross-linked B cells, the subjectis also administered an agent that suppresses CD40: CD40L co-stimulation, for instance antagonistic agents (e.g., antagonistic a-CD40 mAb), an mTOR inhibitor, and an agent that suppresses inflammatory cytokines (e.g., sTNFR), an anti-IL6 agent, for instance an anti-IL-6 mAb (e.g., anti-IL-6R mAb) administered between days -8 and +21.

[0030] FIG. 2 provides an exemplary treatment regimen comprising administering to a recipient a tolerizing composition of AAVrh74 capsid- and microdystrophin-conjugated apoptotic autologous leukocytes as provided herein to tolerize the subject to an AAV gene therapy comprising AAVrh74 and a microdystrophin transgene so as to reduce an adverse immune reaction to said gene therapy vector and microdystrophin as compared to the adverse immune reaction absent administration of the tolerizing composition. In the embodiment illustrated in FIG. 2, B cells derived from the recipient (or differentiated in vitro from stem cells derived from the recipient) are expanded ex vivo, and conjugated to AAVrh74 capsid proteins and microdystrophin by use of a crosslinking agent (e.g., ECDI). The composition of cells is administered intravenously or via local injection to the recipient on days -7 and +1, in relation to a first administration on day 0 of an AAVrh74 gene therapy vector. The administration at day -7 marks the beginning of week -1. In addition to the tolerizing composition of cross-linked B cells, the recipient is also administered an a-CD40 mAb (e.g., DFI105), an mTOR inhibitor (e.g. , Rapamune®), an anti-TNF agent (e.g., Enbrel®), and an anti-IL6 agent (e.g., Actemra®) administered through week 3. A second dose of AAVrh74 gene therapy vector is administered at week 8. Each dose of AAVrh74 gene therapy vector is 2 x 10¹² vector genomes per kilogram of body weight (vg/kg). The recipient is monitored throughout the 8 weeks following the first vector dose and the 8 weeks following the second vector dose.

[0031] FIG. 3 provides an exemplary process of forming AAV- and microdystrophin- conjugated leukocytes, and the uptake of those apoptotic bodies by a host dendritic cell as provided herein. In the embodiment illustrated in FIG. 3, ECDI crosslinking couples full length AAV capsids and full length recombinant microdystrophin to the surface of leukocytes, where the AAV capsid is AAVrh74, AAV9, or AAV2 composed of VP1, VP2, and VP3 proteins. These crosslinked leukocytes become circulating apoptotic bodies when injected into a recipient (intravenously (IV) in the illustrated embodiment). Dendritic cells of the recipient (host) uptake the circulating apoptotic bodies, and maturation of the dendritic cells is arrested after the uptake of apoptotic bodies and treatment of the recipient with anti-a-CD40 mAb (e.g., DFI105) and Rapamycin, thereby inducing tolerance to the AAV and microdystrophin proteins. The anti-a- CD40 mAb blocks activation of the dendritic cells by altering the CD40 receptor.

[0032] FIG. 4 illustrates current rationale for mechanisms that are potentially operative in the induction of tolerance to AAV capsid and transgene product using methods and compositions provided herein. The term TIS refers to transient immunosuppression induced in the subject by providing immunomodulatory agents provided herein. CP stands for classical pathway of complement activation. C3 stands for complement component 3.

[0033] FIG. 5 shows a schematic of immune mechanisms understood to be involved in the induction of tolerance via the administration of tolerizing compositions provided herein under the cover of antagonistic anti-CD40 mAbs.

[0034] FIGS. 6A-6C show graphs of flow cytometry gating for leukocytes treated with or without a crosslinking agent, AAV9, and/or recombinant survival motor neuron 1 (SMN1) protein. FIG. 6A shows cell surface conjugation of AAV protein expression for human B cells that were mixed with 4 x 10⁸ AAV9 capsid particles per cell and treated with or without ECDI (30 mg/ml). FIG. 6B shows graphs and gating of late apoptotic and early apoptotic leukocyte markers for ECDI + AAV9 + SMN1 cells. X-axis: Annexin V, Y-axis: PI. FIG. 6C shows graphs of cell populations expressing AAV9 and/or SMN1 markers. X-axis: SMN1, Y-axis: AAV9.

[0035] FIGS. 7A-7B show graphs of the expression of immuno-inhibitory molecules in monocytes incubated with AAV9 VP1 protein-conjugated AALs. FIG. 7A shows a graph of CD33 mean fluorescence intensity (MFI) of monocytes cultured with media, autologous leukocytes (ALs), VP1 protein (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI, and ALs crosslinked with ECDI and AAV VP 1 protein (0.5 micrograms per 10⁶ B cells). FIG. 7B shows a graph of PD-L1 mean fluorescence intensity (MFI) of monocytes cultured with media, autologous leukocytes (ALs), VP1 protein (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI, and ALs crosslinked with ECDI and AAV VP1 protein (0.5 micrograms per 10⁶ B cells). X-axis: Conditions. Y-axis: MFI.

[0036] FIGS. 8A-8B show graphs of the expression of immuno-inhibitory molecules in monocytes incubated with AAV9 empty capsid-conjugated AALs. FIG. 8A shows a graph of CD33 mean fluorescence intensity (MFI) of monocytes cultured with ALs crosslinked with ECDI, ALs crosslinked with ECDI and empty capsid AAV9 (1.0 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.25 micrograms per 10⁶ B cells), empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV VP1 protein (0.5 micrograms per 10⁶ B cells), and VP1 protein (0.5 micrograms per 10⁶ B cells). FIG. 8B shows a graph of PD- L1 mean fluorescence intensity (MFI) of monocytes cultured with ALs crosslinked with ECDI, ALs crosslinked with ECDI and empty capsid AAV9 (1.0 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.25 micrograms per 10⁶ B cells), empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV VP1 protein (0.5 micrograms per 10⁶ B cells), and VP1 protein (0.5 micrograms per 10⁶ B cells). X-axis: Conditions. Y-axis: MFI.

[0037] FIGS. 9A-9B show graphs of the expression of immuno-inhibitory molecules in monocytes incubated with PKH67-labeled ALs, AAV9 empty capsid-conjugated AALs, unlabeled AAV9 empty capsids, or un-labeled VP1 protein. FIG. 9A shows a graph of CD33 mean fluorescence intensity (MFI) of monocytes cultured with ALs crosslinked with ECDI, ALs crosslinked with ECDI and empty capsid AAV9 (1.0 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.25 micrograms per 10⁶ B cells), empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV VP1 protein (0.5 micrograms per 10⁶ B cells), and VP1 protein (0.5 micrograms per 10⁶ B cells). FIG. 9B shows a graph of PD-L1 mean fluorescence intensity (MFI) of monocytes cultured with ALs crosslinked with ECDI, ALs crosslinked with ECDI and empty capsid AAV9 (1.0 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.25 micrograms), empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV VP1 protein (0.5 micrograms), and VP1 protein (0.5 micrograms per 10⁶ B cells). X-axis: Conditions. Y-axis: MFI.

[0038] FIGS. 10A-10B shows graphs of the expression of immune-inhibitory molecules in CDl lc+ dendritic cells (DCs) cocultured with AALs conjugated with various amounts of empty AAV capsids or VP1 protein. FIG. 10A shows a graph of CD33 mean fluorescence intensity (MFI) of dendritic cells cultured with autologous leukocytes (ALs) alone, ALs crosslinked with ECDI, ALs crosslinked with ECDI and empty capsid AAV9 (1.0 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.25 micrograms per 10⁶ B cells), empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV VP1 protein (0.5 micrograms per 10⁶ B cells), and VP1 protein (0.5 micrograms per 10⁶ B cells). FIG. 10B shows a graph of PD-L1 mean fluorescence intensity (MFI) of dendritic cells cultured with autologous leukocytes (ALs) alone, ALs crosslinked with ECDI, ALs crosslinked with ECDI and empty capsid AAV9 (1.0 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and empty capsid AAV9 (0.25 micrograms), empty capsid AAV9 (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV VP1 protein (0.5 micrograms per 10⁶ B cells), and VP1 protein (0.5 micrograms per 10⁶ B cells). X-axis: Conditions. Y-axis: MFI.

[0039] FIGS. 11A-11B shows graphs of the expression of immune-inhibitory molecules in CDl lc+ dendritic cells (DCs) cocultured with PKH67-labeled ALs, and AAV empty capsid- conjugated AALs. FIG. 11A shows a graph of CD33 mean fluorescence intensity (MFI) of dendritic cells cultured with autologous leukocytes (ALs) alone, ALs crosslinked with ECDI, ALs crosslinked with ECDI and AAV9 empty capsid (1 microgram per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.25 micrograms per 10⁶ B cells), and ALs crosslinked with ECDI and VP1 (0.5 micrograms per 10⁶ B cells). FIG. 11B shows a graph of PD-L1 mean fluorescence intensity (MFI) of dendritic cells cultured with autologous leukocytes (ALs) alone, ALs crosslinked with ECDI, ALs crosslinked with ECDI and AAV9 empty capsid (1 microgram per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.25 micrograms per 10⁶ B cells), and ALs crosslinked with ECDI and VP1 (0.5 micrograms per 10⁶ B cells). X-axis: Conditions. Y-axis: MFI.

[0040] FIGS. 12A-12D show graphs of the fold change in the frequency of T cells when dendritic cells were cultured with AAV9 empty capsid-conjugated ECDLtreated ALs (apoptotic autologous leukocytes, AALs) or VP1 protein-conjugated AALs or AAV9 empty capsid, T cells were stimulated with pre-treated DCs, and T cells were rechallenged with empty AAV9 capsids. FIG. 12A shows a graph of the fold change in frequency of Tregs compared to DC-coculture with AAV9 empty capsid for DCs cocultured with autologous leukocytes (ALs) crosslinked with ECDI and AAV9 (1 microgram per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.25 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and VP1 (0.5 micrograms per 10⁶ B cells), and VP1 protein alone (0.5 micrograms per 10⁶ B cells). FIG. 12B shows a graph of the fold change in frequency of Ki67 positive (Ki67⁺) Tregs compared to DC-coculture with AAV9 empty capsid for DCs cocultured with autologous leukocytes (ALs) crosslinked with ECDI and AAV9 (1 microgram per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.25 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and VP1 (0.5 micrograms per 10⁶ B cells), and VP1 protein alone (0.5 micrograms per 10⁶ B cells). FIG. 12C shows a graph of the fold change in frequency of Tri cells compared to DC-coculture with AAV9 empty capsid for DCs cocultured with autologous leukocytes (ALs) crosslinked with ECDI and AAV9 (1 microgram per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.25 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and VP1 (0.5 micrograms per 10⁶ B cells), and VP1 protein alone (0.5 micrograms per 10⁶ B cells). FIG. 12D shows a graph of the fold change in frequency of Ki67 positive (Ki67⁺) Tri cells compared to DC-coculture with AAV9 empty capsid for DCs cocultured with autologous leukocytes (ALs) crosslinked with ECDI and AAV9 (1 microgram per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.25 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and VP1 (0.5 micrograms per 10⁶ B cells), and VP1 protein alone (0.5 micrograms per 10⁶ B cells). X-axis: Conditions. Y-axis: Fold Change in Frequency.

[0041] FIG. 13 shows a graph of the fold change in frequency of Tri cells compared to monocyte coculture with AAV9 empty capsid for monocytes cocultured with autologous leukocytes (ALs) crosslinked with ECDI and AAV9 (1 microgram per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.5 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and AAV9 empty capsid (0.25 micrograms per 10⁶ B cells), ALs crosslinked with ECDI and VP1 (0.5 micrograms per 10⁶ B cells), and VP1 protein alone (0.5 micrograms per 10⁶ B cells). X-axis: Conditions. Y-axis: Fold Change in Frequency.

[0042] FIG. 14 shows provides an exemplary treatment regimen comprising administering to a recipient a tolerizing composition of adeno-associated virus (AAV) capsid antigen-conjugated apoptotic autologous leukocytes. The exemplary treatment regimen comprises administering an antagonistic anti-CD40 monoclonal antibody (anti-CD40 mAh), an mTOR inhibitor, an sTNFR, and an a-IL-6R monoclonal antibody prior to and after the first dose of a gene therapy and the second dose of a gene therapy.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] The following discussion of the present disclosure has been presented for purposes of illustration and description. The following is not intended to limit the invention to the form or forms disclosed herein. Although the description of the present disclosure has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the present disclosure, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

[0044] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Although various features of the disclosure may be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the disclosure may be provided herein in the context of separate embodiments for clarity, various aspects and embodiments can be implemented in a single embodiment.

Definitions

[0045] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g. , to any commonly owned patent or application. Although any methods and materials similar or equivalent to those provided herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are provided herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0046] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0047] The terms “and/or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.”

[0048] The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0049] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and/or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0050] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0051] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the inventions.

[0052] The term “antibody” as used herein includes IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY, and is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding (Fab) fragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab' and F(ab')2, Fd (consisting of VH and CHI), single-chain variable fragment (scFv), single-chain antibodies, disulfide-linked variable fragment (dsFv) and fragments comprising either a VL or VH domain. The antibodies provided herein can be from any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, can comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CHI, CH2, and CH3 domains. Also included are any combinations of variable region(s) and hinge region, CHI, CH2, and CH3 domains. In some embodiments, the antibodies provided herein are monoclonal, polyclonal, chimeric, humanized, human monoclonal, or polyclonal antibodies.

[0053] The term “antigen” and its grammatical equivalents as used herein can refer to a molecule that contains one or more epitopes capable of being bound by one or more receptors. For example, an antigen can stimulate a host's immune system to make a cellular antigen-specific immune response when the antigen is presented, or a humoral antibody response. An antigen can also have the ability to elicit a cellular and/or humoral response by itself or when present in combination with another molecule. For example, a tumor cell antigen can be recognized by a TCR.

[0054] The term “epitope” and its grammatical equivalents as used herein can refer to a part of an antigen that can be recognized by antibodies, B cells, T cells or engineered cells. For example, an epitope can be a cancer epitope that is recognized by a TCR. Multiple epitopes within an antigen can also be recognized. The epitope can also be mutated.

[0055] The terms “AAV,” “AAV construct,” or “recombinant AAV” or “AAV vector” refer to adeno-associated virus of any of the known serotypes, including AAV-1, AAV-2, AAV-3, AAV- 4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, or AAV-12, scAAV, AAV-rhlO, AAV-rh74, AAV-2i, AAV-DJ, chimeric or hybrid AAV, or any combination, derivative, or variant thereof. As used herein, the terms “recombinant AAV” and “rAAV” are interchangeable. In some embodiments, the rAAV is a hybrid AAV construct that is used in gene therapy. Several AAV vectors and gene therapy products are provided herein, e.g., voretigene neparvovec (Luxtuma®) for Leber congenital amaurosis, onasemnogene abeparvovec (Zolgensma®) for spinal muscular atrophy, or Alipogene tiparvovec (Glybera®) for lipoprotein lipase deficiency. Antigens from any AAV construct can be used in compositions provided herein.

[0056] The terms “recombinant AAV vector” or “AAV vector” or “AAV vector” refer to a vector derived from any of the AAV serotypes mentioned above.

[0057] The terms “AAV virion” or “AAV virion” refer to a virus particle comprising a capsid comprising at least one AAV capsid protein that encapsulates an AAV vector as provided herein, wherein the vector can further comprise a heterologous polynucleotide sequence or a transgene in some embodiments.

[0058] “Improving,” “enhancing,” and their grammatical equivalents as used herein mean any improvement recognized by one of skill in the art. For example, improving gene therapy can mean lessening hyperacute intolerance, which can encompass a decrease, lessening, or diminishing of an undesirable effect or symptom.

[0059] The term “inducing tolerance” refers to change in the level of an immune cell (e.g., increase in number of tolerogenic APC, increase in number of Tregs, increase in number of Tri cells, decrease in CD4+, CD8+ and/or CD20+ cells ), a change in level of immunomodulatory molecules (e.g., increase in IL-10 and TGF-b), or a combination thereof. In some embodiments, modulation of immune response is a suppression of immune response in conjugation with transient immunosuppression. A measure of induction of tolerance in the subject is that T cells in the subject can express markers consistent with T cell exhaustion. Contraction and regulation of immune cells, for instance regulation of memory T cells are also observed as an outcome of tolerance induction in a subject receiving a composition provided herein.

[0060] A “recipient” can be a human or non-human animal that can receive, is receiving, or has received a tolerizing regimen, a preparatory regimen for gene therapy, and/or other compositions provided in the present disclosure. A recipient can also be in need of a preparatory regimen for gene therapy, and/or other compositions provided herein. In some cases, the recipient can be a human or non-human animal that can receive, is receiving, or has received a tolerizing regimen.

In some cases, the recipient can be a human or non-human animal that can receive, is receiving, or has received the presently described tolerizing nanoparticle or preparatory regimen for gene therapy. In some cases, the recipient and the donor can be the same individual.

[0061] In some embodiments, the recipient is a mammal. In some embodiments, the mammal is a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as recipients that represent animal models of gene therapy. In addition, the compositions and methods provided herein can be used in gene therapy in domesticated animals and/or pets.

[0062] A recipient or subject relevant for the instant disclosure can be one who has undergone, is undergoing or will undergo gene therapy. The recipient therefore can be one who is diagnosed with, or currently being treated for, or seeking treatment, from a disease resulting in complications such as organ failure and therefore is a candidate for gene therapy. Non-limiting examples of such diseases include inflammatory disease, autoimmune disease, diabetes and the like. The recipient or subject can be one who is suffering from a cancer.

[0063] The term “non-human animal” and its grammatical equivalents as used herein includes all animal species other than humans, including non-human mammals, which can be a native animal or a genetically modified non-human animal. A non-human mammal includes, an ungulate, such as an even-toed ungulate (e.g., pigs, peccaries, hippopotamuses, camels, llamas, chevrotains (mouse deer), deer, giraffes, pronghorn, antelopes, goat-antelopes (which include sheep, goats and others), or cattle) or an odd-toed ungulate (e.g., horse, tapirs, and rhinoceroses), a non-human primate (e.g., a monkey, or a chimpanzee), a Canidae (e.g., a dog) or a cat.

[0064] The term "genetically modified", “genetically engineered”, "transgenic", “genetic modification” and its grammatical equivalents as used herein refer to having one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism’s genome, For example, genetic modification can refer to alterations, additions, and/or deletion of genes, A genetically modified cell can also refer to a cell with an added, deleted and/or altered gene. A genetically modified cell can be from a genetically modified non-human animal. A genetically engineered cell from a genetically engineered non-human animal can be a cell isolated from such genetically engineered non-human animal. A genetically modified cell from a genetically modified nonhuman animal can be a cell originated from such genetically modified non-human animal. A genetically engineered cell or a genetically engineered animal can comprise a transgene, or other foreign DNA, added or incorporated, or an endogenous gene modified, including, targeted, recombined, interrupted, deleted, disrupted, replaced, suppressed, enhanced, or otherwise altered, to mediate a genotypic or phenotypic effect in at least one cell of the animal, and typically into at least one germ line cell of the animal.

[0065] The term “transgene ” and its grammatical equivalents as used herein refer to a gene or genetic material that can be transferred into an organism. For example, a transgene can be a stretch or segment of DNA containing a gene that is introduced into an organism. The gene or genetic material can be from a different species. The gene or genetic material can be synthetic. When a transgene is transferred into an organism, the organism can then be referred to as a transgenic organism. A transgene can retain its ability to produce RNA or polypeptides (e.g., proteins) in a transgenic organism. A transgene can comprise a polynucleotide encoding a protein or a fragment (e.g., a functional fragment) thereof. The polynucleotide of a transgene can be an exogenous polynucleotide. A fragment (e.g., a functional fragment) of a protein can comprise at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence of the protein. A fragment of a protein can be a functional fragment of the protein. A functional fragment of a protein can retain part or all of the function of the protein. An exogenous polypeptide can encode an exogenous protein or a functional fragment thereof.

[0066] The term “exogenous nucleic acid sequence”, “exogenous polynucleotide” and its grammatical equivalents as used herein can refer to a gene or genetic material that was transferred into a cell or animal that originated outside of the cell or animal. An exogenous nucleic acid sequence can by synthetically produced. An exogenous nucleic acid sequence can be from a different species, or a different member of the same species. An exogenous nucleic acid sequence can be another copy of an endogenous nucleic acid sequence.

[0067] The term “gene knock-out” or “knock-out” can refer to any genetic modification that reduces the expression of the gene being “knocked out.” Reduced expression can include no expression. The genetic modification can include a genomic disruption.

[0068] The term “disrupting” and its grammatical equivalents as used herein can refer to a process of altering a gene, e.g., by deletion, insertion, mutation, rearrangement, or any combination thereof. For example, a gene can be disrupted by knockout. Disrupting a gene can be partially reducing or completely suppressing expression (e.g., mRNA and/or protein expression) of the gene. Disrupting can also include inhibitory technology, such as shRNA, siRNA, microRNA, dominant negative, or any other means to inhibit functionality or expression of a gene or protein. [0069] The term “gene editing” and its grammatical equivalents as used herein can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. For example, gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease). [0070] The term “condition” and its grammatical equivalents as used herein can refer to a disease, event, or change in health status.

[0071] As used herein, a "subject", "patient", "individual" “recipient” and like terms are used interchangeably and refers to a vertebrate, such as a mammal, such as a primate, such as a human. Mammals include, without limitation, humans, primates, rodents, wild or domesticated animals, including feral animals, farm animals, sport animals, and pets. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. The terms, "individual," "patient" and "subject", “recipient” are used interchangeably herein. A subject can be male or female.

[0072] As used herein, the term "in combination" refers to the use of more than one prophylactic and/or therapeutic agent simultaneously or sequentially and in a manner such that their respective effects are additive or synergistic.

[0073] As used herein, the terms "protein", "peptide" and "polypeptide" are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", "peptide" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein", "peptide" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof.

[0074] As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound. In some embodiments, the compositions of the present disclosure are substantially pure. [0075] By "detectable agent" is meant a compound that is linked to a diagnostic agent, therapeutic agent or tolerizing agent (e.g., nanoparticle disclosed herein) to facilitate detection. Such a "detectable agent" may be covalently or non-covalently linked to a nanoparticle, in addition, the linkage may be direct or indirect. Examples of "detectable agents" include, protein purification tags, cytotoxins, enzymes, paramagnetic labels, enzyme substrates, co-factors, enzymatic inhibitors, dyes, radionuclides, chemiluminescent labels, fluorescent markers, growth inhibitors, cytokines, antibodies, and biotin.

[0076] The term “phenotype” and its grammatical equivalents as used herein can refer to a composite of an organism’s observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior. Depending on the context, the term “phenotype” can sometimes refer to a composite of a population’s observable characteristics or traits.

[0077] Some numerical values disclosed throughout are referred to as, for example, “X is at least or at least about 100; or 200.” This numerical value includes the number itself and all of the following: i) X is at least 100; ii) X is at least 200; iii) X is at least about 100; and iv) X is at least about 200.

[0078] All these different combinations are contemplated by the numerical values disclosed throughout. All disclosed numerical values should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary.

[0079] The ranges disclosed throughout are sometimes referred to as, for example, “X is administered on or on about day 1 to 2; or 2 to 3 [or any numerical range].” This range includes the numbers themselves (e.g., the endpoints of the range) and all of the following: i) X being administered on between day 1 and day 2; ii) X being administered on between day 2 and day 3; iii) X being administered on between about day 1 and day 2; iv) X being administered on between about day 2 and day 3; v) X being administered on between day 1 and about day 2; vi) X being administered on between day 2 and about day 3; vii) X being administered on between about day 1 and about day 2; and viii) X being administered on between about day 2 and about day 3. [0080] All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary.

[0081] Provided herein are compositions, systems, and methods for inducing gene therapy tolerance. In particular, the present disclosure relates to conjugated or crosslinked leukocyte and viral antigen compositions and methods useful for modulating an immune response of a subject during gene therapy. Inducing tolerance can include inducing tolerance to a recombinant viral vector, which may comprise a transgene, for therapeutic applications.

Cells

[0082] Provided herein are compositions comprising cells. In some embodiments, the cells are apoptotic cells. In some embodiments, the cells are early apoptotic cells. In some embodiments, the cells are late apoptotic cells. In some embodiments, the cells are living cells. In some embodiments, the cells are cell cycle-arrested cells. In some embodiments, the cells are in Go phase of the cell cycle. In some embodiments, the cells are in mitosis. In some embodiments, the cells are in interphase.

[0083] In some embodiments, the cells are chemically modified. In some embodiments, the cells are leukocytes crosslinked to a bioactive agent. Non-limiting examples of bioactive agents include, e.g., proteins, nucleic acids, vectors, viral vectors, aptamers, and antibodies. Chemical crosslinking of the cells provided herein and bioactive agents that can be used are discussed further below.

[0084] The cells (e.g., leukocytes) provided herein can be obtained from any source, including animals, cells lines, and/or lab-generated cells. For example, leukocyte and lymphocyte cells provided herein can be obtained from a human or non-human animal. In some embodiments, the cells are obtained from a mammal. As but one example, the cells provided herein can be from a cell line (e.g., a human or non-human cell line). Exemplary leukocyte cell lines include but are not limited to: human monocytes, human CD 14+ blood cells, k46 cell, kt-3 cells, kg-1 cells, and plb985 cells.

[0085] Provided herein are compositions comprising cells obtained from a subject. Further provided herein are compositions comprising mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are non-human cells. In some embodiments, the cells are non-human primate cells. In some embodiments, the human cells are human leukocytes. [0086] Cells provided herein can be obtained from living donors or cadaveric donors. In some embodiments, the donor is a living donor. In some cases, the donor is a cadaveric donor. The cadaveric donor may be, for example, a brain dead, heart beating donor (BDD). The cadaveric donor may be, for example, a non-heart beating donor (NHBD). Whether the donor is a living donor or a cadaveric donor (e.g., a BDD or NHBD), the donor can be from any animal, for example, a human or non-human animal. In some embodiments the donor is the same as the recipient of the tolerizing composition and the gene therapy. In some embodiments, the donor is a living or cadaveric donor that is MHC class 2 matched with the recipient of the tolerizing composition and gene therapy.

[0087] Cells can be obtained from a donor animal of any age or stage of development. For example, the donor animal can be a fetal, perinatal, neonatal, pre-weaning, post-weaning, juvenile, young adult, or adult animal. In some cases, cells can be obtained (for example, differentiated) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, and/or mesenchymal stem cells). In some cases, cells used in embodiments provided herein are autologous to a recipient of a composition or a method provided herein.

[0088] In some embodiments, a composition provided herein comprises a population of leukocytes or a mixed population of immune cells. Leukocytes can include, for example, neutrophils, eosinophils, basophils, lymphocytes, monocytes, or a combination thereof. Lymphocytes can include, for example, B lymphocytes (B cells), T lymphocytes (T cells), natural killer (NK) cells, monocytes, or a combination thereof. In some embodiments, the compositions provided herein comprise a population of leukocytes, wherein the population of leukocytes comprise a mixed population of leukocytes. In some embodiments, the mixed population of leukocytes comprises a population of B cells, a population of T cells, a population of monocytes, a population of macrophages, a population of dendritic cells (DCs), and/or a population of natural killer (NK) cells. In some embodiments, the mixed population of leukocytes are enriched for a population of B cells. Methods of enriching for a particular cell type can include, for example flow cytometry sorting, magnetic sorting, or cell capture assays.

[0089] Leukocytes provided herein can be obtained from any source, including, for example, a donor, a cell line, or a differentiated stem cell. In some cases, the donor of the leukocytes or stem cells from which the leukocytes are differentiated, is also the recipient of gene therapy to whom compositions and methods provided herein are administered. Leukocytes can be obtained by ex vivo differentiation of a stem cell, pluripotent cell or induced pluripotent stem cell. Leukocytes obtained from a donor can include leukocytes obtained from a spleen (e.g., splenocytes, splenic B cells); a liver; peripheral blood (including peripheral blood B cells); a lymph node; a thymus; bone marrow; or any other organ, tissue, or bodily fluid; or any combination thereof. In some cases, the tolerizing vaccine or preparatory regimen comprises splenic B cells, peripheral blood B cells, or a combination thereof. In some cases, the tolerizing vaccine or preparatory regimen comprises cells mobilized from the bone marrow to peripheral blood with a mobilization agent, e.g., cells mobilized with granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colonystimulating factor (GM-CSF), Plerixafor (Mozobil®), or a combination thereof. In some embodiments, the leukocytes comprise primary cells, cells expanded ex vivo, or a combination thereof.

[0090] In some embodiments, the leukocytes are pre-apoptotic leukocytes, apoptotic leukocytes, or late apoptotic leukocytes.

[0091] In some embodiments, the pre-apoptotic cells (e.g., pre-apoptotic leukocytes) are characterized by cell shrinkage and pyknosis. Pyknosis is the result of chromatin condensation. In some embodiments, late-stage and necrotic cells are detected by propidium iodide, which binds to DNA and indicates loss of cell membrane integrity. Markers of early apoptosis can be used, for example, markers associated with initiator caspases and inhibition of phosphatidylserine flippase, resulting in exposure of phosphatidylserine on the outer plasma membrane, which can be detected by Annexin 5 staining. A combination of propidium iodide and Annexin 5 can be used to detect dead cells and distinguish early apoptotic leukocytes from dead cells.

[0092] In some embodiments, apoptotic cells (e.g., apoptotic leukocytes) are characterized by plasma membrane blebbing, karyorrhexis, and/or separation of cell fragments into apoptotic bodies or budding. Apoptotic bodies consist of cytoplasm with tightly packed organelles with or without a nuclear fragment. In some embodiments, apoptotic cells (e.g., apoptotic leukocytes) express a caspase. In some embodiments, the caspase proteins are activated, allowing initiation of a protease cascade. Some procaspases aggregate and autoactivate. This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic signaling pathway and leads to rapid cell death.

[0093] In some embodiments, late apoptotic cells (e.g., late apoptotic leukocytes) are characterized as having phosphatidylserine (PS) translocated from the inner to the outer leaflet of the plasma membrane. In some embodiments, the PS is detected by Annexin V. In normal viable cells, PS is located on the inner leaflet of the cytoplasmic membrane.

[0094] A donor of the cells used in a composition provided herein can be genetically modified. Alternatively, or additionally, cells obtained from a donor can be genetically modified ex vivo. In some cases, cell lines are genetically modified to produce cells for use in a tolerizing vaccine or preparatory regimen. The genetically modified donors and/or cells can be produced using any method known in the art, including those provided herein. Regardless of whether the genetically modified cells are isolated from a genetically modified animal, produced in culture, or a combination thereof, the genetically modified cells can be of any animal species, including human and non-human animals. [0095] Genetically modified cells for instance leukocytes used in compositions provided herein, can comprise one or more genetic modifications that reduce or eliminate expression or a gene or gene product (e.g., a protein). The genetic modification(s) can be modifications to the gene whose expression is reduced or eliminated. Such genes can be referred to as disrupted genes. The genetic modification(s) can also be to areas of the genome separate from the gene whose expression is reduced or eliminated (for example, modification to a promoter, enhancer, silencer, transcription factor, etc.). The genetically modified cells can comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more genes whose expression is reduced or eliminated by genetic modification.

[0096] Genetically modified cells, for instance leukocytes for use in compositions provided herein, can comprise, or further comprise, one or more genetic modifications that increase expression of one or more genes or gene products. The increased expression can be from zero expression, e.g., the increased expression can be of a gene or gene product that is not normally expressed in the cell without genetic modification. The increased expression can be compared to a threshold level, e.g., a level normally expressed in the cell without genetic modification. The genetic modification(s) can comprise one or more exogenous polynucleotides encoding a polypeptide (e.g., an endogenous or exogenous polypeptide).

[0097] In some cases, genetic modification of leukocytes used in tolerizing compositions provided herein involves introduction of a transgene into the leukocyte. In certain cases, the transgene is the same as a transgene incorporated in a gene therapy product and the tolerizing composition tolerizes a subject that is the intended recipient of the gene therapy product. In some cases the transgene is a sarcoglycan gene, an RPE gene, CNGB3 gene, a Factor IX, gene or variant thereof, follistatin gene, NF-kB gene, IFN-P gene, ARSA gene or a dystrophin gene designed for use in a gene therapy. [0098] Cells (e.g., leukocytes) can be treated with a fixative or crosslinking agent (e.g., a carbodiimide such as ECDI) in the presence of one or more antigens and/or epitopes. The antigens and/or epitopes can comprise antigens and/or epitopes from a donor, a gene therapy recipient, a third party, a viral antigen of a virus used in a gene therapy, or a combination thereof. These cells can be in a tolerizing vaccine or preparatory regimen. In some cases, the cells in a tolerizing vaccine or preparatory regimen are coupled to viral antigens and/or epitopes.

Gene Therapy Vectors

[0099] Gene therapy is used to introduce genetic material into a subject or cells, often in the treatment of disease or genetic abnormality. Vectors are used as carriers to deliver the genetic materials into cells. Modified viruses may be used as vectors due to their ability to delivery genetic material by infecting a subject. Certain aspects disclosed herein can utilize vectors. Any plasmids and vectors can be used as long as they are replicable and viable in a selected host. Vectors known in the art and those commercially available (and variants or derivatives thereof) can be engineered to include one or more recombination sites for use in the methods. Vectors that can be used include, but not limited to eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-Cl, pPUR, pMAM, pMAMneo, pBHOl, pBH21, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCHl 10, and pKK232- 8 (Pharmacia, Inc.), p3'SS, pXTl, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBa-cHis A, B, and C, pVL1392, pBlueBacl 1, pCDM8, pcDNAl, pZeoSV, pcDNA3, pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.), and variants or derivatives thereof.

[0100] Any vector systems can be used in gene therapies provided herein including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, recombinant herpes simplex virus (HSV) vectors, recombinant poxvirus vectors, recombinant parvovirus vectors, recombinant papillomavirus vectors, recombinant simian virus vectors, recombinant alphavirus vectors, recombinant polyoma virus vectors, recombinant picornavirus vectors, recombinant lentivirus vectors, recombinant retrovirus vectors, recombinant adenovirus vectors, recombinant adenovirus associated virus (AAV) vectors, recombinant flavivirus vectors, recombinant rhabdovirus vectors, recombinant measles virus vectors, recombinant Newcastle disease virus vectors, and a recombinant bacteriophage vectors, etc. The recombinant adenovirus vector can be an adenovirus serotype 5 (Ad5) vector. The recombinant herpes simplex virus vector can be a recombinant herpes simplex virus 1 (HSV1) vector, or a recombinant herpes simplex virus 2 (HSV2) vector. The recombinant retrovirus vector can be a recombinant Moloney murine sarcoma virus (MMSV) vector, or a recombinant murine stem cell virus (MSCV) vector. The recombinant lentivirus vector can be a recombinant human immunodeficiency virus 1 (HIV-1) vector or a recombinant human immunodeficiency virus 2 (HIV-2) vector. The recombinant alphavirus vector can be a recombinant Semliki forest virus (SFV) vector, Sindbis virus (SIN) vector, a recombinant Venezuelan equine encephalitis virus (VEE) vector, or a recombinant alphavirus Ml. The recombinant flavivirus vector can be a recombinant Kunjin virus vector, a recombinant West Nile virus vector, or a recombinant Dengue virus vector. The recombinant rhabdovirus vector can be a recombinant Rabies virus vector, or a recombinant vesicular stomatitis virus vector. The recombinant measles virus vector can be a recombinant MV Edmonston strain (MV-Edm) vector. The recombinant poxvirus vector can be a recombinant vaccinia virus (VV) vector. The recombinant picornavirus vector can be a recombinant Coxsackievirus vector. [0101] Adenovirus associated virus (AAV) vectors can be administered as gene therapy to subjects in methods provided herein. AAV is a small non-enveloped single-stranded DNA virus. They are non-pathogenic parvoviruses and can require helper viruses, such as adenovirus, herpes simplex virus, vaccinia virus, and CMV, for replication. Wild-type AAV is common in the general population, and is not associated with any known pathologies. A hybrid AAV is an AAV comprising a capsid protein of one AAV serotype and genomic material from another AAV serotype. A chimeric AAV comprises genetic and/or protein sequences derived from two or more AAV serotypes, and can include mutations made to the genetic sequences of those two or more AAV serotypes. An exemplary chimeric AAV can comprise a chimeric AAV capsid, for example, a capsid protein with one or more regions of amino acids derived from two or more AAV serotypes. An AAV variant is an AAV comprising one or more amino acid mutations in its genome or proteins as compared to its parental AAV, e.g., one or more amino acid mutations in its capsid protein as compared to its parental AAV. AAV, as used herein, includes avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, wherein primate AAV refers to AAV that infect non-primates, and wherein non-primate AAV refers to AAV that infect non-primate animals, such as avian AAV that infects avian animals. In some cases, the wildtype AAV contains rep and cap genes, wherein the rep gene is required for viral replication and the cap gene is required for the synthesis of capsid proteins. In some cases, an AAV vector can comprise one or more of the AAV wild-type genes deleted in whole or part, such as the rep and/or cap genes, but contains functional elements that are required for packaging and use of AAV virus for gene therapy. For example, functional inverted terminal repeats or ITR sequences that flank an open reading frame or exogenous sequences cloned in are known to be important for replication and packaging of an AAV virion, but the ITR sequences can be modified from the wild-type nucleotide sequences, including insertions, deletions, or substitutions of nucleotides, so that the AAV is suitable for use for the embodiments provided herein, such as a gene therapy or gene delivery system. In some aspects, a self-complementary vector (sc) can be used, such as a self- complementary AAV vector, which can bypass the requirement for viral second-strand DNA synthesis and can lead to higher rate of expression of a transgene protein. In some aspects, AAV vectors can be generated to allow selection of an optimal serotype, promoter, and transgene. In some cases, the vector can be targeted vector or a modified vector that selectively binds or infects immune cells. The recombinant AAV vector can have an AAV serotype of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ. AAV vectors bind to serotype specific receptors in order to enter a cell. The VP1 capsid protein can comprise an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% identical to that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ. The VP2 capsid protein can comprise an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% identical to that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ. The VP3 capsid protein can comprise an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% identical to that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. The empty capsid or nucleocapsid can be any AAV serotype such as AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i.

[0102] Furthermore, any of these vectors can comprise one or more transcription factor, nuclease, and/or transgene. Thus, when one or more CRISPR, TALEN, transposon-based, ZFN, meganuclease, or Mega-TAL molecules and/or transgenes are introduced into the cell, CRISPR, TALEN, transposon-based, ZFN, meganuclease, or Mega-TAL molecules and/or transgenes can be carried on the same vector or on different vectors. When multiple vectors are used, each vector can comprise a sequence encoding one or multiple CRISPR, TALEN, transposon-based, ZFN, meganuclease, or Mega-TAL molecules and/or transgenes.

[0103] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered CRISPR, TALEN, transposon-based, ZFN, meganuclease, or Mega-TAL molecules and/or transgenes in cells (e.g., mammalian cells) and target tissues. Such methods can also be used to administer nucleic acids encoding CRISPR, TALEN, transposonbased, ZFN, meganuclease, or Mega-TAL molecules and/or transgenes to cells in vitro. In some examples, nucleic acids encoding CRISPR, TALEN, transposon-based, ZFN, meganuclease, or Mega-TAL molecules and/or transgenes can be administered for in vivo or ex vivo immunotherapy uses. Non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.

[0104] These vectors can be used to express a gene, e.g., a transgene, or portion of a gene of interest. A gene of portion or a gene can be inserted by using known methods, such as restriction enzyme-based techniques.

Viral Antigens

[0105] A subject that is a recipient of a viral vector for gene therapy may experience a negative immune response due to the use of a virus which produces viral antigens.

[0106] In some embodiments, provided herein are tolerizing compositions that comprise a leukocyte from a subject crosslinked by use of a chemical crosslinking agent to a viral antigen derived from a recombinant viral vector that is used for gene therapy to the subject. A viral antigen used in the compositions provided herein can be any of an antigenic protein or polypeptide or antigenic fragment thereof. In some instances, the viral antigen is a capsid protein an antigenic fragment of a capsid protein, an envelope protein or an antigenic fragment of an envelope protein, or a capsid.

[0107] In case of AAV gene therapy, the viral antigen can be a capsid protein or an antigenic fragment of the capsid protein. Capsid proteins are designated as VP1, VP2, VP3, and VP4. The capsid can be empty or a nucleocapsid. The recombinant AAV vector can have an AAV serotype of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ. In some embodiments, the AAV vector is an AAV9. The recombinant AAV vector of the viral antigen can have at least one serotype in common with the AAV vector of the gene therapy. The VP1 capsid protein can comprise an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% identical to that of an AAV serotype selected from the group consisting of AAV- 1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ. The VP2 capsid protein can comprise an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% identical to that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, - 4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ. The VP3 capsid protein can comprise an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% identical to that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, - 4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. The empty capsid or nucleocapsid can be any AAV serotype such as AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. Exemplary AAV epitopes are provided in Table 1.

Table 1. Exemplary AAV capsid antigenic sequences crosslinked to leukocytes with a chemical crosslinking agent.

Crosslinking Agents

[0108] In some embodiments, the compositions comprise leukocytes crosslinked to viral antigens. Non-limiting examples of crosslinking agents that can be use include, e.g., carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof. In some embodiments, a population of cells provided herein are contacted with a carbodiimide, or a carbodiimide derivative. In some embodiments, treatment with a carbodiimide can chemically crosslink free amine and carboxyl groups, and effectively induce apoptosis in cells, organs, and/or tissues at 37 degrees Celsius. In some embodiments, treatment with ECDI chemically crosslinks an antigen or transgene product provided herein at 4 degrees Celsius.

[0109] Further provided herein are methods of producing a population of apoptotic cells (e.g., leukocytes orB cells). In some embodiments, a population of cells (e.g., leukocytes or B cells) are contacted with a crosslinking agent provided herein to produce a population of cells that are apoptotic. In some embodiments, the population of cells are autologous cells. In some embodiments, a population of leukocytes provided herein are contacted with a crosslinking agent provided herein for at least about 10 minutes up to 6 hours, thereby producing a population apoptotic leukocytes. In some embodiments, the population of leukocytes are contacted with a carbodiimide. In some embodiments, the carbodiimide comprises ethylcarbodiimide; ethylene carbodiimide; N,N'-diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); 1- ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, or ED AC); or a combination thereof. In some embodiments, the carbodiimide comprises ethylcarbodiimide. In some embodiments, the carbodiimide comprises ethylene carbodiimide. In some embodiments, the carbodiimide comprises N,N'-diisopropylcarbodiimide (DIC). In some cases, the carbodiimide comprises N/ N'-dicyclohexylcarbodiimide (DCC). In some embodiments, the carbodiimide comprises l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, or EDAC). In some embodiments, the totalizing vaccine comprises cells treated with EDCI derivatives and/or functionalized EDCI.

[0110] Cells e.g., leukocytes) and optionally the viral antigens provided herein can be contacted with a diimidoester. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. The diimidoester can comprise cyanuric chloride; diisocyanate; diethylpyrocarbonate (DEPC) or diethyl dicarbonate; a maleimide; benzoquinone; or a combination thereof.

[OHl] Cells (e.g. leukocytes), and in some embodiments viral antigens can be contacted with an amine-to- amine crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some embodiments, the amine-to-amine-crosslinker comprises disuccinimidyl glutarate (DSG); disuccinimidyl suberate (DSS); bis(sulfosuccinimidyl)suberate (BS3); tris-(succinimidyl) aminotriacetate (TSAT); BS(PEG)5; BS(PEG)9; dithiobis(succinimidyl propionate) (DSP); 3,3’-dithiobis(sulfosuccinimidyl propionate) (DTSSP); disuccinimidyl tartrate (DST); bis(2- (succinimidooxycarbonyloxy)ethyl)sulfone (BSOCOES); ethylene glycol bis(succinimidyl succinate) (EGS); sulfo-EGS; or any combination thereof. In some embodiments, the amine-to- amine crosslinker comprises an imidoester, such as dimethyl adipimidate (DMA); dimethyl pimelimidate (DMP); dimethyl suberimidate (DMS); dimethyl 3,3’-dithiobispropionimidate (DTBP); or any combination thereof. In some embodiments, the amine-to-amine crosslinker comprises a difluoro, such as l,5-difluoro-2,4- dinitrobenzene (DFDNB).

[0112] Cells e.g. leukocytes) and in some instances viral antigens can be contacted with a sulfhydryl- to-sulfhydryl crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some embodiments, the sulfhydryl-to- sulfhydryl crosslinker comprises a maleimide, such as bismaleimidoethane (BMOE); 1,4- bismaleimidobutane (BMB); bismaleimidohexane (BMH); tris(2-maleimidoethyl)amine (TMEA); BM(PEG)2 (such as 1,8- bismaleimido-di ethyleneglycol); BM(PEG)3 (such as 1,1 1- bismaleimido-tri ethyleneglycol), dithiobismaleimidoethane (DTME); or any combination thereof. [0113] Cells e.g. leukocytes) and in some instances viral antigens can be contacted with an amine- to- sulfhydryl crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some embodiments, the amine-to-sulfhydryl crosslinker comprises a NHS-haloacetyl crosslinker, a NHS-maleimide, a NHS-pyridyldithiol crosslinker, a sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) crosslinker, or any combination thereof. The NHS-haloacetyl crosslinkers can comprise succinimidyl iodoacetate (SIA); succinimidyl 3-(bromoacetamido)propionate (SBAP); succinimidyl (4- iodoacetyl)aminobenzoate (SIAB); sulfo-SIAB; or a combination thereof. The NHS-maleimide can comprise N-a-maleimidoacet-oxysuccinimide ester (AMAS); N-b-maleimidopropyl- oxysuccinimide ester (BMPS); N-g-maleimidobutyryl-oxysuccinimide ester (GMBS); sulfo- GMBS; m-maleimidobenzoyl-N-hydrosuccinimide ester (MBS); sulfo-MBS; SMCC; sulfo- SMCC; N-e-malemidocaproyl-oxysuccinimide ester (EMCS); sulfo-EMCS; succinimidyl 4-(p- maleimidophenyl)butyrate (SMPB); sulfo-SMPB; succinimidyl 6-((beta- maleimidopropionamido)hexanoate) (SMPH); sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxy-(6-amidocaproate) (LC-SMCC); N-K- maleimidoundecanoyl-oxysulfosuccinimide ester (sulfo-KMETS); or a combination thereof. The NHS-pyridyldithiol crosslinker can comprise succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate (LC-SPDP), sulfo-LC- SPDP, or 4- succinimidyloxycarbonyl-alpha-methyl-a(2-pyridyldithio)tolune (SMPT).

[0114] Cells e.g. leukocytes) and in some instances viral antigens can be contacted with a sulfhydryl- to-carbohydrate crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some embodiments, the sulfhydryl-to- carbohydrate crosslinker comprises (N-b-maleimidopropionic acid hydrazide (BMPH), N-e- maleimidocaproic acid hydrazide (EMCH), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), N-K- maleimidoundecanoic acid hydrazide (KMUH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), or any combination thereof.

[0115] In some embodiments, the carboxyl-to-amine crosslinker is dicyclohexylcarbodiimide (DCC),1 -ethyl-3 -(3 -dimethylaminopropyl)-carbodiimide (EDCI, EDC, or ED AC), N- hydroxysuccinimide (NHS), sulfo-NHS, or any combination thereof.

[0116] Cells e.g. leukocytes) and in some instances viral antigens can be contacted with a photoreactive crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some embodiments, the photoreactive crosslinker comprises a NHS ester/aryl azide, a NHS ester/diazirine, or a combination thereof. The NHS ester/aryl azide can comprise N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), sulfo- SANPAH, or a combination thereof. The NHS ester/diazirine can comprise SDA (NHS-diazirine / succinimidyl 4,4’-azipentanoate), sulfo-SDA, LC-SDA (NHS-LC-diazirine / succinimidyl 6- (4,4’- azipentanamido)hexanoate), sulfo-LC-SDA, SDAD (NHS-SS-diazirine / succinimidyl 2- ((4,4’- azipentanamido)ethyl)l,3’ -dithiopropionate), sulfo-SDAD, or a combination thereof.

[0117] Cells e.g. leukocytes) and in some instances viral antigens can be contacted with an in vivo crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. The in vivo crosslinker can comprise BS3, DTSSP, sulfo-EGS, DSG, DSP, DSS, EGS, sulfo-SDA, sulfo-LC-SDA, sulfo-SDAD, SDA, LC-SDA, SDAD, NHS-ester diazirine, or any combination thereof. [0118] In some embodiments, cells e.g. leukocytes) and in some instances viral antigens are treated with a cellular damaging agent or an apoptosis inducer. In some embodiments, the cellular damaging agent induces apoptosis in some or all of the contacted cells. Non-limiting exemplary cellular damaging agents include doxorubicin, staurosporine, etoposide, comptothecin, paclitaxel, vinblastine, or any combination thereof. Non-limiting exemplary apoptosis inducers include marinopyrrole A, maritoclax, (E)-3,4,5,4'-tetramethoxystilbene, 17-(Allylamino)-17- demethoxygeldanamycin, 2,4,3',5'-tetramethoxystilbene, 20HOA, 6,8-bis(benzylthio)-octanoic acid, AT 101, apoptolidin, FET 40 A, ara-G hydrate, aryl quin 1, BAD, BAM7, BAX activator molecule 7, BH3I-1, BID, BMS-906024, BV02, bendamustine, borrelidin, borrelidine, cyclopentanecarboxylic acid, NSC 216128, treponemycin, brassinin, brassinine, brefeldin A, ascotoxin, BFA, cyanein, decumbin, bufalin, CCF642, CCT007093, CD437, CHM-1 hydrate, 2- (2-fhiorophenyl)-6,7-methylenedioxy-2-4-quinolone hydrate, NSC 656158, CIL-102, CP-31398, dihydrochloride hydrate, carnal exin, 3-(Thiazol-2-yl)-lH-indole, carnal exine, carboxyatractyloside, cepharanthine, cepharanthine, cinnabarinic acid, cirsiliol, combretastatin A4, costunolide, DBeQ, DIM-C-pPhtBu, DMXAA, DPBQ, enniatin Al, enniatin A, enniatin Bl, enniatin B, erastin, eupatorin, FADD, fluticasone propionate, fosbretabulin disodium, GO- 201 trifluoroacetate, gambogic acid, HA 14-1, HMBA, hexaminolevulinate (HAL), IMB5046, IMS2186, ikarugamycin, imiquimod, iniparib, kurarinone, LLP-3, lipocalin-2, lometrexol, MI- 4F, ML 210, ML291, mollugin, muristerone A, NA- 17, NID-1, NPC26, NSC59984, Nap-FF, neocarzinostatin, nifetepimine, nitidine chloride, nutlin-3, nutlin-3a, PKF118-310, PRIMA-1, PRT4165, pemetrexed, penta-O-galloyl-P -D-glucose hydrate, phenoxodiol, prodigiosin (PG), psoralidin, pterostilbene, raltitrexed, raptinal, ridaifen-B, rifabutin, roslin 2, s-p- bromobenzylglutathione cyclopentyl diester, SJ-17255, SMBA1, STF-62247, suprafenacine, syrosingopine, talniflumate, taurolidine, temoporfm, temozolomide, tetrazanbigen, thaxtomin A, thiocolchicine, tirapazamine, UCD38B, UML77, undecylprodigiosin, VK3-OCH3, vacquinol-1, violacein, vosaroxin, zerumbone, gAcrp30, gAcrp30/adipolean, or any combination thereof. Cells contacted with a cellular damaging agent or an apoptosis inducer may subsequently be contacted with a fixative or cross-linking agent.

[0119] Cells e.g. leukocytes) and in some instances viral antigens can be fixed for a predetermined amount of time. In some embodiments, the cells and in some embodiments viral antigens or nanoparticles displaying these viral antigens are fixed for a predetermined amount time with the crosslinking agent e.g., ECDI). In some embodiments, the predetermined amount of time is about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours. In some embodiments, the predetermined amount of time is less than an hour. In some embodiments, the predetermined time is at least about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes or 240 minutes. In some embodiments, the predetermined time is at most about 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes or 240 minutes. In some embodiments, the predetermined amount of time is about 1 minute to about 240 minutes, 1 minute to about 10 minutes, 10 minutes to about 240 minutes, about 10 minutes to about 180 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 30 minutes, about 30 minutes to about 240 minutes, about 30 minutes to about 180 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 60 minutes, about 50 minutes to about 240 minutes, about 50 minutes to about 180 minutes, about 50 minutes to about 120 minutes, about 50 minutes to about 90 minutes, about 50 minutes to about 60 minutes, about 10 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 30 minutes to about 40 minutes, about 40 minutes to about 50 minutes, about 50 minutes to about 60 minutes, about 60 minutes to about 70 minutes, about 70 minutes to about 80 minutes, about 80 minutes to about 90 minutes, about 90 minutes to about 100 minutes, about 100 minutes to about 110 minutes, about 110 minutes to about 120 minutes, about 10 minutes to about 30 minutes, about 30 minutes to about 50 minutes, about 50 minutes to about 70 minutes, about 70 minutes to about 90 minutes, about 90 minutes to about 110 minutes, about 110 minutes to about 130 minutes, about 130 minutes to about 150 minutes, about 150 minutes to about 170 minutes, about 170 minutes to about 190 minutes, about 190 minutes to about 210 minutes, about 210 minutes to about 240 minutes, up to about 30 minutes, about 30 minutes to about 60 minutes, about 60 minutes to about 90 minutes, about 90 minutes to about 120 minutes, or about 120 minutes to about 150 minutes. In some embodiments, a population of cells (e.g., leukocytes or B cells) provided herein are contacted with a crosslinking agent provided herein for at least about 10 minutes up to 6 hours. In some embodiments, a population of cells (e.g., leukocytes or B cells) is contacted with a crosslinking agent provided herein for about 1 hour. In some embodiments, population of cells (e.g., leukocytes or B cells) are contacted with a crosslinking agent provided herein for about 10 minutes up to 6 hours.

[0120] The contacting can be at any temperature. In some embodiments the contacting is performed on ice (e.g., at 4 °C). In other embodiments, the contacting is performed at room temperature. In some embodiments, the contacting is performed at a temperature of at least about 0 °C, 2 °C, 4 °C, 8 °C, 15 °C, 20 °C, 25, 30 °C, 35 °C, or 37 °C. In some embodiments, the contacting is performed at a temperature of at most about 4 °C, 8 °C, 15 °C, 20 °C, 25, 30 °C, 35 °C, 37 °C, or 40 °C. In some embodiments, the contacting is performed at a temperature of about 0 °C to about 37 °C, about 0 °C to about 25 °C, about 0 °C to about 15 °C, about 0 °C to about 10 °C, about 0 °C to about 8 °C, about 0 °C to about 6 °C, about 0 °C to about 4 °C, about 0 °C to about 2 °C, about 2 °C to about 10 °C, about 2 °C to about 8 °C, about 2 °C to about 6 °C, about 4 °C to about 25 °C, about 4 °C to about 10 °C, about 15 °C to about 37 °C, about 15 °C to about 25 °C, about 20 °C to about 40 °C, about 20 °C to about 37 °C, or about 20 °C to about 30 °C. [0121] In some embodiments, following the contacting with the crosslinking agent, the cells (e.g., leukocytes) provided herein are stored at a temperature of about 4°C or less for a period of time until the cells (e.g., apoptotic leukocytes) provided herein are administered to a subject. In some embodiments, a composition provided herein comprise trace amounts of the crosslinking agent (e.g., less than 10% of the total composition (w/v)). In some embodiments, the purity of a composition provided herein is at least about 90% or more, 95% or more, 99% or more, 99.5% or more, up to 100% purity. Purity can be determined, for example, by mass spectrometry, or liquid chromatography/mass spectrometry (LC/MS).

Gene Therapies and Transgenes

[0122] Provided herein are methods of administering a gene therapy to a subject. In some embodiments, the method comprises, administering to the subject a tolerizing composition provided herein and a gene therapy composition. The gene therapy can be any gene therapy for the treatment of a disease, condition or disorder. Non-limiting examples of gene therapies that can be used in combination with the compositions provided herein include, for example, idecabtagene vicleucel, lisocabtagene maraleucel, talimogene laherparepvec, voretigene neparvovec, onasemnogene abeparvovec, alipogene tiparvovec, atidarsagene autotemcel, brexucabtagene autoleucel, axicabtagene ciloleucel, betibeglogene autotemcel, cambiogenplasmid, elivaldogene autotemcel, gendicine, tisagenlecleucel, and valoctocogene roxaparvovec. In some embodiments, the gene therapy comprises a transgene encoding a-CD40 mAb, an mTOR inhibitor (e.g., Rapamune®), an anti-TNF agent (e.g., Enbrel®), or an anti-IL6 agent (e.g., Actemra®).

[0123] In some embodiments, the gene therapy composition comprises a viral vector and a transgene. Transgenes are routinely delivered by use of viral vectors as gene therapy to a subject in need thereof. Compositions provided herein can incorporate one or more transgenes that are crosslinked with leukocytes and viral antigens provided herein to induce tolerance in a recipient to the transgene and the viral antigen, thereby inducing tolerance to the gene therapy composition. [0124] In some embodiments, transgenes or antigenic fragments thereof are recombinantly expressed and contacted with the leukocyte and viral antigen, for instance AAV capsid, in the presence of a crosslinking agent. In some embodiments, a crosslinking agent crosslinks the viral antigen, an antigenic fragment, or a variant thereof to the leukocyte. [0125] Provided below in Table 2 is a list of exemplary transgenes that can be incorporated in compositions provided herein via a crosslinking agent with a leukocyte and AAV antigen such as AAV capsid of one or more serotypes or an antigenic fragment thereof by use of a crosslinking agent provided herein.

[0126] In some embodiments, a composition provided herein comprises a transgene or a transgene product in Table 2 or a fragment thereof. In some embodiments, the compositions provided herein reduce an immune response to a transgene listed in Table 2 as compared with the transgene administered without the composition provided herein.

Table 2. Exemplary transgenes for crosslinking with leukocytes and viral antigens in embodiments provided herein

Carriers

[0127] Further provided herein are compositions comprising a particle or a carrier. A carrier or a particle provided herein can be used to deliver a composition, a cell, a viral vector, a viral antigen, a transgene, or a combination thereof to a target organ. In some embodiments, the particle is a nanoparticle. A nanoparticle can be any particle between about 10 nm and about 1000 nm in diameter. In suitable embodiments, the diameter of the nanoparticles of the present disclosure is less than about 900 nm in diameter, and more suitably about 500 nm in diameter. In certain such embodiments, the nanoparticles of the present disclosure is between about 10 nm and about 1000 nm, between 20 and about 900 nm, between 30 nm and about 800 nm, between 40 nm and about 700 nm between, 50 and about 600 nm, between 60 nm and about 500 nm, between 100 nm and about 500 nm, between about 30 nm and about 100 nm, or between about 40 nm and about 80 nm in diameter.

[0128] Non-limiting examples of nanoparticles include solid lipid nanoparticles (lipids that are in solid phase at room temperature and surfactants for emulsification, the mean diameters of which range from 50 nm to 1000 nm for colloid drug delivery applications), lipid nanoparticle, nanoemulsions (oil-in-water emulsions done on a nano-scale), albumin nanoparticles, and polymeric nanoparticles, micelles. Nanoparticles can be surface coated to modulate their stability, solubility, and targeting. A coating that is multivalent or polymeric confers high stability. A nonlimiting example includes coating with hydrophilic polymer such as polyethylene glycol or polysorbate-80. The nanoparticle can be, for example, solid-lipid nanoparticles (SLNs), polymeric nanoparticles, or oil-in-water nanoemulsions. Solid — lipid nanoparticles are surfactant-stabilized aqueous colloidal dispersions of lipid nanoparticles that solidify upon cooling. They contain a lipid phase dispersed in an aqueous environment. Polymeric nanoparticles are solid colloidal particles created from polymeric systems.

[0129] In some embodiments, the nanoparticle is a lipid nanoparticle. Lipid nanoparticles can be used as a transfer vehicle and comprise one or more lipids (e.g., cationic lipids, non-cationic lipids, arid PEG-modified lipids) e.g., a liposome.

[0130] Further provided herein are compositions comprising a liposome. Liposomes are vesicular structures having lipid-containing membranes enclosing an aqueous interior. In cell biology, a vesicular structure is a hollow, lamellar, spherical structure, and provides a small and enclosed compartment, separated from the cytosol by at least one lipid bilayer. Liposomes can have one or more lipid membranes. Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 100 nm. Liposomes with several nonconcentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are termed multivesicular vesicles. Liposomes can further comprise one or more additional lipids and/or other components such as sterols, e.g., cholesterol. Additional lipids can be included in the liposome compositions for a variety of purposes, such as to prevent lipid oxidation, to stabilize the bilayer, to reduce aggregation during formation or to attach ligands onto the liposome surface. Any of a number of additional lipids and/or other components can be present, including amphipathic, neutral, cationic, anionic lipids, and programmable fusion lipids. Such lipids and/or components can be used alone or in combination. One or more components of the liposome can comprise a ligand, e.g., a targeting ligand.

[0131] Liposome compositions can be prepared by a variety of methods, for example, solvent dispersion, micro-emulsification, membrane extrusion, drying reconstituted vesicles, sonication, or French pressure cell extrusion.

[0132] Liposomes are vesicular structures with an aqueous core surrounded by a hydrophobic lipid membrane. In some embodiments, a peptide provided herein is encapsulated within a liposome. In some embodiments, a peptide provided herein is in complex with a liposome. Liposomes are contemplated to have either a single layer (uni-lamellar), or multiple phospholipid bilayer membranes (multilamellar structure). In some embodiments, a peptide or a nucleic acid encoding a peptide provided herein is encapsulated in a noisome. Niosomes are non-ionic surfactant-based synthetic vesicles that are not phospholipid based and have properties and function like liposomes.

[0133] Examples of suitable lipids for lipid nanoparticle include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Also contemplated is the use of polymers as transfer vehicles, whether alone or in combination with other transfer vehicles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide- polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate., collagen, chitosan, cyclodextrins, dendrimers and polyethylenimine (PEI).

[0134] In other embodiments, the compositions and methods provided herein are directed to lipid nanoparticles comprising one or more cleavable lipids, such as, for example, one or more cationic lipids or compounds that comprise a cleavable disulfide (S-S) functional group.

[0135] Provided herein are compositions comprising lipid nanoparticles, wherein the lipid nanoparticles comprise a cationic lipid. Cationic lipids are lipid species that carry a net positive charge at a selected pH, such as physiological pH. In some embodiments, the cationic lipid nanoparticle comprises phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates, dialkyl trimethylammonium-propanes, (e.g., DOTAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g., DC-Chol), or derivatives thereof.

[0136] Lipid nanoparticles can be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and PEG- modified lipids. Several cationic lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the compositions and methods include, e.g., C12-200. In some embodiments, the compositions and methods provided herein employ a lipid nanoparticle comprising an ionizable cationic lipid such as, e.g., (15Z, 18Z)~N,N-dimeihyl- 6-(9Zs 12Z)-octadeca-9., 12-dien- 1 -yl)tetracosa- 15,18-dien- 1 - amine (HGT5000), (15Z,18Z)- N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-l - yl)tetracosa-4,l 5,18-trien-l -amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6- ((9Z,12Z)-octadeca-9,12-dien-l-yl)tetracosa-:5,15 8-trien-l - amine (HGT5002).

[0137] In some embodiments, the cationic lipid comprises N-f l-(2,3-dioleyloxy)propyl]-N,NjN- trimethylammonium chloride or "DOTMA" is used. DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or "DOPE" or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, 5- carboxyspermylglycinedioctadecylamide or "DOGS," 2,3 -di oleyloxy -N- [2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium or "DOSPA" l,2-Dioleoyl-3- Dimethylammonium-Propane or "DODAP", L,2-Dioleoyl-3- Trimemylammonium-Propane or "DOTAP". Contemplated cationic lipids also include l,2-distearyloxy-N,N-dimethyl-3- aminopropane or "DSDMA", 1,2- dioleyloxy-N,N-dimethyl-3-aminopiOpane or "DODMA", 1,2- dilinoleyloxy-N,N- dimethyl-3 -aminopropane or "DLinDMA", l,2-dilinolenyloxy-N,N-dimethyl- 3- aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammomum chloride or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB". N-(:l,2- dimyristyloxyprop-3-yl)- N,N-dimethyl-N-hydroxyethyl ammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5- en-3-beta-oxybutan-4-oxy)-l-(ci s,cis-9,12- octadecadienoxy)propane or "CLinDMA", 2-[5'- (cholest-5-en-3-beta-oxy)-3 oxapentoxy)-3-dimethy l-l-(cis,cis-9', 1-2'- octadecadienoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-di oleyloxybenzylamine or "DMOBA", 1,2-N,N'- dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3- Dilmoleoyloxy- N,N-dimethylpropylamine or "DLinDAP", l,2-N,N'-Dilinoleylcai-bamyI-3- dimethylaminopropane or "DLincarbDAP", l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl- [l.,3]-dioxolane or "DLin- -DMA", 2,2- dilinoleyl-4-dimethylaminoethyl-[ 1 ,3]- dioxolane or "DLin- -XTC2-DMA", and 2-(2,2- di((9Z,12Z)-octadeca-9,12-dien-l- yl)-l ,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2- DMA)) or mixtures thereof. The use of cholesterol-based cationic lipids is also contemplated by the present disclosure. Such cholesterol-based cationic lipids can be used, either alone or • in combination with other cationic or non-cationic lipids. Suitable cholesterol-based, cationic lipids include, for example, DC-Choi (N,N-dimethyl-N- ethylcarbbxamidocholesterol), 154-bis(3-N- oleylamino-propyl)piperazine or ICE.

[0138] The use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl- Sphingosine-1- [Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention, either alone or preferably in combination with other lipids together which comprise the transfer vehicle (e.g., a lipid nanoparticle). Contemplated PEG-modified lipids include, but is not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. The addition of such components may prevent aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, or they are selected to rapidly exchange out of the formulation in vivo.

[0139] Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or CIS). The PEG-modified phospholipid and derivatized lipids of the present disclosure may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal transfer vehicle.

[0140] The present disclosure also contemplates the use of non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearqylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphOsphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), di ol eoy Iphosphati dy 1 ethanol amine (DOPE), palmitoyloleoylphosphatidylchdline (POPC), palmitoy i ol eoy 1 - phosphati dy 1 ethanol amine

(POPE), dioleoyl-phosphatidylethanolamine 4-(N~ maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphati dyl -ethanol amine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl

PE, 18-1- trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), cholesterol, or a mixture thereof. Such non-cationic lipids may be used alone, or in combination with other excipients, for example, cationic lipids. When used in combination with a cationic lipid, the noncationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10 % to about 70% of the total lipid present in the transfer vehicle.

[0141] In some embodiments, a lipid nanoparticle is prepared by combining multiple lipid and/or polymer components. For example, a transfer vehicle may be prepared using Cl 2-200, DOPE, chol, DMG-PEG2K at a molar ratio of 40:30:25:5, or DODAP, DOPE, cholesterol, DMG-PEG2K at a molar ratio of 18:56:20:6, or HGT5000, DOPE, chol, DMG-PEG2 at a molar ratio of

40:20:35:5, or HGTSOO1, DOPE, chol, DMG-PEG2K at a molar ratio of 40:20:35:5. The, selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the peptides to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly. For example, in embodiments, the percentage of cationic lipid in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. The percentage of non-cationic lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of cholesterol in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of PEG-modified lipid in the lipid nanoparticle may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.

[0142] In some embodiments, the lipid nanoparticle comprises at least one of the following cationic lipids: DLinKC2DMA, C12-200, DLin- C2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In embodiments, the lipid nanoparticle comprises cholesterol and/or a PEG-modified lipid. In some embodiments, the nanoparticle comprises DMG-PEG2K. In some embodiments,, the nanoparticle comprises one of the following lipid formulations: CI 2-200. DOPE, chol, DMG- PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, chol, DMG-PEG2K, HGT5001, DOPE, chol, DMG-PEG2K.

[0143] Nanoparticles disclosed herein may or may not contain PEG. In addition, certain embodiments can be directed towards copolymers containing poly(ester-ether)s, e.g., polymers having repeat units joined by ester bonds (e.g., R — C(O) — O — R' bonds) and ether bonds (e.g., R — O — R' bonds). In some embodiments of the invention, a biodegradable polymer, such as a hydrolyzable polymer, containing carboxylic acid groups, may be conjugated with polyethylene glycol) repeat units to form a poly(ester-ether). It is contemplated that PEG may include a terminal end group, for example, when PEG is not conjugated to a ligand. For example, PEG may terminate in a hydroxyl, a methoxy or other alkoxyl group, a methyl or other alkyl group, an aryl group, a carboxylic acid, an amine, an amide, an acetyl group, a guanidino group, or an imidazole. Other contemplated end groups include azide, alkyne, maleimide, aldehyde, hydrazide, hydroxylamine, alkoxyamine, or thiol moieties.

[0144] In some embodiments, the nanoparticle is further conjugated with a peptide tag, detecting agent, or a therapeutic agent.

[0145] In some embodiments, the nanoparticle is a micelle formed from lipid-associated peptides disclosed herein, e.g., peptides of the present disclosure conjugated to at least one amphiphilic carrier, in which the micelles have an average diameter of, for example, less than about 1000 nm, preferably. In some embodiments, micelles have an average diameter less than about 500 nm, and in some embodiments, micelles have an average diameter less than about 100 nm, or even less than about 20 nm. Micelles are a type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all hydrophobic portions on the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

[0146] Provided herein are nanoparticles and/or leukocytes comprising or displaying one or more peptides derived from a MHC class II molecule. In some embodiments, the nanoparticles or leukocytes further comprise one or more peptides derived from a MHC class I molecule.

[0147] In some embodiments, the peptides conjugated on the surface of nanoparticles are derived from a MHC molecule. An MHC molecule is a molecule comprising Major Histocompatibility Complex (MHC) glycoprotein protein sequences. A Major Histocompatibility Complex (MHC) is a set of gene loci specifying major histocompatibility complex glycoprotein antigens including the human leukocyte antigen (HLA). Human Leukocyte Antigens (HLA) are the major histocompatibility antigens found in humans. HLA is the human form of MHC and therefore can be used interchangeably. Examples of HLA proteins that can be utilized in accordance with the presently disclosed and claimed inventive concept(s) include, but are not limited to, an HLA class I a chain, an HLA class II a chain and an HLA class II b chain. Specific examples of HLA class II a and/or b proteins that may be utilized in accordance with the presently disclosed and claimed inventive concept(s) include, but are not limited to, those encoded at the following gene loci: HLA-DRA; HLA-DRB1; HLA-DRB3, HLA-DRB4, HLA-DRB5; HLA- DQA; HLA-DQB; HLA-DPA; and HLA-DPB. MHC class II glycoproteins, HLA-DR, HLA-DQ, and HLA-DP (encoded by alleles at the HLA-DR, DP, and DQ loci) have a domain structure, including antigen binding sites, similar to that of Class I. MHC class II molecules are heterodimers, consist of two nearly homologous subunits; a and P chains, both of which are encoded in the MHC. Accordingly, in some embodiments, the MHC class II molecule refers to a heterodimer of MHC class II a chain and MHC class II P chain (e.g., HLA-DQ, HLA-DR, HLA- DP). In some embodiments, the MHC class II molecule can be a subunit of the heterodimer. In some embodiments the MHC class II molecule can be MHC class II a chain (e.g., HLA-DPA, HLA-DQA, or HLA-DRA), or MHC class II p chain (e.g. , HLA-DPB, HLA-DQB, or HLA-DRB), or domains thereof. In some embodiments, the MHC class II molecule is HLA-DRB.

[0148] The HLA-DRB is encoded by four gene loci in human (HLA-DRB 1, HLA-DRB3, HLA- DRB4 and HLA-DRB4), however no more than 3 functional loci are present in a single individual, and no more than two on a single chromosome. In some embodiments, the MHC class II molecule that is HLA-DRB is encoded by HLA-DRB1, HLA-DRB3, HLA-DRB4 or HLA-DRB4 gene locus. In some embodiments, the MHC class II molecule is encoded by HLA-DRB 1*01, HLA- DRBl*03, HLA-DRB1*O4, HLA-DRB1*O7 HLA-DRB1*11, HLA-DRB1*15, or HLA- DRB 1*16 . The HLA-DRB 1 locus is ubiquitous and encodes a very large number of functionally variable gene products (HLA-DR1 to HLA-DR17). The HLA-DRB3 locus encodes the HLA- DR52 specificity, is moderately variable and is variably associated with certain HLA-DRB 1 types. The HLA-DRB4 locus encodes the HLA-DR53. In some embodiments, the MHC class II molecule that is HLA-DRB is selected from HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, or HLA-DR5. In some embodiments, the peptide derived from HLA-DR3 can comprise a sequence selected from Table 3 or Table 4. In some embodiments, the peptide derived from HLA-DR4 can comprise a sequence derived from Table 5.

[0149] Selected MHC class I and II peptides that bind with high affinity to HLA DR3 or DR4 are presented below. Table 3. Table 3 lists exemplary peptides derived from HLA DR3 that are capable of binding HLA DR3 complex.

Table 4. Table 4 lists exemplary peptides derived from HLA DR3 that are capable of binding HLA DR4 complex.

Table 5. Table 5 lists exemplary peptides derived from HLA DR4 that are capable of binding HLA DR3 complex

Immunomodulatory agents

[0150] In some embodiments, the methods and compositions for tolerizing a recipient to gene therapy further comprise administering to the recipient an effective amount of one or more immunomodulatory molecule. In some embodiments, the one or more immunomodulatory molecule are encapsulated into a nanoparticle provided herein, or conjugated to a nanoparticle, viral antigen or leukocyte provided herein. In some embodiments, the one or more immunomodulatory molecule is an anti-CD40 agent, anti-CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor, IFNy, an NFKB inhibitor, vitamin D3, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, alpha 1 anti-trypsin inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), or any combination thereof.

[0151] In some embodiments, the NF-kB inhibitor is curcumin, triptolide, Bay-117085, or a combination thereof. In some embodiments, the one or more immunomodulatory molecule is a JAK3 inhibitor, a recombinant IL-37, a recombinant V-domain immunoglobulin suppressor of T cell activation (VISTA), belatacept, a CTLA4-immunoglobulin, or an CD28 antagonist.

[0152] In some embodiments, the anti-CD40 agent is CD40 siRNA. In some embodiments, the anti-CD40 agent is a CD40 binding peptide inhibitor, anti-CD40 monoclonal antibody, a Fab’ anti- CD40 monoclonal antibody fragment, FcR-engineered, Fc silent anti-CD40 monoclonal domain antibody, anti-CD40 siRNA, a CD40L-binding fusion protein.

[0153] In some embodiments, the anti CD40L agent is an anti-CD40 L monoclonal antibody, a Fab’ anti-CD40L monoclonal antibody fragment CDP7657, a FcR-engineered, Fc silent anti- CD40L monoclonal domain antibody, a Fab’ anti-CD40L antibody, CD40 binding peptides or an Fc-engineered anti-CD40L antibody.

[0154] In some embodiments, the anti-CD40 or the anti-CD40L antibody or antibody fragment comprises: dapirolizumab pegol, dazodalibep, iscalimab, tegoprubart (AT-1501), SAR441344, KPL-404, letolizumab, or APB-A1.

In some embodiments, the immunomodulatory molecules can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), CD40L (CD 154), CD40, inducible costimulatory (ICOS), IL-2, TNF-a, IL-6, IL-7, CD2, CD20, CD52, a-4 integrin, mTOR, DNA synthesis, molecules in pro-inflammatory pathways (e.g., cytokines, al -antitrypsin, NFkB , or any combination thereof. In some embodiments, the immunomodulatory molecule is an NFkB inhbitor (e.g. dehydroxymethylepoxy quinomicin (DHMEQ)). In some embodiments, the one or more immunomodulatory molecule can target B-cell, (e.g., B-cell depleting biologies, for example, a biologic targeting CD20, CD 19, or CD22, and/or B-cell modulating biologic, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1). In some embodiments, the B cell targeting biologic can be anti-CD20 mAb (such as rituximab) or other B- cell depleting antibody. In some embodiments, the immunomodulatory molecules can be a MHC/TCR interaction blockade, a nonselective depleting agent, calcineurin inhibitor, costimulatory signal blockade, cytokine blockade, B cell modulating agent, lymphocyte depleting agent, cell adhesion inhibitor, IL-2 signaling inhibitor, cell cycle blocker, or any combination thereof. For example, the MHC/TCR interaction blockade can be anti-abTCR mAb T10B9. For example, the nonselective depleting agent can be anti-CD3 mAb (OKT3) or antithymocyte globulin (ATG). For example, the calcineurin inhibitor can be cyclosporine or tacrolimus. For example, the costimulatory signal blockade can be anti-CELA-4 mAb, abatacept, ipilimumab, anti-PD-1 (such as pembrolizumab), anti-PD-Ll (such as MPDL3280A), anti-CD154 mAb, Fc- engineered anti-CD40L antibodies, anti-CD40 mAb, or anti-ICOS mAb. For example, the cytokine blockade can be anti-CD25 mAb (such as daclizumab or basiliximab), anti-TNF (infliximab), anti-IL-6 mAb (such as ALD518, tocilizumab), or anti -IL-7 mAb. For example, the lymphocyte depleting agent can be anti-CD2 mAb, fusion protein with IgGl (such as alefacept), anti-CD20 mAb (such as rituximab), or anti-CD52 mAb (such as alemtuzumab). For example, the cell adhesion inhibitor can be anti -very large antigen 4 (VLA4) (such as natalizumab). For example, the mTOR inhibitor can be sirolimus (rapamycin) or everolimus or any other mTOR inhibitor. For example, the cell cycle blocker can be my cophenolate mofetil (MMF) or azathioprine. In some embodiments, the immunomodulatory molecules can be T cell recirculation inhibitors (e.g., FTY720 and other sphingosine 1 -phosphate (SIP) receptor agonists.

[0155] In some embodiments, a composition such as a tolerizing composition or a tolerizing regimen provided herein can be administered with or without one or more immunomodulatory molecules that inhibit T cell activation. The immunomodulatory molecules that inhibit T cell activation can be an anti-CD40 or anti-CD40L (CD 154) agent. The anti-CD40 or anti-CD40L agent can be an antibody, for example, an antagonistic antibody. The anti-CD40 or anti-CD40L antibody can be a Fab’ anti-CD40L monoclonal antibody fragment CDP7657. The anti-CD40 or anti-CD40L antibody can be a FcR-engineered, Fc silent anti-CD40L monoclonal domain antibody, a Fab’ anti-CD40L antibody, or an otherwise Fc-engineered anti-CD40L antibody. In some embodiments, a composition, a tolerizing regimen or a preparatory regimen can further be administered with one or more additional immunomodulatory molecules provided herein; for example, with one or more of a B-cell targeting biologic (e.g., B cell depleting biologic, for example, a biologic targeting CD20, CD 19, or CD22, and/or B cell modulating biologic, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, al -antitrypsin, a nitrogen mustard alkylating agent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, IFNy, an NFKB inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein (e.g., CD46, CD55, or CD59), any combination thereof, or any fragment thereof. In some cases, the NFKB inhibitor is curcumin, triptolide, Bay-117085, or a combination thereof. Nonlimiting examples of B-cell targeting biologic include Rituximab, anti-CD20 antibody. In some embodiments, a composition, a tolerizing regimen or a preparatory regimen can be administered with a B-cell depleting antibody. In some embodiments, a composition, a tolerizing regimen or a preparatory regimen is not required to be administered with a B-cell depleting antibody.

[0156] In some embodiments, a tolerizing regimen or preparatory regimen provided herein can also include a complement C3 or C5 inhibitor. Some non-limiting examples of such inhibitors include Compstatin or analogs thereof such as pegcetacoplan and AMY-101, APL-02, Eculizumab (Soliris®), Berinert, Cinryze, Avacopan, LNP023, OMS721 etc.

[0157] In some embodiments, the one or more immunomodulatory molecules comprise MMF (my cophenolate mofetil (Cellcept®)), ATG (anti -thymocyte globulin), anti-CD154 (CD40L), alemtuzumab (Campath®), B-cell targeting agent (e.g., B cell depleting biologies, for example, a biologic targeting CD20, CD 19, or CD22, and/or B cell modulating biologic, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), anti-IL-6R antibody (tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab), CTLA4-Ig (Abatacep®t/Orencia®), belatacept (LEA29Y), sirolimus (Rapamune®), tacrolimus (Prograf®), daclizumab, basiliximab (Simulect®), infliximab (Remicade®), cyclosporin, deoxyspergualin, soluble complement receptor 1, cobra venom factor, compstatin, anti C5 antibody (eculizumab/Soliris®), methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody, human anti-CD154 monoclonal antibody, CD40 antagonist, and CD40L (CD154) antagonist. Non-limiting examples of B-cell targeting biologic include rituximab, anti- CD20 antibody.

Methods of Use and Dosing

[0158] The compositions of the present disclosure can take the form of a tolerogenic composition, a tolerizing regimen or a preparatory regimen. In some embodiments, the tolerogenic composition, tolerizing regimen, and/or preparatory regimen provided herein modulates an immune response to a recombinant viral vector when administered to a subject.

[0159] Further provided herein are the use of a tolerizing regimen to induce immune tolerance to a gene therapy. In some embodiments, the tolerizing regimen is used for AAV gene therapy. An AAV vector used in the tolerizing regimen and the AAV vector used in the gene therapy have at least one serotype in common.

[0160] In some embodiments, the AAV capsid protein-conjugated ex vivo expanded B cells derived from the patient/subject, or AAV capsid protein-recombinantly expressed transgene- conjugated, ex vivo expanded B cells derived from the patient/subject, are administered intravenously or via local injection on days -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1 and +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +16, +17, +18, +19, +20, or +21 relative to the first administration of an AAV gene therapy vector of the same AAV serotype as the AAV capsid protein-conjugated, ex vivo expanded B cells. In this embodiment, immunomodulatory agents provided herein, including, for example, an agent that suppresses CD40:CD40L costimulation, for instance antagonistic agents (e.g., antagonistic a-CD40 mAb), a protein kinase inhibitor (e.g., mTOR inhibitor), and an agent that suppresses inflammatory cytokines (e.g., sTNFR), for instance an anti-IL6 agent, for instance an anti-IL-6 mAb (e.g., anti-IL-6R mAb) are administered between days -8 and +21.

[0161] In some embodiments, AAV capsid proteins of a given AAV serotype are encapsulated in nanoparticles particles optionally in the presence of recombinantly expressed transgene. These nanoparticles can be, for example, PLGA and have a diameter of about 500 nm. MHC class II DRB peptides are encapsulated in negatively-charged nanoparticles, which may also be PLGA with a diameter of about 500 nm. Both the AAV capsid nanoparticles and the MHC class II nanoparticles can be co-administered to a patient on days -7 and +1 relative to the first administration of the AAV gene therapy vector of the same AAV serotype on day 0. The administration may coincide with the administration of immunomodulatory agents. The immunomodulatory agents may contain an agent that suppresses CD40:CD40L co-stimulation, for instance antagonistic agents (e.g., antagonistic a-CD40 mAb), a protein kinase inhibitor (e.g., mTOR inhibitor), and an agent that suppresses inflammatory cytokines (e.g., sTNFR), for instance an anti-IL6 agent, for instance an anti-IL-6 mAb (e.g., anti-IL-6R mAb). The immunomodulatory agents may be administered between days -8 and +21 in some instances.

[0162] Provided herein are methods of inducing tolerance to a gene therapy. As used herein, the term “tolerance” or “immune tolerance” refers to a state of unresponsiveness of the immune system to substances or tissues that have the capacity to elicit an immune response. Compositions of the disclosure are useful for achieving tolerance or partial tolerance against the gene therapy upon administration of said gene therapy. As used herein, a “partial tolerance” is a partial immune tolerance results in a reduced immune response. As used herein, the term “immune response” includes T cell mediated and/or B cell mediated immune responses. The B cell response can be B- cell activation, B-cell proliferation, and production of neutralizing antibodies specific for the viral antigen. The B cell response can be B- cell activation, B-cell proliferation, and production of antibodies specific for the viral antigen. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity, in addition, the term immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. The T cell response can be T cell activation or proliferation, generation of memory T cells, generation of T cell effector function, generation of T cell effector function involving cytokines or cytolytic mechanisms. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4+, CD8+, Thl and Th2 cells); antigen presenting cells (e.g. professional antigen presenting cells such as dendritic cells); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0163] Thus, treated subjects with the compositions disclosed herein in comparison with untreated subjects, display, for example, : a) a decreased level of an immune response against the gene therapy (thought to be mediated at least in part by B cell mediated immune responses, more particularly donor-specific antibodies); b) a delay in the onset or progression of an immune response against the gene therapy; c) a reduced risk of the onset or progression of an immune response against the gene therapy, or induction of regulatory T cells, Tri cells, Breg cells, BIO cells, myeloid derived suppressor cells or induction of immunosuppressive cytokines. The effect of the use of a composition of this disclosure can be seen by observation of a change in the level of an immune cell (e.g, increase in number of tolerogenic APC, increase in number of Tregs, increase in number of Tri cells, decrease in CD4+, CD8+ and/or CD20+ cells).

[0164] Methods of this disclosure can include modulating immune response to a gene therapy in a recipient. This method can include administering to the recipient any composition of this disclosure in an amount effective to modulate immune response generated by contacting the recipient with a recombinant viral vector.

[0165] Methods of this disclosure can include a method for sustained expression of a transgene in a recipient. This method can include administering to the recipient a composition of this disclosure prior to, simultaneously and/or subsequent to administering the recombinant viral vector comprising the transgene.

[0166] In embodiments provided herein, one or more doses of the composition or the preparatory regimen or the tolerizing regimen can be administered to a gene therapy recipient. The one or more dose(s) of the composition, preparatory regimen or tolerizing regimen can be administered before and/or during and/or after the gene therapy. The day of gene therapy can be referred to as day 0. Preceding days relative to day 0 (the day the recipient receives the gene therapy) can be referred to by negative numbers. For example, a composition or preparatory regimen administered 7 days before the gene therapy, can be designated as being administered on day -7. Similarly, days following the day the recipient receives the gene therapy can be referred to by positive numbers. For example, a composition or preparatory regimen administered 7 days after the gene therapy, can be designated as being administered on day 7 or day +7.

[0167] In some cases, a dose of a composition, a preparatory regimen or a tolerizing regimen is administered at least or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,

21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days,

32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days,

43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days,

54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days prior to gene therapy.

[0168] In some cases, a dose of a nanoparticle composition, a preparatory regimen or a tolerizing regimen is administered on the same day the recipient receives the gene therapy (e.g., the dose is administered on day 0). A dose administered on day 0 can be administered concurrently with the gene therapy, or within 24 hours of the gene therapy. For example, the dose of the composition or preparatory regimen can be administered at -23, -22, -21, -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours relative to the gene therapy.

[0169] In some cases, a dose of a composition, a preparatory regimen, or a tolerizing regimen is administered at least or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,

54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days after gene therapy.

[0170] A preparatory regimen and/or a tolerizing regimen can comprise multiple doses of a composition before, and/or during and/or after gene therapy. The multiple doses can be referred to as comprising an initial dose and one or more booster doses. In some embodiments, the methods disclosed herein comprise administering an initial dose of a composition, a preparative regimen, or a tolerizing regimen and subsequently administering a booster dose of a composition, a preparative regimen, or a tolerizing regimen. Typically, the initial dose occurs prior to or concurrently with the gene therapy. The booster dose(s), when administered, occur after the initial dose. Depending upon when the initial dose of the compositions disclosed herein is administered, one or more booster doses can be administered before, and/or concurrently with, and/or after the gene therapy. [0171] Subsequent (e.g., booster) dose(s) of a composition, preparative regimen, tolerizing regimen disclosed herein can be administered in any interval of time following a preceding dose (e.g., an initial dose). For example, the subsequent dose can be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days,

27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days,

38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days,

49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days,

60 days, 90 days, 120 days, 150 days, or 180 days after the preceding dose. Depending upon when the initial dose is administered subsequent (booster) dose(s) can be administered before, concurrently with, or after the gene therapy. In some cases, the preparatory regimen and/or the tolerizing regimen comprises at least one dose of the compositions disclosed herein prior to gene therapy. In some cases, the preparatory regimen and/or the tolerizing regimen comprises at least two doses of the compositions prior to gene therapy (e.g., an initial dose and a booster dose). In some cases, the preparatory regimen and/or the tolerizing regimen comprises at least three doses of composition prior to gene therapy (e.g., an initial dose and two booster doses). In some cases, the preparatory regimen and/or tolerizing regimen comprises an initial dose of composition prior to gene therapy and at least one dose of booster composition concurrently with or after the gene therapy.

[0172] In some cases, the methods disclosed herein comprises administering to a recipient at least two doses of the compositions or the tolerizing regimens. For example, the first dose can be administered on day -12 relative to gene therapy on day 0. For example, the second dose can be administered on day -4 relative to gene therapy on day 0. For example, the first dose can be administered on day -11, -12, -13, or -14 relative to gene therapy on day 0. For example, the second dose can be administered on day -3, -4, -5, or -6 relative to gene therapy on day 0. In some cases, a second dose of the composition disclosed herein (e.g., a booster dose) can be administered on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to gene therapy on day 0. For example, the second dose of the composition disclosed herein (e.g., a booster dose) can be administered on 1 day after (e.g., day 1) gene therapy.

[0173] In some cases, a third dose of the composition disclosed herein (e.g., a booster dose) can be administered on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to gene therapy on day 0. For example, the composition can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to gene therapy on day 0.

[0174] In some cases, a fourth dose of the composition (e.g., a booster composition) can be administered on day 600, day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to gene therapy on day 0. For example, the composition can be administered on or on about day 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to gene therapy on day 0.

[0175] In some cases, a fifth dose of the composition (e.g., a booster composition) can be administered on day 1,000, day 900, day 800, day 700, day 600, day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to gene therapy on day 0. For example, the composition can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to gene therapy on day 0. In some cases, a second dose of a booster dose is not required. In some cases, a second dose of a booster composition is given concomitantly on day 0 with gene therapy. [0176] In some cases, the long term tolerance is for a period of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least thirteen months, at least fourteen months, at least fifteen months, at least sixteen months, at least seventeen months, at least eighteen months, at least nineteen months, at least twenty months, at least twenty-one months, at least twenty -two months, at least twenty -three months, or at least twenty-four months. In some cases, the long term tolerance is for a period of at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some cases, the long term tolerance is achieved in the absence of a booster dose or booster regimen. In some cases, the long term tolerance is achieved with an administration of a booster dose or booster regimen in one or multiple doses. In some cases, one or more booster doses are administered on the day of, or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days,

33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days,

44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days,

55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days,

90 days, 95 days, 100 days, 105 days, 110 days, 115 days, 120 days, 125 days, 130 days, 135 days, 140 days, 145 days, 150 days, 155 days, 160 days, 165 days, 170 days 175 days, 180 days, 185 days, 190 days, 195 days, 200 days, 205 days, 210 days, 215 days, 220 days, 230 days or 240 days after the gene therapy. In certain specific cases, one or more (for instance three) doses of a preparatory regimen is administered prior to gene therapy, and one or more booster vaccine doses are provided 1, 7, 14, 21, 90, or up to 180 days after gene therapy.

[0177] The compositions and the tolerizing regimens disclosed herein for each dose of administration can be suspended in a volume suitable for transfusion. For example, the compositions can be suspended in a volume of about: 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 41 ml, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml, 49 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, or 500 ml. For example, the compositions and the tolerizing regimens disclosed herein for each dose of administration can be suspended in a volume of about: 0.1 ml to 1 ml; 1 ml to 10 ml; 10 ml to 50 ml; 50 ml to 100 ml; 100 ml to 200 ml; 200 ml to 300 ml; 300 ml to 400 ml; or 400 ml to 500 ml.

[0178] In some embodiments, the compositions and tolerizing regimens provided herein are administered (e.g., by intravenous infusion) in a volume that varies depending upon the weight of the recipient. For example, the composition, the tolerizing regimens and/or the preparatory regimens can be given intravenously in a volume of at least or at least about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 20 ml, 30 ml, 40 ml or 50 ml per kg recipient body weight, e.g., at least or at least about 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to 40; or 40 to 50 ml per kg recipient body weight. In some embodiments, the composition (e.g., comprising nanoparticle) is given intravenously in a volume of about 7 ml per kg recipient body weight. Booster doses of a composition can comprise lower dose than an initial dose. For example, a booster or subsequent dose of the composition can be lower by about: 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75% or more than the initial or preceding dose of the compositions or preparatory regimen disclosed herein. Pharmaceutical Compositions and Formulations

[0179] Provided herein are pharmaceutical compositions comprising an effective amount of a composition comprising of any one of aspects provided herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0180] The compositions provided herein can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like, including those adapted for the following: (1) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (2) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (3) intravaginally or intrarectally, for example, as a pessary, cream or foam; (4) ocularly (e.g., intravitreally); (5) transdermally; (6) transmucosally; or (7) nasally. The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0181] The compositions disclosed herein can comprise a preservative. A preservative is a compound which can be added to the diluent to essentially reduce bacterial action in the reconstituted formulation, thus facilitating the production of a multi-use reconstituted formulation, for example. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride.

[0182] A pharmaceutical composition provided herein is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, oral, otic, and rectal administration. In some embodiments, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, ocular, otic administration, or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. [0183] If any compositions or immunomodulatory agents disclosed herein are to be formulated for oral administration, compositions can be formulated orally in the form of tablets, capsules, cachets, gel caps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g, sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).

[0184] In some embodiments, the methods of the disclosure can comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0185] In some embodiments, pharmaceutical compositions provided herein or immunomodulatory agents provided herein are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. Preferably, the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid form should be stored at between 2° C. and 8° C. in its original container. [0186] The pharmaceutical compositions can be administered in various ways, depending on the preference for local or systemic treatment, and on the area to be treated. Administration may be done topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, subdural, intramuscular or intravenous injection, or via an implantable delivery device. Formulations for topical administration may include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Compositions for oral administration include powders or granules, suspensions or solutions in water or nonaqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable. Formulations for parenteral administration may include, but are not limited to, sterile solutions, which may also contain buffers, diluents and other suitable additives. Formulations for implantable delivery devices may similarly include, but are not limited to, sterile solutions, which may also contain buffers, diluents and other suitable additives.

Therapeutic applications

Methods for reducing adverse events associated with gene therapy

[0187] With FDA and EMA approvals for Zolgensma®, Luxturna®, and Glybera®, recombinant adeno-associated viruses (rAAVs) are considered efficient and successful vectors for the treatment of inherited disorders. However, the immunotoxicities associated with AAV gene therapy and immune-mediated injury of transduced cells are affecting the clinical development of AAV gene therapies for other disorders. FDA-CBER convened a Cellular, Tissue and Gene Therapies Advisory Committee (CTGTAC) in September of 2021 to discuss the toxicity of AAV vector-based gene therapy products.

[0188] Hepatotoxicity is the most common adverse event associated with intravenous (systemic) administration of AAV vectors. Severe hepatotoxicity has been reported by subjects receiving high doses of recombinant AAV vectors to treat spinal muscular atrophy (SMA type I), X-linked Myotubular Myopathy (XLMTM), and Duchenne’s Muscular Dystrophy (DMD). Hepatotoxicity presented in these clinical trials as elevated liver enzymes, drug-induced liver injury, hepatic failure, and/or death. Accordingly, in embodiments provided herein are compositions and methods for reducing hepatotoxicity in a recipient of AAV gene therapy encoding a transgene, wherein the compositions and methods comprise a tolerizing composition comprising B cells from the recipient or B cells MHC class 2 matched to the recipient that are crosslinked with an AAV antigen such as an AAV capsid and recombinantly expressed transgene, wherein the AAV capsid shares a serotype with the AAV used in the gene therapy. In some embodiments, compositions provided herein are administered to a recipient of a gene therapy to reduce or treat one or more of elevated liver enzymes, drug-induced liver injury, hepatic failure as compared to the recipient in the absence of said administering. In some embodiments, additional immunomodulatory compositions are administered to the subject as part of a tolerizing regimen provided herein.

[0189] To identify strategies for preventing the acute immunotoxicity associated with AAV gene therapy, it is necessary to understand the underlying root cause triggers and the resulting innate, humoral, and cellular immune responses to AAV vectors. A myriad of immune responses against the rAAV vector have been demonstrated in several preclinical studies and clinical trials, and additional mechanisms contribute to immune responses to the transgene product.

[0190] Vector manufacturing and purification methods are a possible contributor to innate immune activation and resulting adverse effects. The innate immune response can prime and augment adaptive immunity against rAAVs. The elicited immune responses are known to be rAAV vector dose dependent; the first 3 deaths in the ASPIRO XLMTM trial all occurred in the high- dose cohort (3 x 10^A14 vg/kg). In some embodiments, provided herein are methods of improving tolerance for high dose rAAV vector gene therapy by administering tolerizing compositions provided herein, for instance a tolerizing composition comprising B cells from the recipient or B cells MHC class 2 matched to the recipient that are crosslinked with an AAV antigen such as an AAV capsid and recombinantly expressed transgene, wherein the AAV capsid shares a serotype with the rAAV used in the gene therapy. In some embodiments,, additional immunomodulatory compositions are administered to the subject as part of a tolerizing regimen provided herein.

[0191] Provided herein are compositions for use as a tolerizing composition, vaccine, or preparatory regimen to mitigate or reduce immune response in a recipient of a gene therapy. A cell crosslinked by use of a chemical crosslinking agent to a viral antigen such as an AAV capsid can be given to a recipient prior to, concurrently with, or after receiving gene therapy with an AAV vector. In some embodiments, the AAV shares a serotype with an AAV capsid. The cell can be a leukocyte, and can be the subject’s own cells. The cells can be ex vivo expanded. Alternatively, the viral antigen such as the AAV capsid may be encapsulated in, or conjugated to the surface of, a nanoparticle, which may be a lipid or polymeric nanoparticle.

[0192] The approaches provided herein mitigate humoral and cellular immune responses to AAV gene therapy vectors. In some embodiments, a transgene product or fragment or variant thereof is also delivered in conjunction with one or more compositions provided herein by, for example, conjugating the transgene product or fragment or variant thereof to the surface of a cell or other delivery system. Inducing immune tolerance to the AAV capsid and the transgene product by one or more intravenous infusions of autologous leukocytes crosslinked to an AAV capsid antigen and/or a transgene product prevents reactivation of memory T and B cell responses and elicitation of de novo antibody and T cell immune responses to a subsequent gene therapy and allows re-administration of AAV gene therapy to achieve and maintain long-term transgene expression, thereby greatly improving patient satisfaction, outcomes, and enhancing the efficacy of gene and cell therapies. Further, inducing tolerance to the AAV capsid prior to the first administration of AAV gene therapy by administering compositions provided herein prevents (re)activation of viral antigen- and capsid-specific T cells and the immunotoxicities associated with the cytolytic response by these T cells to transduced cells that express the processed viral capsid antigen on their surface by their MHC class I molecules. In some embodiments,, patient/recipient-type MHC class II DRA or DRB peptides or variants or derivatives thereof are delivered with the viral antigen and/or transgene product, which can enhance the regulatory responses by providing copious amounts of recipient-specific MHC DRB peptides for presentation on the recipient’s MHC class II DRB molecules, thus providing thymic (t) T regulatory cell activation signals (tTregs). These activated tTregs can exhibit a substantial increase in regulatory potency and can promote the expansion of T regulatory type 1 (Tri) cells with direct or indirect specificity for the AAV capsid antigens and expansion of other cells with regulatory phenotypes and functions such as Breg and BIO cells.

[0193] The compositions and methods provided herein can be used to prevent unwanted immune responses to an antigen such as a transgene product or gene therapy vector, enable repeat AAV gene therapy dosing, and allow for greater and long lasting transgene expression. The full length protein or fragment or derivative of the transgene product can be crosslinked to the surface of the leukocyte or to the surface of nanoparticles. In some embodiments, the leukocytes are transduced with the AAV vector carrying the transgene that includes the transgene product prior to crosslinking. The leukocytes transcribe and translate the transgene protein. Once the leukocytes are given to the recipient, the apoptotic bodies contain antigenic protein epitopes from the full length transgene product.

[0194] Further provided herein are methods of suppressing one or more immune responses against rAAV vector gene therapies in a subject, for instance as described in Table 6, by administering to the subject tolerizing compositions provided herein, for instance a tolerizing composition comprising B cells from the recipient or B cells MHC class 2 matched to the recipient that are crosslinked with an AAV antigen such as an AAV capsid and recombinantly expressed transgene, wherein the AAV capsid shares a serotype with the rAAV used in the gene therapy. In some embodiments,, additional immunomodulatory compositions are administered to the subject as part of a tolerizing regimen provided herein. Table 6. Immune responses against rAAV vector gene therapies

[0195] In some embodiments, acute immunotoxicity of AAV gene therapy is mediated by capsid antigen-specific cytotoxic T lymphocytes (CTLs). In some embodiments, acute immunotoxicity of AAV gene therapy is mediated by the complement system (e.g., classical complement pathway activation). Further provided herein are methods of suppressing or reducing acute immunotoxicity mediated by CTLs or by the complement system against rAAV vector gene therapies in a subject, the methods comprising: administering to a subject a tolerizing compositions provided herein. In some embodiments, the tolerizing composition comprises B cells from the recipient or B cells MHC class 2 matched to the recipient that are crosslinked with an AAV antigen such as an AAV capsid and recombinantly expressed transgene. In some embodiments, the AAV capsid shares a serotype with the rAAV used in the gene therapy. In some embodiments,, additional immunomodulatory compositions are administered to the subject as part of a tolerizing regimen provided herein.

CD8+ T Cell-Mediated Immunity Against Transduced Cells:

[0196] Immune mechanistic studies analyzing the underlying factors contributing to liver injury in the rAAV2-Factor IX trial in hemophilia and in subsequent trials in other indications suggest that cellular immune responses to the vector contributed to the recently experienced severe adverse events (SAEs) following high-dose rAAV gene therapy.

[0197] Memory T cells with specificity for AAV capsid antigens are generated during childhood after natural infections. Subjects participating in rAAV gene therapy trials carry capsid-specific CD8+ memory T cells against AAVs and the elicited immune response is dose-dependent. Following systemic administration of rAAV vector gene therapy, transgene and capsid peptides are cross-presented via MHC class I molecules by transduced cells (e.g., hepatocytes) to CD8+ memory T cells and to naive CD8+ T cells. Transfected dendritic cells are likely even more potent in reactivating memory CD8+ T cells and in priming naive CD8+ T cells. These anti-AAV CD8+ T cells can mediate severe hepatotoxicity and transduced cell clearance within days after gene therapy. In a considerable fraction of subjects participating in high-dose AAV gene therapy trials the cytotoxicity of capsid-specific CD8+ T cells is not effectively countered by concomitant administration of glucocorticosteroids.

[0198] Further provided herein are methods of suppressing or reducing cytotoxicity of capsidspecific CD8+ T cells against rAAV vector gene therapies in a subject, the method comprising: administering to the subject a tolerizing composition provided herein. In some embodiments, the tolerizing composition comprises B cells from the subject in need of a gene therapy. In some embodiments, the B cells are MHC class II matched to the subject that are crosslinked with an AAV antigen such as an AAV capsid and recombinantly expressed transgene. In some embodiments, the AAV capsid shares a serotype with the rAAV used in the gene therapy. In some embodiments, an additional immunomodulatory composition is administered to the subject as part of a tolerizing regimen provided herein. Complement Activation-Associated Host Cell Damage

[0199] Complement activation following high dose rAAV administration can amplify immune responses against capsid antigens and facilitate host cell damage. The acute kidney injury, hemolysis and thrombocytopenia (hemolytic uremic syndrome) experienced by three participants in DMD gene therapy trials (NCT03362502 and NCT03368742) treated with rAAV9-mini- dystrophin gene therapy were accompanied by activation of the complement system and was associated - in at least one subject - with a rapid antibody response. The inflammatory toxi cities associated with high dose rAAV administration resulted in part from classical pathway complement activation triggered by binding of pre-existing or newly formed antibodies to the AAV capsid, with soluble C3a recruiting macrophages and neutrophils to the target tissues and membrane-bound C3b opsonizing antigens, leading to the formation of the membrane attack complex and cell death. Complement activation fragments bound to antigens provide a powerful costimulation signal to B cells, thereby increasing the amplitude of the antibody response. Thus, besides interfering with vector transduction in AAV gene therapy, preformed and newly formed antibodies to rAAV capsid antigens can elicit complement activation and cause adverse events.

[0200] In some embodiments, provided herein are methods of suppressing or reducing complement activation and cause adverse events associated with complement activation against rAAV vector gene therapies in a subject, by administering to the subject tolerizing compositions provided herein, for instance a tolerizing composition comprising B cells from the recipient or B cells MHC class 2 matched to the recipient that are crosslinked with an AAV antigen such as an AAV capsid and recombinantly expressed transgene, wherein the AAV capsid shares a serotype with the rAAV used in the gene therapy. In some embodiments,, additional immunomodulatory compositions are administered to the subject as part of a tolerizing regimen provided herein.

Strategies to Control Immune Responses to AA V Vectors and Transgene Products

[0201] Several lines of evidence suggest that the immune tolerance platform technology provided herein, for instance tolerizing compositions and regiments provided herein mitigate and prevent the cytopathic response mediated by capsid antigen-specific CD8+ T cells. Preemptive presentation of antigen conjugated onto apoptotic leukocytes to the immune system under the cover of transient immunotherapy with antagonistic anti-CD40 mAb, rapamycin (sirolimus), sTNFR and anti-IL-6R mAb can effectively and safely induce immune tolerance to gene therapy. Mechanisms of immune tolerance by compositions and methods provided herein can include anergy, deletion of antigen-specific T cells, expansion of an antigen-specific regulatory network, suppression of effector T cell expansion and function, and exhaustion of antigen-specific, T cells (e.g., PD-1 positive T cells). Apoptotic donor leukocytes (ADLs) in tolerizing compositions provided herein combined with anti-CD40 antibody can demonstrate efficacy in suppressing the elicitation of gene therapy-specific antibodies, suppress the expansion of CD8+ effector memory T (Tern) cells, and promote the expansion of FoxP3 -positive Tregs and additional immune regulatory cell subsets in recipients.

[0202] Preemptive delivery of AAV capsid antigens via apoptotic autologous leukocytes (AAV- AALs) as part of tolerizing compositions provided herein under the cover of transient immunotherapy with anti-CD40 mAb, rapamycin (sirolimus), sTNFR and anti-IL-6R mAb, can induce deletion, regulation, and exhaustion of AAV capsid antigen-specific T cells before and shortly after AAV gene therapy (Figure 4). The resulting immune tolerance to AAV capsid antigens is expected to be robust and long-lasting; thereby markedly mitigating if not eliminating the severe immunotoxicity associated with high dose rAAV vector therapy reported in recent clinical trials in subjects immunosuppressed with corticosteroids. Corticosteroids initiated prior to vector administration and continued for at least 30 days is part of the administration protocol for Zolgensma and similar protocols are used for many AAV products currently in clinical development. The use of corticosteroids has improved outcomes in AAV clinical trials but often fails to prevent the acute immunotoxicity mediated by reactivated memory CD8+ T cells and the loss of transgene expression and presents risks associated with generalized immunosuppression.

[0203] In embodiments provided herein is a method of contraction of the clone size of AAV capsid-specific T cells shortly after the first and second intravenous administration of tolerizing compositions provided herein under the cover of tolerizing regimen including anti-CD40, rapamycin, sTNFR and anti-IL-6R. The contraction of the capsid-specific T cell pool prior to AAV gene therapy would profoundly reduce the risk of acute immunotoxicity mediated by AAV- specific T cells.

[0204] Naturally occurring regulatory T cells (nTregs), which express the transcription factor FoxP3, play indispensable roles in the maintenance of immunological self-tolerance and homeostasis. The majority of nTregs are thymus-derived (thymus-derived Tregs [tTregs]) and a subset is generated in the periphery from conventional T (Tconv) T cells (peripherally derived Tregs [pTregs]). While these CD4+ Treg cells are the most thoroughly studied regulatory cell type, several other immune cells contribute to peripheral tolerance and immune homeostasis. Infusions of compositions provided herein under transient immunosuppression can expand several regulatory cell subsets in NHPs, including Treg, Tri, Natural Suppressor, and Breg cells and their persistence can be associated with significant reduction of adverse effects in gene therapy.

[0205] Clinical trials of intramuscular gene therapy indicate that rAAV gene transfer may be able to initiate a regulatory T cell response that allows - albeit at reduced levels - ongoing transgene expression, thereby highlighting the potential of tolerance to AAV capsids and transgene products. However, regulatory T cell responses have not been observed in clinical trials following intravenous gene therapy. Inducing an antigen-specific regulatory network by the infusion of antigen-carrying apoptotic cells, as demonstrated in preclinical transplant studies, could mitigate immunity to transduced cells following systemic AAV gene therapy and facilitate stable transgene expression at clinically relevant levels. The in vivo expansion and activation of antigen-specific Tregs and Tri cells with the proposed strategy would overcome the challenges of identification of the relevant antigens, in vivo stability, and migratory behavior of Treg cells that are associated with adoptive transfer of Tregs, including the transfer of capsid specific chimeric antigen receptor T regulatory cells.

[0206] Mechanisms by which in vivo generated/expanded and activated Tregs and Tri cells could protect from the immunotoxicities associated with AAV gene therapy are multifaceted. Firstly, regulatory T cells are expected to suppress the expansion and effector functions of memory and de novo CD8+ T cells through an antigen presenting cell (APC)-dependent pathway or an APC-independent pathway involving inhibitory cytokines or Granzyme/perforin cytolytic mechanisms. Secondly, regulatory T cells could mediate exhaustion in non-deleted, capsidspecific CD8+ T cells and maintain these cells in an exhausted state. Finally, by tolerizing capsidspecific CD4+ T helper cells and restraining their “help (CD40L)”, regulatory T cells would restrict effector functions of capsid-specific CD8+ T cells, B cells (and their antibody production and class switching), and inflammatory cells.

[0207] T cell exhaustion describes a state of deteriorating T cell function in response to chronic antigen stimulation in the settings of chronic viral infection and cancer. These cells co-express multiple inhibitory receptors (e.g., PD-1), have functional defects (e.g., compromised ability to secrete cytokines) and develop an altered transcriptional, epigenetic, metabolic, and differentiation program. Mimicking a chronic viral infection, exhausted T cells have been detected in situ in muscle biopsies of patients in whom long-term capsid and transgene persistence were demonstrated after intramuscular AAV gene therapy. In some embodiments, intravenous AAV capsid antigen delivery via apoptotic autologous cells as part of a tolerizing composition provided herein induces a state of exhaustion in capsid-specific T cells following intravenous AAV gene therapy. First, infusions of a large dose of AAV capsid antigen on days -7 and +1 relative to the first vector administration on day 0 can provide a high load of capsid antigen. Second, the infusion of antigen-carrying apoptotic leukocytes can cause a rapid and extensive proliferation of antigenreactive CD4+ and CD8+ T cells. Third, splenic marginal zone macrophages (MZMs) can express IL- 10 following IV infusion of leukocyte compositions provided herein, and both MZMs and splenic DCs can demonstrate an IL-10 dependent increase in the expression of PD-L1 following internalization of ADLs. Fourth, the percentages of circulating PD-1+ CD4+ and CD8+ T cells can significantly increase following infusions of leukocyte compositions provided herein. Fifth, concomitant therapy with antagonistic anti-CD40 mAbs can interfere with CD4+ help, thereby promoting CD8+ T cell exhaustion. CD40:CD40L costimulation blockade with anti-CD40 also lowers the exposure of T cells to IL- 12, thereby increasing their susceptibility to exhaustion. Sixth, the antigen-specific regulatory network induced by infusions of apoptotic donor leukocyte compositions provided herein, including IL- 10 secreting Tri cells, can play a role in maintaining antigen-specific T cells in an exhausted state. Finally, the capsid antigen persistence in a tolerized subject is expected to promote the continued exhaustion of T cells.

[0208] The AAV capsid antigens and the antigen encoded by the transgene are delivered via crosslinking with apoptotic leukocytes under the cover of transient immunotherapy to induce tolerance to the AAV capsid and the transgene product via mechanisms described above.

Strategies to Avert Complement Activation

[0209] The concomitant administration of inhibitors of upstream complement components (e.g., the complement C3 modulator APL-9) and the above-referenced immune tolerance regime are predicted to synergize against the deleterious immune responses to the AAV capsid.

[0210] In some embodiments,, targeting C3 and blocking its cleavage as part of a tolerizing regiment provided herein has several benefits in AAV gene therapy. Limiting the formation of soluble C3a is expected to reduce the recruitment of macrophages and neutrophils to target tissues and their inflammatory reactions. The C3 -inhibitor may impede the interaction of the viral capsid with iC3b fragments that can lead to phagocytosis and macrophage activation and uptake by dendritic cells. C3 inhibition at the time of AAV vector administration would also limit C3d- opsonization of AAV particles and their recognition by CR2 on B cell surfaces, which lowers the threshold for B cell expansion. Thus, the administration of inhibitors of upstream complement components is predicted to complement the benefits associated with tolerance induction, i.e., the prevention of de novo antibodies to the capsid and their activation of the classical complement pathway upon binding to target tissues.

Methods of Treatment

[0211] Provided herein are methods of treating a disease or a condition, wherein the methods comprise: administering to a subject in need thereof a composition provided herein.

[0212] In some embodiments, the disease, disorder, or condition is not treatable by a small molecule. In some embodiments, the subject has an allergic immune response to a therapeutic agent. In some embodiments, the disease or condition is selected from the group consisting of Cystic Fibrosis, Parkinson's disease, Alzheimer's disease, Alpha- 1 -antitrypsin deficiency, Arthritis, Leber congenital amaurosis, Hemophilia B, Late infantile neuronal lipofuscinosis, a muscular dystrophy (e.g., Duchenne Muscular Dystrophy), heart failure, prostate cancer, epilepsy, retinal dystrophy, macular degeneration, familial lipoprotein lipase deficiency, choroideremia, melanoma, optic neuropathy, limb ischemia, limb girdle muscular dystrophy, amyotrophic lateral sclerosis, Canavan disease, COPD and liver disease, rheumatoid arthritis, galactosialidosis, spinal muscular atrophy, limb-girdle muscular dystrophy, Giant Axonal Neuropathy, LOPD, multiple myeloma, cancer, metachromatic leukodystrophy, beta thalassemia, sickle cell anemia, lung cancer, cardiomyopathy, cardiac hypertrophy, myocardial fibrosis, progeria syndrome, blindness, hearing impairment, multiple sclerosis, diabetes, and pre-term birth. In some embodiments, the subject has, is at risk of developing, or is diagnosed with a disease or condition provided herein. [0213] Provided herein are methods of tolerizing a subject to a gene therapy composition. Further provided herein is a method of treating a disease or condition with a gene therapy, the method comprising: (a) contacting a population of leukocytes with a crosslinking agent; and a plurality of viral antigens, antigenic fragments, or variants thereof, wherein at least one viral antigen, antigenic fragment, or variant thereof is conjugated or cross-linked to a leukocyte within the population of leukocytes via the crosslinking agent, thereby producing a tolerizing composition; (b) administering to a subject the tolerizing composition; and (c) administering a gene therapy to the subject, thereby treating the disease or the condition. In some embodiments, the tolerizing composition reduces the immune response to the gene therapy. In some embodiments, the tolerizing composition increases the proliferation of Tri cells in the subject. Type 1 regulatory T (Tri) cells are classified as a distinct subset of T cells, and they secret high levels of IL-10 but lack the expression of the forkhead box P3 (FoxP3). Tri cells act as key regulators in the immune network, and play a central role in maintaining immune homeostasis. [0214] Provided herein are methods of tolerizing a population of immune cells to a gene therapy composition. In some embodiments, the methods comprise contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: viral capsid, a fragment, or a variant thereof. In some embodiments, the modified leukocyte comprises a protein. In some embodiments, the modified leukocyte comprises a viral capsid, a fragment, or a variant thereof crosslinked or conjugated to the modified leukocyte. In some embodiments, the modified leukocyte comprises a protein crosslinked or conjugated to the modified leukocyte. In some embodiments, the methods further comprise contacting the population of immune cells with the gene therapy composition. In some embodiments, the gene therapy composition comprises a viral vector. In some embodiments, the gene therapy composition comprises a protein. In some embodiments, the contacting is in vitro or ex vivo. In some embodiments, the contacting increases the level of CD33 expressing immune cells in the population of immune cells relative to immune cells that have not been contacted with a modified leukocyte provided herein. In some embodiments, the contacting increases the level of PD-L1 expressing immune cells in the population of immune cells relative to immune cells that have not been contacted with a modified leukocyte provided herein.

[0215] In some embodiments, the contacting results in at least about a 5 fold increase in the frequency of Tri cells relative to immune cells that have not been contacted with a modified leukocyte provided herein. In some embodiments, the level of CD33 expressing immune cells is elevated in a subject that has been administered a composition provided herein. In some embodiments, the level of PD-L1 expressing immune cells is elevated in a subject that has been administered a composition provided herein. In some embodiments, the frequency of Tri cells is elevated be at least about 10% in a subject that has been administered a composition provided herein relative the frequency of Tri cells prior to administration of a composition provided herein. In some embodiments, the frequency of Tri cells is increased in a population of immune cells or a subject that has been administered a composition provided herein at least about 5-fold relative to a comparable population of immune cells.

[0216] In some embodiments, the contacting results in activation of a T cell. In some embodiments, the T cell is a regulatory T cell. In some embodiments, the T cell is a Tri cell or a Treg cell. In some embodiments, the contacting results in the retention of T cells (e.g., antigenspecific T cells) in a secondary lymphoid organ in a subject relative to the population of T cells in the secondary lymphoid organ prior to contacting. In some embodiments, the T cells are antigenspecific T cells. In some embodiments, the contacting results in the retention of B cells in a secondary lymphoid organ in a subject relative to the population of B cells in the secondary lymphoid organ prior to contacting. In some embodiments, the B cells are antigen-specific B cells.

Additional embodiments

[0217] The memory and de novo CD8+ T cell responses to the AAV capsid antigens are mediators of deleterious immunotoxicity to transduced cells in recent high-dose AAV gene therapy trials. In embodiments provided herein, the presentation of AAV capsid antigen via crosslinked leukocyte tolerizing compositions provided herein under the cover of transient immunotherapy both 1 week before and 1 day after the first vector administration can serve as a safe and highly efficacious strategy to delete antigen-specific T cells, expand and activate in vivo an antigenspecific regulatory network, and trigger exhaustion of antigen-specific T cells, thereby protecting the subject from the acute immunotoxicity associated with intravenous AAV gene therapy and facilitating sustained expression of the transgene in transduced cells. If deleterious immune responses to transduced cells and transgene products can be prevented by tolerance induction, lower doses of vectors could achieve sustained and therapeutic transgene expression. As the epitopes on the AAV capsid that are recognized by CD8+ cells are conserved across AAV serotypes, it is conceivable that tolerance induced to one AAV serotype could protect a subject from immunotoxicity to other AAV serotypes.

[0218] Thus, it is reasonable to assume that the concerted action of deletion, regulation, and exhaustion of capsid antigen-specific T cells induced by AAV capsid and transgene antigen delivery via tolerizing compositions comprising AAV capsid crosslinked leukocytes under the cover of transient immunosuppression with anti-CD40, rapamycin, sTNFR, anti-IL-6R, and APL- 9 can profoundly increase both the safety and efficacy of AAV gene therapy trials.

[0219] Provided herein are methods of delivering a gene therapy and a population of cells provided herein or a tolerizing composition provided herein. In some embodiments, the method comprises administering a viral capsid antigen conjugated to an autologous B cell. In some embodiments, the method comprises administering a nanoparticle conjugated to a viral capsid antigen. In some embodiments, the administering is prior to the first dose of a viral gene therapy. In some embodiments, the administering increases the efficacy of the gene therapy. The efficacy of the gene therapy can be determined by several parameters, including but not limited to: the size of capsid antigen-specific T and/or B cell clones, inducing exhaustion in antigen-specific T cells, and modulating the specificity of an immune cell for a capsid antigen in advance of the first dose of the viral gene therapy. In some embodiments, a transgene product or fragment or derivative thereof is conjugated to the recipient-derived B cells or incorporated into the nanoparticles, thereby also establishing immune regulation with specificity for the transgene product delivered by the subsequent gene therapy. Indeed, the use of chemically crosslinked recipient-derived, apoptotic leukocytes or B cells as carriers of antigen can confer tolerance to any peptide or protein antigens. [0220] Further provided herein are methods for inducing tolerance to one or more viral antigens or antigenic fragments thereof (e.g., CPI). In some embodiments, the method comprises promoting linked suppression. Linked suppression occurs when tolerance induced to a single T cell epitope inhibits the response to all epitopes in the same protein. In some embodiments, tolerance can be induced to multiple viral antigens by conjugating apoptotic autologous cells (e.g., leukocytes) with the antigen, as well as conjugating with one or more transgene products or derivatives or variants thereof. Linked suppression can suppress immune responses to other viral antigens and transgene product antigens, as tolerance induced to a single antigen can inhibit the response to other antigens presented by the same antigen presenting cell in the recipient.

[0221] Further provided herein are compositions, wherein the compositions comprise: (a) a leukocyte; and (b) a viral antigen. In some embodiments, the viral antigen is conjugated or crosslinked to the leukocyte by use of a cross-linking agent for instance a chemical crosslinking agent such as a carbodiimide crosslinking agent. In some embodiments, the viral antigen is selected from the group consisting of: an antigen or an antigenic fragment, a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a capsid. In some embodiments, the viral antigen is an AAV antigen that shares a serotype with a recombinant AAV viral vector that is used to deliver a therapeutic transgene to a subject that is the recipient of gene therapy. In some embodiments, the composition comprises a recombinant protein as a transgene product. In some embodiments, the transgene product is a protein expressed from said transgene.

[0222] Further provided herein are compositions, wherein the compositions comprise: (a) a plurality of leukocytes; or (b) a plurality of nanoparticles formulated for delivering at least one of a viral antigen and/or a transgene product. In some embodiments, the viral antigen and/or transgene product is crosslinked to a leukocyte or a nanoparticle with a cross-linking agent, for instance, a chemical crosslinking agent such as a carbodiimide crosslinking agent. In some embodiments, the nanoparticles comprise: an MHC class II molecule, or one or more peptides derived from the MHC class II molecule; an MHC class I molecule; a viral antigen; or a combination thereof. In some embodiments, the viral antigen is selected from the group consisting of: an antigen or an antigenic fragment, a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, and a capsid. In some embodiments, the MHC class II molecule, or one or more peptides derived from the MHC class II molecule can be encapsulated in, conjugated, or crosslinked to the nanoparticle. In some embodiments, the nanoparticles can be lipid nanoparticles.

[0223] Crosslinking agents can facilitate crosslinking of protein antigens to the surface of the leukocytes. For example, ECDI treatment of leukocytes incubated with full length AAV particles comprising assembled VP1, VP2, and VP3 capsid proteins can crosslink the AAV capsids to the surface of the leukocyte. In this way, the AAV epitopes crosslinked to the leukocyte can be complementary to the AAV epitopes of the gene therapy, which can eliminate the need for knowledge of precise epitopes needed to facilitate immunotolerance to the gene therapy. Use of assembled AAV particles can increase stability leading to greater incorporation of viral capsid proteins.

[0224] In some embodiments, the AAV particles comprise an empty capsid. The viral vector of DNA is not needed in all contexts, including, for example, providing viral protein antigens. AAV particles can further comprise multiple serotypes.

[0225] In some embodiments, the transgene that encodes a transgene product (e.g., a protein or an RNA) can be delivered by an AAV gene therapy vector, and the same transgene product can be delivered by the fixed leukocytes to facilitate immunotolerance to the foreign protein epitopes. Protein transgenes can include microdystrophin, RPE65, Human FVIII, Cas9, Factor IX, Survival Motor Neuron protein, Myotubularin, sarcoglycan, or any other transgene product corresponding to a transgene product delivered by a gene therapy, some of which have been described in more detail in other parts of this disclosure.

Exemplary Embodiments

[0226] Provided herein are compositions, wherein the compositions comprise: (a) a leukocyte; (b) a crosslinking agent; and (c) a viral antigen or an antigenic fragment or variant thereof, wherein the viral antigen or antigenic fragment or variant thereof is conjugated or cross-linked to the leukocyte. Further provided herein are compositions, wherein the compositions comprise: (a) a leukocyte; (b) optionally, a crosslinking agent; and (c) a viral antigen or an antigenic fragment or variant thereof, wherein the viral antigen or antigenic fragment or variant thereof is conjugated or cross-linked to the leukocyte. Further provided herein are compositions, wherein the compositions comprise: (a) a population of leukocytes; (b) optionally, a crosslinking agent; and (c) a viral antigen or an antigenic fragment or variant thereof, wherein the viral antigen or antigenic fragment or variant thereof is conjugated or cross-linked to a leukocyte within the population of leukocytes. Further provided herein are compositions, wherein the viral antigen or antigenic fragment or variant thereof is selected from the group consisting of a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a viral capsid. Further provided herein are compositions, wherein the viral antigen or antigenic fragment or variant thereof is from a recombinant viral vector. Further provided herein are compositions, wherein said recombinant viral vector is selected from the group consisting of a recombinant herpes simplex virus (HSV) vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picornavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector. Further provided herein are compositions, wherein the viral antigen comprises an empty capsid or a nucleocapsid. Further provided herein are compositions, wherein the recombinant viral vector further comprises a transgene. Further provided herein are compositions, further comprising a transgene product or fragment thereof. Further provided herein are compositions, wherein the crosslinking agent comprises a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof. Further provided herein are compositions, wherein said diimidoester comprises cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof. Further provided herein are compositions, wherein the crosslinking agent comprises a carbodiimide that comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof. Further provided herein are compositions, wherein the crosslinking agent comprises l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (ECDI). Further provided herein are compositions, wherein the leukocyte comprises an apoptotic leukocyte or a pre-apoptotic leukocyte. Further provided herein are compositions, wherein the leukocyte comprises a mammalian leukocyte. Further provided herein are compositions, wherein the mammalian leukocyte comprises a human leukocyte. Further provided herein are compositions, wherein the leukocyte comprises a cadaveric leukocyte. Further provided herein are compositions, wherein the leukocyte comprises a stem cell derived leukocyte. Further provided herein are compositions, wherein the cadaveric leukocyte comprises from a non-heart beating donor, or a brain-dead donor. Further provided herein are compositions, wherein the leukocyte comprises from a living donor. Provided herein are compositions for use in tolerizing a living donor to a viral antigen. Further provided herein are compositions for use, wherein said use comprises administering to said living donor a composition provided herein. Further provided herein are compositions, wherein the leukocyte is obtained by ex vivo differentiation of a stem cell, pluripotent cell or induced pluripotent stem cell. Further provided herein are compositions, wherein the leukocyte is isolated from a spleen, a lymph node, a secondary lymphoid organ, a tissue, bone marrow, or peripheral blood. Further provided herein are compositions, wherein the leukocyte comprises an ex vivo expanded leukocyte. Further provided herein are compositions, wherein the leukocyte comprises a B-lymphocyte. Further provided herein are compositions, wherein the leukocyte is fixed with said crosslinking agent for a pre-determined amount of time. Further provided herein are compositions, wherein said predetermined amount of time comprises at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes. Further provided herein are compositions for use in tolerizing a recipient, wherein the leukocyte expresses a MHC class II molecule that is matched with that of the recipient, and wherein said use comprises administering said composition to said recipient. Further provided herein are compositions, wherein the leukocyte further comprises a MHC class II molecule or one or more peptides derived from the MHC class II molecule, wherein the MHC class II molecule or the one or more peptides derived from the MHC class II molecule is conjugated with the leukocyte. Further provided herein are compositions for use in tolerizing a recipient, wherein the MHC class II molecule is matched with that of the recipient, and wherein said use comprises administering the composition to the recipient. Further provided herein are compositions, wherein the MHC class II molecule comprises HLA-DP, HLA-DQ, or HLA-DR. Further provided herein are compositions, wherein the MHC class II molecule HLA-DP comprises HLA-DPA (a chain), or HLA-DPB (P chain). Further provided herein are compositions, wherein the MHC class II molecule HLA-DQ comprises HLA-DQA, or HLA-DQB. Further provided herein are compositions, wherein the MHC class II molecule HLA-DR comprises HLA-DRA, or HLA-DRB. Further provided herein are compositions, wherein the MHC class II molecule HLA- DRB is selected from HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, or HLA-DR5. Further provided herein are compositions, wherein the MHC class II molecule is encoded by HLA- DRBl*01, HLA-DRBl*03, HLA-DRB1*O4, HLA-DRB1*O7 HLA-DRB1*11, HLA-DRB1*15, or HLA-DRB 1*16 allele of the recipient. Further provided herein are compositions, wherein the one or more peptides derived from the MHC class II molecule comprises a sequence from a hypervariable region of the MHC class II molecule. Further provided herein are compositions, wherein the one or more peptides derived from the MHC class II molecule is at least 10 to 30 amino acid residues in length. Further provided herein are compositions, wherein the one or more peptides derived from the MHC class II molecule are synthesized or recombinant. Further provided herein are compositions, wherein the viral antigen comprises an adenovirus associated virus (AAV) antigen. Further provided herein are compositions, wherein the AAV antigen is from a recombinant adenovirus associated virus (AAV) vector that has an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i, AAV- DJ or any combination thereof. Further provided herein are compositions, wherein said capsid protein comprises an AAV VP1, VP2, or VP3 capsid protein. Further provided herein are compositions, wherein said VP1, VP2 or VP3 capsid protein comprises an amino acid sequence that is at least 60% identical to the corresponding capsid protein of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ. Further provided herein are compositions, wherein the empty capsid or the nucleocapsid comprises that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, - 6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i, and AAV-DJ. Further provided herein are compositions, wherein the viral antigen is from a recombinant adenovirus vector which is an AAV vector of AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i, AAV-DJ or any chimera or combination thereof. Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant herpes simplex virus vector which comprises a recombinant herpes simplex virus 1 (HSV1) vector, or a recombinant herpes simplex virus 2 (HSV2) vector. Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant retrovirus vector which comprises a recombinant Moloney murine sarcoma virus (MMSV) vector, or a recombinant murine stem cell virus (MSCV) vector. Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant lentivirus vector which comprises a recombinant human immunodeficiency virus 1 (HIV-1) vector or a recombinant human immunodeficiency virus 2 (HIV-2) vector. Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant alphavirus vector which comprises a recombinant Semliki forest virus (SFV) vector, Sindbis virus (SIN) vector, a recombinant Venezuelan equine encephalitis virus (VEE) vector, or a recombinant alphavirus Ml . Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant flavivirus vector which comprises a recombinant Kunjin virus vector, a recombinant West Nile virus vector, or a recombinant Dengue virus vector. Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant rhabdovirus vector which comprises a recombinant Rabies virus vector, or a recombinant vesicular stomatitis virus vector. Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant measles virus vector which comprises a recombinant MV Edmonston strain (MV-Edm) vector. Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant poxvirus vector which comprises a recombinant vaccinia virus (VV) vector. Further provided herein are compositions, wherein the recombinant viral vector comprises a recombinant picomavirus vector which comprises a recombinant Coxsackievirus vector.

[0227] Provided herein are compositions, wherein the compositions comprise: (a) a nanoparticle, (b) an MHC class II molecule, or one or more peptides derived from the MHC class II molecule; and (c) a viral antigen, wherein the viral antigen is selected from the group consisting of a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a capsid, and wherein (b) and (c) are encapsulated in, conjugated, or crosslinked to the nanoparticle. Further provided herein are compositions, wherein the viral antigen is derived from a recombinant viral vector. Further provided herein are compositions, wherein the compositions further comprise a protein corresponding to a transgene product or fragment thereof. Further provided herein are compositions, wherein the recombinant viral vector is selected from the group consisting of a recombinant herpes simplex virus (HSV) vector, recombinant alphavirus vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picomavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated vims (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector. Further provided herein are compositions, wherein the capsid comprises an empty capsid or a nucleocapsid. Further provided herein are compositions, wherein the nanoparticle comprises a lipid nanoparticle. Further provided herein are compositions, wherein the lipid nanoparticle comprises one or more cationic lipids. Further provided herein are compositions, wherein the recombinant viral vector further comprises a transgene. Further provided herein are compositions, wherein the lipid nanoparticle comprises one or more noncationic lipids. Further provided herein are compositions, wherein the lipid nanoparticle comprises one or more PEG modified lipids. Further provided herein are compositions, wherein the lipid nanoparticle comprises C12-200. Further provided herein are compositions, wherein the lipid nanoparticle comprises DLin-KC2-DMA, CHOL, DMGPEG2K, DOPE, and DMG-PEG-2000. Further provided herein are compositions, wherein the lipid nanoparticle comprises a cleavable lipid. Further provided herein are compositions, wherein the nanoparticle comprises a polymer nanoparticle. Further provided herein are compositions, wherein the polymer nanoparticle comprises a polymer that is biodegradable. Further provided herein are compositions, wherein the nanoparticle comprises solid-lipid nanoparticle. Further provided herein are compositions, wherein the nanoparticle comprises a micelle. Further provided herein are compositions, wherein the micelle comprises a polymer comprises an amphiphilic polymer. Further provided herein are compositions, wherein the micelle comprises a water soluble micelle. Further provided herein are compositions, wherein the micelle coats a solid core. Further provided herein are compositions, wherein the core comprises a traceable inorganic material selected from the group consisting of iron oxide, CdSe/CdS/ZnS, silver and gold. Further provided herein are compositions, wherein the diameter of the core is about 5 to 30 nm. Further provided herein are compositions, wherein the nanoparticle is negatively charged. Further provided herein are compositions, wherein the nanoparticle comprises a zeta potential from about -100 mV to about 0 mV. Further provided herein are compositions, wherein the nanoparticle comprises a zeta potential from about -60 mV to about -40 mV. Further provided herein are compositions, wherein the nanoparticle surface comprises a functionalized surface group. Further provided herein are compositions, wherein the functionalized surface group comprises a hydroxyl group, amine group, a thiol group, an alcohol group, or a carboxylic acid group. Further provided herein are compositions, wherein the polymer comprises a synthetic polymer selected from group consisting of poly(maleic anhydride-alt-l-octa-decene), poly(maleic anhydride-alt-1 -tetradecene), and polyisoprene-block poly-ethylene-oxide block copolymer, polylactide-polyglycolide copolymers, polyacrylates, polycaprolactones, poly( D , L -lactide), polycyanoacrylate and poly(lactic-co- glycolic acid) (PLGA) or poly(lactic acid), and poly(ethyl methacrylate) (PEMA).Further provided herein are compositions, wherein the polymer comprises PLGA modified with PEMA as a surfactant. Further provided herein are compositions, wherein the polymer comprises a natural polymer selected from a group consisting of albumin, gelatin, alginate, collagen, chitosan, and dextran. Further provided herein are compositions, wherein the nanoparticle is formulated for targeting to a splenic marginal zone antigen presenting cell or a non-splenic marginal zone macrophage, a dendritic cell, a liver sinusoidal endothelial cell, or an antigen presenting cell in vitro or in vivo. Further provided herein are compositions, wherein the nanoparticle comprises a diameter in the range of 10-1000 nm. Further provided herein are compositions, wherein the nanoparticle comprises a diameter in the range of 20-900 nm.

[0228] Further provided herein are compositions, wherein the nanoparticle comprises a diameter of 500 nm. Further provided herein are compositions, wherein the nanoparticle is coated with polyethylene glycol. Further provided herein are compositions, wherein said composition is for use in tolerizing a recipient, wherein the MHC class II molecule is matched with that of said recipient, and said tolerizing comprises administered said composition to said recipient. Further provided herein are compositions, wherein the MHC class II molecule comprises HLA-DP, HLA- DQ, or HLA-DR. Further provided herein are compositions, wherein the HLA-DP comprises HLA-DPA (a chain), or HLA-DPB (P chain). Further provided herein are compositions, wherein the HLA-DQ comprises HLA-DQA, or HLA-DQB. Further provided herein are compositions, wherein the HLA-DR comprises HLA-DRA, or HLA-DRB. Further provided herein are compositions, wherein the HLA-DR comprises HLA-DRB, and wherein said HLA-DRB is selected from HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, or HLA-DR5. Further provided herein are compositions, wherein said composition is for use in tolerizing a recipient, wherein the MHC class II molecule is encoded by HLA-DRBl*01, HLA-DRBl*03, HLA-DRB1*O4, HLA- DRB 1*07 HLA-DRB1*11, HLA-DRB1*15, or HLA-DRB 1*16 allele of said recipient, and wherein said tolerizing comprises administering the composition to said recipient. Further provided herein are compositions, wherein the one or more peptides derived from the MHC class II molecule comprises a sequence from a hypervariable region of the MHC class II molecule. Further provided herein are compositions, wherein the one or more peptides derived from the MHC class II molecule is at least 10 to 30 amino acid residues in length. Further provided herein are compositions, wherein the one or more peptides derived from the MHC class II molecule are synthetic or recombinant. Further provided herein are compositions, wherein the recombinant viral vector comprises the recombinant adenovirus associated virus (AAV) vector. Further provided herein are compositions, wherein the recombinant adenovirus associated virus (AAV) vector has an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, - 11, -rh74, -rhlO, AAV-2i, chimera or combinations thereof. Further provided herein are compositions, wherein said capsid protein comprises a VP1, VP2, or VP3 capsid protein. Further provided herein are compositions, wherein the VP1 capsid protein comprises an amino acid sequence that is at least 60% identical to that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i, wherein the VP2 capsid protein comprises an amino acid sequence that is at least 60% identical to that of the AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i, and wherein the VP3 capsid protein comprises an amino acid sequence that is at least 60% identical to that of the AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. Further provided herein are compositions, wherein the empty capsid or the nucleocapsid comprises that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i. Further provided herein are compositions, wherein the recombinant adenovirus vector comprises an adenovirus serotype 5 (Ad5) vector. Further provided herein are compositions, wherein the recombinant herpes simplex virus vector comprises a recombinant herpes simplex virus 1 (HSV1) vector, or a recombinant herpes simplex virus 2 (HSV2) vector. Further provided herein are compositions, wherein the recombinant retrovirus vector comprises a recombinant Moloney murine sarcoma virus (MMSV) vector, or a recombinant murine stem cell virus (MSCV) vector. Further provided herein are compositions, wherein the recombinant lentivirus vector comprises a recombinant human immunodeficiency virus 1 (HIV-1) vector or a recombinant human immunodeficiency virus 2 (HIV-2) vector. Further provided herein are compositions, wherein the recombinant alphavirus vector comprises a recombinant Semliki forest virus (SFV) vector, Sindbis virus (SIN) vector, a recombinant Venezuelan equine encephalitis virus (VEE) vector, or a recombinant alphavirus Ml. Further provided herein are compositions, wherein the recombinant flavivirus vector comprises a recombinant Kunjin virus vector, a recombinant West Nile virus vector, or a recombinant Dengue virus vector. Further provided herein are compositions, wherein the recombinant rhabdovirus vector comprises a recombinant Rabies virus vector, or a recombinant vesicular stomatitis virus vector. Further provided herein are compositions, wherein the recombinant measles virus vector comprises a recombinant MV Edmonston strain (MV-Edm) vector. Further provided herein are compositions, wherein the recombinant poxvirus vector comprises a recombinant vaccinia virus (VV) vector. Further provided herein are compositions, wherein the recombinant picornavirus vector comprises is a recombinant Coxsackievirus vector. Further provided herein are compositions, wherein the recombinant adenovirus vector comprises an AAV chimera. Further provided herein are compositions, wherein the recombinant adenovirus vector comprises the AAV chimera AAV-DJ. Further provided herein are compositions, wherein the nanoparticle further comprises a peptide tag, detecting agent, a therapeutic agent, a one or more immunomodulatory agents or a combination thereof encapsulated in, or conjugated with the nanoparticle.

[0229] Further provided herein are compositions, further comprising an immunomodulatory agent, wherein said immunomodulatory agent is an anti-CD40 agent, anti-CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor, IFNy, an NFKB inhibitor, vitamin D3, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, alpha 1 antitrypsin inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), or any combination thereof. Further provided herein are compositions, wherein the one or more immunomodulatory agents blocks CD40:CD40L co-stimulation. Further provided herein are compositions, wherein the NFkB inhibitor comprises curcumin, triptolide, Bay-117085, or a combination thereof. Further provided herein are compositions, wherein the anti-CD40 agent comprises CD40 siRNA. Further provided herein are compositions, wherein the anti-CD40 agent comprises a CD40 binding peptide inhibitor, anti-CD40 monoclonal antibody, a Fab’ anti-CD40 monoclonal antibody fragment, FcR- engineered, Fc silent anti-CD40 monoclonal domain antibody. Further provided herein are compositions, wherein the anti-CD40 agent comprises an anti-CD40 L monoclonal antibody, a Fab’ anti-CD40L monoclonal antibody fragment CDP7657, a FcR-engineered, Fc silent anti- CD40L monoclonal domain antibody, a Fab’ anti-CD40L antibody, CD40 binding peptides, anti- CD40 siRNA, CD40L-binding fusion protein or an Fc-engineered anti-CD40L antibody.

[0230] Provided herein are tolerogenic compositions, wherein the tolerogenic compositions comprise: two or more compositions provided herein, wherein the tolerogenic composition is capable of modulating an immune response to the recombinant viral vector in a recipient that is administered said composition. Provided herein are tolerogenic compositions, wherein the tolerogenic compositions comprise: (a) a first composition, wherein the first composition comprises: (i) a leukocyte; (ii) optionally, a crosslinking agent; and (iii) a viral antigen or an antigenic fragment or variant thereof, wherein the viral antigen or antigenic fragment or variant thereof is conjugated or cross-linked to the leukocyte; and (b) a second composition, wherein the second composition comprises: (i) a nanoparticle, (ii) an MHC class II molecule, or one or more peptides derived from the MHC class II molecule; and (iii) a viral antigen, wherein the viral antigen is selected from the group consisting of: a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a capsid, and wherein (ii) and (iii) are encapsulated in, conjugated, or crosslinked to the nanoparticle, wherein the tolerogenic composition is capable of modulating an immune response to the recombinant viral vector in a recipient that is administered said composition. Further provided herein are tolerogenic compositions, wherein the leukocyte is an apoptotic leukocyte or a pre-apoptotic leukocyte and expresses a MHC class II molecule that is matched with that of the recipient. Further provided herein are tolerogenic compositions, wherein the leukocyte comprises the MHC molecule or one or more peptides derived from the MHC molecule that is matched with that of the recipient. Further provided herein are tolerogenic compositions, wherein the leukocyte comprises the MHC molecule in combination with one or more peptides derived from the MHC molecule that is matched with that of the recipient. Further provided herein are tolerogenic compositions, wherein the tolerogenic composition comprises the leukocyte derived from the recipient. Further provided herein are tolerogenic compositions, wherein the nanoparticle comprises the MHC class II molecule that is matched with that of the recipient. Further provided herein are tolerogenic compositions, wherein the recombinant viral vector further comprises a transgene. Further provided herein are tolerogenic compositions, wherein the transgene encodes a nucleic acid or a polypeptide. Further provided herein are tolerogenic compositions, wherein modulating immune response comprises inhibiting immune response to the viral antigen. Further provided herein are tolerogenic compositions, wherein inhibiting immune response comprises: inhibiting a B- cell response, inhibiting a T cell response, inhibiting B-cell activation, inhibiting T-cell proliferation, inhibiting T cell migration, inhibiting B-cell proliferation, inhibiting B-cell migration, inhibiting macrophage activation, inhibiting production of one or more cytokines, inhibiting production of antibodies specific for the viral antigen or a combination thereof. Further provided herein are tolerogenic compositions, wherein modulating immune response comprises: in vivo generation, expansion and/or activation of Treg cells CD4+ Tregs, CD8+ Tregs, CD4+ Tri cells, CD8+ Natural Suppressor cells, Breg cells, BIO cells, myeloid derived suppressor cells or other immune regulatory subsets in the recipient. Further provided herein are tolerogenic compositions, wherein the modulating immune response comprises contraction of CD4+ and/or CD8+ T cells specific to said viral antigen in said recipient as compared to corresponding amounts of said CD4+ and/or CD8+ T cells absent administration of the tolerogenic composition. Further provided herein are tolerogenic compositions, wherein the modulating immune response comprises exhaustion of CD4+ and/or CD8+ T cells specific to the viral antigen in said recipient as compared to corresponding amounts of said CD4+ and/or CD8+ T cells absent administration of the tolerogenic composition.

[0231] Provided herein are methods for inducing tolerance to a recombinant viral vector comprising a transgene in a recipient, wherein the methods comprise: administering to a recipient a tolerogenic composition provided herein in an amount effective to induce tolerance to the recombinant viral vector comprising the transgene. Further provided herein are methods, wherein the methods further comprise administering to said recipient an immunomodulatory agent, wherein said immunomodulatory agent comprises an anti-CD40 agent, anti-CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor, IFNy, an NFKB inhibitor, vitamin D3, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, an alpha 1 anti-trypsin inhibitor, dehydroxymethylepoxy quinomycin (DHMEQ), or any combination thereof. Further provided herein are methods, wherein said administering is performed intravenously, intraocularly, intravitreally, via otic administration, via intracardiac injection, or intramuscularly. Further provided herein are methods, wherein the transgene encodes an autoantigen. Further provided herein are methods, wherein the autoantigen comprises an islet cell autoantigen selected from the group comprising GAD65, ZnT8, IGRP, or preproinsulin.

[0232] Provided herein are methods of tolerizing a recipient of an AAV vector to said AAV vector, wherein the methods comprise: administering to said recipient a tolerizing regimen that comprises: a population of leukocytes from said recipient or a population of leukocytes differentiated in vitro from stem cells extracted from said recipient; a crosslinking agent; and an AAV viral antigen or an antigenic fragment or variant thereof, wherein the AAV viral antigen or antigenic fragment or variant thereof shares a serotype with said AAV vector, and wherein said AAV viral antigen is conjugated or cross-linked to the leukocyte. Provided herein are methods of tolerizing a recipient of an AAV vector to said AAV vector, wherein the methods comprise: administering to said recipient a tolerizing regimen that comprises: a population of leukocytes from said recipient or a population of leukocytes differentiated in vitro from stem cells extracted from said recipient; optionally, a crosslinking agent; and an AAV viral antigen or an antigenic fragment or variant thereof, wherein the AAV viral antigen or antigenic fragment or variant thereof shares a serotype with said AAV vector, and wherein said AAV viral antigen is conjugated or cross-linked to the leukocyte. Further provided herein are methods, wherein said methods further comprise administering to the recipient an agent that block the binding of CD40 and CD40L, a mTOR inhibitor, and an inhibitor of at least one pro-inflammatory cytokine. Further provided herein are methods, wherein said inhibitor of at least one pro-inflammatory cytokine comprises a TNF-alpha inhibitor and/or an IL-6 inhibitor. Further provided herein are methods, wherein the tolerogenic regimen is administered intravenously on days -7 and +1 relative to a first administration of said AAV vector. Further provided herein are methods, wherein the administering is performed prior to, simultaneously and/or subsequent to administering the recombinant viral vector to the recipient. Further provided herein are methods, wherein the administering of said composition inhibits a B- cell response, a T cell response, macrophage activation, cytokine production, or a combination thereof in said recipient, thereby inducing tolerance. Further provided herein are methods, wherein the B cell response comprises at least one of B- cell activation, B-cell proliferation, and production of neutralizing antibodies specific for the viral antigen. Further provided herein are methods, wherein the T cell response comprises at least one of T cell activation, T cell proliferation, generation of memory T cells, and generation of T cell effector function involving cytokines or cytolytic mechanisms. Further provided herein are methods, wherein the administering induces in vivo generation, expansion and/or activation of Treg cells, CD4+ Tregs, CD8+ Tregs, CD4+ Tri cells, CD8+ Natural Suppressor cells, Breg cells, BIO cells, myeloid derived suppressor cells or other immune regulatory subsets in the recipient, thereby inducing tolerance.

[0233] Provided herein are methods of modulating an immune response to a transduced cell in a recipient, the methods comprising: administering to the recipient the tolerogenic composition provided herein, in an amount effective to modulate immune response to the transduced cell, wherein the transduced cell is generated by contacting a cell with the recombinant viral vector. Further provided herein are methods for sustained expression of a transgene in a recipient, wherein the methods comprise: administering to the recipient the tolerogenic composition provided herein, prior to, simultaneously and/or subsequent to administering the recombinant viral vector comprising the transgene.

[0234] Provided herein are compositions for tolerizing a subject to a viral gene therapy vector and an associated transgene that encodes for a transgene product, the compositions comprising: a population of leukocytes conjugated by a crosslinking agent to (i) a component of the viral gene therapy vector, and (ii) the transgene product or a fragment or derivative thereof. Further provided herein are compositions, wherein the viral gene therapy vector comprises AAV. Further provided herein are compositions, wherein the transgene product comprises a nucleic acid. Further provided herein are compositions, wherein the nucleic acid comprises a ribonucleic acid. Further provided herein are compositions, wherein the transgene product comprises a recombinantly expressed protein or a polypeptide fragment or derivative thereof. Further provided herein are compositions, wherein the transgene product is a transgene listed in Table 2. Further provided herein are compositions, wherein the component of the viral gene therapy vector is a viral antigen, or antigenic fragment thereof. Further provided herein are compositions, wherein the viral antigen is a viral capsid or antigenic fragment, domain, peptide or variant thereof. Further provided herein are compositions, wherein the capsid is empty. Further provided herein are compositions, wherein the viral gene therapy vector comprises one or more AAV serotypes. Further provided herein are compositions, wherein the AAV serotype is selected from AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, - 10, -11, -rh74, -rhlO and AAV-2i. Further provided herein are compositions, wherein the transgene product or fragment thereof comprises microdystrophin, sarcoglycan, RPE65, Human F VIII, Cas9, or similar transgene product associated with a gene therapy. Further provided herein are compositions, wherein said population of leukocytes are derived from the subject. Further provided herein are compositions, wherein said population of leukocytes are derived from a donor that is MHC Class 2 matched to the subject. Further provided herein are compositions, wherein said population of leukocytes comprises a population of apoptotic leukocytes. Further provided herein are compositions, wherein said population of leukocytes comprises a population of pre- apoptotic leukocytes. Further provided herein are compositions, wherein said crosslinking agent comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof. [0235] Provided herein are methods of reducing hepatotoxicity in a recipient of a gene therapy encoding a transgene, wherein the methods comprise: administering to the recipient an effective amount of a composition provided herein, a tolerizing composition provided herein, a gene therapy composition provided herein or any combination thereof.

[0236] Provided herein are methods of reducing or treating one or more of elevated liver enzymes, drug-induced liver injury and hepatic failure in a recipient of a gene therapy encoding a transgene, wherein the methods comprise: administering to the recipient an effective amount of a composition provided herein, a tolerizing composition provided herein, a gene therapy composition provided herein or any combination thereof, wherein said administering results in a reduction or treatment of said elevated liver enzymes, drug-induced liver injury and hepatic failure as compared to the recipient in the absence of said administering.

[0237] Provided herein are methods of improving tolerance to a high dose rAAV vector gene therapy in a subject, wherein the methods comprise: administering to the subject an effective amount of a composition provided herein, a tolerizing composition provided herein, a gene therapy composition provided herein or any combination thereof.

[0238] Provided herein are methods of suppressing or reducing acute immunotoxicity mediated by CTLs or by the complement system against rAAV vector gene therapy in a subject, wherein the methods comprise: administering to the subject an effective amount of a composition provided herein, a tolerizing composition provided herein, a gene therapy composition provided herein or any combination thereof.

[0239] Provided herein are methods of suppressing or reducing cytotoxicity of capsid-specific CD8+ T cells against rAAV vector gene therapy in a subject, wherein the methods comprise: administering to the subject an effective amount of a composition provided herein, a tolerizing composition provided herein, a gene therapy composition provided herein or any combination thereof.

[0240] Provided herein are methods of suppressing or reducing complement activation and adverse events associated with complement activation against rAAV vector gene therapies in a subject, wherein the methods comprise: administering to the subject an effective amount of administering to the subject an effective amount of a composition provided herein, a tolerizing composition provided herein, a gene therapy composition provided herein or any combination thereof.

[0241] Provided herein are methods of producing a tolerizing composition, wherein the methods comprise: contacting a leukocyte with a crosslinking agent; and a viral antigen, an antigenic fragment, or variant thereof, wherein the viral antigen, the antigenic fragment, or variant thereof is conjugated or cross-linked to the leukocyte via the crosslinking agent, thereby producing a tolerizing composition. Further provided herein are methods, wherein the contacting is at least about 10 minutes up to 6 hours. Further provided herein are methods, wherein the viral antigen or antigenic fragment or variant thereof is selected from the group consisting of: a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a viral capsid. Further provided herein are methods, wherein the viral antigen or antigenic fragment or variant thereof is from a recombinant viral vector. Further provided herein are methods, wherein said recombinant viral vector is selected from the group consisting of: a recombinant herpes simplex virus (HSV) vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picornavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector. Further provided herein are methods, wherein the viral antigen comprises an empty capsid or a nucleocapsid. Further provided herein are methods, wherein the tolerizing composition further comprises a transgene. Further provided herein are methods, wherein the tolerizing composition further comprises a transgene product or fragment thereof. Further provided herein are methods, wherein the crosslinking agent comprises a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof. Further provided herein are methods, wherein said diimidoester comprises cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof. Further provided herein are methods, wherein the crosslinking agent comprises a carbodiimide that comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof. Further provided herein are methods, wherein the leukocyte comprises at least one of a late apoptotic leukocyte or a pre-apoptotic leukocyte. Further provided herein are methods, wherein the leukocyte comprises a mammalian leukocyte. Further provided herein are methods, wherein the mammalian leukocyte comprises a human leukocyte. Further provided herein are methods, wherein the leukocyte comprises a cadaveric leukocyte. Further provided herein are methods, wherein the leukocyte comprises a stem cell derived leukocyte. Further provided herein are methods, wherein the cadaveric leukocyte comprises from a non-heart beating donor, or a braindead donor. Further provided herein are methods, wherein the leukocyte is derived from a living donor.

[0242] Provided herein are tolerizing compositions produced by the methods provided herein.

[0243] Provided herein are methods of producing a gene therapy tolerization composition, wherein the methods comprise: contacting a population of leukocytes for at least about 10 minutes up to 6 hours with: (a) a crosslinking agent; (b) a viral antigen, an antigenic fragment, or variant thereof, wherein the viral antigen, an antigenic fragment, or variant thereof is conjugated or crosslinked to a leukocyte; and (c) a bioactive agent, wherein the bioactive agent is conjugated or crosslinked to the leukocyte, thereby producing a gene therapy tolerization composition. Further provided herein are methods, wherein the bioactive agent comprises a nucleic acid, a transgene, a transgene product, a protein or a functional fragment thereof, an antibody or an antibody fragment, a nucleic acid encoding an antibody or an antibody fragment, a viral vector, or a combination thereof. Further provided herein are methods, wherein the transgene product comprises survival motor neuron 1 (SMN1), a microdystrophin, a sarcoglycan family protein, a RPE65 protein, Human FVIII, or a Cas protein. Further provided herein are methods, wherein the Cas protein is Cas9. Further provided herein are methods, wherein the nucleic acid encoding an antibody or an antibody fragment comprises a sequence encoding for an anti-CD40 antibody or a fragment thereof; or an anti-CD20 antibody or a fragment thereof. Further provided herein are methods, wherein the antibody or an antibody fragment comprises a sequence encoding for an anti-CD40 antibody or a fragment thereof; or an anti-CD20 antibody or a fragment thereof. Further provided herein are methods, wherein the nucleic acid is a DNA, an RNA, a messenger RNA (mRNA), a microRNA (miRNA), a small non-coding RNA, a long non-coding RNA, or an aptamer. Further provided herein are methods, wherein the viral antigen or antigenic fragment or variant thereof is selected from the group consisting of: a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a viral capsid. Further provided herein are methods, wherein the viral antigen or antigenic fragment or variant thereof is from a recombinant viral vector. Further provided herein are methods, wherein said recombinant viral vector is selected from the group consisting of a recombinant herpes simplex virus (HSV) vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picornavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector. Further provided herein are methods, wherein the viral antigen comprises an empty capsid or a nucleocapsid. Further provided herein are methods, wherein the recombinant viral vector further comprises a transgene. Further provided herein are methods, further comprising a transgene product or fragment thereof. Further provided herein are methods, wherein the crosslinking agent comprises a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof. Further provided herein are methods, wherein said diimidoester comprises cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof. Further provided herein are methods, wherein the crosslinking agent comprises a carbodiimide that comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof. Further provided herein are methods, wherein the leukocyte comprises a late apoptotic leukocyte or a pre-apoptotic leukocyte. Further provided herein are methods, wherein the leukocyte comprises a mammalian leukocyte. Further provided herein are methods, wherein the mammalian leukocyte comprises a human leukocyte. Further provided herein are methods, wherein the leukocyte comprises a cadaveric leukocyte. Further provided herein are methods, wherein the leukocyte comprises a stem cell derived leukocyte. Further provided herein are methods, wherein the cadaveric leukocyte comprises from a non-heart beating donor, or a brain-dead donor. Further provided herein are methods, wherein the leukocyte comprises from a living donor.

[0244] Provided herein are gene therapy tolerization compositions made by the methods provided herein. Further provided herein are gene therapy compositions that comprise the gene therapy tolerization composition provided herein; and a viral vector. Further provided herein are gene therapy compositions, wherein the viral vector is an adenovirus associated viral (AAV) vector, a recombinant AAV (rAAV), a lentiviral vector, a retroviral vector, or an alphaviral vector. Further provided herein are gene therapy compositions, wherein the gene therapy compositions, further comprise a transgene.

[0245] Provided herein are methods of treating a disease or condition in a subject, wherein the methods comprise: (a) contacting a population of leukocytes with a crosslinking agent; and a plurality of viral antigens, antigenic fragments, or variants thereof, wherein at least one viral antigen, antigenic fragment, or variant thereof is conjugated or cross-linked to a leukocyte within the population of leukocytes via the crosslinking agent, thereby producing a tolerizing composition; (b) administering to a subject the tolerizing composition; and (c) administering a gene therapy composition to the subject, thereby treating the disease or the condition. Further provided herein are methods, wherein the tolerizing composition further comprises a bioactive agent, wherein the bioactive agent is conjugated or cross-linked to a leukocyte. Further provided herein are methods, wherein the bioactive agent comprises a nucleic acid, a transgene, a transgene product, a protein or a functional fragment thereof, an antibody or an antibody fragment, or a nucleic acid encoding an antibody or an antibody fragment. Further provided herein are methods, wherein the transgene product comprises survival motor neuron 1 (SMN1), a microdystrophin, a sarcoglycan family protein, a RPE65 protein, or Human FVIII. Further provided herein are methods, wherein the subject has, is suspected of having, or is diagnosed with diabetes, blindness, hearing impairment, multiple sclerosis (MS), Parkinson's disease, Alzheimer's disease, alpha-1- antitrypsin deficiency, arthritis, rheumatoid arthritis, Leber congenital amaurosis, hemophilia B, late infantile neuronal lipofuscinosis, muscular dystrophy, Duchenne muscular dystrophy, heart failure, cancer, epilepsy, retinal dystrophy, macular degeneration, familial lipoprotein lipase deficiency, choroideremia, neuropathy, limb ischemia, limb girdle muscular dystrophy, amyotrophic lateral sclerosis, Canavan disease, liver disease, kidney disease, chronic obstructive pulmonary disease (COPD), galactosialidosis, spinal muscular atrophy, limb-girdle muscular dystrophy, giant axonal neuropathy, or late-onset Pompe disease (LOPD). Further provided herein are methods, wherein the contacting produces a population of apoptotic leukocytes, wherein the population of apoptotic leukocytes are conjugated or crosslinked to the plurality of viral antigens, antigenic fragments, or variants thereof. Further provided herein are methods, wherein the contacting produces a mixed population of leukocytes, wherein the mixed population of leukocytes comprise at least about 20% apoptotic leukocytes. Further provided herein are methods, wherein the gene therapy composition comprises: (a) a viral vector, viral antigen, viral antigenic fragment, or variant thereof; and (b) optionally, a transgene. Further provided herein are methods, wherein the gene therapy composition comprises a transgene, and wherein the transgene comprises survival motor neuron 1 (SMN1), a microdystrophin, a sarcoglycan family protein, a RPE65 protein, or Human FVIII. Further provided herein are methods, wherein the gene therapy composition comprises: idecabtagene vicleucel, lisocabtagene maraleucel, talimogene laherparepvec, voretigene neparvovec, onasemnogene abeparvovec, alipogene tiparvovec, atidarsagene autotemcel, brexucabtagene autoleucel, axicabtagene ciloleucel, betibeglogene autotemcel, cambiogenplasmid, elivaldogene autotemcel, gendicine, tisagenlecleucel, and valoctocogene roxaparvovec. [0246] Provided herein are methods of totalizing a population of immune cells to a gene therapy composition, wherein the methods comprise: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: (a) viral vector capsid, a fragment, or a variant thereof; and (b) a protein or a fragment thereof, wherein (a) and (b) are conjugated or crosslinked to the modified leukocyte via a crosslinking agent, thereby tolerizing the population of immune cells to the gene therapy composition. Further provided herein are methods, wherein the method further comprises contacting the population of immune cells with a gene therapy composition. Further provided herein are methods, wherein the gene therapy composition comprises a viral vector; and a transgene encoding the protein in (b). Further provided herein are methods, wherein the gene therapy composition comprises a viral vector comprising the viral vector capsid, the fragment, or the variant thereof in (a). Further provided herein are methods, wherein the viral vector is an adeno-associated viral vector (AAV), or a recombinant adeno- associated viral vector (rAAV). Further provided herein are methods, wherein the AAV comprises an AAV2, an AAV8, an AAV5, an AAV9, or an AAVrh47. Further provided herein are methods, wherein the gene therapy composition comprises a protein, wherein the protein comprises spinal motor neuron 1 (SMN1), RPE65, Hemoglobin subunit beta, alpha sarcoglycan, or a microdystrophin. Further provided herein are methods, wherein the contacting is in vitro, in vivo, or ex vivo. Further provided herein are methods, wherein the contacting is for a period of at least about 10 minutes up to 6 hours. Further provided herein are methods, wherein the contacting is for a period of at least about 1 hour up to 4 hours. Further provided herein are methods, wherein the population of immune cells comprise a population of monocytes. Further provided herein are methods, wherein the population of immune cells comprise a population of dendritic cells. Further provided herein are methods, wherein the population of dendritic cells comprise a population of CDl lc positive (CDl lc+) dendritic cells. Further provided herein are methods, wherein the population of immune cells comprise a population of T cells. Further provided herein are methods, wherein the population of immune cells comprise a mixed population of monocytes, dendritic cells, and T cells. Further provided herein are methods, wherein the crosslinking agent comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'-diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof. Further provided herein are methods, wherein the contacting increases the level of CD33 in a population of monocytes as compared to the level of CD33 in a population of immune cells that have not been contacted with the modified leukocyte. Further provided herein are methods, wherein the contacting increases the level of PD-L1 in a population of monocytes as compared to the level of PD-L1 in a population of immune cells that have not been contacted with the modified leukocyte. Further provided herein are methods, wherein the contacting increases the level of CD33 in a population of CD11c positive (CDl lc+) dendritic cells as compared to the level of CD33 in a population of immune cells that have not been contacted with the modified leukocyte. Further provided herein are methods, wherein the contacting increases the level of PD-L1 in a population of CD1 lc+ dendritic cells as compared to the level of PD-L1 in a population of immune cells that have not been contacted with the modified leukocyte. Further provided herein are methods, wherein after contacting the population of immune cells with a modified leukocyte and the gene therapy composition, the population of immune cells have at least a 5 fold increase in the frequency of Tri cells. Further provided herein are methods, wherein after contacting the population of immune cells with a modified leukocyte and the gene therapy composition, the population of immune cells have at least an 8-fold increase in the frequency of Tri cells.

[0247] Provided herein are methods of tolerizing a population of immune cells to an adeno- associated virus (AAV) vector and a spinal motor neuron 1 protein, wherein the methods comprise: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: (a) an AAV capsid, a fragment, or a variant thereof; and (b) a spinal motor neuron 1 (SMN1) protein or a fragment thereof, wherein (a) and (b) are conjugated or crosslinked to the modified leukocyte via a crosslinking agent, thereby tolerizing the population of immune cells to the AAV vector and the SMN1 protein. Further provided herein are methods, wherein the contacting is in vitro, in vivo, or ex vivo. Further provided herein are methods, wherein the contacting is for at least about 10 minutes up to 6 hours. Further provided herein are methods, wherein the AAV capsid is derived from an AAV2, an AAV5, an AAV8, an AAV9, or an AAVrh74. Further provided herein are methods, wherein the AAV capsid comprises an AAV9 VP1. Further provided herein are methods, wherein the population of immune cells comprise a population of monocytes. Further provided herein are methods, wherein the population of immune cells comprise a population of dendritic cells. Further provided herein are methods, wherein the population of dendritic cells comprise a population of CDl lc positive (CDl lc+) dendritic cells. Further provided herein are methods, wherein the population of immune cells comprise a population of T cells. Further provided herein are methods, wherein the population of immune cells comprise a mixed population of monocytes, dendritic cells, and T cells. Further provided herein are methods, further comprising, contacting the immune cells with an SMN1 protein. Further provided herein are methods, wherein the crosslinking agent comprises l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (ECDI); N,N'-diisopropylcarbodiimide (DIC); N,N'- dicyclohexylcarbodiimide (DCC); or a combination thereof.

[0248] Provided herein are methods of tolerizing a subject to a gene therapy composition, wherein the methods comprise: (a) administering to a subject a population of modified leukocytes, wherein the population of modified leukocytes comprise: (i) a viral vector capsid, a fragment, or variant thereof; and (ii) a protein or a fragment thereof, wherein (i) and (ii) are conjugated or crosslinked to a modified leukocyte via a crosslinking agent, (b) administering to a subject a gene therapy composition, wherein the gene therapy composition comprises a viral vector and a transgene encoding the protein crosslinked or conjugated to the modified leukocyte, wherein the administering of population of modified leukocytes tolerizes the subject to the gene therapy composition. Further provided herein are methods, wherein the level of CD33 expressing immune cells is increased relative to immune cells from a subject that has not been administered the population of modified leukocytes. Further provided herein are methods, wherein the level of PD- L1 expressing immune cells is increased relative to immune cells from a subject that has not be administered the population of modified leukocytes. Further provided herein are methods, wherein the subject has at least about a 5 fold increase in the frequency of Tri cells relative to immune cells from a subject that has not be administered the population of modified leukocytes.

[0249] Provided herein are methods of increasing Type 1 regulatory T (Tri) cell proliferation, wherein the methods comprise: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: (a) a viral antigen, antigenic fragment, or variant thereof; or (b) a viral vector, wherein (a) or (b) is conjugated or crosslinked to the modified leukocyte, thereby increasing Tri cell proliferation within the population of immune cells relative to a comparable population of immune cells that have not been contacted with the modified leukocyte.

[0250] Provided herein are methods of increasing the level of CD33 and the level of PD-L1 in a monocyte, wherein the methods comprise: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises: (a) a viral antigen, antigenic fragment, or variant thereof; or (b) a viral vector, wherein (a) or (b) is conjugated or crosslinked to the modified leukocyte, and wherein the population of immune cells comprise a population of monocytes, thereby increasing the level of CD33 and the level of PD-L1 in a monocyte relative to a comparable population of immune cells that have not been contacted with the modified leukocyte.

[0251] While preferred embodiments of the present disclosure have been shown and provided herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure provided herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. EXAMPLES

EXAMPLE 1. Suppression of T Cell Activation with a Tolerizing Regimen or Preparatory Regimen

[0252] To determine whether the tolerizing regimen can suppress the immune response by a recipient, the tolerizing regimen (e.g., apoptotic leukocytes ECDI-crosslinked to viral antigen) is administered to the recipient before and after gene therapy. After the gene therapy and administration of tolerizing regimen, T cell activation in the recipient’s PBMCs is examined.

[0253] Leukocytes prepared from gene therapy recipients are administered to the respective recipient at different time points, for instance: (1) 7 day before the gene therapy; (2) 7 day before the gene therapy and concomitantly with the gene therapy on day 0; or (3) 7 day before and 1 day after the gene therapy. PBMCs are collected from recipients before the gene therapy, and 7, 14, 28, 49, 77, and 91 days after the gene therapy. Direct and indirect T cell activation in the PBMCs are examined by ELISPOT. PBMCs from recipients without tolerizing regimen are used as a negative control. T cell activation in following groups are analyzed: (1) recipients without gene therapy or tolerizing regimen; (2) recipients who received gene therapy + tolerizing regimen; (3) recipients who received gene therapy + no tolerizing regimen. It is expected that administration of the tolerizing regimen can significantly reduce T cell activation induced by the gene therapy.

EXAMPLE 2. Inducing Gene Therapy Tolerance in a Gene Therapy Recipient

[0254] Methods for tolerizing a gene therapy recipient for instance, mammalian subjects (e.g., human or a non-human primate) with the preparatory regimen provided herein (e.g., apoptotic leukocytes ECDI-crosslinked to viral antigen) are provided.

[0255] Cells (e.g., leukocytes) from the gene therapy recipient are fixed by ECDI and used to suppress immune response in the recipient. The tolerizing vaccine or preparatory regimen (e.g, apoptotic leukocytes ECDI-crosslinked to viral antigen) is administered to the recipient before and after gene therapy to determine whether the tolerizing vaccine or preparatory regimen can suppress intolerance by the recipient. T cell activation in the recipient’s PBMCs is examined after the gene therapy and administration of the tolerizing vaccine or preparatory regimen, as described in Example 1.

[0256] Human recipients in need of gene therapy are treated with ECDI fixed cells crosslinked to a viral antigen from the viral vector used for the gene therapy to tolerize the recipient to the gene therapy.

[0257] The ECDI fixed cells can be given to the recipient about 7 days before gene therapy, concomitantly with the gene therapy on day 0, and/or again at about 1 day after gene therapy. A dose of a CD40:CD40L pathway blocking agent (e.g., antagonistic anti-CD40 antibody, antagonistic anti-CD 154 (CD40L) antibody, antagonistic anti-CD40 mAb antibody, Fc- engineered antagonistic anti-CD40L antibodies, antagonistic anti-gp39 antibody, 2C10, 2C10R4, ASKP1240, 4D11, bleselumab, BI-655064, HCD122, CFZ533, DFI105, (or precursor anitbodies ch5D12, PG102, FFP104, etc.), CDP7657, BG9588, ruplizumab, toralizumab, IDEC-131, dapirolizumab, letolizumab, BMS-986004, VIB4920, or MEDI4920), rapamycin, compstatin, a- IL-6R, sTNFR, or any combination thereof can also be given to the recipient about 8 days before gene therapy and 7 and 14 days after gene therapy. The dose of the CD40:CD40L pathway blocking agent can be at least about 30 mg antagonistic anti-CD40 antibody per kg recipient body weight. In some cases, the dose of the CD40:CD40L pathway blocking agent can be about 5-10 mg/kg.

[0258] Optionally, a B-cell targeting agent, (e.g., a B-cell depleting biologic, for example, a biologic targeting CD20, CD 19, or CD22, and/or B cell modulating biologic, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1) or any combination thereof can also be given to the recipient about 8 days before gene therapy and 7 and 14 days after gene therapy. A B-cell targeting biologic can be, for example, Rituximab, or anti-CD20 antibody.

EXAMPLE 3. Inducing Immunotolerance Towards an AAV Vector + Transgene (e.g. Microdystrophin or Sarcoglycan)

[0259] In this example, the compositions and methods of the present disclosure are applied to AAV gene therapy with a specific AAV vector and a transgene.

[0260] In this example, non-human primates (NHPs) lacking pre-existing antibodies to an adeno-associated viral vector, for instance, AAV2, AAV5, AAV8, AAV9, or AAVrh74, undergo infusion of a tolerizing regimen comprising an AAV and transgene (e.g. microdystrophin or sarcoglycan)-conjugated apoptotic autologous leukocytes + anti-CD40 antibody prior to and after the first intravenous delivery of an AAV gene therapy vector (e.g., AAV2, AAV5, AAV8, AAV9, or AAVrh74), which carries the transgene.

[0261] The effect of the tolerizing regimen are examined by monitoring AAV response (parameters discussed below) and liver enzymes in the recipient over 8 weeks and monitoring the frequency of circulating and tissue CD8+ T cells with specificity for peptides derived from capsid proteins of the AAV serotype, in this case AAV-rh74 (for instance by loading MHC class I tetramers with said peptides). After 8 weeks, the AAV vector is given to the recipient, and a necropsy is performed 8 weeks after the second dose.

[0262] A number of parameters are observed before, during, and after the above described infusions. These include total and neutralizing anti-AAV capsid & anti-microdystrophin antibodies; cytokine production and complement activation; T Cell (Circulating & Splenic) Immunity; Capsid Antigen-/transgene-Specific IFN-y, TNF-a secreting T Cells, CD4/CD8 Tem/Tex, CD4/CD8 Treg & CD4 Tri Cells; and transgene expression. To monitor the safety of the infusions, liver enzymes (e.g., AST, ALT, GGT, GLDH, total bilirubin and ALP) can be assessed.

[0263] As set forth in Table 7 below, the assay comprises three groups: a control group which receive only the AAV gene therapy (containing 3 NHPs), a group which receive the AAV gene therapy and induction immunotherapy (containing 3 NHPs), and a group which receive the tolerizing regimen, the AAV gene therapy, and induction immunotherapy (containing 3-6 NHPs). The control group receive 7 x 10¹³ viral genomes (vg)/kg recipient bodyweight AAV + transgene e.g. microdystrophin or sarcoglycan) as AAV gene therapy at weeks 0 and 8, no tolerizing regimen, and no induction therapy. The induction therapy group receives 2 x 10¹² vg/kg recipient bodyweight AAV + transgene as AAV gene therapy at weeks 0 and 8, no tolerizing regimen, and anti-CD40 antibody + Rapamune ®, Enbrel ®, and Actemra® as induction immunotherapy. The tolerizing regimen group receives 7 x 10¹³ vg/kg recipient body weight AAV + transgene as AAV gene therapy at weeks 0 and 8, 5 x 10⁹ AAV- and transgene (e.g. microdystrophin or sarcoglycan)- conjugated apoptotic autologous leukocytes, and anti-CD40 antibody + Rapamune, Enbrel, and Actemra as induction immunotherapy. This scheme is shown in Table 7.

Table 7. Organization of NHP Study of AAV Gene Therapy

EXAMPLE 4. Preclinical Evaluation of ToleraCell Apoptotic Autologous Leukocytes and Transient Immunosuppression with anti-CD40 antibody, Rapamycin, Etanercept, and Tocilizumab for Inducing Tolerance to Repeat Administrations of AAV Transgene Gene Therapy in Male Cynomolgus Macaques

PROTOCOL SYNOPSIS

EXAMPLE 5. Production of tolerizing compositions comprising apoptotic autologous leukocytes conjugated to AA V capsid antigens and optionally a transgene (AA V-AALs)

[0264] Provided is a method of making a composition provided herein, namely an AAV-AALs for tolerizing a recipient to an AAV gene therapy vector that encodes a transgene product.

[0265] The amount of viral antigen or AAV capsid protein is determined. The viral antigen or AAV is conjugated to a leukocyte by crosslinking 0.1- 1 mg of capsid protein per 1 x 10⁹ leukocyte cells autologous to the recipient of the gene therapy. In addition, 0.1-1 mg recombinant transgene product is expressed, and conjugated by crosslinking to the same 10⁹ leukocyte cells (AAV/Transgene-AALs). AAV particles with a given titer shall be used, (as opposed to mass of AAV), conjugation is based on particle number and/or protein content. Accordingly, 1 x 10¹⁵ AAV vector particles contains ~6.5 mg of AAV capsid protein and ~3.5 mg of vector DNA = 10 mg. Approximately 0.1 - 0.9 mg of AAV particles per 1 x 10⁹ leukocyte cells are crosslinked with the leukocytes by contacting leukocytes and AAV particles in the presence of ECDI for a sufficient period of time (e.g., approximately 1-4 hours). For the transgene, e.g. recombinant human survival motor neuron 1 (SMN1) protein are purified (e.g., SEQ ID NO: 45), and 0.1-1 mg of purified protein are crosslinked by use of ECDI to the 1 x 10⁹ leukocyte cells for sufficient time. EXAMPLE 6. Antigen conjugation to apoptotic autologous leukocytes following ECDI fixation [0266] Optimization of the antigen conjugation step to achieve the targeted amount of cell surface AAV9 capsid protein on apoptotic autologous leukocytes (AALs) after fixation with ECDI is described.

[0267] Western blot analysis of the AAV9-coupled apoptotic autologous leukocytes are performed following quantification of the amount of empty capsid protein conjugated to apoptotic autologous B cells.

[0268] The amount of capsid protein present on the conjugated leukocyte after fixation with ECDI is determined. Initially, one concentration of 0.5 microgram per 1 X 10⁶ B cells are conjugated to apoptotic autologous leukocytes and the protein amount retained is determined by Western blot.

[0269] Based on the findings, subsequent studies are used to determine the amount of protein provided for conjugation to retain 0.5 microgram of capsid protein per 1 X 10⁶ apoptotic B cells.

EXAMPLE 7. Antigen Conjugation to Apoptotic Autologous Leukocytes Following ECDI Fixation

[0270] Optimization to determine the concentration of empty AAV9 capsid antigen conjugated to apoptotic autologous leukocytes (AALs) to induce a tolerogenic signature in antigen presenting cells is described. Assays to evaluate in vitro responses of monocytes and dendritic cells (DCs) to the empty AAV9 vector capsid after conjugation to AALs is performed. The AALs are labelled with a lipophilic membrane labeling dye, Vybrant™ Dil Cell-Labeling Solution or PKH67, prior to ECDI treatment to track and analyze the specific monocytes or dendritic cells that have taken up (also referred to herein as internalize or internalization) AALs.

Analysis of tolerogenic potential of AAV9 -conjugated AALs in human DCs

[0271] Briefly, recipient monocytes from cynomolgus macaques are stimulated for 4 hrs and 24 hrs with expanded autologous B cells in Table 8 below.

Table 8. Conditions.

[0272] After in vitro stimulation of monocytes, expression levels of CD80, CD86, CD33, MARCO, PD-L1, and IL-10 are analyzed by multiparametric flow cytometry. Tolerogenic monocytes, especially myeloid-derived suppressor cells (MDSCs) are generated and analyzed. The presence of the lipophilic dye in the monocytes is utilized in the gating strategy to specifically determine the population of monocytes that have, and have not, internalized the AALs.

[0273] The results of the in vitro studies are interpreted as follows. First, a 2-fold increase in the expression of the inhibitory receptors (CD33, PD-L1), (intracellular) IL- 10, and MARCO along with a reduction in the expression of costimulatory molecules (CD80 and CD86) on monocytes stimulated with AALs conjugated with optimal concentrations of empty AAV9 (and recombinant AAV9-VP1 protein for reference) are considered a tolerance signature.

Analysis of tolerogenic potential of AA V9-conjugated apoptotic autologous leukocytes in human DCs

[0274] Human DCs are left untreated or are stimulated at 37°C with AALs coupled with AAV9 with and without anti-CD40 antibody treatment. DCs are analyzed for the expression of MHC class II, CD80, CD86, and CD83 (maturation marker). DCs are analyzed for p65 activation (inflammatory DCs) and TNFR1 (inflammatory) and TNFR2 (DC survival) expression.

[0275] The presence of the lipophilic dye in the DCs is utilized in the gating strategy to specifically analyze the monocytes that have, and have not, internalized the AALs. To determine whether DCs are maturation-arrested after internalization of apoptotic bodies, DCs are washed thoroughly and co-cultured with allogeneic (stimulatory) PBMCs at a ratio of 2 * 10⁴ DC to 1 x 10⁵ T cells for 3 days. PBMCs are analyzed for proliferation of CD4+ and CD8+ T cells by Ki67 staining.

Effect of timing of anti-CD40 antibody administration on DC maturation

[0276] Human DCs are incubated for 3 hrs or 24 hrs with an anti-CD40 antibody. DCs are left untreated or stimulated at 37°C with recombinant sTNF (50 ng/ml). DCs are then be analyzed for the expression of MHC class II, CD80, CD86, and CD83 (maturation marker). DCs are analyzed for p65 activation (inflammatory DCs) and TNFR1 (inflammatory) and TNFR2 (DC survival) expression.

[0277] DCs are washed thoroughly and cocultured with allogeneic (stimulatory) PBMCs at a ratio of 2 x 10⁴ DC to 1 ^x 10⁵ T cells for 3 days. PBMCs are analyzed for proliferation of CD4+ and CD8+ T cells by Ki67 staining.

Molecular signature of tolerance in monocytes

[0278] Monocytes are stimulated with AALs coupled with empty capsid for 4 hrs. Monocytes are collected at 30, 45, 60, 90, 120, and 180 minutes after AAL stimulation for analysis by Droplet Digital PCR. Whole transcriptome analysis is performed on for monocyte markers including CD33 and PD-L1. The kinetics of the monocyte molecular signature is used to predict the tolerance potential of AAL products.

Effect of timing of anti-CD40 antibody administration on CD40 signaling in B cells and monocytes [0279] B cells and - in separate wells - monocytes are incubated for 3 hrs or 24 hrs with an anti- CD40 antibody.

[0280] Cells are left untreated or stimulated at 37°C with multimeric CD40L. Cells are analyzed for the expression of MHC class II, CD80, CD86, and CD83 (maturation marker). Cell proliferation is analyzed by Ki67 staining.

EXAMPLE 8. Optimization to determine the concentration of empty AAV9 capsid antigen conjugated to apoptotic autologous leukocytes to induce a tolerogenic signature in T and B cells [0281] Monocytes generated above are used to stimulate monocyte-depleted PBMCs in a 7-day in vitro culture to study their effect on induction, expansion, and development of effector, memory, and exhaust CD4+ and CD8+ T cell subsets. Different ratios of responders to stimulators (1 : 1, 1 :2, 1 :5) are tested to assess T cell proliferation, T cell signatures, and B cell proliferation.

[0282] Next, PBMCs are challenged with empty AAV9 empty capsids in 5,6-carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reactions (CFSE-MLR). Multiparametric flow cytometry is performed to test the potency of tolerance induction.

[0283] To facilitate the identification of a T cell signature by challenge with AAV9 empty capsid conjugated apoptotic autologous leukocytes (AALs), the functional state of the effector, regulatory and exhaust CD4+ and CD8+ T cells are analyzed to determine the response to rechallenge with AAV9 empty capsids by multiparametric flow cytometry. The frequency of proliferating effector memory T cells (TEM) and regulatory populations of CD4+ and CD8+ T cells and B cells are analyzed.

[0284] Aliquots of cells are stored in SmartTubes for subsequent analysis by CyTOF is also performed. When compared with PBMCs initially stimulated with empty AAV9 capsid (or soluble VP1 recombinant protein), a greater than 50% increase in exhausted (CD4+ or CD8+ T cells expressing PD1+, or TIGIT+ or TIM-3+) or regulatory subsets (Treg or Tri cells) and/or a greater than 2-fold increase in the expression of SH2D2 in regulatory T cell subsets as a marker of their activation in response to stimulation with AALs coupled with AAV9 empty capsid (or AAV9 VP1 protein for reference) is considered a tolerance signature. When compared with PBMCs initially stimulated with AAV9 empty capsid (or soluble VP1 protein), a greater than 50% reduction in the T cells with effector phenotypes (CD4⁺ CD2^, CD8⁺ CD2^hi) that proliferate in response to rechallenge with AAV9 empty capsid (or AAV9 VP1 protein for reference) is considered a tolerance signature.

EXAMPLE 9. ECDI Fixation of AA V capsids and transgene proteins

[0285] Human B cells were mixed with 4xl0⁸ AAV capsid particles per cell and treated with or without ECDI (30mg/ml) for 1 hour at 4°C followed by incubation for 4 hours at 37°C. Fixation of capsid to the cell surface was detected by pan-AAV-specific antibody and measured by flow cytometry (FIG. 6A).

[0286] Apoptosis 4 hours post-ECDI fixation consistently produced 70-90% apoptotic B cells.

[0287] Fixation of AAV9 capsids and proteins to the B cell surface was performed. Conditions are shown in Table 9.

Table 9. ECDI fixation of AAV capsids and transgene proteins.

[0288] Fixation of recombinant transgene protein, SMN1, to the B cell surface was performed. The sequence of human SMN1 is found, e.g., on the NCBI database as Gene ID: 6606 (SEQ ID NO: 44) The amino acid sequence for the recombinant human SMN1 is:

MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGKPKTTPKRKPAKK NKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVWYTGYGNREEQNLSDLLSPICEVANNIEQ NAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSC WLPPFPSGPPI I PPPPPICPDSLDDADALGSMLI SWYMSGYHTGYYMGFRQNQKEGRCSHSLN (SEQ ID NO: 45).

Additional isoforms include survival motor neuron protein isoform a (NCBI Ref: NP 001284644.1, SEQ ID NO: 46), survival motor neuron protein isoform b (NCBI Ref: NP 075012.1, SEQ ID NO: 47), and survival motor neuron protein isoform d (NCBI Ref: NP_000335.1, SEQ ID NO: 48).

[0289] The AAV9 capsid VP1 sequence (e.g., GenBank Ref: AY530579.1) is:

>AY530579 . 1 Adeno-as sociated virus 9 isolate hu . 14 capsid protein VP1 ( cap ) gene , complete cds ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGG CTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCT TCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCG GCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACA ACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGC AGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCT GGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTG CACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACC AATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCA GTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAAT GGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTA CAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCC TGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACA ACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGA CAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTAT CAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGA TTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCT GGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTA CCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACT TGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGG ACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCA ACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGAC GTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTT GTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATA ACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACC ACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTG GCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCAC CCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTG TACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGG CCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAG TACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAAC CCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA (SEQ ID NO: 49) [0290] The amino acid sequence for the AAV9 VP1 capsid used is:

>NCBI Ref: AAS99264.1 capsid protein VP 1 [Adeno-associated virus 9]

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQI SNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMI PQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYI PGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLI FGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKI PHTDGNFH PSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQ YTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 50)

[0291] Late apoptotic cells showed higher levels of conjugated AAV than early apoptotic cells (FIGS. 6B-6C) When B cells were fixed with both AAV capsids and a transgene encoding a protein, the majority of the PH7 Annexin V+ cells expressed AAV in combination with a transgene protein (SMN1) on the cell surface.

EXAMPLE 10. Analysis of monocyte activation by apoptotic leukocytes coupled with AA V.

[0292] AAV9 VP1 protein-conjugated AALs were generated. Monocytes (0.3 xlO^A6/well) from

3 cynomolgus macaques were stimulated in vitro with AAV9 VP1 protein conjugated to AALs for

4 hours. Incubation of monocytes with autologous leukocytes treated with ECDI (apoptotic autologous leukocytes, AALs), with and without conjugated VP1, increased expression of immuno-inhibitory molecules CD33 and PD-L1 as compared with autologous leukocytes and VP 1 protein alone, indicating the induction of a tolerogenic phenotype (FIGS. 7A-7C).

[0293] AAV9 empty capsid-conjugated AALs were generated. Monocytes (0.3 xlO^A6/well) from 4 cynomolgus macaques were stimulated in vitro with AAV9 empty capsid- or VP1 protein- conjugated AALs or with control antigen (un-conjugated AAV9 empty capsid or VP1 protein) for 4 hrs.

[0294] Incubation of monocytes with autologous leukocytes treated with ECDI (apoptotic autologous leukocytes, AALs) with and without conjugated AAV9 empty capsid or VP1, increased their expression of immuno-inhibitory molecules CD33 and PD-L1 compared with controls, indicating the induction of a tolerogenic phenotype (FIGS. 8A-8B).

[0295] Monocytes (0.3 xlO^A6/well) from 4 cynomolgus macaques were stimulated in vitro with PKH67-labeled autologous leukocytes, AAV empty capsid-conjugated AALs, un-labeled AAV empty capsids, or un-labeled VP1 protein for 4 hrs.

[0296] Incubation of monocytes with AALs conjugated with various amounts of empty AAV capsids or VP1 protein increased the expression of CD33 and PD-L1 in monocytes that have internalized PKL67-labeled autologous leukocytes or AALs compared with controls (unconjugated AAV9 empty capsid or VP1 protein alone), indicating the induction of a tolerogenic phenotype (FIGS. 9A-9B). EXAMPLE 11. Analysis of dendritic cell phenotypes.

[0297] AAV9 empty capsid-conjugated AALs were generated. Dendritic cells (DCs) (0.3 xlO^A6/well) from 3 human subjects were stimulated in vitro with Als, AALs, AAV9 empty capsid- conjugated AALs, AAV9 empty capsids, or VP1 protein for 4 hrs.

[0298] Incubation of CD1 lc+ DCs with AALs conjugated with various amounts of empty AAV capsids or VP1 protein increased their expression of CD33 and PD-L1 compared with controls (autologous leukocytes, AAV9 empty capsid, or VP1 protein alone), indicating the induction of a tolerogenic phenotype (FIGS. 10A-10B).

[0299] AAV9 empty capsid-conjugated AALs were generated. DCs (0.3 xlO^A6/well) from 3 human subjects were stimulated in vitro with PKH67-labeled autologous leukocytes and AAV empty capsid-conjugated AALs for 4 hrs.

[0300] Incubation of DCs with ECDI-treated apoptotic autologous leukocytes (AALs) conjugated with various amounts of empty AAV capsids or VP1 protein increased the expression of CD33 and PD-L1 in DCs that have internalized PKL67-labeled Als or AALs compared with controls (autologous leukocytes not treated with ECDI), indicating the induction of a tolerogenic phenotype (FIGS. 11A-11B).

EXAMPLE 12. Analysis of T cell phenotypes

[0301] AAV9 empty capsid-conjugated AALs were generated. DCs (0.3 xlO^A6/well) from 3 human subjects were stimulated in vitro with AAV9 empty capsid-conjugated ECDI-treated autologous leukocytes (apoptotic autologous leukocytes, AALs) or VP1 protein-conjugated AALs for 4 hrs.

[0302] Subsequently, the antigen-pulsed DCs were used to stimulate autologous T cells (1.5 xlO^A6/well) in vitro for 5 days. T cells (1.0 xlO^A6/well) were then re-challenged with empty AAV9 capsids for 5 days.

[0303] Presentation of antigen (AAV9 empty capsid or VP1) conjugated to AALs - compared to incubation with AAV9 empty capsids - increased the expansion of Tregs and Tri cells (both fold-change frequency of Tregs (FIG. 12A) and Tri (FIG. 12C) cells and the frequency ofKi67+, proliferating Tregs (FIG. 12B) and Tri (FIG. 12D) cells) after rechallenge with AAV9 empty capsid antigen.

[0304] AAV9 empty capsid-conjugated AALs were generated. Monocytes (0.3 xlO^A6/well) from 4 cynomolgus macaques were stimulated in vitro with AAV9 empty capsid-conjugated ECDI-treated autologous leukocytes (apoptotic autologous leukocytes, AALs), VP1 protein- conjugated AALs, or unconjugated VP1 for 4 hrs. [0305] Subsequently, these antigen-pulsed DCs were used to stimulate autologous T cells (1.5 xlO^A6/well) in vitro for 5 days. T cells (1.0 xlO^A6/well) were then re-challenged with empty AAV9 capsids for 5 days.

[0306] Presentation of antigen (AAV9 empty capsid or VP1) conjugated to AALs - compared to incubation with AAV9 empty capsids - increased the expansion of Tri cells (fold-change frequency of Tri cells) after rechallenge with empty AAV9 capsid antigen (FIG. 13).

EXAMPLE 13. Treatment regimen for inducing tolerance to a gene therapy in cynomolgus macaques.

[0307] The tolerogenic efficacy and safety of apoptotic autologous leukocyte (AALs) provided herein and transient immunosuppression including an anti-CD40 antibody, and/or other immunosuppressive agents is to be determined by the methods below. An exemplary protocol for the treatment regimen is shown in FIG. 14.

Study Design:

[0308] The assays provided herein are performed in healthy Mauritius cynomolgus macaques (MCMs) with baseline titers of total antibodies against AAV9 of <1 :25 using a validated enzyme- linked immunosorbent assay (ELISA).

[0309] Three animals are assigned to Groups A and B. All test subjects in Groups A and B are administered at least one dose of AAV9-survival motor neuron (SMN1) gene therapy comprised of AAV9 vector packaged with a human SMN1 gene that incorporates a FLAG Tag sequence and is expressed from a is expressed from a cytomegalovirus enhancer/chicken-P-actin hybrid promoter (CAB) promoter (scAAV9.CAB.hSMNl-FLAG). The vector construct is the same, except for the included FLAG tag, as that used in commercially available AAV9 human SMN1 gene therapy vector known as ZOLGENSMA®. Group A subjects receive a single vector dose. Qualifying Group B subjects receive a second vector dose if criteria are met as outlined below.

[0310] Group A test subjects do not receive any tolerance inducing immunotherapy, as the control group. Group B test subjects receive a AAL infusion before and after the first dose of gene therapy and transient immunotherapy including an anti-CD40 antibody, sirolimus, etanercept, and tocilizumab. The primary endpoints are assessed at 16 weeks after the administration of the vector on Day 0 or at end of study, if the study is terminated early.

[0311] If Group B subjects maintain total anti-AAV9 antibody levels below 1 :400 at both week 4 and 7, then at week 8, those qualifying animals are administered a second dose of AAV9-survival motor neuron (SMN1) gene therapy under transient immunotherapy but without a concomitant AAL infusion and followed up for an additional 8 weeks. [0312] scAAV9. CAB. hSMNl-FLAG gene therapy is infused intravenously on day 0 into all test subjects at a dose of 7.4 x 10¹³ viral genomes (vg) per kilogram subject body weight. Qualifying Group B subjects receive a second dose of 7.4 x 10¹³ vg/kg 8 weeks apart from the first dose. Group A receive gene therapy dosing only and serve as a control group for immune activation after a single dose of gene therapy.

[0313] AAL product are infused intravenously in test subjects in Group B at a dose of 0.18 - 0.20 xlO⁹ cells per kilogram subject body weight twice during the study, once 7 days prior to and once 1 day after the first gene therapy administration on day 0. The AAL infusion comprises ex- vivo expanded AALs enriched for B cells, which are conjugated with AAV9 empty capsid protein antigens.

[0314] The anti-CD40 antibody is infused intravenously in all test subjects in Group B at a dose of 70 mg/kg on days -9, -8, -1, 7, 14, 28, 42, 55, 70 and 84 relative to first gene therapy administration on day 0.

[0315] Etanercept and tocilizumab are administered to all test subjects in Group B between days -7 and +21 and in those test subjects that receive a second dose of gene therapy on day +56, etanercept and tocilizumab are administered between days +49 and +77. Sirolimus is administered to all test subjects in Group B between days -9 and +84 relative to the first gene therapy administration on day 0.

Follow-up Period:

[0316] 16 weeks after first and 8 weeks after second gene therapy administration (total follow up of 16 weeks).

Primary Endpoint:

[0317] Proportion of subjects with total anti- AAV9 antibody titer of <1 : 100 at 16 weeks after the first intravenous administration of scAAV9.CAB.hSMNl-FLAG (corresponding to 8 weeks after the second gene therapy in Group B test subjects).

Secondary Endpoints:

[0318] Proportion of subjects with total antibody titers of <1 : 100 against the AAV9 capsid at 4, 8, and 12 weeks after the first intravenous administration of scAAV9.CAB.hSMNl-FLAG (the latter time point corresponding to 4 weeks after the second gene therapy in Group B test subjects). [0319] Proportion of subjects with serum neutralizing antibody titers of <1 :25 against the AAV9 capsid at 4, 8, 12, and 16 weeks after the first intravenous administration of scAAV9.CAB.hSMNl-FLAG (the latter two time points corresponding to 4 and 8 weeks after the second gene therapy in Group B test subjects).

[0320] Titers of total antibodies against the AAV9 capsid at 4, 8, 12, and 16 weeks after the first intravenous administration of scAAV9.CAB.hSMNl-FLAG (the latter two time points corresponding to 4 and 8 weeks after the second gene therapy in Group B test subjects).

[0321] Titers of neutralizing antibodies against the AAV9 capsid at 4, 8, 12, and 16 weeks after the first intravenous administration of scAAV9.CAB.hSMNl-FLAG (the latter two time points corresponding to 4 and 8 weeks after the second gene therapy in Group B test subjects).

[0322] Quantity of hSMNl-FLAG protein expression in muscle biopsies at week 6 and 16, as measured by Western Blot.

[0323] Quantity of hSMNl-FLAG protein expression in muscle biopsies and dorsal root ganglion at week 6 and 16, as measured by immunofluorescence.

[0324] Levels [IgG and IgM; pg/ml] of serum antibodies to hSMNl-FLAG at 8 weeks and 16 weeks after the first intravenous administration of scAAV9.CAB.hSMNl-FLAG.

[0325] Percentages of distinct, circulating T and B cell (incl. naive, exhausted, effector, and various regulatory subsets) and myeloid cell clusters identified at single-cell level by high dimensional mass cytometry.

[0326] Percentage of CD45RA+CD45-CCR7- AAV9-specific CD8+ TEMRA cells of total circulating CD8+ T cells (phenotyping utilizing MHC class I tetramers loaded with AAV9-derived peptides remains to be established) before and at 4, 8, 12, and 16 weeks after the first intravenous administration of scAAV9.CAB.hSMNl-FLAG (the latter two time points corresponding to 4 and 8 weeks after the second gene therapy in Group B test subjects).

[0327] Number of fFN-y-secreting T cells (SFC/10^A6 cells) in response to AAV9-derived peptides by ELISPOT before and at intervals after the first and second administration of scAAV9.CAB.hSMNl-FLAG.

[0328] Number of TNF-a-secreting T cells (SFC/10^A6 cells) in response to AAV9-derived peptides by ELISPOT before and at intervals after the first and second administration of scAAV9.CAB.hSMNl-FLAG.

[0329] Percentages of circulating T cell subsets with distinct secretory proteomes (incl. IFN-y TNF-a, IL- 10 cytokines) without prior and after stimulation with AAV9-derived peptides using the IsoLight® Platform.

[0330] Levels of serum cytokines before and after each intravenous administration of scAAV9.CAB.hSMNl-FLAG.

[0331] Levels of Type I interferons before and after each intravenous administration of scAAV9.CAB.hSMNl-FLAG. [0332] Levels of serum complement activation markers (Bb, C4a, C3a, and sC5b-9) before and after each intravenous administration of scAAV9.CAB.hSMNl-FLAG.

[0333] Incidence and severity of Test Article-Emergent Adverse Events (TEAE) and Adverse Events of Special Interest (AESI, i.e., events related to the administration of scAAV9.CAB.hSMNl-FLAG suggestive of hepatotoxicities, thrombotic microangiopathies or peripheral nerve toxi cities [Time Frame: Baseline up to week 16],

[0334] As one of skill in the art will readily appreciate, this disclosure has been presented for purposes of illustration and description. The disclosure above is not intended to limit the invention to the form or forms disclosed herein. Although the description of the disclosure has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the present disclosure, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

Claims

CLAIMS What Is Claimed Is:

1. A composition that comprises:

(a) a leukocyte;

(b) a crosslinking agent; and

(c) a viral antigen or an antigenic fragment or variant thereof, wherein the viral antigen or antigenic fragment or variant thereof is conjugated or cross-linked to the leukocyte.

2. The composition of claim 1, wherein the viral antigen or antigenic fragment or variant thereof is selected from the group consisting of a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a viral capsid.

3. The composition of any one of claims 1 and 2, wherein the viral antigen or antigenic fragment or variant thereof is from a recombinant viral vector.

4. The composition of claim 3, wherein said recombinant viral vector is selected from the group consisting of: a recombinant herpes simplex virus (HSV) vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picomavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector.

5. The composition of any one of claims 1-3, wherein the viral antigen comprises an empty capsid or a nucleocapsid.

6. The composition of any one of claims 3-5, wherein the recombinant viral vector further comprises a transgene.

7. The composition of claims 1-6, further comprising a transgene product or fragment thereof.

8. The composition of any one of claims 1-7, wherein the crosslinking agent comprises a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof.

9. The composition of claim 8, wherein said diimidoester comprises cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof.

10. The composition of claim 8, wherein the crosslinking agent comprises a carbodiimide that comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof.

11. The composition of any one of claims 1-10, wherein the leukocyte comprises an apoptotic leukocyte or a pre-apoptotic leukocyte.

12. The composition of any one of claims 1-11, wherein the leukocyte comprises a mammalian leukocyte.

13. The composition of claim 12, wherein the mammalian leukocyte comprises a human leukocyte.

14. The composition of any one of claims 1-13, wherein the leukocyte comprises a cadaveric leukocyte.

15. The composition of any one of claims 1-13, wherein the leukocyte comprises a stem cell derived leukocyte.

16. The composition of claim 14, wherein the cadaveric leukocyte comprises from a non-heart beating donor, or a brain-dead donor.

17. The composition of any one of claims 1-16, wherein the leukocyte is from a living donor.

18. A composition for use in tolerizing said living donor of claim 17 to a viral antigen, said use comprising administering to said living donor the composition of claim 17.

19. The composition of any one of claims 1-18, wherein the leukocyte is obtained by ex vivo differentiation of a stem cell, a pluripotent cell, or an induced pluripotent stem cell.

20. The composition of any one of claims 1-19, wherein the leukocyte is isolated from a spleen, a lymph node, a secondary lymphoid organ, a tissue, bone marrow, or peripheral blood.

21. The composition of any one of claims 1-20, wherein the leukocyte comprises an ex vivo expanded leukocyte.

22. The composition of any one of claims 1-21, wherein the leukocyte comprises a B- lymphocyte.

23. The composition of any one of claims 1-22, wherein the leukocyte is fixed with said crosslinking agent for a pre-determined amount of time.

24. The composition of claim 23, wherein said pre-determined amount of time comprises at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes.

25. The composition of any one of claims 1 -24 for use in tolerizing a recipient, wherein the leukocyte expresses a MHC class II molecule that is matched with that of the recipient, and wherein said use comprises administering said composition to said recipient.

26. The composition of any one of claims 1-25, wherein the leukocyte further comprises a MHC class II molecule or one or more peptides derived from the MHC class II molecule, wherein the MHC class II molecule or the one or more peptides derived from the MHC class II molecule is conjugated with the leukocyte.

27. The composition of claim 26 for use in tolerizing a recipient, wherein the MHC class II molecule is matched with that of the recipient, and wherein said use comprises administering the composition to the recipient.

28. The composition of any one of claims 25-27, wherein the MHC class II molecule comprises HLA-DP, HLA-DQ, or HLA-DR.

29. The composition of claim 28, wherein the MHC class II molecule HLA-DP comprises HLA-DP A (a chain), or HLA-DPB (P chain).

30. The composition of claim 28, wherein the MHC class II molecule HLA-DQ comprises HLA-DQA, or HLA-DQB.

31. The composition of claim 28, wherein the MHC class II molecule HLA-DR comprises HLA-DRA, or HLA-DRB.

32. The composition of claim 31, wherein the MHC class II molecule HLA-DRB is selected from HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, or HLA-DR5.

33. The composition of any one of claims 25-32, wherein the MHC class II molecule is encoded by HLA-DRBl*01, HLA-DRBl*03, HLA-DRB1*O4, HLA-DRB1*O7 HLA- DRB1*11, HLA-DRB1*15, or HLA-DRB 1*16 allele of the recipient.

34. The composition of any one of claims 26-33, wherein the one or more peptides derived from the MHC class II molecule comprises a sequence from a hypervariable region of the MHC class II molecule.

35. The composition of any one of claims 26-33, wherein the one or more peptides derived from the MHC class II molecule is at least 10 to 30 amino acid residues in length.

36. The composition of any one of claims 26-33, wherein the one or more peptides derived from the MHC class II molecule are synthesized or recombinant.

37. The composition of any one of claims 1-36, wherein the viral antigen comprises an adenovirus associated virus (AAV) antigen.

38. The composition of claim 37, wherein the AAV antigen is from a recombinant adenovirus associated virus (AAV) vector that has an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i, AAV-DJ or any combination thereof.

39. The composition of any one of the preceding claims, wherein said capsid protein comprises an AAV VP1, VP2, or VP3 capsid protein.

40. The composition of claim 39, wherein said VP1, VP2 or VP3 capsid protein comprises an amino acid sequence that is at least 60% identical to the corresponding capsid protein of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, - 10, -11, -rh74, -rhlO, AAV-2i and AAV-DJ.

41. The composition of any one of the preceding claims, wherein the empty capsid or the nucleocapsid comprises that of an AAV serotype selected from the group consisting of AAV- 1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i, and AAV-DJ.

42. The composition of any one of claims 1-36, wherein the viral antigen is from a recombinant adenovirus vector which is an AAV vector of AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i, AAV-DJ or any chimera or combination thereof.

43. The composition of any one of claims 3-36, wherein the recombinant viral vector comprises a recombinant herpes simplex virus vector which comprises a recombinant herpes simplex virus 1 (HSV1) vector, or a recombinant herpes simplex virus 2 (HSV2) vector.

44. The composition of any one of claims 3-36, wherein the recombinant viral vector comprises a recombinant retrovirus vector which comprises a recombinant Moloney murine sarcoma virus (MMSV) vector, or a recombinant murine stem cell virus (MSCV) vector.

45. The composition of any one of claims 3-36, wherein the recombinant viral vector comprises a recombinant lentivirus vector which comprises a recombinant human immunodeficiency virus 1 (HIV-1) vector or a recombinant human immunodeficiency virus 2 (HIV-2) vector.

46. The composition of any one of claims 3-36, wherein the recombinant viral vector comprises a recombinant alphavirus vector which comprises a recombinant Semliki forest virus (SFV) vector, Sindbis virus (SIN) vector, a recombinant Venezuelan equine encephalitis virus (VEE) vector, or a recombinant alphavirus Ml.

47. The composition of any one of claims 3-36, wherein the recombinant viral vector comprises a recombinant flavivirus vector which comprises a recombinant Kunjin virus vector, a recombinant West Nile virus vector, or a recombinant Dengue virus vector.

48. The composition of any one of claims 3-36, wherein the recombinant viral vector comprises a recombinant rhabdovirus vector which comprises a recombinant Rabies virus vector, or a recombinant vesicular stomatitis virus vector.

49. The composition of claim 3, wherein the recombinant viral vector comprises a recombinant measles virus vector which comprises a recombinant MV Edmonston strain (MV- Edm) vector.

50. The composition of any one of claims 3-36, wherein the recombinant viral vector comprises a recombinant poxvirus vector which comprises a recombinant vaccinia virus (VV) vector.

51. The composition of any one of claims 3-36, wherein the recombinant viral vector comprises a recombinant picornavirus vector which comprises a recombinant Coxsackievirus vector.

52. A composition that comprises:

(a) a nanoparticle,

(b) an MHC class II molecule, or one or more peptides derived from the MHC class II molecule; and

(c) a viral antigen, wherein the viral antigen is selected from the group consisting of: a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a capsid, wherein (b) and (c) are encapsulated in, conjugated, or crosslinked to the nanoparticle.

53. The composition of claim 52, wherein the viral antigen is derived from a recombinant viral vector.

54. The composition of claim 52, further comprising a protein corresponding to a transgene product or fragment thereof.

55. The composition of claim 53, wherein the recombinant viral vector is selected from the group consisting of a recombinant herpes simplex virus (HSV) vector, recombinant alphavirus vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picornavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector.

56. The composition of any one of claims 52-55, wherein the capsid comprises an empty capsid or a nucleocapsid.

57. The composition of any one of claims 52-56, wherein the nanoparticle comprises a lipid nanoparticle.

58. The composition of claim 57, wherein the lipid nanoparticle comprises one or more cationic lipids.

59. The composition of any one of claims 53-58, wherein the recombinant viral vector further comprises a transgene.

60. The composition of any one of claims 57-59, wherein the lipid nanoparticle comprises one or more noncationic lipids.

61. The composition of any one of claims 57-60, wherein the lipid nanoparticle comprises one or more PEG modified lipids.

62. The composition of any one of claims 57-61, wherein the lipid nanoparticle comprises C12-200.

63. The composition of any one of claims 57-62, wherein the lipid nanoparticle comprises DLin-KC2-DMA, CHOL, DMGPEG2K, DOPE, and DMG-PEG-2000.

64. The composition of any one of claims 57-63 wherein the lipid nanoparticle comprises a cleavable lipid.

65. The composition of any one of claims 52-56, wherein the nanoparticle comprises a polymer nanoparticle.

66. The composition of claim 65, wherein the polymer nanoparticle comprises a polymer that is biodegradable.

67. The composition of any one of claims 52-56, wherein the nanoparticle comprises solid-lipid nanoparticle.

68. The composition of any one of claims 52-56, wherein the nanoparticle comprises a micelle.

69. The composition of claim 68, wherein the micelle comprises a polymer comprises an amphiphilic polymer.

70. The composition of claim 68 or claim 69, wherein the micelle comprises a water soluble micelle.

71. The composition of any one of claims 68-70, wherein the micelle coats a solid core.

72. The composition of claim 71, wherein the core comprises a traceable inorganic material selected from the group consisting of iron oxide, CdSe/CdS/ZnS, silver and gold.

73. The composition of claim 71 or claim 72, wherein the diameter of the core is about 5 to 30 nm.

74. The composition of any one of claims 52-73, wherein the nanoparticle is negatively charged.

75. The composition of claim 74, wherein the nanoparticle comprises a zeta potential from about -100 mV to about 0 mV.

76. The composition of claim 74, wherein the nanoparticle comprises a zeta potential from about -60 mV to about -40 mV.

77. The composition of any one of claims 52-76, wherein the nanoparticle surface comprises a functionalized surface group.

78. The composition of claim 77, wherein the functionalized surface group comprises a hydroxyl group, amine group, a thiol group, an alcohol group, or a carboxylic acid group.

79. The composition of any one of claims 65-66, wherein the polymer comprises a synthetic polymer selected from group consisting of poly(maleic anhydride-alt-l-octa-decene), poly(maleic anhydride-alt-1 -tetradecene), and polyisoprene-block poly-ethylene-oxide block copolymer, polylactide-polyglycolide copolymers, polyacrylates, polycaprolactones, poly( D , L -lactide), polycyanoacrylate and poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid), and poly(ethyl methacrylate) (PEMA).

80. The composition of claim 79, wherein the polymer comprises PLGA modified with PEMA as a surfactant.

81. The composition of any one of claims 65-66, wherein the polymer comprises a natural polymer selected from a group consisting of albumin, gelatin, alginate, collagen, chitosan, and dextran.

82. The composition of any one of claims 52-81, wherein the nanoparticle is formulated for targeting to a splenic marginal zone antigen presenting cell or a non-splenic marginal zone macrophage, a dendritic cell, a liver sinusoidal endothelial cell, or an antigen presenting cell in vitro or in vivo.

83. The composition of any one of claims 52-82, wherein the nanoparticle comprises a diameter in the range of 10-1000 nm.

84. The composition of claim 83, wherein the nanoparticle comprises a diameter in the range of 20-900 nm.

85. The composition of claim 84, wherein the nanoparticle comprises a diameter of 500 nm.

86. The composition agent of any one of claims 52-85, wherein the nanoparticle is coated with polyethylene glycol.

87. The composition of any one of claims 52-86, wherein said composition is for use in tolerizing a recipient, wherein the MHC class II molecule is matched with that of said recipient, and said tolerizing comprises administered said composition to said recipient.

88. The composition of any one of claims 52-87, wherein the MHC class II molecule comprises HLA-DP, HLA-DQ, or HLA-DR.

89. The composition of claim 88, wherein the HLA-DP comprises HLA-DPA (a chain), or HLA-DPB (P chain).

90. The composition of claim 88, wherein the HLA-DQ comprises HLA-DQA, or HLA-DQB

91. The composition of claim 88, wherein the HLA-DR comprises HLA-DRA, or

HLA-DRB.

92. The composition of claim 91, wherein the HLA-DR comprises HLA-DRB, and wherein said HLA-DRB is selected from HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, or HLA- DR5.

93. The composition of any one of claims 52-92, wherein said composition is for use in tolerizing a recipient, wherein the MHC class II molecule is encoded by HLA-DRB 1*01, HLA- DRBl*03, HLA-DRB1*O4, HLA-DRB1*O7 HLA-DRB1*11, HLA-DRB1*15, or HLA- DRB 1*16 allele of said recipient, and wherein said tolerizing comprises administering the composition to said recipient.

94. The composition of any one of claims 52-93, wherein the one or more peptides derived from the MHC class II molecule comprises a sequence from a hypervariable region of the MHC class II molecule.

95. The composition of any one of claims 52-94, wherein the one or more peptides derived from the MHC class II molecule is at least 10 to 30 amino acid residues in length.

96. The composition of any one of claims 52-95, wherein the one or more peptides derived from the MHC class II molecule are synthetic or recombinant.

97. The composition of any one of claims 55-96, wherein the recombinant viral vector comprises the recombinant adenovirus associated virus (AAV) vector.

98. The composition of claim 97, wherein the recombinant adenovirus associated virus (AAV) vector has an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, - 6, - 7, -8, -9, -10, -11, -rh74, -rhlO, AAV-2i, chimera or combinations thereof.

99. The composition of any one of claims 52-98, wherein said capsid protein comprises a VP1, VP2, or VP3 capsid protein.

100. The composition of claim 99, wherein the VP1 capsid protein comprises an amino acid sequence that is at least 60% identical to that of an AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i, wherein the VP2 capsid protein comprises an amino acid sequence that is at least 60% identical to that of the AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i, and wherein the VP3 capsid protein comprises an amino acid sequence that is at least 60% identical to that of the AAV serotype selected from the group consisting of AAV-1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i

101. The composition of any one of claims 52-96, wherein the empty capsid or the nucleocapsid comprises that of an AAV serotype selected from the group consisting of AAV-1, - 2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i.

102. The composition of any one of claims 55-96, wherein the recombinant adenovirus vector comprises an adenovirus serotype 5 (Ad5) vector.

103. The composition of any one of claims 55-96, wherein the recombinant herpes simplex virus vector comprises a recombinant herpes simplex virus 1 (HSV1) vector, or a recombinant herpes simplex virus 2 (HSV2) vector.

104. The composition of any one of claims 55-96, wherein the recombinant retrovirus vector comprises a recombinant Moloney murine sarcoma virus (MMSV) vector, or a recombinant murine stem cell virus (MSCV) vector.

105. The composition of any one of claims 55-96, wherein the recombinant lentivirus vector comprises a recombinant human immunodeficiency virus 1 (HIV-1) vector or a recombinant human immunodeficiency virus 2 (HIV-2) vector.

106. The composition of any one of claims 55-96, wherein the recombinant alphavirus vector comprises a recombinant Semliki forest virus (SFV) vector, Sindbis virus (SIN) vector, a recombinant Venezuelan equine encephalitis virus (VEE) vector, or a recombinant alphavirus Ml .

107. The composition of any one of claims 55-96, wherein the recombinant flavivirus vector comprises a recombinant Kunjin virus vector, a recombinant West Nile virus vector, or a recombinant Dengue virus vector.

108. The composition of any one of claims 55-96, wherein the recombinant rhabdovirus vector comprises a recombinant Rabies virus vector, or a recombinant vesicular stomatitis virus vector.

109. The composition of any one of claims 55-96, wherein the recombinant measles virus vector comprises a recombinant MV Edmonston strain (MV-Edm) vector.

110. The composition any one of claims 55-96, wherein the recombinant poxvirus vector comprises a recombinant vaccinia virus (VV) vector.

111. The composition any one of claims 55-96, wherein the recombinant picornavirus vector comprises is a recombinant Coxsackievirus vector.

112. The composition of any one of claims 55-96, wherein the recombinant adenovirus vector comprises an AAV chimera.

113. The composition of any one of claims 55-96, wherein the recombinant adenovirus vector comprises the AAV chimera AAV-DJ.

114. The composition of any one of claims 52-113, wherein the nanoparticle further comprises a peptide tag, detecting agent, a therapeutic agent, a one or more immunomodulatory agents or a combination thereof encapsulated in, or conjugated with the nanoparticle.

115. The composition of any one of claims 1-114, further comprising an immunomodulatory agent, wherein said immunomodulatory agent is an anti-CD40 agent, anti- CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor, IFNy, an NFKB inhibitor, vitamin D3, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, alpha 1 anti-trypsin inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), or any combination thereof.

116. The composition of claim 115, wherein the one or more immunomodulatory agents blocks CD40:CD40L co-stimulation.

117. The composition of claim 115, wherein the NF-kB inhibitor comprises curcumin, triptolide, Bay- 117085, or a combination thereof.

118. The composition of claim 115, wherein the anti-CD40 agent comprises CD40 siRNA.

119. The composition of claim 115, wherein the anti-CD40 agent comprises a CD40 binding peptide inhibitor, anti-CD40 monoclonal antibody, a Fab’ anti-CD40 monoclonal antibody fragment, FcR-engineered, Fc silent anti-CD40 monoclonal domain antibody.

120. The composition of claim 115, wherein the anti-CD40 agent comprises an anti- CD40 L monoclonal antibody, a Fab’ anti-CD40L monoclonal antibody fragment CDP7657, a FcR-engineered, Fc silent anti-CD40L monoclonal domain antibody, a Fab’ anti-CD40L antibody, CD40 binding peptides, anti-CD40 siRNA, CD40L-binding fusion protein or an Fc-engineered anti-CD40L antibody.

121. A tolerogenic composition that comprises:

(a) the composition of any one of claims 1-51; and/or

(b) the composition of any one of claims 52-120, wherein the tolerogenic composition is capable of modulating an immune response to the recombinant viral vector in a recipient that is administered said composition.

122. The tolerogenic composition of claim 121, wherein the leukocyte is an apoptotic leukocyte or a pre-apoptotic leukocyte and expresses a MHC class II molecule that is matched with that of the recipient.

123. The tolerogenic composition of claim 121 or claim 122, wherein the leukocyte comprises the MHC molecule or one or more peptides derived from the MHC molecule that is matched with that of the recipient.

124. The tolerogenic composition of claim 121 or claim 122, wherein the leukocyte comprises the MHC molecule in combination with one or more peptides derived from the MHC molecule that is matched with that of the recipient.

125. The tolerogenic composition of any one of claims 121-123, wherein the tolerogenic composition comprises the leukocyte derived from the recipient.

120

126. The tolerogenic composition of any one of claims 121-123, wherein the nanoparticle comprises the MHC class II molecule that is matched with that of the recipient.

127. The tolerogenic composition of any one of claims 121-125, wherein the recombinant viral vector further comprises a transgene.

128. The tolerogenic composition of claim 127, wherein the transgene encodes a nucleic acid or a polypeptide.

129. The tolerogenic composition of any one of claims 121-127, wherein modulating immune response comprises inhibiting immune response to the viral antigen.

130. The tolerogenic composition of claim 129, wherein inhibiting immune response comprises: inhibiting a B- cell response, inhibiting a T cell response, inhibiting B-cell activation, inhibiting T-cell proliferation, inhibiting T cell migration, inhibiting B-cell proliferation, inhibiting B-cell migration, inhibiting macrophage activation, inhibiting production of one or more cytokines, inhibiting production of antibodies specific for the viral antigen or a combination thereof.

131. The tolerogenic composition of any one of claims 121-130, wherein modulating immune response comprises: in vivo generation, expansion and/or activation of Treg cells CD4+ Tregs, CD8+ Tregs, CD4+ Tri cells, CD8+ Natural Suppressor cells, Breg cells, BIO cells, myeloid derived suppressor cells or other immune regulatory subsets in the recipient.

132. The tolerogenic composition of any one of claims 121-130, wherein the modulating immune response comprises contraction of CD4+ and/or CD8+ T cells specific to said viral antigen in said recipient as compared to corresponding amounts of said CD4+ and/or CD8+ T cells absent administration of the tolerogenic composition.

133. The tolerogenic composition of any one of claims 121-130, wherein the modulating immune response comprises exhaustion of CD4+ and/or CD8+ T cells specific to the viral antigen in said recipient as compared to corresponding amounts of said CD4+ and/or CD8+ T cells absent administration of the tolerogenic composition.

134. A method for inducing tolerance to a recombinant viral vector comprising a transgene in a recipient, the method comprising: administering to the recipient the tolerogenic composition of any one of claims 121-127, in an amount effective to induce tolerance to the recombinant viral vector comprising the transgene.

135. The method of claim 134, further comprising administering to said recipient an immunomodulatory agent, wherein said immunomodulatory agent comprises an anti-CD40 agent, anti-CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor,

121 IFNy, an NFKB inhibitor, vitamin D3, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, an alpha 1 anti-trypsin inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), or any combination thereof.

136. The method of claim 134 or 135, wherein said administering is performed intravenously, intraocularly, intravitreally, via otic administration, via intracardiac injection, or intramuscularly.

137. The method of claim 134, wherein the transgene encodes an autoantigen.

138. The method of claim 137, wherein the autoantigen comprises an islet cell autoantigen selected from the group comprising GAD65, ZnT8, IGRP, or preproinsulin.

139. A method of tolerizing a recipient of an AAV vector to said AAV vector comprising administering to said recipient a tolerizing regimen that comprises: a population of leukocytes from said recipient or a population of leukocytes differentiated in vitro from stem cells extracted from said recipient, wherein the population of leukocytes have been contacted with a crosslinking agent; and an AAV viral antigen or an antigenic fragment or variant thereof, wherein the AAV viral antigen or antigenic fragment or variant thereof shares a serotype with said AAV vector, and wherein said AAV viral antigen is conjugated or cross-linked to the leukocyte.

140. The method of claim 139, wherein said method further comprises administering to the recipient an agent that block the binding of CD40 and CD40L, a mTOR inhibitor, and an inhibitor of at least one pro-inflammatory cytokine.

141. The method of claim 140, wherein said inhibitor of at least one pro-inflammatory cytokine comprises a TNF-alpha inhibitor and/or an IL-6 inhibitor.

142. The method of claim 139 wherein the tolerogenic regimen is administered intravenously on days -7 and +1 relative to a first administration of said AAV vector.

143. The method of any one of claims 139 -142, wherein the administering is performed prior to, simultaneously and/or subsequent to administering the recombinant viral vector to the recipient.

144. The method of any one of claims 139 -143, wherein the administering of said composition inhibits a B- cell response, a T cell response, macrophage activation, cytokine production, or a combination thereof in said recipient, thereby inducing tolerance.

145. The method of claim 144, wherein the B cell response comprises at least one of B- cell activation, B-cell proliferation, and production of neutralizing antibodies specific for the viral antigen.

146. The method of claim 144, wherein the T cell response comprises at least one of T cell activation, T cell proliferation, generation of memory T cells, and generation of T cell effector function involving cytokines or cytolytic mechanisms.

147. The method of claim 139, wherein the administering induces in vivo generation, expansion, and/or activation of Treg cells, CD4+ Tregs, CD8+ Tregs, CD4+ Tri cells, CD8+ Natural Suppressor cells, Breg cells, BIO cells, myeloid derived suppressor cells or other immune regulatory subsets in the recipient, thereby inducing tolerance.

148. A method to modulate immune response to a transduced cell in a recipient, the method comprising: administering to the recipient the tolerogenic composition of claim 121, in an amount effective to modulate immune response to the transduced cell, wherein the transduced cell is generated by contacting a cell with the recombinant viral vector.

149. A method for sustained expression of a transgene in a recipient comprising: administering to the recipient the tolerogenic composition of any one of claims 121-133, prior to, simultaneously and/or subsequent to administering the recombinant viral vector comprising the transgene.

150. A composition for tolerizing a subject to a viral gene therapy vector and an associated transgene that encodes for a transgene product, the composition comprising: a population of leukocytes conjugated by a crosslinking agent to (i) a component of the viral gene therapy vector, and (ii) the transgene product or a fragment or derivative thereof.

151. The composition of claim 150, wherein the viral gene therapy vector comprises AAV.

152. The composition of claim 150, wherein the transgene product comprises a nucleic acid.

153. The composition of claim 152, wherein the nucleic acid comprises a ribonucleic acid.

154. The composition of claim 150, wherein the transgene product comprises a recombinantly expressed protein or a polypeptide fragment or derivative thereof.

155. The composition of claim 154, wherein the transgene product is a transgene listed in Table 2.

156. The composition of claim 150, wherein the component of the viral gene therapy vector is a viral antigen, or antigenic fragment thereof.

157. The composition of claim 156, wherein the viral antigen is a viral capsid or antigenic fragment, domain, peptide or variant thereof.

158. The composition of claim 157, wherein the capsid is empty.

159. The composition of claim 151, wherein the viral gene therapy vector comprises one or more AAV serotypes.

160. The composition of claim 159, wherein the AAV serotype is selected from AAV- 1, -2, -3, -4, -5, -6, - 7, -8, -9, -10, -11, -rh74, -rhlO and AAV-2i.

161. The composition of claim 150, wherein the transgene product or fragment thereof comprises microdystrophin, sarcoglycan, RPE65, Human FVIII, Cas9, or similar transgene product associated with a gene therapy.

162. The composition of any one of claims 150-161, wherein said population of leukocytes are derived from the subject.

163. The composition of any one of claims 150-162, wherein said population of leukocytes are derived from a donor that is MHC Class 2 matched to the subject.

164. The composition of any one of claims 150-163 wherein said population of leukocytes comprises a population of apoptotic leukocytes.

165. The composition of any one of claims 150-163 wherein said population of leukocytes comprises a population of pre-apoptotic leukocytes.

166. The composition of any one of claims 150-165, wherein said crosslinking agent comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof.

167. A method of reducing hepatotoxicity in a recipient of a gene therapy encoding a transgene, comprising administering to the recipient an effective amount of the composition of any one of claims 150-166.

168. A method of reducing or treating one or more of elevated liver enzymes, drug- induced liver injury and hepatic failure in a recipient of a gene therapy encoding a transgene, comprising administering to the recipient an effective amount of the composition of any one of claims 150-166, wherein said administering results in a reduction or treatment of said elevated liver enzymes, drug-induced liver injury and hepatic failure as compared to the recipient in the absence of said administering.

169. A method of improving tolerance for high dose rAAV vector gene therapy in a subject comprising administering to the subject an effective amount of the composition of any one of claims 150-166.

170. A method of suppressing or reducing acute immunotoxicity mediated by CTLs or by the complement system against rAAV vector gene therapy in a subject, comprising administering to the subject an effective amount of the composition of any one of claims 150-166.

124

171. A method of suppressing or reducing cytotoxicity of capsid-specific CD8+ T cells against rAAV vector gene therapy in a subject, comprising administering to the subject an effective amount of the composition of any one of claims 150-166.

172. A method of suppressing or reducing complement activation and adverse events associated with complement activation against rAAV vector gene therapies in a subject, comprising administering to the subject an effective amount of the composition of any one of claims 150-166.

173. A method of producing a tolerizing composition, the method comprising: contacting a leukocyte with a crosslinking agent; and a viral antigen, an antigenic fragment, or variant thereof, wherein the viral antigen, the antigenic fragment, or variant thereof is conjugated or cross-linked to the leukocyte via the crosslinking agent, thereby producing a tolerizing composition.

174. The method of claim 173, wherein the contacting is at least about 10 minutes up to 6 hours.

175. The method of claim 173 or claim 174, wherein the viral antigen or antigenic fragment or variant thereof is selected from the group consisting of: a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a viral capsid.

176. The method of any one of claims 173 to 175, wherein the viral antigen or antigenic fragment or variant thereof is from a recombinant viral vector.

177. The method of claim 176, wherein said recombinant viral vector is selected from the group consisting of: a recombinant herpes simplex virus (HSV) vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picomavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector.

178. The method of any one of claims 173 to 177, wherein the viral antigen comprises an empty capsid or a nucleocapsid.

179. The method of any one of claims 176 to 178, wherein the tolerizing composition further comprises a transgene.

180. The method of any one of claims 173 to 179, wherein the tolerizing composition further comprises a transgene product or fragment thereof.

125

181. The method of any one of claims 173 to 180, wherein the crosslinking agent comprises a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof.

182. The method of claim 181, wherein said diimidoester comprises cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof.

183. The method of claim 181, wherein the crosslinking agent comprises a carbodiimide that comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof.

184. The method of any one of claims 173 to 183, wherein the leukocyte comprises at least one of a late apoptotic leukocyte or a pre-apoptotic leukocyte.

185. The method of any one of claims 173 to 184, wherein the leukocyte comprises a mammalian leukocyte.

186. The method of claim 185, wherein the mammalian leukocyte comprises a human leukocyte.

187. The method of any one of claims 173 to 186, wherein the leukocyte comprises a cadaveric leukocyte.

188. The method of any one of claims 173 to 186, wherein the leukocyte comprises a stem cell derived leukocyte.

189. The method of claim 187, wherein the cadaveric leukocyte comprises from a nonheart beating donor, or a brain-dead donor.

190. The method of any one of claims 173 to 189, wherein the leukocyte is derived from a living donor.

191. A totalizing composition produced by the method of any one of claims 173-190.

192. A method of producing a gene therapy tolerization composition, the method comprising: contacting a population of leukocytes for at least about 10 minutes up to 6 hours with:

(a) a crosslinking agent;

(b) a viral antigen, an antigenic fragment, or variant thereof, wherein the viral antigen, an antigenic fragment, or variant thereof is conjugated or cross-linked to a leukocyte; and

(c) a bioactive agent, wherein the bioactive agent is conjugated or crosslinked to the leukocyte, thereby producing a gene therapy tolerization composition.

126

193. The method of claim 192, wherein the bioactive agent comprises a nucleic acid, a transgene, a transgene product, a protein or a functional fragment thereof, an antibody or an antibody fragment, a nucleic acid encoding an antibody or an antibody fragment, a viral vector, or a combination thereof.

194. The method of claim 193, wherein the transgene product comprises survival motor neuron 1 (SMN1), a microdystrophin, a sarcoglycan family protein, a RPE65 protein, Human FVIII, or a Cas protein.

195. The method of claim 194, wherein the Cas protein is Cas9.

196. The method of claim 193, wherein the nucleic acid encoding an antibody or an antibody fragment comprises a sequence encoding for an anti-CD40 antibody or a fragment thereof; or an anti-CD20 antibody or a fragment thereof.

197. The method of claim 193, wherein the antibody or an antibody fragment comprises a sequence encoding for an anti-CD40 antibody or a fragment thereof; or an anti-CD20 antibody or a fragment thereof.

198. The method of any one of claims 192 to 197, wherein the nucleic acid is a DNA, an RNA, a messenger RNA (mRNA), a microRNA (miRNA), a small non-coding RNA, a long non-coding RNA, or an aptamer.

199. The method of any one of claims 192 to 198, wherein the viral antigen or antigenic fragment or variant thereof is selected from the group consisting of: a capsid protein or an antigenic fragment of the capsid protein, an envelope protein or an antigenic fragment of the envelope protein, or a viral capsid.

200. The method of any one of claims 192 to 199, wherein the viral antigen or antigenic fragment or variant thereof is from a recombinant viral vector.

201. The method of claim 200, wherein said recombinant viral vector is selected from the group consisting of a recombinant herpes simplex virus (HSV) vector, recombinant poxvirus vector, recombinant parvovirus vector, recombinant papillomavirus vector, recombinant simian virus vector, recombinant alphavirus vector, recombinant polyoma virus vector, recombinant picomavirus vector, recombinant lentivirus vector, recombinant retrovirus vector, recombinant adenovirus vector, recombinant adenovirus associated virus (AAV) vector, recombinant flavivirus vector, recombinant rhabdovirus vector, recombinant measles virus vector, recombinant Newcastle disease virus vector, and a recombinant bacteriophage vector.

202. The method of any one of claims 192 to 201, wherein the viral antigen comprises an empty capsid or a nucleocapsid.

203. The method of any one of claims 192 to 202, wherein the recombinant viral vector further comprises a transgene.

127

204. The method of any one of claims 192 to 203, further comprising a transgene product or fragment thereof.

205. The method of any one of claims 192 to 204, wherein the crosslinking agent comprises a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof.

206. The method of claim 205, wherein said diimidoester comprises cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof.

207. The method of claim 205, wherein the crosslinking agent comprises a carbodiimide that comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof.

208. The method of any one of claims 192 to 207, wherein the leukocyte comprises a late apoptotic leukocyte or a pre-apoptotic leukocyte.

209. The method of any one of claims 192 to 208, wherein the leukocyte comprises a mammalian leukocyte.

210. The method of claim 209, wherein the mammalian leukocyte comprises a human leukocyte.

211. The method of any one of claims 192 to 210, wherein the leukocyte comprises a cadaveric leukocyte.

212. The method of any one of claims 192 to 211, wherein the leukocyte comprises a stem cell derived leukocyte.

213. The method of claim 211, wherein the cadaveric leukocyte comprises from a nonheart beating donor, or a brain-dead donor.

214. The method of any one of claims 192 to 213, wherein the leukocyte comprises from a living donor.

215. A gene therapy tolerization composition made by the method of any one of claims 192 to 214.

216. A gene therapy composition that comprises the gene therapy tolerization composition of claim 215; and a viral vector.

217. The gene therapy composition of claim 216, wherein the viral vector is an adenovirus associated viral (AAV) vector, a recombinant AAV (rAAV), a lentiviral vector, a retroviral vector, or an alphaviral vector.

218. The gene therapy composition of any one of claims 216-217, further comprising a transgene.

128

219. A method of treating a disease or condition in a subject, the method comprising:

(a) contacting a population of leukocytes with a crosslinking agent; and a plurality of viral antigens, antigenic fragments, or variants thereof, wherein at least one viral antigen, antigenic fragment, or variant thereof is conjugated or cross-linked to a leukocyte within the population of leukocytes via the crosslinking agent, thereby producing a tolerizing composition;

(b) administering to a subject the tolerizing composition; and

(c) administering a gene therapy composition to the subject, thereby treating the disease or the condition.

220. The method of claim 219, wherein the tolerizing composition further comprises a bioactive agent, wherein the bioactive agent is conjugated or cross-linked to a leukocyte.

221. The method of claim 220, wherein the bioactive agent comprises a nucleic acid, a transgene, a transgene product, a protein or a functional fragment thereof, an antibody or an antibody fragment, or a nucleic acid encoding an antibody or an antibody fragment.

222. The method of claim 221, wherein the transgene product comprises survival motor neuron 1 (SMN1), a microdystrophin, a sarcoglycan family protein, a RPE65 protein, or Human FVIII.

223. The method of any one of claims 219 to 222, wherein the subject has, is suspected of having, or is diagnosed with diabetes, blindness, hearing impairment, multiple sclerosis (MS), Parkinson's disease, Alzheimer's disease, alpha- 1 -antitrypsin deficiency, arthritis, rheumatoid arthritis, Leber congenital amaurosis, hemophilia B, late infantile neuronal lipofuscinosis, muscular dystrophy, Duchenne muscular dystrophy, heart failure, cancer, epilepsy, retinal dystrophy, macular degeneration, familial lipoprotein lipase deficiency, choroideremia, neuropathy, limb ischemia, limb girdle muscular dystrophy, amyotrophic lateral sclerosis, Canavan disease, liver disease, kidney disease, chronic obstructive pulmonary disease (COPD), galactosialidosis, spinal muscular atrophy, limb-girdle muscular dystrophy, giant axonal neuropathy, or late-onset Pompe disease (LOPD).

224. The method of any one of claims 219 to 223, wherein the contacting produces a population of apoptotic leukocytes, wherein the population of apoptotic leukocytes are conjugated or crosslinked to the plurality of viral antigens, antigenic fragments, or variants thereof.

225. The method of claim 219 to 223, wherein the contacting produces a mixed population of leukocytes, wherein the mixed population of leukocytes comprise at least about 20% apoptotic leukocytes.

226. The method of any one of claims 219 to 225, wherein the gene therapy composition comprises:

(a) a viral vector, viral antigen, viral antigenic fragment, or variant thereof; and

129 (b) optionally, a transgene.

227. The method of claim 226, wherein the gene therapy composition comprises a transgene, and wherein the transgene comprises survival motor neuron 1 (SMN1), a microdystrophin, a sarcoglycan family protein, a RPE65 protein, or Human FVIII.

228. The method of any one of claims 219 to 226, wherein the gene therapy composition comprises: idecabtagene vicleucel, lisocabtagene maraleucel, talimogene laherparepvec, voretigene neparvovec, onasemnogene abeparvovec, alipogene tiparvovec, atidarsagene autotemcel, brexucabtagene autoleucel, axicabtagene ciloleucel, betibeglogene autotemcel, cambiogenplasmid, elivaldogene autotemcel, gendicine, tisagenlecleucel, and valoctocogene roxaparvovec.

229. A method of tolerizing a population of immune cells to a gene therapy composition, the method comprising: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises:

(a) viral vector capsid, a fragment, or a variant thereof; and

(b) a protein or a fragment thereof, wherein (a) and (b) are conjugated or crosslinked to the modified leukocyte via a crosslinking agent, thereby tolerizing the population of immune cells to the gene therapy composition.

230. The method of claim 229, wherein the method further comprises contacting the population of immune cells with a gene therapy composition.

231. The method of claim 229 or claim 230, wherein the gene therapy composition comprises a viral vector; and a transgene encoding the protein in (b).

232. The method of any one of claims 229 to 231, wherein the gene therapy composition comprises a viral vector comprising the viral vector capsid, the fragment, or the variant thereof in (a).

233. The method of any one of claims 232, wherein the viral vector is an adeno- associated viral vector (AAV), or a recombinant adeno-associated viral vector (rAAV).

234. The method of claim 233, wherein the AAV comprises an AAV2, an AAV8, an AAV5, an AAV9, or an AAVrh47.

235. The method of any one of claims 229 to 234, wherein the gene therapy composition comprises a protein, wherein the protein comprises spinal motor neuron 1 (SMN1), RPE65, Hemoglobin subunit beta, alpha sarcoglycan, or a microdystrophin.

236. The method of any one of claims 229 to 235, wherein the contacting is in vitro, in vivo, or ex vivo.

130

237. The method of any one of claims 229 to 236, wherein the contacting is for a period of at least about 10 minutes up to 6 hours.

238. The method of any one of claims 229 to 236, wherein the contacting is for a period of at least about 1 hour up to 4 hours.

239. The method of any one of claims 229 to 238, wherein the population of immune cells comprise a population of monocytes.

240. The method of any one of claims 229 to 239, wherein the population of immune cells comprise a population of dendritic cells.

241. The method of claim 240, wherein the population of dendritic cells comprise a population of CD11c positive (CD1 lc⁺) dendritic cells.

242. The method of any one of claims 229 to 241, wherein the population of immune cells comprise a population of T cells.

243. The method of any one of claims 229 to 242, wherein the population of immune cells comprise a mixed population of monocytes, dendritic cells, and T cells.

244. The method of any one of claims 229 to 243, wherein the crosslinking agent comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof.

245. The method of any one of claims 229 to 244, wherein the contacting increases the level of CD33 in a population of monocytes as compared to the level of CD33 in a population of immune cells that have not been contacted with the modified leukocyte.

246. The method of any one of claims 229 to 245, wherein the contacting increases the level of PD-L1 in a population of monocytes as compared to the level of PD-L1 in a population of immune cells that have not been contacted with the modified leukocyte.

247. The method of any one of claims 229 to 246, wherein the contacting increases the level of CD33 in a population of CD11c positive (CD1 lc⁺) dendritic cells as compared to the level of CD33 in a population of immune cells that have not been contacted with the modified leukocyte.

248. The method of any one of claims 229 to 247, wherein the contacting increases the level of PD-L1 in a population of CDl lc⁺ dendritic cells as compared to the level of PD-L1 in a population of immune cells that have not been contacted with the modified leukocyte.

249. The method of any one of claims 229 to 248, wherein after contacting the population of immune cells with a modified leukocyte and the gene therapy composition, the population of immune cells have at least a 5 fold increase in the frequency of Tri cells.

250. The method of any one of claims 229 to 248, wherein after contacting the population of immune cells with a modified leukocyte and the gene therapy composition, the population of immune cells have at least an 8-fold increase in the frequency of Tri cells.

131

251. A method of tolerizing a population of immune cells to an adeno-associated virus

(AAV) vector and a spinal motor neuron 1 protein, the method comprising: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises:

(a) an AAV capsid, a fragment, or a variant thereof; and

(b) a spinal motor neuron 1 (SMN1) protein or a fragment thereof, wherein (a) and (b) are conjugated or crosslinked to the modified leukocyte via a crosslinking agent, thereby tolerizing the population of immune cells to the AAV vector and the SMN1 protein.

252. The method of claim 251, wherein the contacting is in vitro, in vivo, or ex vivo.

253. The method of claim 251 or claim 252, wherein the contacting is for at least about 10 minutes up to 6 hours.

254. The method of any one of claims 251 to 253, wherein the AAV capsid is derived from an AAV2, an AAV5, an AAV8, an AAV9, or an AAVrh74.

255. The method of any one of claims 251 to 254, wherein the AAV capsid comprises an AAV9 VP1.

256. The method of any one of claims 251 to 255, wherein the population of immune cells comprise a population of monocytes.

257. The method of any one of claims 251 to 256, wherein the population of immune cells comprise a population of dendritic cells.

258. The method of claim 257, wherein the population of dendritic cells comprise a population of CD11c positive (CD1 lc⁺) dendritic cells.

259. The method of any one of claims 251 to 258, wherein the population of immune cells comprise a population of T cells.

260. The method of any one of claims 251 to 259, wherein the population of immune cells comprise a mixed population of monocytes, dendritic cells, and T cells.

261. The method of any one of claims 251 to 260, further comprising, contacting the immune cells with an SMN1 protein.

262. The method of any one of claims 251 to 261, wherein the crosslinking agent comprises l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N'- diisopropylcarbodiimide (DIC); N,N'-dicyclohexylcarbodiimide (DCC); or a combination thereof.

263. A method of tolerizing a subject to a gene therapy composition, the method comprising:

(a) administering to a subject a population of modified leukocytes, wherein the population of modified leukocytes comprise:

132 (i) a viral vector capsid, a fragment, or variant thereof; and

(ii) a protein or a fragment thereof, wherein (i) and (ii) are conjugated or crosslinked to a modified leukocyte via a crosslinking agent,

(b) administering to a subject a gene therapy composition, wherein the gene therapy composition comprises a viral vector and a transgene encoding the protein crosslinked or conjugated to the modified leukocyte, wherein the administering of population of modified leukocytes tolerizes the subject to the gene therapy composition.

264. The method of claim 263, wherein the level of CD33 expressing immune cells is increased relative to immune cells from a subject that has not been administered the population of modified leukocytes.

265. The method of claim 263 or claim 264, wherein the level of PD-L1 expressing immune cells is increased relative to immune cells from a subject that has not be administered the population of modified leukocytes.

266. The method of claim 264 or claim 265, wherein the subject has at least about a 5 fold increase in the frequency of Tri cells relative to immune cells from a subject that has not be administered the population of modified leukocytes.

267. A method of increasing Type 1 regulatory T (Tri) cell proliferation, the method comprising: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises:

(a) a viral antigen, antigenic fragment, or variant thereof; or

(b) a viral vector, wherein (a) or (b) is conjugated or crosslinked to the modified leukocyte, thereby increasing Tri cell proliferation within the population of immune cells relative to a comparable population of immune cells that have not been contacted with the modified leukocyte.

268. A method of increasing the level of CD33 and the level of PD-L1 in a monocyte, the method comprising: contacting a population of immune cells with a modified leukocyte, wherein the modified leukocyte comprises:

(a) a viral antigen, antigenic fragment, or variant thereof; or

(b) a viral vector, wherein (a) or (b) is conjugated or crosslinked to the modified leukocyte,

133 and wherein the population of immune cells comprise a population of monocytes, thereby increasing the level of CD33 and the level of PD-L1 in a monocyte relative to a comparable population of immune cells that have not been contacted with the modified leukocyte.

134