Recombinant adeno-associated virus vectors lacking immunodominant T cell epitopes and uses thereof

Abstract

Recombinant adeno-associated virus (AAV) vectors encoding modified VP1 proteins lacking immunodominant T cell epitopes, as well as AAV vector particles containing modified VP1 proteins, are described. The use of recombinant AAV vectors and vector particles as improved gene therapy vectors with reduced immunogenicity is also described. An isolated VP1 peptide containing immunodominant T cell epitopes and its use are further described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 486,299, filed February 22, 2023, which is incorporated herein by reference in its entirety.

[0002] Field The present disclosure relates to recombinant adeno-associated virus (AAV) vectors that express a modified form of the capsid protein VP1 that lacks immunodominant CD4 T cell epitopes. The disclosure further relates to the use of the recombinant AAV vectors, such as in gene therapy applications. Incorporation of electronic sequence listings The Electronic Sequence Listing submitted herewith as an XML file named 9531-109705-02.xml (41,488 bytes), created on February 9, 2024, is hereby incorporated by reference in its entirety. [Background technology]

[0003] background Gene therapy using recombinant adeno-associated virus (AAV) vectors is one of the most promising approaches for treating a wide range of inherited and acquired diseases (Mingozzi and High, Nat Rev Genet 12:341-355, 2011). However, the safety and efficacy of AAV vector-mediated gene transfer in humans is affected by immune responses to AAV capsid proteins, and some cases result in acute toxicity and loss of efficacy (Mingozzi et al., Nat Med 13:419-422, 2007; Verdera et al., Mol Ther 28:723-746, 2020). Several approaches have been explored to modify or prevent the immune response; for example, pharmacological immunosuppressants to block the immune response (Verdera et al., Mol Ther 28:723-746, 2020), vector engineering to reduce therapeutic doses by maximizing vector potency (Ogden et al., Science 366:1139-1143, 2019), codon optimization to interfere with toll-like receptor (TLR) target binding (Wright, Mol Ther 28:1756-1758, 2020), and removal of pre-existing anti-AAV neutralizing antibodies (Leborgne et al., Nat Med 26:1096-1101, 2020; Monteilhet et al., Mol Ther 19:2084-2091, 2011). However, these approaches have their own inherent toxicities (George et al., N Engl J Med 385:1961-1973, 2021) and do not necessarily eliminate immune-related toxicities, especially when high doses of viral vectors are used (High-dose AAV gene therapy deaths, Nat Biotechnol 38:910, 2020). Thus, there is a need for improved AAV gene therapy vectors with reduced immunogenicity and enhanced safety. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Mingozzi and High, Nat Rev Genet 12:341-355, 2011 [Non-patent document 2] Mingozzi et al., Nat Med 13:419-422, 2007 [Non-patent document 3] Verdera et al., Mol Ther 28:723-746, 2020 [Non-patent document 4] Ogden et al., Science 366:1139-1143, 2019 [Non-Patent Document 5] Wright, Mol Ther 28:1756-1758, 2020 [Non-patent document 6] Leborgne et al., Nat Med 26:1096-1101, 2020 [Non-Patent Document 7] Monteilhet et al., Mol Ther 19:2084-2091, 2011 [Non-patent document 8] George et al., N Engl J Med 385:1961-1973, 2021 Summary of the Invention [Means for solving the problem]

[0005] overview Disclosed herein is the identification of a promiscuous, immunodominant CD4 T cell epitope in AAV9 virion protein 1 (VP1) and its elimination in AAV vectors by rationally designed chimerization. Recombinant AAV vectors engineered to lack the T cell epitope maintained their function and efficacy, including yield, cell specificity, in vitro and in vivo transduction efficacy, and biodistribution in mice, without eliciting any cellular immune response. Thus, the present disclosure addresses the unmet need for AAV vectors with reduced immunogenicity and improved safety.

[0006] Provided herein is an isolated nucleic acid molecule encoding a modified AAV virion VP1 protein that lacks a native CD4 T cell epitope. In some embodiments, the modified AAV VP1 protein has an amino acid sequence that is at least 75% identical to the wild-type AAV serotype 9 (AAV9) VP1 protein set forth as SEQ ID NO: 1, and includes one or more amino acid substitutions resulting in a valine at position 315 and an isoleucine at position 317 of SEQ ID NO: 1; an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO: 1; an arginine at position 314 and a valine at position 315 of SEQ ID NO: 1; an arginine at position 314 and an isoleucine at position 317 of SEQ ID NO: 1; or an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO: 1. In some examples, the modified VP1 comprises a valine at position 315 and an isoleucine at position 317 of SEQ ID NO: 1. In other examples, the modified VP1 comprises an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO: 1.

[0007] Also provided is a vector comprising a nucleic acid molecule encoding the modified VP1 disclosed herein.In some embodiments, the vector is an AAV vector, such as an AAV1, AAV2, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV13 vector.In some examples, the AAV vector is an AAV9 vector.In some examples, the AAV vector further comprises a heterologous open reading frame (ORF), such as a therapeutic gene.

[0008] Further provided is a host cell comprising an isolated nucleic acid molecule or vector disclosed herein.

[0009] Also provided are recombinant AAV vector particles comprising a modified AAV VP1 protein lacking a native CD4 T-cell epitope. In some embodiments, the modified AAV VP1 protein has an amino acid sequence at least 75% identical to the wild-type AAV9 VP1 protein set forth as SEQ ID NO:1, and includes one or more amino acid substitutions resulting in a valine at position 315 and an isoleucine at position 317 of SEQ ID NO:1; an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO:1; an arginine at position 314 and a valine at position 315 of SEQ ID NO:1; an arginine at position 314 and an isoleucine at position 317 of SEQ ID NO:1; or an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO:1. In some examples, the modified VP1 comprises a valine at position 315 and an isoleucine at position 317 of SEQ ID NO: 1. In other examples, the modified VP1 comprises an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO: 1. The AAV vector particle can be, for example, an AAV1, AAV2, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV13 vector particle. In specific examples, the AAV vector particle further comprises an AAV genome, such as a recombinant AAV genome comprising a heterologous ORF (e.g., a therapeutic gene).

[0010] Also provided are compositions comprising a pharmaceutically acceptable carrier and a recombinant AAV vector or recombinant AAV vector particle disclosed herein.

[0011] Further provided herein is a method of administering a therapeutic gene to a subject by administering to the subject a recombinant AAV vector particle disclosed herein, wherein the vector particle comprises a recombinant AAV genome containing the therapeutic gene.

[0012] Also provided herein are isolated VP1-derived peptides containing CD4 T cell epitopes. In some embodiments, the isolated peptides are 40 amino acids or less in length and contain the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. Kits are also provided that include the isolated peptides disclosed herein. Such kits can be used, for example, in immune monitoring assays.

[0013] Further provided is a method for inducing immune tolerance to AAV in a subject by administering to the subject an isolated VP1 peptide disclosed herein. In some embodiments, the method further comprises administering to the subject one or more immunomodulatory agents.

[0014] The foregoing and other features of the present disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1-1]Rational design of chimeric AAV9 gene therapy vectors. (Figure 1A) Responses of PBMCs obtained from 52 donors to a peptide pool derived from the AAV9 VP1 protein were measured by interleukin (IL)-2 and interferon (IFN)-γ ELISpot assays. PBMCs from healthy donors were stimulated with empty AAV9 capsids for 14 days, followed by restimulation with a peptide pool spanning the amino acid sequence of AAV9 VP1. 24 hours later, IL-2 and IFN-γ-producing cells were detected by ELISpot assays. PBMCs were considered responders if the peptide induced a >3-fold change in IL-2 or a >2.1-fold change in IFN-γ. (Figure 1B) Immunodominant peptides (SEQ ID NOS: 3-5) were identified in pool 9. (Figure 1C) Using intracellular flow cytometry staining, TNF-α and IFN-γ-producing cells were measured within gated CD4 or CD8 T cells. (Figure 1D) CD4 or CD8 T cells in AAV9-stimulated PBMCs were depleted using microbeads and stimulated with peptides 103–105 or phytohemagglutinin (PHA). Next, the number and size of spots for IFN-γ were analyzed using an ELISpot assay. (Figure 1E) VP1 peptides 103–105 in AAV serotypes 1–13 were aligned using DNAstar software and showed a high degree of conservation, with the exception of amino acids R312, L313, N314, F315, and L317. (Figure 1F) The IEDB T cell epitope prediction tool was used to predict the binding affinity of peptides (SEQ ID NOs: 19–27) for 27 human leukocyte antigen (HLA) class II alleles. Predictions were made using the IEDB consensus method. The number of alleles was counted if their percentile ranks predicted by the IEDB method were below 10%. (Figure 1G) Location of modified amino acids in the AAV9 VP1 protein and AAV9 capsid (DiMattia et al., J Virol 86:6947-6958, 2012) (PBD-3UX1, PMID: 22496238). Amino acids K311 to V326, which contain the immunodominant epitope, and the five amino acids modified into the AAV5 sequence are indicated. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above.

[0016] [Figure 2-1]Characterization of chimeric AAV9 variants. (Figures 2A-B) HEK293T cells were transduced with chimeric AAV9 variants carrying the GFP gene at the indicated multiplicity of infection (MOI; viral genomes (vg) / cell). (Figure 2A) The percentage of GFP-positive cells was determined by flow cytometry. (Figure 2B) A four-parameter curve fit was calculated for each vector, and the area under the curve (AUC) was calculated. Values ​​are presented as mean ± SD (n = 3). (Figures 2C-D) Chimeric AAV9 variants expressing NanoLuc were incubated with pooled human serum at the indicated concentrations for 1 hour. HEK293T cells were transduced with the vectors at an MOI of 50,000 vg / cell. Transgene expression was determined by luminescence and expressed as transduction efficiency (%). Complete transduction (100%) was defined based on the relative light units (RLU) obtained from incubation of AAV vectors with fetal bovine serum. Inhibition of vector transduction by neutralizing antibodies is expressed as a percentage of transduction. (Figure 2C) ND50 values ​​were calculated as the dilution required for 50% neutralization of vector transduction. (Figure 2D) AUC was calculated for Figure 2C. Values ​​are presented as mean ± SD (n = 3). *p < 0.05 and **p < 0.01. P values ​​were determined by ANOVA with Tukey's multiple comparison test. (Figures 2E-H) NanoLuc-expressing chimeric AAV9 variants (1 × 10 vg / mouse) were intravenously injected into Balb / c mice. (Figure 2E) Representative images of NanoLuc expression in mice 8 days after vector administration. (Figure 2F) Quantification of NanoLuc signal in vector-injected mice on days 8, 17, and 29 after vector administration. (Figures 2G-2H) Representative images (Figure 2G) and quantification (Figure 2H) of luciferase signal in various organs of vector-injected mice on day 29. (Figure 2I) Quantification of viral genomes in various organs of vector-injected mice on day 29. (Figure 2J) PBMCs were stimulated with the indicated AAV vectors for 14 days. Cells were restimulated with individual peptides (X-axis; SEQ ID NOs: 19-27, from left to right), and IFN-γ-producing cells were detected by ELISpot assay. Values ​​are presented as mean ± SD.(Figures 2K-2L) PBMCs were stimulated with the indicated AAV vectors for 14 days. Cells were restimulated with individual peptides (SEQ ID NO: 19, SEQ ID NO: 23, and SEQ ID NO: 27, from top to bottom), and IFN-γ (Figure 2K) and IL-2 (Figure 2L)-producing cells were measured by ELISpot assay. Fold changes compared to medium controls were analyzed (number of spots × size for IFN-γ and number of spots for IL-2). Data are means ± sem. Data were evaluated by two-tailed unpaired t-test. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above.

[0017] [Figure 3-1] Representative patterns of IL-2 and IFN-γ secretion in response to AAV9-derived peptides. (Figure 3A-B) Healthy PBMCs were stimulated with empty AAV9 capsids for 14 days, followed by restimulation with the AAV9 pool. 24 hours later, IFN-γ (Figure 3A) and IL-2 (Figure 3B) producing cells were detected by ELISpot assay. (Figure 3C-D) IFN-γ (Figure 3C) and IL-2 (Figure 3D) responses to the 12 peptides (97-108) that comprise Pool 9. Values ​​are presented as mean ± SD. [Figure 3-2] Same as above.

[0018] [Figure 4] Representative gating strategy. (Figure 4A) Single cells (FSC-A / FSC-H). FSC-A versus viability dye within single cells allows for detection of live cells. CD8+ or CD4+ cells within the CD3+TCRγδ- population allow for identification of helper CD4 T cells or cytotoxic CD8 T cells. (Figure 4B) Populations of CD4 and CD8 T cells analyzed in Figure 1D.

[0019] [Figure 5]Epitopes 103-105 are HLA-DP restricted. PBMCs were stimulated with empty AAV9 capsids for 14 days and then restimulated with peptides 103-105 in the presence of HLA-blocking antibodies. 24 hours later, IFN-γ-producing cells were detected by ELISpot assay. The inhibition rate was normalized by the isotype control (0%). Each dot represents an individual donor, and each bar shows the mean ± SEM.

[0020] [Figure 6] Comparison of IFN-γ secretion in response to AAV5 and AAV9 peptides. (Figure 6A) Table listing the peptides derived from AAV5 or AAV9 used in these assays. (Figures 6B-6C) Healthy PBMCs were stimulated with empty AAV5 (Figure 6B) or AAV9 (Figure 6C) capsids for 14 days, followed by restimulation with the indicated peptides. 24 hours later, IFN-γ-producing cells were detected by ELISpot assay. Fold change in spot number × size was calculated by comparing with the respective medium-only control. Each bar represents the mean ± SD.

[0021] [Figure 7] Epitopes 103-105 are not found in functionally important amino acid positions. Functionally important amino acid residues in AAV9 VP1 (SEQ ID NO: 1) are highlighted based on previously published data (Adachi et al., Nat Commun 5:3075, 2014). Black boxes indicate epitopes 103-105.

[0022] [Figure 8-1]Comparison of the transduction efficacy of chimeric AAV9 variants in other cell lines. HeLa cells (Figures 8A and 8C), HEK293T cells (Figure 8C), and A375 cells (Figures 8B and 8C) were transduced with AAV9, AAV9-VI, AAV9-RSRVI, or AAV5 vectors at the indicated MOIs. The percentage of GFP-positive cells was determined by flow cytometry. Each bar represents the mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. P values ​​were determined by ANOVA with Tukey's multiple comparison test. (Figure 8C) Representative GFP fluorescence images of HeLa, HEK293T, and A375 cells 2 days after transduction with individual AAV vectors. Images were captured at 20x magnification. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0023] [Figure 9-1] Comparison of transduction efficacies of chimeric AAV9 variants with the NanoLuc transgene. HeLa cells (FIG. 9A), HEK293T cells (FIG. 9B), and A375 cells (FIG. 9C) were transduced with AAV9, AAV9-VI, AAV9-RSRVI, or AAV5 vectors expressing NanoLuc at the indicated MOIs. The following day, the bioluminescence intensity of the cells was determined using a luminometer. Each bar represents the mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. P values ​​were determined by ANOVA with Tukey's multiple comparison test. [Figure 9-2] Same as above. [Figure 9-3] Same as above.

[0024] [Figure 10-1]Neutralizing antibody analysis of chimeric AAV9 variants. Chimeric AAV9 variants expressing NanoLuc were incubated with the indicated concentrations of human serum for 1 hour. HeLa (Figure 10A) and A375 (Figure 10B) cells were transduced with the vectors at an MOI of 50,000, and the AUC was calculated. Transgene expression was determined by luminescence and expressed as transduction efficiency (%). Complete transduction (100%) was defined based on the RLU results obtained from incubation of AAV vectors with fetal bovine serum. Inhibition of vector transduction by neutralizing antibodies is expressed as a percentage of transduction. The ND50 value was calculated as the dilution required to neutralize 50% vector transduction. Values ​​are presented as mean ± SD (n = 3). Each bar represents the mean ± SD. *p < 0.05. P values ​​were determined by ANOVA with Tukey's multiple comparison test. [Figure 10-2] Same as above.

[0025] [Figure 11] Chimeric AAV9 variants do not create new T cell epitopes. Representative responses to 20 peptide pools after stimulation with the indicated vectors and restimulation with AAV9 peptide pools. Data from two separate experiments are combined.

[0026] [Figure 12] Table showing HLA class II alleles for the 12 responders.

[0027] [Figure 13] Table showing predicted binding of modified AAV9 peptides (SEQ ID NOs: 19-27) to IEDB major histocompatibility complex (MHC) class II alleles.

[0028] [Figure 14]The second most prevalent epitope in pool 18 is not a good candidate for chimera design. (Figures 14A-B) Healthy PBMCs were stimulated with empty AAV9 capsids for 14 days, followed by restimulation with individual peptides (205-216) in pool 18 (Figure 14A) or AAV5 peptides aligned with the AAV9 peptides (Figure 14B). 24 hours later, IFN-γ-producing cells were detected by ELISpot assay. The spot number × size fold change was calculated by comparing with the respective medium-only control. (Figure 14C) The IEDB T cell epitope prediction tool was used to predict the binding affinity of peptides to 27 HLA class II alleles. Predictions were made using the "IEDB consensus method." The number of alleles was counted if their percentile rank predicted by the IEDB method was below 10%.

[0029] [Figure 15-1] Representative silver staining of WT and chimeric AAVs.

[0030] [Figure 15-2] Representative UV chromatograms displaying normalized UV intensity (solid line) with an overlaid scatter plot showing the Full-Total Ratio (Vg / Cp) of WT and chimeric constructs. DETAILED DESCRIPTION OF THE INVENTION

[0031] array The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and single-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. In the accompanying sequence listing:

[0032] SEQ ID NO: 1 is the amino acid sequence of AAV9 VP1. Residues 307-327, corresponding to peptides 103-105, are underlined. [ka]

[0033] SEQ ID NO: 2 is the amino acid sequence of AAV5 VP1. [ka]

[0034] SEQ ID NO: 3 is the amino acid sequence of peptide 103 (GFRPKRLNFKLFNIQ).

[0035] SEQ ID NO: 4 is the amino acid sequence of peptide 104 (PKRLNFKLFNIQVKE).

[0036] SEQ ID NO: 5 is the amino acid sequence of peptide 105 (LNFKLFNIQVKEVTD).

[0037] SEQ ID NOs: 6 to 18 are the amino acid sequences of the peptides shown in FIG. 1E. [Table 5-1] [Table 5-2]

[0038] SEQ ID NOs: 19 to 27 are the amino acid sequences of the peptides shown in FIG. 1F, FIG. 2J, and FIG. [Table 6]

[0039] SEQ ID NOs: 3 to 5 and 28 to 34 are the amino acid sequences of the peptides shown in FIG. 6A. [Table 7]

[0040] SEQ ID NO: 35 is the amino acid sequence of a modified AAV9 VP1 protein with F315V and L317I substitutions (AAV9-VI; substitutions are indicated by bold underlining). [ka] [ka]

[0041] SEQ ID NO: 36 is the amino acid sequence of a modified AAV9 VP1 protein (AAV9-RSRVI; substitutions are indicated by bold underlines) with K311R, R312S, N314R, F315V, and L317I substitutions. [ka]

[0042] SEQ ID NO: 37 is the nucleic acid sequence encoding the AAV9-VI protein (codon changes compared to WT AAV9 VP1 are indicated by bold underlining). [ka] [ka]

[0043] SEQ ID NO: 38 is the nucleic acid sequence encoding the AAV9-RSRVI protein (codon changes compared to WT AAV9 VP1 are indicated by bold underlining). [ka] [ka]

[0044] Detailed Description I. Introduction This disclosure describes research to identify and eliminate immunodominant T cell epitopes within the AAV capsid. Elimination of capsid T cell epitopes allows for the development of improved AAV gene therapy vectors with reduced immunogenicity. In the first clinical trial for the treatment of hemophilia B, circulating AAV capsid-specific T cells increased concomitantly with liver enzymes, resulting in the loss of transgene expression within several weeks (Mingozzi et al., Nat Med 13:419-422, 2007). Rational design of AAV vectors by replacing components containing epitopes for T cell recognition has direct benefits for many gene therapies. To reduce or eliminate the immunogenicity of AAV vectors, it is important that methods for eliminating MHC-binding epitopes do not disrupt vector structure and function. Disclosed herein are rationally designed chimeric AAV vectors that use the integration of ex vivo T cell assays, MHC epitope prediction (Martini et al., Immunogenetics 72:57-76, 2020) and sequence conservation analysis in AAV phylogeny.

[0045] II. Abbreviations AAV adeno-associated virus Area under the AUC curve DLS Dynamic Light Scattering HLA human leukocyte antigen IFN Interferon IL Interleukin ITR inverted terminal repeat MHC major histocompatibility complex MOI Multiplicity of infection ND50 50% neutralizing dose ORF Open Reading Frame PBMC peripheral blood mononuclear cells PHA Phytohemagglutinin RLU Relative Light Units TCR T cell receptor TLR toll-like receptor TNF tumor necrosis factor vg viral genome VP1 virion protein 1 WT wild type

[0046] III. Terminology Overview Unless otherwise stated, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's Genes XII, published by Jones & Bartlett Learning, 2017. As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" refer to both the singular and the plural. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." It should be further understood that, unless otherwise indicated, any and all base or amino acid sizes and any molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for convenience. While many methods and materials similar or equivalent to those described herein can be used, certain preferred methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

[0047] Adeno-associated virus (AAV): A small, replication-deficient, non-enveloped virus that infects humans and some other primate species. AAV is not thought to cause disease and induces a mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can persist extrachromosomally without integrating into the host cell genome. These features make AAV an attractive viral vector for gene therapy. Currently, there are 13 recognized natural serotypes of AAV (AAV1-13). In the context of this disclosure, "AAV vector" refers to a vector based on the nucleic acid AAV. In some embodiments, the disclosed AAV vectors contain 5' and 3' inverted terminal repeats (ITRs), a heterologous promoter, and / or a heterologous ORF (e.g., a therapeutic gene). In the context of this disclosure, "AAV vector particle" refers to a viral particle composed of AAV capsid proteins, including virion protein 1 (VP1), VP2, and VP3. In some embodiments of the present disclosure, the AAV vector particle comprises a VP1 protein lacking immunodominant T cell epitopes. In some examples, the AAV vector particle further comprises an AAV genome, such as a genome comprising 5' and 3' ITRs, a heterologous promoter, and / or a heterologous ORF (e.g., a therapeutic gene).

[0048] Administration: To provide or give an agent, such as a therapeutic agent (e.g., a recombinant AAV), to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intratumoral, or renal vein injection), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0049] Codon-optimized: Nucleic acid molecules encoding proteins (such as modified AAV VP1) can be codon-optimized for expression in a particular organism by including the codon most likely to encode a particular amino acid at each position in the sequence. Codon usage bias is the difference in the frequency of synonymous codons (which encode the same amino acid) in coding DNA. A codon is a sequence of three nucleotides (triplets) that encodes a specific amino acid residue in a polypeptide chain or terminates translation. While there are 20 different naturally occurring amino acids, there are 64 different codons (61 codons that encode amino acids, plus three stop codons). Thus, degeneracy exists because an amino acid can be encoded by more than one codon. By assessing the codon usage bias in an organism and selecting the codon most likely to encode a particular amino acid, a nucleic acid sequence can be optimized for expression in a particular organism (such as humans) of interest. Multivariate statistical methods, such as correspondence analysis and principal component analysis, are widely used to analyze variation in codon usage. Computer programs, such as Codon W, GCUA, and INCA, are available for performing statistical analyses of codon usage.

[0050] Degenerate variant: A polynucleotide encoding a protein (e.g., a modified VP1 protein) that contains a sequence that is degenerate as a result of the genetic code. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Thus, any degenerate nucleotide sequence is included as long as the amino acid sequence of the T cell receptor (TCR) or portion thereof encoded by the nucleotide sequence is unchanged.

[0051] Epitope: Antigenic determinant. This is a particular chemical group or peptide sequence on a molecule that is antigenic (elicits a specific immune response). A TCR or antibody specifically binds to a particular antigenic epitope on a polypeptide.

[0052] Heterologous: Originating from a different genetic source. In the context of this disclosure, a heterologous ORF refers to an ORF that is not native to the AAV.

[0053] Host cell: A cell in which a vector can be propagated and its nucleic acid can be expressed. This term includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell, since there may be mutations that occur during replication. However, when the term "host cell" is used, such progeny are included. The cell can be a prokaryotic or eukaryotic cell, such as a mammalian cell, yeast cell, insect cell, or bacterial cell. In some embodiments, the cell is a human cell.

[0054] Human leukocyte antigen (HLA): A protein encoded by the MHC gene complex. HLA from MHC class I includes the HLA-A, HLA-B, and HLA-C genes. HLA from MHC class II includes the HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR genes. HLA genes are highly variable, with up to several hundred variant alleles at several loci.

[0055] Immune tolerance: prevention or inhibition of the immune response to a particular antigen.

[0056] Immunomodulator: A drug that stimulates or suppresses the immune system. Immunosuppressants can be used to reduce immune responses to foreign antigens, transplanted tissues / organs, and to treat some types of autoimmune diseases. Exemplary immunosuppressants include, for example, cyclosporine A, tacrolimus, sirolimus, prednisone, dexamethasone, azathioprine, cyclophosphamide, and certain types of monoclonal antibodies. Conversely, immunostimulatory agents enhance the immune system, such as to promote immune responses against infectious agents and tumors. Exemplary immunostimulatory agents include BCG, LPS, recombinant cytokines (e.g., IL-2, IL-1, IL-12, and IFN-γ), and antigen-specific antibodies (e.g., tumor-specific antibodies, e.g., 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab Mertansine). mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, catumaxomab, CC49, cetuximab, sitatuzumab bogatox, cizutumumab, clivatuzumab tetraxetan Tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab Ozogamicin, Girentuximab, Glembatumumab Vedotinvedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Rumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentan, ofatumumab, olaratumumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, robatumumab, satumomab pendetide pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab, TGN1412, Ticilimumab (Tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab, and Zalutumumab.

[0057] Inverted terminal repeats (ITRs): Symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs function as replication origins for viral DNA synthesis and are cis-components for generating AAV integrating vectors.

[0058] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, virus, or cell) has been substantially separated or purified from other biological components, such as other chromosomal and extrachromosomal DNA and RNA, proteins, and cells, such as cells or tissues of the organism in which it occurs or the organism itself. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins. Isolated does not require absolute purity and can include proteins, peptides, nucleic acids, viruses, or cells that are at least 50% pure, e.g., at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% pure.

[0059] Open reading frame (ORF): A nucleic acid molecule (e.g., DNA or cDNA) that does not contain a stop codon when translated into amino acids.

[0060] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0061] Pharmaceutically acceptable carriers: Remington: The Science and Practice of Pharmacy, 22 nd(ed., London, UK: Pharmaceutical Press, 2013) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules, or agents (e.g., AAV vectors). Generally, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations typically comprise an injectable fluid that includes a pharmaceutically and physiologically acceptable fluid, such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like, as a vehicle. In addition to a biologically neutral carrier, the pharmaceutical composition to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, and the like, for example, sodium acetate or sorbitan monolaurate.

[0062] Promoter: A region of DNA that directs / initiates transcription of a nucleic acid (e.g., a gene). A promoter contains necessary nucleic acid sequences near the transcription start site. Typically, a promoter is placed near the gene it transcribes. A promoter also optionally contains distal enhancer or repressor elements, which can be located as far as several thousand base pairs from the transcription start site. Promoters can be constitutive, inducible, tissue-specific, and / or ubiquitous.

[0063] Recombinant: A recombinant nucleic acid molecule is a nucleic acid molecule having a sequence that does not occur in nature or that has a sequence that has been created by the artificial combination of two otherwise separated sequence segments. This artificial combination can be achieved by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, e.g., by genetic engineering techniques. Similarly, a recombinant virus is a virus that contains a sequence (such as a genomic sequence) that does not occur in nature or that has been created by the artificial combination of at least two sequences of different origins. The term "recombinant" also includes nucleic acids, proteins, and viruses that have been altered solely by the addition, substitution, or deletion of portions of naturally occurring nucleic acid molecules, proteins, or viruses.

[0064] Sequence identity: The identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity / similarity when aligned using standard methods.

[0065] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed discussion of sequence alignment methods and homology calculations.

[0066] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI), and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found on the NCBI website.

[0067] Serotype: A group of closely related microorganisms (e.g., viruses) distinguished by a characteristic set of antigens. AAV has at least 13 known natural serotypes, designated AAV1 through AAV13.

[0068] Subject: Living multi-cellular vertebrate organisms, a category that includes humans and non-human mammals (such as mice, rats, cats, dogs, rabbits, sheep, horses, cows, goats, pigs, and non-human primates).

[0069] Synthetic: Produced by artificial means in a laboratory; for example, synthetic nucleic acids can be chemically synthesized in a laboratory.

[0070] Therapeutic gene: A nucleic acid sequence (e.g., a DNA or cDNA sequence) that encodes a protein (or functional fragment thereof) or an inhibitory nucleic acid molecule, e.g., an inhibitory RNA molecule (e.g., a microRNA or short hairpin RNA (shRNA)), that is useful in the treatment or prevention of a disease, disorder, or condition. For example, a therapeutic gene can inhibit, reduce, or eliminate one or more signs or symptoms of a disease, disorder, or condition, or can increase the survival and / or life expectancy of a subject being treated with the therapeutic gene. Therapeutic genes are further described in Section VI.

[0071] Vector: A vector is a nucleic acid molecule that allows the insertion of an exogenous nucleic acid without destroying the vector's ability to replicate and / or integrate in a host cell. A vector can contain a nucleic acid sequence that allows the vector to replicate in a host cell, such as an origin of replication. A vector can also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow the transcription and translation of the inserted gene(s). In some embodiments herein, the vector is an AAV vector.

[0072] Virion protein 1 (VP1): One of the three capsid proteins of AAV. The AAV cap gene encodes VP1, VP2, and VP3, which assemble to form a protein coat of 60 subunits.

[0073] IV. Recombinant Nucleic Acids, AAV Vectors, and AAV Vector Particles Despite the high safety profile demonstrated in clinical trials, adeno-associated virus (AAV)-mediated gene therapy still faces significant obstacles due to its immunogenicity. To address the unmet need for AAV vectors with reduced immunogenicity, the identification of a promiscuous, immunodominant CD4 T cell epitope in the AAV VP1 protein and its elimination by rationally designed chimerization are described herein. The data described herein demonstrate that the recombinant vector maintained its function and efficacy, including yield, cell specificity, in vitro and in vivo transduction efficacy, and biodistribution in mice, without eliciting any cellular immune response.

[0074] Disclosed herein is an isolated nucleic acid molecule encoding a modified AAV VP1 protein that lacks a native CD4 T cell epitope. In some embodiments, the modified AAV VP1 protein has an amino acid sequence that is at least 75% identical to the wild-type AAV9 VP1 protein set forth herein as SEQ ID NO:1, and includes one or more amino acid substitutions resulting in a valine at position 315 and an isoleucine at position 317 of SEQ ID NO:1; an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO:1; an arginine at position 314 and a valine at position 315 of SEQ ID NO:1; an arginine at position 314 and an isoleucine at position 317 of SEQ ID NO:1; or an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO:1. In some examples, the modified VP1 comprises a valine at position 315 and an isoleucine at position 317 of SEQ ID NO: 1. In other examples, the modified VP1 comprises an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO: 1. For VP1 proteins from AAV serotypes that have native VP1 protein lengths that differ from AAV9 VP1, references to positions 311, 312, 314, 315, and 317 refer to the positions corresponding to positions 311, 312, 314, 315, and 317 of SEQ ID NO: 1.

[0075] In some embodiments, the AAV is AAV1, AAV2, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV13. In some examples, the AAV is AAV9. In other examples, the AAV vector is a hybrid of two or more AAV serotypes (including, but not limited to, AAV2 / 1, AAV2 / 7, AAV2 / 8, or AAV2 / 9). The choice of AAV serotype will depend, in part, on the cell type(s) to be targeted for gene therapy.

[0076] In some embodiments, the amino acid sequence of the modified VP1 protein is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, having a valine at position 315 and an isoleucine at position 317 (and can also have an arginine at position 311, a serine at position 312, and / or an arginine at position 314, e.g., one, two, or all three of these), all with reference to SEQ ID NO: 1. In some examples, the amino acid sequence of the modified VP1 protein comprises or consists of SEQ ID NO: 35 (AAV9-VI) or SEQ ID NO: 36 (AAV9-RSRVI).

[0077] In other embodiments, the amino acid sequence of the modified VP1 protein is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 and has an arginine at position 314 of SEQ ID NO:1; an arginine at position 314 and a valine at position 315 of SEQ ID NO:1; an arginine at position 314 and an isoleucine at position 317 of SEQ ID NO:1; or an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO:1.

[0078] In some embodiments, the nucleic acid molecule is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:37 or SEQ ID NO:38, and encodes a valine at position 315 and an isoleucine at position 317 (and may also encode an arginine at position 311, a serine at position 312, and / or an arginine at position 314, e.g., one, two, or all three of these), all with reference to SEQ ID NO:1. In some examples, the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO:37 (or a degenerate variant thereof) or SEQ ID NO:38 (or a degenerate variant thereof).

[0079] In some aspects, the nucleic acid sequence is codon-optimized for expression in mammalian cells, such as human cells, canine cells, porcine cells, feline cells, or non-human primate cells.

[0080] Also disclosed herein are vectors comprising the isolated nucleic acid molecules described herein. In some embodiments, the isolated nucleic acid molecules are operably linked to a promoter. In some examples, the promoter is a tissue-specific promoter. Exemplary tissue-specific promoters include, for example, the thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, mucin-2 promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, retinoschisin promoter, K12 promoter, CC10 promoter, pulmonary surfactant protein C (SP-C) promoter, PRCl promoter, RRM2 promoter, uroplakin 2 (UPII) promoter, or lactoferrin promoter. In other examples, the promoter is a constitutive promoter, such as a Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, or an EF1α promoter. In other examples, the promoter is an inducible promoter. Examples of inducible promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, a tetracycline-repressible system, a tetracycline-inducible system, an RU486-inducible system, and a rapamycin-inducible system. In some instances, the vector further comprises other regulatory sequences, such as one or more enhancers.

[0081] In some embodiments, the vector is an AAV vector, such as an AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV13 vector. In some examples, the AAV vector is an AAV9 vector. In other examples, the AAV vector is a hybrid of two or more AAV serotypes (including, but not limited to, AAV2 / 1, AAV2 / 7, AAV2 / 8, or AAV2 / 9). In some examples, the AAV vector comprises 5' and 3' ITRs. In some examples, the AAV vector further comprises a heterologous open reading frame (ORF), such as a therapeutic gene. The therapeutic gene can be any nucleic acid sequence (e.g., a DNA or cDNA sequence) encoding a protein or inhibitory nucleic acid molecule (e.g., an inhibitory RNA molecule, such as a microRNA or shRNA) that is useful in treating or preventing a disease, disorder, or condition. Non-limiting examples of therapeutic genes are provided in Section VI.

[0082] Further described herein is an isolated host cell comprising the nucleic acid molecule or vector disclosed herein.In some embodiments, the isolated host cell is a cell (or cell line) suitable for producing recombinant AAV.In some examples, the host cell is a mammalian cell, such as HEK-293, BHK, Vero, RD, HT-1080, A549, Cos-7, ARPE-19 or MRC-5 cell.In certain examples, the cell is a 293 cell or its derivative (e.g., FREESTYLE 293-F cell).

[0083] Also provided herein are recombinant AAV vector particles comprising a nucleic acid molecule encoding the VP1 protein disclosed herein.

[0084] Further provided herein are recombinant AAV vector particles comprising a modified VP1 protein having an amino acid sequence at least 75% identical to SEQ ID NO: 1, wherein the modified VP1 protein comprises one or more amino acid substitutions resulting in: a valine at position 315 and an isoleucine at position 317 of SEQ ID NO: 1; an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315 and an isoleucine at position 317 of SEQ ID NO: 1; an arginine at position 314 and a valine at position 315 of SEQ ID NO: 1; an arginine at position 314 and an isoleucine at position 317 of SEQ ID NO: 1; or an arginine at position 314, a valine at position 315 and an isoleucine at position 317 of SEQ ID NO: 1. In some examples, the modified VP1 comprises a valine at position 315 and an isoleucine at position 317 of SEQ ID NO: 1. In another example, the modified VP1 comprises an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO: 1. For VP1 proteins from AAV serotypes that have native VP1 protein lengths that differ from AAV9 VP1, references to positions 311, 312, 314, 315, and 317 refer to positions corresponding to positions 311, 312, 314, 315, and 317 of SEQ ID NO: 1.

[0085] In some embodiments, the AAV is AAV1, AAV2, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV13. In some examples, the AAV is AAV9. In other examples, the AAV vector is a hybrid of two or more AAV serotypes (including, but not limited to, AAV2 / 1, AAV2 / 7, AAV2 / 8, or AAV2 / 9). The choice of AAV serotype will depend, in part, on the cell type(s) to be targeted for gene therapy.

[0086] In some embodiments, the amino acid sequence of the modified VP1 protein is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, having a valine at position 315 and an isoleucine at position 317 (and can also have an arginine at position 311, a serine at position 312, and / or an arginine at position 314, e.g., one, two, or all three of these), all with reference to SEQ ID NO: 1. In some examples, the amino acid sequence of the modified VP1 protein comprises or consists of SEQ ID NO: 35 (AAV9-VI) or SEQ ID NO: 36 (AAV9-RSRVI).

[0087] In other embodiments, the amino acid sequence of the modified VP1 protein is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 and has an arginine at position 314 of SEQ ID NO:1; an arginine at position 314 and a valine at position 315 of SEQ ID NO:1; an arginine at position 314 and an isoleucine at position 317 of SEQ ID NO:1; or an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO:1.

[0088] In some embodiments, the recombinant AAV vector particle further comprises an AAV genome. In some examples, the AAV genome comprises 5' and 3' ITRs. In some examples, the AAV vector further comprises a heterologous ORF, such as a therapeutic gene. The therapeutic gene can be any nucleic acid sequence (e.g., a DNA or cDNA sequence) that encodes a protein or an inhibitory nucleic acid molecule (e.g., an inhibitory RNA molecule, such as a microRNA or shRNA) that is useful in treating or preventing a disease, disorder, or condition. Non-limiting examples of therapeutic genes are provided in Section VI.

[0089] As disclosed herein, further provided is a method of administering a therapeutic gene to a subject by administering to the subject recombinant AAV vector particles containing an AAV genome into which the therapeutic gene has been inserted. In some embodiments, the therapeutic gene is a therapeutic gene listed in Section VI. In some embodiments, the recombinant AAV vector particles are about 1 x 10 10 ~Approx. 1×10 14 In some instances, the AAV vector particles are administered at a dose of about 1 x 10 viral particles (vp) / kg. 11 ~Approx. 1×10 13 vp / kg dose or approximately 1 × 10 12 vp / kg. The recombinant AAV vector particles can be administered in a single dose or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as needed for the desired therapeutic result.

[0090] Also provided is a composition comprising the disclosed recombinant AAV vector particle and a pharmaceutically acceptable carrier, such as water or saline.In some examples, the composition is liquid, frozen, or lyophilized.In some embodiments, the composition is formulated for intravenous, transdermal, intraocular, intrathecal, intracerebral, oral, subcutaneous, intranasal, inhalation, or intramuscular administration.Suitable pharmaceutical formulations for administering AAV can be found, for example, in US Patent Application Publication No. 2012 / 0219528.

[0091] Further provided are isolated peptides containing the immunodominant CD4 T cell epitopes (or portions thereof) disclosed herein. In some embodiments, the isolated peptides are 40 amino acids or less in length and comprise the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In some examples, the peptides are 39 or less, 38 or less, 37 or less, 36 or less, 35 or less, 34 or less, 33 or less, 32 or less, 31 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, or 21 or less amino acids in length. In some examples, the isolated peptides are 21-40, 25-40, 30-40, or 35-40 amino acids in length. In particular examples, the amino acid sequence of the peptide consists of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0092] Further provided is a method for inducing immune tolerance to AAV in a subject. In some embodiments, the method comprises administering one or more of the isolated peptides disclosed herein to the subject. The peptide can be administered in multiple doses, for example, 2, 3, 4, or 5 doses. In some examples, the method further comprises administering one or more immunomodulatory agents to the subject. In certain examples, the immunomodulatory agent is cyclosporin A, tacrolimus, sirolimus, prednisone, dexamethasone, azathioprine, cyclophosphamide, or a monoclonal antibody. In some examples, the peptide(s) are encapsulated in nanoparticles or microparticles.

[0093] Also provided herein is a kit comprising one or more isolated peptides disclosed herein. Such kits can be used, for example, for immune monitoring assays. In some examples, the kit comprises one or more recombinant AAV vector particles disclosed herein, for example, for use in the therapeutic methods provided herein. In some embodiments, the kit further comprises solid support(s), buffer(s), syringe(s), container(s), and / or instruction(s). The instruction(s) can be written, electronic, or visual (such as video files).

[0094] V. Recombinant AAV for Gene Therapy Applications AAV belongs to the family Parvoviridae and the genus Dependovirus. AAV is a small, non-enveloped virus that packages a linear single-stranded DNA genome. Both the sense and antisense strands of AAV DNA are packaged into AAV capsids with equal frequency.

[0095] The AAV genome is characterized by two inverted terminal repeats (ITRs) flanking two open reading frames (ORFs). In the AAV2 genome, for example, the first 125 nucleotides of the ITR are a palindrome that folds back on itself to maximize base pairing, forming a T-shaped hairpin structure. The other 20 bases of the ITR, called the D sequence, remain unpaired. The ITRs are cis-acting sequences important for AAV DNA replication; they are the origin of replication and function as primers for second-strand synthesis by DNA polymerase. The double-stranded DNA formed during this synthesis, called replicative monomers, is used in a second round of self-priming replication to form replicative dimers. These double-stranded intermediates are processed via a strand displacement mechanism, resulting in single-stranded DNA used for packaging and double-stranded DNA used for transcription. Rep binding elements and terminal resolution sites (TRSs) are located within the ITRs. These features are used by the viral regulatory protein Rep during AAV replication to process double-stranded intermediates. In addition to their role in AAV replication, the ITRs are also required for AAV genome packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008).

[0096] The left ORF of AAV contains the Rep gene, which encodes four proteins - Rep78, Rep68, Rep52, and Rep40. The right ORF contains the Cap gene, which produces three viral capsid proteins (VP1, VP2, and VP3). The AAV capsid contains 60 viral capsid proteins arranged in icosahedral symmetry. VP1, VP2, and VP3 exist in a 1:1:10 molar ratio (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008).

[0097] AAV is a virus that is frequently used for gene therapy.AAV infects humans and some other primate species, but is not thought to cause disease, and induces mild immune response.The gene therapy vector that utilizes AAV can infect both dividing cells and quiescent cells, and can persist in extrachromosomal state without being integrated into the genome of host cell.

[0098] AAV has some desirable characteristics for gene therapy vectors, including the ability to bind and enter target cells, enter the nucleus, be expressed in the nucleus for a long period of time, and relatively low toxicity.However, the small size of the AAV genome limits the size of the heterologous DNA that can be incorporated.To minimize this obstacle, AAV vectors that do not code for Rep and integration efficiency element (IEE) have been constructed.Because ITR is the cis signal required for packaging, it is retained (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008).

[0099] Methods for producing rAAV suitable for gene therapy are well known (see, e.g., U.S. Patent Application Nos. 2012 / 0100606; 2012 / 0135515; 2011 / 0229971; and 2013 / 0072548; U.S. Patent No. 11,578,340; and Ghosh et al., Gene Ther 13(4):321-329, 2006) and may be utilized with the recombinant nucleic acid molecules, vectors, and methods disclosed herein.

[0100] VI. Therapeutic Genes The recombinant AAV vector disclosed herein can optionally contain a heterologous ORF, such as a therapeutic gene, for use in gene therapy.The therapeutic gene can encode any protein (or functional fragment thereof) that is useful in treating, inhibiting, gene editing, or preventing a disease, disorder, or condition.Alternatively, the therapeutic gene can encode an inhibitory nucleic acid molecule, such as an inhibitory RNA (e.g., microRNA or shRNA).Non-limiting examples of therapeutic genes that can be used in the AAV vectors and vector particles disclosed herein are listed below (see also U.S. Patent No. 11,578,340).

[0101] In some embodiments, the therapeutic gene encodes a growth factor, an interleukin, an interferon, an anti-apoptotic factor, a cytokine, an anti-diabetic factor, an anti-apoptotic agent, a clotting factor, or an anti-tumor factor. In some examples, the therapeutic gene is BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, or IL-18 gene. In some examples, the therapeutic gene encodes an inhibitory nucleic acid molecule that modulates (eg, inhibits) the expression of one of the above-listed genes.

[0102] In some embodiments, the therapeutic gene is a gene for treating a disease associated with reduced, lost, or dysfunctional expression of a gene, such as glucose-6-phosphatase, phosphoenolpyruvate-carboxykinase, galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase, branched-chain alpha-keto acid dehydrogenase, fumarylacetoacetate hydrolase, methylmalonyl-CoA mutase, medium-chain acyl-CoA dehydrogenase, ornithine transcarbamylase, argininosuccinate synthetase, low-density lipoprotein receptor protein, UDP-glucuronosyltransferase, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, biotinidase, beta- Genes encoding glucocerebrosidase, beta-glucuronidase, peroxisomal membrane protein 70 kDa, porphobilinogen deaminase, alpha-1 antitrypsin, erythropoietin, vascular endothelial growth factor, angiopoietin-1, fibroblast growth factor, thrombomodulin, tissue factor pathway inhibitor, aromatic amino acid decarboxylase (AADC), tyrosine hydroxylase (TH), beta-adrenergic receptor, sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), cardiac adenylyl cyclase, p53, dystrophin, minidystrophin, utrophin, miniutrophin, and insulin are included. In some examples, the therapeutic gene encodes an inhibitory nucleic acid molecule that modulates (e.g., inhibits) the expression of one of the above-listed genes.

[0103] In some embodiments, the therapeutic gene is useful in treating diseases, conditions, or disorders related to the central nervous system. In some examples, the therapeutic gene is DRD2, GRIA1, GRIA2, GRIN1, SLC1A1, SYP, SYT1, CHRNA7, 3 Rtau / 4 rTUS, APP, BAX, BCL-2, GRIK1, GFAP, IL-1, AGER, UCH-L1, SKP1, EGLN1, Nurr-1, BDNF, TrkB, gstm1, S106β, IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, atrophin1, FTL, TITF-1, FXN, ASPA, DMD, SMN1, UBE1, or DYNC1H1.

[0104] In some embodiments, the therapeutic gene is a gene useful in treating diseases, disorders or conditions related to the cardiovascular system.In some examples, the gene is VEGF, FGF, SDF-1, connexin 40, connexin 43, SCN4a, HIF1α, SERCa2a, ADCY1 or ADCY6.In some examples, the therapeutic gene encodes an inhibitory nucleic acid molecule that modulates (e.g., inhibits) the expression of one of the above-listed genes.

[0105] In some embodiments, the therapeutic gene is a gene useful in treating a disease, disorder, or condition associated with the pulmonary system. In some examples, the gene is TNFα, TGFβ1, SFTPA1, SFTPA2, SFTPB, SFTPC, HPS1, HPS3, HPS4, ADTB3A, IL1A, IL1B, LTA, IL6, CXCR1, or CXCR2. In some examples, the therapeutic gene encodes an inhibitory nucleic acid molecule that modulates (e.g., inhibits) the expression of one of the above-listed genes.

[0106] In some embodiments, the therapeutic gene is a gene encoding a gene encoding a gene for the liver (e.g., α1-AT, HFE, ATP7B, fumarylacetoacetate hydrolase (FAH), glucose-6-phosphatase, NCAN, GCKR, LYPLAL1, or PNPLA3), kidney (e.g., PKD1, PKD2, PKHD1, NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, TRPC6, WT1, LMX1B, SMARCAL1), or tumor necrosis factor receptor 1 (TNF-α, T ... , COQ2, PDSS2, SCARB3, FN1, COL4A5, COL4A6, COL4A3, COL4A4, FOX1C, RET, UPK3A, BMP4, SIX2, CDC5L, USF2, ROBO2, SLIT2, EYA1, MYOG, SIX1, SIX5, FRAS1, FREM2, GATA3, KAL1, PAX2, TCF2, or SALL1), eye (e.g., CFH, C3, MT-N D2, ARMS2, TIMP3, CAMK4, FMN1, RHO, USH2A, RPGR, RP2, TMCO, SIX1, SIX6, LRP12, ZFPM2, TBK1, GALC, myocilin, CYP1B1, CAV1, CAV2, optineurin or CDKN2B), gastrointestinal tract (e.g., CYP2C19, CCL26, APC, IL12, IL10 or IL-18), pancreas (e.g., PRSS 1, SPINK1, STK11, MLH1, KRAS2, p16, p53, or BRAF), the urinary tract (e.g., HSPA1B, CXCR1&2, TLR2, TLR4, TGF-1, FGFR3, RB1, HRAS, TP53, or TSC1), or the uterus (e.g., DN-ER, MLH1, MSH2, MSH6, PMS1, or PMS). In some examples, the therapeutic gene encodes an inhibitory nucleic acid molecule that modulates (e.g., inhibits) the expression of one of the above-listed genes.

[0107] In some embodiments, the therapeutic gene is a gene useful in the treatment of cancer. In some examples, the gene is AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2, BHLHB2, BLMH, BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9, C DKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, CO L6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1, CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8 , DLG3, DVL1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2, ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES, FGFR1, FGR, FKBP8, FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB, FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2, GNB2L1, GPR39, GRB2, GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HER2, HMMR, HPRT1, HRB, HSPA4, HSPA5, HSPA8, HSPB1, HSPH1, HYAL1, HYOU1, ICAM1, ID1, ID2, IDUA,IER3, IFITM1, IGF1R, IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1, IRF3, ITGA3, ITGA6, ITGB4, JAK1, JARID1A, JUN, JUNB, JUND, K-ALPHA-1, KIT, KI TLG、KLK10、KPNA2、KRAS2、KRT18、KRT2A、KRT9、LAMB1、LAMP2、LCK、LCN2、LE P、LITAF、LRPAP1、LTF、LYN、LZTR1、MADH1、MAP2K2、MAP3K8、MAPK12、MAPK13、 MAPKAPK3, MAPRE1, MARS, MAS1, MCC, MCM2, MCM4, MDM2, MDM4, MET, MGST1, MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA, MSH2, MSH6, MT3, MYB, MYBL1 、MYBL2、MYC、MYCL1、MYCN、MYD88、MYL9、MYLK、NEO1、NF1、NF2、NFKB1、NFKB2 、NFSF7、NID、NINJ1、NMBR、NME1、NME2、NME3、NOTCH1、NOTCH2、NOTCH4、NPM1、 NQO1、NR1D1、NR2F1、NR2F6、NRAS、NRG1、NSEP1、OSM、PA2G4、PABPC1、PARP1、 PCNA、PCTK1、PCTK2、PCTK3、PDGFA、PDGFB、PDGFRA、PDPK1、PEA15、PFDN4、PFD N5、PGAM1、PHB、PIK3CA、PIK3CB、PIK3CG、PIM1、PKM2、PKMYT1、PLK2、PPARD、 PPARG、PPIH、PPP1CA、PPP2R5A、PRDX2、PRDX4、PRKAR1A、PRKCBP1、PRNP、PRSS 15、PSMA1、PTCH、PTEN、PTGS1、PTMA、PTN、PTPRN、RAB5A、RAC1、RAD50、RAF1、 RALBP1、RAP1A、RARA、RARB、RASGRF1、RB1、RBBP4、RBL2、REA、REL、RELA、RELB 、RET、RFC2、RGS19、RHOA、RHOB、RHOC、RHOD、RIPK1、RPN2、RPS6KB1、RRM1、SAR S、SELENBP1、SEMA3C、SEMA4D、SEPP1、SERPINH1、SFN、SFPQ、SFRS7、SHB、SHH、SIAH2, SIVA, SIVA TP53, SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, SMPD1, SNAI2, SND1, SNRPB2, SOCS1, SOCS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STAT2, STAT3, S TAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, TK1, TLE1, TNF, TNFRSF10A, TNFRSFlOB, TNFRSFlA, TNFRSFlB, TNFRSF6, TNFSF7, TNK1, TOB1, TP53, TP53BP2, TP53I3, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL, VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB, YWHAZ, ZAP70, or ZNF9. In some examples, the therapeutic gene encodes an inhibitory nucleic acid molecule that modulates (e.g., inhibits) the expression of one of the above-listed genes.

[0108] Other types include RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3. BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L1 0, BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2, BIRC3, XIAP, BIRC5, BIRC6, BIR C7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L, BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1, CARD6, CARD S, CARDS, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA, CI DEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, GADD45A, GDNF, HRK, IGF1R, LTA, LTBR, ​​MCL1, NOL3, PYCARD, RIPK 1, RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRSF12A, TNFRSF14, TNFRSF19. TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25, CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18 CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 DRD2, GRIA1, GRIA2, GRIN1, SLC1A1, SYP, SYT1, CHRNA7, 3 Rtau / 4 rTUS, APP, BAX, BCL-2, GRIK1, GFAP, IL-1, AGER, UCH-L1, SKP1, EGLN1, Nurr-1, BDNF, TrkB, gstm1, S106β, IT15, PRNP, JPH3 TBP, ATXN1, ATXN2, ATXN3, FTL, TITF-1, FXN, ASPA The DMD, SMN1, UBE1, and DYNC1H1 are the most commonly used skeletal muscles.In some examples, the therapeutic gene encodes an inhibitory nucleic acid molecule that modulates (eg, inhibits) the expression of one of the above-listed genes. [Example]

[0109] The following examples are provided to illustrate particular features of certain embodiments of the present disclosure, but the claims should not be limited to those exemplified features.

[0110] Example 1 material and method This example describes the materials and experimental procedures used in the study described in Example 2.

[0111] Plasmids and DNA mutagenesis For rAAV production, the plasmids pAAV2 / 9n (112865, Addgene, Watertown, MA), pAAV2 / 5n (104964, Addgene), pHelper vector (340202, Cell Biolabs, San Diego, CA), pscAAV-GFP (AAV-410, Cell Biolabs), and pAAV-CMV-NanoLuc-Halotag (Promega, Madison, WI) were used. Empty capsids were produced by transfecting pHelper and pAAV2 / 9n or pAAV2 / 5n. Plasmid mutagenesis was performed by Gene Universal (Newark, DE). The accuracy of nucleotide changes was verified by DNA sequence analysis immediately after production and again after plasmid expansion.

[0112] Recombinant vector production Empty AAV9 and AAV5 capsids were produced using a slightly modified protocol described by Kimura et al. (Zincarelli et al., Mol Ther 16:1073-1080, 2008). Briefly, FreeStyle 293F cells (Thermo Fisher Scientific, San Jose, CA) were transfected using PEI MAX (Polysciences, Warrington, PA), lysed in acidic buffer, the homogenate was cleared from debris by centrifugation, and the pH was neutralized using HEPES buffer. Subsequently, AAV particles in the lysate and medium were recovered by polyethylene glycol (PEG) precipitation.

[0113] A standard triple transfection method was used to generate GFP- and NanoLuc-expressing AAV (Bing et al., Mol Ther Methods Clin Dev 24:255-267, 2022). Briefly, virus-producing cells 2.0 (Thermo Fisher Scientific) were transfected and harvested 72 hours later. After three freeze-thaw cycles, sonication, and BENZONASE™ treatment, AAV was purified using two sequential rounds of ultracentrifugation on an iodixanol gradient. For sham controls, all procedures were identical except for the presence of the Rep / Cap plasmid. Capsid protein in the vectors was quantified using an AAV5 or AAV9 ELISA kit (Progen, Wayne, PA). Vector genome copy titers were determined using Taqman quantitative PCR (qPCR) as previously described (Bing et al., Mol Ther Methods Clin Dev 24:255-267, 2022).

[0114] Peptide synthesis A total of 242 15-mer peptides overlapping by 12 amino acids, as well as chimeric and AAV5 VP1-derived peptides, were purchased from GenScript Biotech (Piscataway, NJ). The peptides spanned the entire sequence of VP1 from the AAV9 capsid. Peptide purity was >95% as determined by high-performance liquid chromatography. Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO) at 50 mg / mL and diluted with RPMI medium. For initial screening, peptides were pooled into groups of 12 consecutive peptides, except for pools 19 and 20, which each contained 13 peptides.

[0115] Human PBMC samples Peripheral blood mononuclear cells (PBMCs) were collected from apheresis samples of 52 healthy donors. Samples were isolated using gradient-density separation with Ficoll-Hypaque (GE Healthcare, Chicago, IL) according to the manufacturer's instructions and stored frozen in liquid nitrogen until assayed. HLA typing was performed as previously described by Scisco Genetics (Seattle, WA) (Puig et al., Front Immunol 11:629399, 2020).

[0116] In vitro expansion of AAV-specific PBMC cells PBMCs were thawed and cultured at 5 × 10 in RPMI medium containing 5% heat-inactivated human serum, 1% GLUTAMAX™ (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), MEM non-essential amino acids (Thermo Fisher Scientific), and 1% penicillin / streptomycin (Thermo Fisher Scientific). 6 PBMCs were incubated with heated empty AAV9 capsids, empty AAV5 capsids, or mutated AAVs (2 × 10 10Cells were stimulated with 20 units of IL-2 (MilliporeSigma, Burlington, MA), 5 μg / mL of IL-7 (Biolegend, San Diego, CA), and 25 μg / mL of IL-15 (Biolegend) every 3–4 days after initial antigen stimulation. On day 14, cells were harvested and screened for reactivity to AAV-specific peptide pools or individual peptides.

[0117] ELISpot assay IL-2 and IFN-γ secretion was analyzed using an ELISpot assay according to the manufacturer's recommendations (Mabtech, Cincinnati, OH). After in vitro expansion, cells were harvested and incubated with either peptide pools or individual peptides (10 μg / mL) at a density of 100,000 cells / well in plates precoated with anti-human IL-2 or IFN-γ antibodies. Negative controls were treated with medium, and positive controls were treated with CEF, CEFT (PANATecs, Baden-Wuerttemberg, Germany), or PHA (MilliporeSigma). After 24 hours, spots were developed using biotin-conjugated anti-IL-2 or anti-IFN-γ antibodies (Mabtech, Nacka Strand, Sweden), streptavidin alkaline phosphate (Mabtech), and nitroblue tetrazolium and 5-bromo-4-chloro-3'-indolyl phosphate (BCIP / NBT) substrate (KPL, Thermo Fisher Scientific). Spot-forming cells (SFCs) were enumerated using computer software (Immunospot 7.0; Cellular Technology Limited, Cleveland, OH). For IFN-γ analysis, spot size was incorporated into the analysis by multiplying the spot count by the average spot size per well. Responses were considered positive if the magnitude of the response exceeded 2.1 (IFN-γ) or 3 (IL-2) times the background level of the negative control.

[0118] For experiments utilizing CD4 or CD8 T cell depletion, CD4 T cells were purified by negative selection using magnetic beads and LD columns (Miltenyi Biotec, Bergisch Gladbach, Germany), and CD8 T cells were isolated by positive selection using magnetic beads and LS columns (Miltenyi Biotec) according to the manufacturer's protocol.

[0119] For HLA-restriction assays, AAV-expanded PBMCs were incubated with 20 μg / mL monoclonal Abs against HLA class I (W6 / 32), HLA-DR (G46-6), HLA-DQ (SPV-L3), or HLA-DP (B7 / 21) for 30 min before peptide addition. Cytokine production in response to positive peptides was then measured by ELISpot assay as described above.

[0120] FACS After in vitro expansion, cells were restimulated with 10 μg / mL peptide for 24 hours at 37°C. Cytokine secretion in cell cultures was blocked by adding GolgiPlug / GolgiStop (BD Biosciences, San Jose, CA) for 5 hours before cell harvesting and staining. Cells were stained for surface markers CD3 (clone UCHT1), CD4 (clone SK3), CD8 (clone RPA-T8), CD56 (clone HCD56), and TCRγδ (clone 11F2). After washing, cells were fixed and permeabilized using CYTOFIX / CYTOPERM solution (BD Biosciences) and then stained for IFN-γ (clone B27) and TNF-α (clone MAb11). Acquisition was performed using a Cytek Aurora (Cytek Biosciences, Fremont, CA), and analysis was performed using Flowjo (Treestar, Ashland, OR) software.

[0121] In silico HLA binding prediction Peptide binding affinities for HLA class I and class II were predicted using binding prediction tools available in the Immune Epitope Database (IEDB) (online at iedb.org; Reynisson et al., Nucleic Acids Res 48:W449-w454, 2020; Wang et al., BMC Bioinformatics 11:568, 2010). HLA binding rankings were predicted for 9-mer peptides with 27 HLA class I alleles (Weiskopf et al., Proc Natl Acad Sci USA 110:E2046-2053, 2013) and 15-mer peptides with 27 HLA class II alleles (Wang et al., BMC Bioinformatics 11:568, 2010). These alleles represent binding specificities shared by the majority of the world's population (Wang et al., BMC Bioinformatics 11:568, 2010; Weiskopf et al., Proc Natl Acad Sci USA 110:E2046-2053, 2013). Percentile ranks provide a consistent measure that allows comparison between various predictors. Higher affinity is indicated by lower percentile rank values. Initial cutoffs of less than 1% or 10% percentile ranks for HLA class I or class II alleles, respectively, were used to identify potential binders in AAV9 VP1.

[0122] In vitro transduction and neutralization assays HeLa, HEK293T, or A375 cells were grown at 2 x 10 4Cells were seeded at a final density of 100 cells / well in DMEM (Thermo Fisher) medium containing 10% fetal bovine serum (FBS, Millipore Sigma) and penicillin / streptomycin. After 24 hours, the medium was replaced with DMEM + 2% FBS medium, and virus was added at MOIs of 10, 100, 1,000, 10,000, and 100,000 vg / cell. GFP expression was analyzed 48 hours post-transduction by direct fluorescence imaging and flow cytometry. To detect NanoLuc luciferase, furimazine (Promega) was added to the cultures 24 hours post-transduction, and the results were immediately evaluated using a luminometer.

[0123] Neutralization assays were performed as previously described (Meliani et al., Hum Gene Ther Methods 26:45-53, 2015). Briefly, serial dilutions of pooled heat-inactivated human serum (MilliporeSigma) were mixed with vectors expressing GFP or NanoLuc and incubated for 1 hour. After incubation, samples were added to cells, and reporter gene activity was measured 24 hours later.

[0124] Dynamic Light Scattering (DLS) AAV particles were added to the resuspension buffer at 2 x 10 11 The solution was diluted to a concentration of 10 ...

[0125] SEC-MALS Size-exclusion separation was performed using an Agilent 1260 Infinity II high-performance liquid chromatography (HPLC) system (Agilent, Santa Clara, CA). The system consisted of a binary pump, a multisampler maintained at 4 °C, a multicolumn thermostat maintained at 25 °C, and a diode array detector. A DAWN® multiangle light scattering (MALS) detector and an OPTILAB® differential refractive index (RI) detector (Waters | Wyatt Technology) were installed in series with the HPLC. The MALS detector was attached to an internal dynamic light scattering fiber (detection angle 135°) for simultaneous in-line DLS measurements. The system was equipped with an XBridge Premier GTx BEH size-exclusion column (7.8 mm × 30 cm, 2.5 μm particle size, 450 Å pore size, Waters Corporation, Milford, MA). The mobile phase used for all injections was 10 mM sodium phosphate, 350 mM sodium chloride, 0.001 vol / vol% Pluronic® F-68, pH 7.4. The flow rate was maintained at 0.5 mL / min for all injections. Injections were performed using VISION® 3.2.0.67 (Waters|Wyatt Technology). MALS, UV, and RI data were collected and analyzed using ASTRA® 8.1.2.1 (Waters|Wyatt Technology).

[0126] In vivo bioluminescence imaging of NanoLuc Six- to eight-week-old BALB / c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). NanoLuc-expressing rAAV vector was diluted in 200 μL of PBS and then transferred to 10 11The mice were injected intravenously via the tail vein at a dose of 1000 mg / mouse (4-6 mice / group). On days 8, 17, and 29, mice were injected with 0.44 μmol of fluorofurimazine (Promega), anesthetized with isoflurane, and imaged 3 ± 1 min later using an IVIS Spectrum Imager (PerkinElmer, Waltham, MA). On day 29, mouse organs were removed, rinsed with PBS, and then analyzed using the IVIS Imager. Signal quantification in specific regions of interest (ROIs) was corrected for background by subtracting signals from the same organs of sham controls.

[0127] Example 2 Identification of immunodominant epitopes in the AAV9 capsid To identify immunodominant epitopes in the AAV9 capsid, we used 242 overlapping peptides spanning the sequence of AAV9 VP1 and human PBMCs from a cohort of 52 donors with a distribution of HLA alleles comparable to that in the North American population. Peptides were pooled into pools containing 12 overlapping peptides each. Only resulting pools that tested positive in IL-2 and IFN-γ ELISpot assays were deconvoluted to determine which specific peptides contained the epitope.

[0128] Figure 1A shows a heat map of responses from 52 donors. IL-2 and IFN-γ response rates were 17% and 23%, respectively, for pool 9; and 23% and 19%, respectively, for pool 18. Stimulation with the individual peptides comprising these pools (peptides 97–108 in pool 9 and peptides 205–216 in pool 18) revealed that pool 9 contained the most immunodominant epitope in the AAV9 capsid protein (peptides 103–105) (Figure 1A and Figures 3A–3D), while pool 18 contained the second and third most immunodominant epitopes (Figure 14A and Table 2). The immunodominant epitope in AAV9 was identified as containing amino acids 307–327 (Figure 1B), and immune responses to this epitope were found in 23% (12 of 52) of donor samples (Table 1). Table 1. IL-2 and IFN-γ secretion in response to peptides 103–105 in 12 donors [Table 1-1] [Table 1-2]

[0129] Table 2. Top 3 T cell epitopes in AAV9 VP1 [Table 2] 1 Responders were experimentally defined if the peptide had a >3-fold change in IL-2 SFC / 1E5 or a >2-fold change in IFN-γ SFC / 1E5. 2 Responding T cell types are defined by intracellular flow cytometry when the number of IL-2, IFN-γ, or TNF-α-producing cells exceeds a 1.5-fold change compared to the medium-only control and confirmed by ELISpot assay. 3 HLA binding predictions were made using IEDB class I and class II prediction tools.

[0130] To determine the phenotype of this epitope, cells were restimulated with peptide 103-105 and intracellular cytokine flow cytometry was used to distinguish between CD4 and CD8 activation, as shown in Figure 4A. It was found that peptide 103-105 stimulated IFN-γ and TNF-α secretion in CD4 but not CD8 T cells (Figure 1C). Furthermore, in ELISpot assays, the increase in IFN-γ secretion upon stimulation with peptide 103-105 was no longer observed in CD4-depleted cells, whereas peptide 103-105 activated CD8-depleted cells (Figure 1D and Figure 4B). These findings indicate that the immunodominant epitope 307-327 (peptide 103-105) is recognized by CD4 T cells.

[0131] Furthermore, anti-HLA-DP antibodies inhibited IFN-γ secretion, whereas HLA-DR and HLA-DQ-targeting antibodies did not (Figure 5). The 12 donors who responded to epitope 307-327 (peptides 103-105) carried various HLA-DP alleles, indicating the promiscuity of this epitope (Figure 12). Using the Immune Epitope Database (IEDB) binding algorithm, binding of epitope 307-327 (peptides 103-105) to 27 MHC class II alleles was predicted, which was shown to provide maximum coverage of all potential binding cores (Martini et al., Immunogenetics 72:57-76, 2020; Greenbaum et al., Immunogenetics 63:325-335, 2011). The percentile ranks of the three HLA-DR alleles, one HLA-DQ allele, and all six HLA-DP alleles submitted to the IEDB were below 10, indicating a promiscuous binder (Figure 13). This in silico data is consistent with the HLA restriction data and HLA distribution of the 12 donors, further suggesting that epitope 307-327 (peptides 103-105) is HLA-DP restricted and promiscuous.

[0132] Sequence alignment of the epitope 307–327 (peptides 103–105) region to 12 native AAV serotypes revealed that this epitope is conserved (86% conservation) with the exception of amino acids R312, L313, N314, F315, and L317 (Figure 1E). Non-conserved amino acids were found in the AAV5 serotype, which is most distantly related to AAV9 in the AAV phylogenetic tree (R299, S300, R312, V313, and I315). To test whether this region in AAV5 induces an immune response, PBMCs (three donors randomly selected from 12 donors) were expanded with AAV5 empty capsids and restimulated with AAV5 peptides aligned to epitope 307–327 (peptides 103–105) in AAV9. The AAV5 peptide did not stimulate cells to produce IFN-γ, indicating that epitope 307-327 is not present in AAV5 (Figures 6A-6C).

[0133] The IEDB was used to predict the binding affinity of AAV5 sequences to candidate amino acids responsible for immune silencing of AAV9 immunodominant epitopes. The number of HLA alleles with the modified sequences AAV9-F315V / L317I (AAV9-VI) and AAV5 (Figure 1F and Figure 13) with percentile ranks below 10 was reduced to 4 and 5, respectively. Based on the predicted data, site-directed mutagenesis was used to generate two variants of AAV9 (AAV9-VI and AAV9-RSRVI). In the AAV9-VI variant, two residues were replaced with the corresponding AAV5 amino acids (F315V and L317I), and AAV9-RSRVI was designed by replacing five residues with the AAV5 sequence (K311R, R312S, N314R, F315V, and L317I). Because they are neither located on the surface of the AAV9 capsid nor at known functionally important amino acids, these mutations pose a low risk of affecting transduction efficacy or cell interactions (Figure 1G and Figure 7).

[0134] When compared with unmodified AAV5 and AAV9 capsids containing the same transgene cassette (GFP or NanoLuc luciferase), amino acid mutations in the two AAV9 variants had no effect on production yield, particle size, particle thermostability, percentage of empty capsids, or any change in the ratio between viral genome concentration and total viral particles when produced by triple transfection of HEK293 cells (Table 3, Table 4, and Figures 15A-B). Table 3. Titers of chimeric AAV9 variants [Table 3] *Data from 3 batches Table 4. Characteristics of chimeric AAV9 variants [Table 4]

[0135] Next, we examined the transduction profiles of the two chimeric AAV vectors at different MOIs in HEK293T cells. Using flow cytometry and fluorescence microscopy, we analyzed the transduction efficiency of each chimeric AAV vector and compared it with wild-type (WT) AAV9-GFP and AAV5-GFP vectors. AAV9, AAV9-VI, and AAV9-RSRVI transduced cells with similar efficiencies and significantly outperformed the AAV5 vector with an 80% higher AUC (Figures 2A, 2B, and 8C), indicating that the rational design of AAV9-VI and AAV9-RSRVI did not impair the vectors' ability to transduce cells. Similar results were observed in other cell lines (HeLa and A375) or using a different transgene (Hall et al., ACS Chem Biol 7:1848-1857, 2012) (NanoLuc) (Figures 8A-8B and 9A-9C). Next, we investigated whether AAV9 mutations affected vector neutralization by anti-AAV antibodies. Before addition to cultured cells, vectors containing the NanoLuc transgene were incubated with serial dilutions of pooled human serum to determine the 50% neutralizing dose (ND). 50 ) was calculated. ND between AAV9 and mutant vectors 50 The results showed that the mutations had no effect on vector neutralization (Figures 2C-D and 10A-B). To evaluate the transduction efficiency of the mutated vectors in vivo, Balb / c mice were intravenously injected with the vectors carrying the NanoLuc transgene or a sham control. The levels of luciferase expression in mice injected with the mutated vectors, AAV9-VI, and AAV9-RSRVI, were comparable to those in AAV9-injected mice, indicating that the mutations had no effect on transduction efficiency in vivo (Figure 2E). Transgene expression in all groups remained stable after 29 days (Figure 2F).

[0136] Because AAV tropism is an important and sensitive parameter in capsid engineering (Adachi et al., Nat Commun 5:3075, 2014), we also analyzed the biodistribution of the mutated vectors in mice. AAV9 showed strong expression in the liver, heart, muscle, and thymus, consistent with previously published data (Zincarelli et al., Mol Ther 16:1073-1080, 2008). Meanwhile, expression by AAV5 was weaker and largely restricted to the liver and lung (Figure 2G). Further analysis of viral genome biodistribution using droplet digital PCR (ddPCR) demonstrated strong infection of AAV ITRs in the liver, spleen, kidney, and thymus in the AAV9 group (Figure 2I). Contrary to the transgene expression data, AAV9-RSRVI transduced less in the liver and spleen, while AAV9-VI did not show any differences in genome expression in the tissues (Figure 2I). Although AAV9-RSRVI showed reduced transduction efficiency in the liver and spleen, AAV9-VI still had similar biodistribution of transgenes and vector genomes to the parental AAV vector (AAV9), indicating that rational design had no effect on vector tropism (Figures 2G-I).

[0137] Finally, we investigated whether the rational design eliminated the immunodominant epitope 307–327 (peptides 103–105). Restimulation of PBMCs with the mutated peptide after expansion with the mutated vector did not activate the cells to produce IFN-γ, in contrast to the AAV9-expanded group, suggesting that the rational design successfully silenced the immunodominant epitope in the AAV9 capsid (Figure 2K–L). One potential concern when removing immunodominant epitopes is the emergence of subdominant epitopes that were not identified when the immunodominant epitope was present (Liu et al., J Immunol 151:1852–1858, 1993). To investigate this concern, PBMCs were stimulated with the mutated vector and then restimulated with a peptide library spanning all AAV9 sequences used to identify the epitope in Figure 1A or with peptides corresponding to mutations in AAV9 VP1. Figure 11 shows that none of the peptide pools activated PBMCs when the cells were expanded with the mutated chimeric vector, indicating that immune silencing resulted in complete elimination of the epitope without generating a subdominant epitope.

[0138] Further studies investigated the manipulation of the second most prevalent epitope in pool 18 using a chimera design strategy. Deconvolution of individual peptides in pool 18 revealed two distinct and strong epitopes, peptides 215-216 and 205-206 (Figure 14A and Table 2). However, the equivalent peptide in AAV5 (AAV5_211-212) aligned to the second epitope (peptide 215-216) stimulated cells to produce cytokines as strongly as the AAV9 peptide (AAV9_215-216) (Figure 14B). In silico predictions also showed that the binding affinity of the AAV5 sequence aligned to the second epitope in AAV9 was similar to that of the AAV9 sequence (Figure 14C).

[0139] Collectively, these studies identified T cell epitopes in the AAV9 viral capsid protein, including promiscuous immunodominant epitopes, that can be eliminated by rational chimerization without compromising its function and efficacy. Such design may result in safer and more effective gene delivery vectors by reducing T cell-mediated toxicity and preventing T cell-mediated death of transduced cells, potentially resulting in longer-lasting transgene expression.

[0140] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure, and applicants claim all such modifications and variations that come within the scope and spirit of the following claims.

Claims

1. 1. An isolated nucleic acid molecule encoding a modified adeno-associated virus (AAV) VP1 protein having an amino acid sequence that is at least 75% identical to SEQ ID NO:1, wherein the modified VP1 protein comprises: Valine at position 315 and isoleucine at position 317 of SEQ ID NO:1; Arginine at position 311, serine at position 312, arginine at position 314, valine at position 315, and isoleucine at position 317 of SEQ ID NO: 1; Arginine at position 314 of SEQ ID NO: 1; Arginine at position 314 and valine at position 315 of SEQ ID NO: 1; Arginine at position 314 and isoleucine at position 317 of SEQ ID NO: 1; or Arginine at position 314, valine at position 315 and isoleucine at position 317 of SEQ ID NO:1 An isolated nucleic acid molecule comprising:

2. 2. The isolated nucleic acid molecule of claim 1, wherein the AAV is AAV serotype 1 (AAV1), AAV2, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV13.

3. 3. The isolated nucleic acid molecule of claim 1 or claim 2, wherein the modified VP1 protein comprises a valine at position 315 and an isoleucine at position 317 of SEQ ID NO:

1.

4. 3. The isolated nucleic acid molecule of claim 1, wherein the modified VP1 protein comprises an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO:

1.

5. The isolated nucleic acid molecule according to any one of claims 1 to 4, wherein the AAV is AAV9.

6. The isolated nucleic acid molecule of any one of claims 1 to 5, wherein the amino acid sequence of the modified VP1 protein is at least 80% identical to SEQ ID NO:

1.

7. 7. The isolated nucleic acid molecule of any one of claims 1 to 6, wherein the amino acid sequence of the modified VP1 protein is at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

1.

8. 8. The isolated nucleic acid molecule of any one of claims 1 to 7, wherein the amino acid sequence of the modified VP1 protein comprises or consists of SEQ ID NO: 35 (AAV9-VI) or SEQ ID NO: 36 (AAV9-RSRVI).

9. SEQ ID NO: 37 or a degenerate variant thereof; or SEQ ID NO: 38 or a degenerate variant thereof 9. The isolated nucleic acid molecule of claim 1, comprising:

10. 10. The isolated nucleic acid molecule of any one of claims 1 to 9, wherein the nucleic acid sequence is codon-optimized for expression in mammalian cells.

11. A vector comprising the isolated nucleic acid molecule of any one of claims 1 to 10.

12. The vector of claim 11 , wherein the isolated nucleic acid molecule is operably linked to a promoter.

13. 13. The vector of claim 11 or claim 12, which is an AAV vector.

14. 14. The vector of claim 13, wherein the AAV vector is an AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV13 vector.

15. The vector of claim 14, wherein the AAV vector is an AAV9 vector.

16. The vector of any one of claims 11 to 15, further comprising a heterologous open reading frame (ORF).

17. The vector of claim 16 , wherein the heterologous ORF is a therapeutic gene.

18. An isolated host cell comprising an isolated nucleic acid molecule according to any one of claims 1 to 10 or a vector according to any one of claims 11 to 17.

19. A recombinant AAV vector particle comprising the nucleic acid molecule of any one of claims 1 to 10.

20. 1. A recombinant adeno-associated virus (AAV) vector particle comprising a modified VP1 protein having an amino acid sequence that is at least 75% identical to SEQ ID NO:1, wherein the modified VP1 protein comprises: Valine at position 315 and isoleucine at position 317 of SEQ ID NO:1; Arginine at position 311, serine at position 312, arginine at position 314, valine at position 315, and isoleucine at position 317 of SEQ ID NO: 1; Arginine at position 314 of SEQ ID NO: 1; Arginine at position 314 and valine at position 315 of SEQ ID NO: 1; Arginine at position 314 and isoleucine at position 317 of SEQ ID NO: 1; Arginine at position 314, valine at position 315 and isoleucine at position 317 of SEQ ID NO:1 A recombinant AAV vector particle comprising:

21. The recombinant AAV vector particle of claim 20, wherein the AAV is AAV1, AAV2, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV13.

22. 22. The recombinant AAV vector particle of claim 20 or claim 21, wherein the modified VP1 protein comprises a valine at position 315 and an isoleucine at position 317 of SEQ ID NO:

1.

23. 22. The recombinant AAV vector particle of claim 20 or claim 21, wherein the modified VP1 protein comprises an arginine at position 311, a serine at position 312, an arginine at position 314, a valine at position 315, and an isoleucine at position 317 of SEQ ID NO:

1.

24. The recombinant AAV vector particle according to any one of claims 20 to 23, wherein the AAV is AAV9.

25. The recombinant AAV vector particle of any one of claims 20 to 24, wherein the amino acid sequence of the modified VP1 protein is at least 80% identical to SEQ ID NO:

1.

26. 26. The recombinant AAV vector particle of any one of claims 20 to 25, wherein the amino acid sequence of the modified VP1 protein is at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

1.

27. 27. The recombinant AAV vector particle of any one of claims 20 to 26, wherein the amino acid sequence of the modified VP1 protein comprises or consists of SEQ ID NO: 35 or SEQ ID NO:

36.

28. The recombinant AAV vector particle of any one of claims 20 to 27, further comprising an AAV genome.

29. 29. The recombinant AAV vector particle of claim 28, wherein the AAV genome comprises a heterologous ORF.

30. 30. The recombinant AAV vector particle of claim 29, wherein the heterologous ORF is a therapeutic gene.

31. 31. A method of administering a therapeutic gene to a subject, comprising administering to the subject the recombinant AAV vector particle of claim 30.

32. A composition comprising the recombinant AAV vector particle of any one of claims 20 to 30 and a pharmaceutically acceptable carrier.

33. An isolated peptide that is 40 amino acids or less in length and comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

34. 34. The isolated peptide of claim 33, which is no more than 35, 30, 25 or 21 amino acids in length.

35. 35. The isolated peptide of claim 33 or claim 34, wherein the amino acid sequence of the peptide consists of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

36. An isolated peptide according to any one of claims 33 to 35; and Solid Support, Buffers and / or Instructions Includes a kit.

37. 36. A method of inducing immune tolerance to AAV in a subject, comprising administering to the subject an isolated peptide according to any one of claims 33 to 35.

38. 37. The method of claim 36, further comprising administering to the subject one or more immunomodulatory agents.

39. 39. The method of claim 37 or claim 38, wherein the peptide is encapsulated in a nanoparticle or microparticle.

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