Nanoparticles for use with adeno-associated virus particles

Abstract

The present disclosure relates to nanoparticles comprising an AAV capsid peptide and / or a transgene product peptide. Also disclosed are methods of inducing tolerance (e.g., inducing tolerance of CD4+ T cells and / or CD8+ T cells) to (i) an AAV particle, and / or (ii) a polypeptide encoded by an AAV-vectored transgene by administering a nanoparticle provided herein. In addition, the present disclosure provides methods of reducing a humoral immune response to an AAV particle and / or a polypeptide encoded by a transgene by administering a nanoparticle provided herein. In yet another aspect, provided herein are methods of increasing and / or prolonging expression of a transgene in a subject in need thereof by administering a nanoparticle provided herein.

Description

NANOPARTICLES FOR USE WITH ADENO-ASSOCIATED VIRUS PARTICLESCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of EP 24306311.2 filed August 2, 2024, the disclosure of which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14779-006-227_SEQLISTING.xml”, was created on November 4, 2024, and is 103,699 bytes in size.1. FIELD

[0002] The present disclosure relates, in part, to nanoparticles for use with adeno-associated virus (AAV) particles. In certain aspects, the present disclosure relates, in part, to nanoparticles for use in tolerizing or reducing an immune response to AAV-mediated gene therapy.2. BACKGROUND

[0003] Gene transfer using adeno-associated viral (AAV) vectors represent a safe and efficient means to transfer transgenes in vivo and have been widely used in preclinical and clinical gene therapy. However, immune responses against AAV capsid antigens and / or transgene product(s) still represent a major obstacle, which limits long-term therapeutic efficacy and preclude AAV- redosing. In particular, subjects injected with AAV vectors can mount T and B cell responses against the AAV capsid and / or the transgene product. Such immune responses can lead to rejection of transduced cells, or production of AAV neutralizing antibodies that prevent successful reapplication of the vector. Accordingly, provided herein are nanoparticles for use with AAV vectors that reduce or prevent immune responses against the AAV capsid antigens and / or transgene product.3. SUMMARY

[0004] In one aspect, provided herein is a nanoparticle comprising: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to afragment of a transgene product, wherein the peptide is 8 to 50 amino acids long. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the transgene product. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the transgene product. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the transgene product. In some embodiments, the transgene product is suitable for use in a gene therapy. In some embodiments, the transgene product is encoded by human F8. In some embodiments, the transgene product is not an autoantigen. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs: 17-47.

[0005] In another aspect, provided herein is a nanoparticle comprising: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of an adeno-associated viral (AAV) capsid polypeptide, wherein the peptide is 8 to 50 amino acids long. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs: 10-15.

[0006] In some embodiments, the peptide provided herein is 8 to 11 amino acids long. In some embodiments, the peptide is 13 to 25 amino acids long.

[0007] In some embodiments, the peptide provided herein comprises an MHC-I epitope. In some embodiments, the peptide comprises an MHC-II epitope.

[0008] In some embodiments, the nanoparticle provided herein comprises a linker at the N- terminus of the peptide. In some embodiments, the nanoparticle has a hydrodynamic diameter (z- average) in the range of 10 to 100 nm, as determined by Dynamic Light Scattering (DLS). In some embodiments, the nanoparticle has a zeta potential between about -20 and -50 mV, asmeasured at pH 6 to pH 7. In some embodiments, the nanoparticle provided herein has a hydrodynamic diameter (z-average) between 10 and 100 nm, as measured by DLS.

[0009] In another aspect, provided herein is a pharmaceutical composition comprising a nanoparticle of the present disclosure, and a pharmaceutically acceptable carrier.

[0010] In another aspect, provided herein is a kit comprising: a nanoparticle of the present disclosure, and an AAV particle comprising the AAV capsid and / or the transgene.

[0011] In another aspect, provided herein is a method of inducing tolerance to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the method comprises administering a nanoparticle of the present disclosure to the subject.

[0012] In another aspect, provided herein is a method of inducing tolerance of CD4+ T cells to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0013] In another aspect, provided herein is a method of inducing tolerance of CD4+ T cells to a polypeptide encoded by an AAV-vectored transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by thetransgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0014] In another aspect, provided herein is a method of inducing tolerance of CD 8+ T cells to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0015] In another aspect, provided herein is a method of inducing tolerance of CD 8+ T cells to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0016] In another aspect, provided herein is a method of inducing tolerance of CD4+ T cells to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the AAV vector comprises an AAV capsid, and wherein the method comprises administering to thesubject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV- DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs:10-18.

[0017] In another aspect, provided herein is a method of inducing tolerance of CD4+ T cells to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the AAV vector comprises an AAV capsid, and wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV- DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs: 10-18.

[0018] In another aspect, provided herein is a method of inducing tolerance of CD 8+ T cells to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the AAV vector comprises an AAV capsid, and wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) apeptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV- DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs:10-18.

[0019] In another aspect, provided herein is a method of inducing tolerance of CD 8+ T cells to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the AAV vector comprises an AAV capsid, and wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV- DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs: 10-18.

[0020] In another aspect, provided herein is a method of inducing tolerance to an AAV particle in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle of the present disclosure.

[0021] In another aspect, provided herein is a method of inducing tolerance of CD4+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV- DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs: 10-18.

[0022] In another aspect, provided herein is a method of inducing tolerance of CD4+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV- DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs: 10-18.

[0023] In another aspect, provided herein is a method of inducing tolerance of CD 8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV- DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs: 10-18.

[0024] In another aspect, provided herein is a method of inducing tolerance of CD 8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0025] In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOs: 10-18.

[0026] In another aspect, provided herein is a method of inducing tolerance of CD4+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0027] In another aspect, provided herein is a method of inducing tolerance of CD4+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0028] In another aspect, provided herein is a method of inducing tolerance of CD 8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope. In some embodiments, the peptide comprises an aminoacid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0029] In another aspect, provided herein is a method of inducing tolerance of CD 8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises: (a) an amphiphilic polymer; and (b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0030] In some embodiments, tolerance is maintained for at least 30 days after administration of the AAV particle. In some embodiments, tolerance is maintained for at least 50 days after administration of the AAV particle. In some embodiments, tolerance is maintained for at least 60 days after administration of the AAV particle. In some embodiments, tolerance is determined by IFN^ ELISPOT.

[0031] In some embodiments, the method further comprises administering the AAV vector to the subject.

[0032] In another aspect, provided herein is a method of reducing a humoral immune response to a polypeptide encoded by a transgene in a subject in need thereof, wherein the method comprises: (a) administering a nanoparticle to the subject; and (b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene, wherein the nanoparticle comprises: (i) an amphiphilic polymer; and (ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein thehumoral immune response is reduced relative to a method comprising step (b) but not step (a). In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8.

[0033] In another aspect, provided herein is a method of reducing a humoral immune response to an AAV particle in a subject in need thereof, wherein the method comprises: (a) administering a nanoparticle to the subject; and (b) administering the AAV particle to the subject, wherein the AAV particle comprises an AAV capsid, wherein the nanoparticle comprises: (i) an amphiphilic polymer; and (ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the humoral immune response is reduced relative to a method comprising step (b) but not step (a). In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid polypeptide. In some embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, a synthetic AAV, combinations, engineered, or modified versions thereof. In some embodiments, the AAV capsid is AAV1 or AAV8.

[0034] In another aspect, provided herein is a method of reducing a humoral immune response to a polypeptide encoded by a transgene in a subject in need thereof, wherein the method comprises: (a) administering a nanoparticle to the subject; and (b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene, wherein the nanoparticle comprises: (i) an amphiphilic polymer; and (ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the humoral immune response is reduced relative to a method comprisingstep (b) but not step (a). In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0035] In another aspect, provided herein is a method of reducing a humoral immune response to an AAV particle in a subject in need thereof, wherein the method comprises: (a) administering a nanoparticle to the subject; and (b) administering the AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the nanoparticle comprises: (i) an amphiphilic polymer; and (ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the humoral immune response is reduced relative to a method comprising step (b) but not step (a). In some embodiments, the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene. In some embodiments, the transgene is human F8.

[0036] In some embodiments, the humoral immune response is measured by detecting absolute units of an anti-transgene IgG titer. In some embodiments, the humoral immune response is measured by detecting absolute units of an anti- AAV capsid IgG titer. In some embodiments, the humoral immune response is reduced by more than about 60%, relative to a method comprising step (b) but not step (a). In some embodiments, the humoral immune response is reduced by more than about 70%, relative to a method comprising step (b) but not step (a). In some embodiments, the humoral immune response is reduced by more than about 80%, relative to a method comprising step (b) but not step (a). In some embodiments, the humoral immune response is reduced by more than about 90%, relative to a method comprising step (b) but not step (a).

[0037] In some embodiments, the method further comprises administering Temsirolimus.

[0038] In another aspect, provided herein is a method of increasing and / or prolonging expression of a transgene in a subject in need thereof, wherein the method comprises: (a) administering a nanoparticle to the subject; and (b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene, wherein the nanoparticle comprises: (i) an amphiphilic polymer; and (ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the expression is increased and / or prolonged relative to a method comprising step (b), but not step (a). In some embodiments, expression of the transgene is increased relative to a method comprising step (b), but not step (a). In some embodiments, the expression of the transgene is increased by about 2-fold, relative to a method comprising step (b), but not step (a). In some embodiments, the expression of the transgene is increased by about 4-fold, relative to a method comprising step (b), but not step (a). In some embodiments, the expression of the transgene is increased by more than 5-fold, relative to a method comprising step (b), but not step (a). In some embodiments, the expression of the transgene is prolonged for more than 30 days after administering the AAV particle. In some embodiments, the expression of the transgene is prolonged for more than 40 days after administering the AAV particle. In some embodiments, the expression of the transgene is prolonged for more than 50 days after administering the AAV particle. In some embodiments, the expression of the transgene is prolonged for more than 60 days after administering the AAV particle.

[0039] In some embodiments, administering the nanoparticle particle is performed intravenous. In some embodiments, administering the AAV particle is performed intravenous or intramuscular. In some embodiments, the nanoparticle is administered at least once prior to administration of the AAV particle. In some embodiments, the nanoparticle is administered one day prior to administration of the AAV particle. In some embodiments, the nanoparticle is administered at least twice prior to administration of the AAV particle. In some embodiments, the nanoparticle is administered five days prior and one day prior to administration of the AAV particle The nanoparticle of any one of embodiments. In some embodiments, the nanoparticle is administered at least once after administration of the AAV particle. In some embodiments, the nanoparticle is administered at least twice after administration of the AAV particle.

[0040] In another aspect, provided herein is the use of the nanoparticle of the present disclosure in the manufacture of a medicament for reducing an immune response to the AAV capsid and / or the transgene.

[0041] In another aspect, provided herein is a nanoparticle of the present disclosure or a pharmaceutical composition of the present disclosure for use in reducing an immune response to the AAV capsid and / or the transgene.4. BRIEF DESCRIPTION OF THE FIGURES

[0042] FIG. 1A-FIG. IE illustrate a time course of Ova-specific CD8+ T cells following injection with nanoparticles. Mice were treated with multiple injections of Ova-I nanoparticles, Ova-I / Ova-II nanoparticles, Ova-I / PL8 / ML8 nanoparticles (MHC class I restricted viral peptides), Ova-I / Ova-II / PL8 / RK15 nanoparticles (MHC class I and II restricted viral peptides), empty nanoparticles (“TPCs”), or vehicle (Tris-Mannitol buffer). Bar plots show % of H- 2Kb / Ova+ on CD 8+ T cells in blood collected at day 14 (FIG. 1A), day 21 (FIG. IB), day 53 (FIG. 1C), and day 67 (FIG. ID) post AAV8-CMV-sOVA injection, as measured by flow cytometry. A time course of Ova-specific CD8+ T cells followed in the blood by dextramer staining upon intramuscular (i.m.) injection of AAV8-CMV-sOVA (2.5xlOnvg / kg) in both gastrocnemii of female C57BL / 6 mice (FIG. IE).

[0043] FIG. 2A-FIG. 2M illustrate that nanoparticles comprising Ova-P-II peptides inhibit Ova-mediated cellular responses in muscle gene transfer and allow persistence of transgene expression. Female C57BL / 6 mice were i.m. injected at day 0 with an AAV8-CMV-sOVA (2.5xlOnvg / kg) in both gastrocnemii and received mixture of TPC-Ova257-264 and TPC-Ova323- 339 (TPC-Ova-P-II) in comparison to empty nanoparticles (TP) intravenous (i.v.) at days -1, 3, 7, 12 and 19 post-AAV injection (p.i.); n=8 / group) (FIG. 2A). Frequency of Ova-specific CD8+ T cells in the blood was determined by flow cytometry using H-2Kb / Ova-I dextramers staining (gated Ova-specific CD8+ T cells) at indicated timepoints after AAV injection of TP (top line) or TPC-Ova-F-II (bottom line) (FIG. 2B). Representative flow cytometric panel (FIG. 2C) and frequency of Ova-specific CD8+ T cells in the blood at day 21 p.i. (FIG. 2D). Mean fluorescence intensity of PD-1 expression on gated Ova-specific CD8+ T cells at day 39 p.i. (FIG. 2E). Frequency of CX3CRlhlghcells on gated Ova-specific CD8+ T cells in the blood at day 67 p.i. (FIG. 2F). Frequency of KLRG-1+ cells on gated Ova-specific CD 8+ T cells in the blood at day67 p.i. (FIG. 2G). Frequency of CDR45RBhlghcells on gated Ova-specific CD8+ T cells in the spleen at day 67 p.i. (FIG. 2H). Frequency of CD73+FR4+ cells on gated Ova-specific CD8+ T cells in the spleen at day 67 p.i. (FIG. 21). Splenocytes were collected at day 67 p.i. and analyzed by ex vivo enzyme-linked immunospot (ELISpot) assays for their capability to secrete IFNy after restimulation with Ova-I (FIG. 2J). Female C57BL / 6 mice were i.v. injected at day 0 with an AAV8-CMV-sOVA (5xl0nvg / kg) and received mixture of TPC-Ova-I / -II, TPC-PL8 / RK15 peptide and TPC-Ova-I / -II- PL8 / RK15 peptide in comparison to empty nanoparticles (TP) i.v. at days -1, 3, 7, 12 and 25 post-AAV injection (p.i.; n=8 / group). Mean fluorescence intensity of PD-1 expression (FIG. 2K) and frequencies of CD45RBhlgh(FIG. 2L) and CD73+FR4+ (FIG. 2M) on gated Ova-specific CD8+ T cells at day 27 from blood sample. Graphs show means ± S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001 by Mann- Whitney test.

[0044] FIG. 3A-FIG. 3E illustrate Ova-specific CD8+ T cell frequencies and kinetics in the liver. Ova-specific CD8+ T cells frequencies in the liver at day 12 upon i.v. injection of AAV8- CMV-sOVA (day 0) in mice (n=4) treated with TPC Ova-I / Ova-II on days -5, -1 and 5, measured by flow cytometry (FIG. 3A). Kinetic of Ova-specific CD8+ T cells in the liver (FIG. 3B) and spleen (FIG. 3C) after three applications of TPC Ova-I / Ova-II or empty nanoparticles (“empty TPCs”). Kinetic of Ova-specific CD8+ T cells in the liver after five applications of nanoparticles and adoptive transfer of CD45.1+ CD4 and CD8 T cells (OT-I and OT-II) (FIG. 3D and 3E). Kinetic of Ova-specific CD45.1+ CD8+ T cells (FIG. 3D). Kinetic of total Ova- specific CD8+ T cells (endogenous + OT-I transferred cells) (FIG. 3E).

[0045] FIG. 4A - FIG. 4C illustrate Ova transgene expression between different treatment groups. Ovalbumin transgene expression upon i.v. injection of AAV8-CMV-sOVA between mice treated with Ova-I nanoparticles, Ova-I / Ova-II nanoparticles, Ova-I / PL8 / ML8 nanoparticles (MHC class I restricted viral peptides), Ova-POva-II / PL8 / RK15 nanoparticles (MHC class I and II restricted viral peptides), empty nanoparticles (“empty TPCs” or “TP”), or vehicle (Tris-Mannitol buffer), measured by qRT-PCR. Transgene expression on day 67 p.i. in gastrocnemius muscles (FIG. 4A). Transgene expression on day 62 p.i. in the liver of mice treated with Ova-I nanoparticles, Ova-II nanoparticles, or Ova-I / Ova-II nanoparticles (FIG. 4B). Transgene expression on day 67 p.i. in gastrocnemius muscles of mice treated with Ova-I / Ova-II nanoparticles (FIG. 4C). Graphs show means ± S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001 by Mann- Whitney test.

[0046] FIG. 5A - FIG. 5E illustrate Ova-specific T cells evaluated by ELISpot assays for their capability to secrete IFNy after restimulation with Ova-I (for CD8) or Ova-II (for CD4) peptides upon i.m. injection of AAV8-CMV-sOVA (5xl09vg / mouse) in mice treated with Ova- I / Ova-II nanoparticles, PL8 / RK15 nanoparticles (MHC class I and II restricted viral peptides), or with Ova-l / Ova-II / PL8 / RK15 nanoparticles, in comparison to positive control, i.e. AAVl-Ova and AAV8-Ova At days -1, 3, 7, 12 and 19. Ova-specific CD8+ (FIG. 5A) and CD4+ T cells (FIG. 5B) on day 60 p.i.. Ova-specific CD4+ T cells on day 67 p.i. (FIG. 5C). Female C57BL / 6 mice were i.v. injected at day 0 with an AAV8-CMV-sOVA (5x 10" vg / kg) and received mixture of nanoparticle-Ova257-264 and nanoparticle-Ova323-339 (TPC-Ova-I / -II) in comparison to empty nanoparticles (TP) i.v. at days -5, -1, 5 and 10 post- AAV injection (p.i.; n=8 / group). Frequency of activated CD25+ Foxp3+ CD4+ Tregs (FIG. 5D) and mean fluorescence intensity of Ki67 expression on CD4+ Tregs (FIG. 5E) in the liver on day 16 p.i., measured by flow cytometry. Graphs show means ± S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001 by Mann-Whitney test.

[0047] FIG. 6A - FIG. 6G illustrate the efficiency of nanoparticles comprising peptides derived from AAV8 capsid to inhibit cellular immune responses. AAV-specific CD8+ (FIG. 6A) and CD4+ T cells (FIG. 6B) evaluated by IFNy ELISpots 67 days upon i.m. injection of AAV8- CMV-sOVA in mice treated with Ova-I nanoparticles (third column from left, triangles), Ova- I / Ova-II nanoparticles (fifth column from left, diamond), Ova-I / PL8 / ML8 nanoparticles (MHC class I restricted viral peptides) (fourth column from left, upside down triangle), Ova-I / Ova- IPPL8 / RK15 nanoparticles (MHC class I and II restricted viral peptides) (sixth column from left, circle), empty TPCs (second column from left, square) and vehicle (Tris-Mannitol buffer) (first column from left, circle). AAV capsid-specific T cells evaluated by ELISpot assays 60 days after i.m. injection of AAV8-CMV-sOVA in mice treated with Ova-I / Ova-II nanoparticles, PL8 / RK15 nanoparticles (MHC class I and II restricted viral peptides), or with Ova-I / Ova-II / PL8 / RK15 nanoparticles, empty TPCs, and positive control, i.e. AAVl-Ova and AAV8-Ova after restimulation with AAV capsid MHC-I epitope PL8 (FIG. 6C), MHC-I epitope ML8 (FIG. 6D), MHC-II epitope EL15 (FIG. 6E), MHC-II epitope RK15 (FIG. 6F), or MHC-II epitope QN15 (FIG. 6G) peptides.

[0048] FIG. 7A-FIG. 7J illustrate that the nanoparticles display a bystander tolerization effect. Female C57BL / 6 mice were i.m. injected at day 0 with an AAV8-Ova (2.5xlOnvg / kg) inboth gastrocnemii and received nanoparticle-Ova-I or nanoparticle-Ova-I / PL8 / ML8 or empty nanoparticle (TP) i.v. at days -1, 3, 7, 12 and 19 p.i. (n=8 / group) (FIG. 7A). Splenocytes were collected at 67 p.i. and analyzed by ex vivo ELISpot assays for their capability to secrete IFNy after restimulation with Ova-I (FIG. 7B), Ova-II (FIG. 7C), AAV capsid MHC-I epitope PL8 (FIG. 7D), or MHC-II epitope RK15 (FIG. 7E) peptides. C57BL / 6 mice were i.v. injected at day 0 with an AAV8-Ova (2.5x10" vg / kg) and received nanoparticle-Ova-I / -II. or nanoparticle- PL8 / Rkl5 i.v. at days -5, -1, 5, 20 and 26 p.i. (n=8 / group) (FIG. 7F). Splenocytes were collected at 60 p.i. and analyzed by ex vivo ELISpot assays for their capability to secrete IFNy after restimulation with Ova-I (FIG. 7G), Ova-II (FIG. 7H), AAV capsid MHC-I epitope PL8 (FIG. 71), or MHC-II epitope RK15 (FIG. 7J) peptides. Graphs show means ± S.E.M. ****p < 0.0001 by One-way ANOVA.

[0049] FIG. 8A- FIG. 8H illustrate that the nanoparticles do not impede vaccination against a tumor (Third Party Antigen Trp2) and tumor control. C57BL / 6 mice were i.v. injected at day 0 with an AAV8-Ova (5x10" vg / kg) and received nanoparticle-Ova-I / -II i.v. at days -5, -1, 5, 12 and 19 p.i.. Vaccinated mice were i.m. injected in both gastrocnemii at days 9 and 16 p.i. with 200 pg of deltaV Trp2 peptide together with 100 pg of poly(I:C). At day 33 p.i., 5xl05B16 melanoma cells were subcutaneous (s.c.) inoculated (FIG. 8A). Frequency of Trp2-specific CD8+ T cells was determined in the blood by flow cytometry using H-2Kb / Trp2180-188 dextramers staining at day 21 p.i. (FIG. 8B). Representative flow cytometric panel of Trp2+ CD8+ T cells (FIG. 8C). At day 35 p.i., PBMCs were collected and analyzed by ex vivo ELISpot assays for their capability to secrete IFNy after restimulation with Trp2180-188 (FIG. 8D), Ova-I (FIG. 8E), and Ova-II peptides (FIG. 8F). Mean tumor volumes (FIG. 8G) and survival curves (FIG. 8H) of groups of mice injected with B 16 melanoma cells, p < 0.05 with the logrank test for trend in survival analysis. Graphs show means ± S.E.M. ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001 by Mann- Whitney test.

[0050] FIG. 9A - FIG. 9D illustrate the time course of anti-Ova IgG and anti-AAV8 IgG after i.m. injection of AAV8-Ova (2.5x10" vg / kg) in both gastrocnemii and administering Ova- I / Ova-II TPCs i.v. at days -5, -1, 5, 10 and 20, combined with the application of Temsirolimus (0.4 mg / kg), injected intraperitoneally (i.p.) every other day from days -2 to day 20, in comparison to groups tolerized with TPCs, but without co-application of Temsirolimus. IgG levels without or with 0.4 mg / kg Temsirolimus were measured at days 19, 27, 41, 57 and 106including anti-Ova IgG levels (FIG. 9 A and FIG. 9C) and anti-AAV8 IgG levels (FIG. 9B and FIG. 9D).

[0013] FIG. 10A - FIG. 10D illustrate ELISpot results at day 21 p.i. with AAV8-CMV-F8 (3x1011 vg / kg), and after multiple application of TPC coupled with pooled F8 peptides (three CD4+ T cell epitopes and two CD8+ T cell epitopes) at day -5, -1, 5, 10 and 20. Splenocytes were collected at day 21 and analyzed by ex vivo ELISpot assays for their capability to secrete IFNy after restimulation with F8 peptides. Heatmaps show quantification of the response to antigen stimulation, higher values representative of stronger immune response. Heatmap shows mean SFC (FIG. 10A), median SFC (FIG. 10B), and frequency of mice responding to antigen stimulation (FIG. 10C). Scatter plot shows anti-F8 IgG titer after day 21-23 p.i. (FIG. 10D). The groups received TP (empty nanoparticles) (Fl.l) or TPC-conjugated with two F8- peptides (CD8+ T cell epitopes) (Fl .2) or TPC-conjugated with three F8- peptides (CD4+ T cell epitopes) (Fl.3) or a pool of these five peptides (F1.4). *p < 0.05 by Kruskal Wallis tests - Dunn’s Multiple comparison test.FIG. 11A - FIG. 11C illustrate heatmaps of ELISpot results at day 56 p.i. after immunization with AAV8-CMV-F8 (3x1011 vg / kg), and after multiple application of TPC coupled with pooled F8 peptides (three CD4+ T cell epitopes and two CD8+ T cell epitopes) on day -5, -1, 5, 10 and 20. Splenocytes were collected at day 56 and analyzed by ex vivo ELISpot assays for their capability to secrete IFNy after restimulation with F8 peptides. Heatmap shows quantification of the response to antigen stimulation, higher values representative of stronger immune response with mean SFC (FIG. 11 A), median SFC (FIG. 1 IB), and frequency of mice responding to antigen stimulation (FIG. 11C). The groups received TP (empty nanoparticles) (F2.4) or TPC- conjugated with two F8- peptides (CD8+ T cell epitopes) (F2.1) or TPC-conjugated with three F8- peptides (CD4+ T cell epitopes) (F2.2) or a pool of these five peptides (F2.3).

[0051] FIG. 12A - FIG. 12E illustrate scatter plots show anti-F8 titer at day 14, 28, 42, and 56 p.i. (FIG. 12A-D) after the same groups as shown in Fig. 11 have received the same immunization and tolerization protocol as described in Fig. 11. Heatmap shows quantification of anti-F8 IgG titer (FIG. 12E). *p < 0.05 by Kruskal Wallis tests - Dunn’s Multiple comparison test.5. DETAILED DESCRIPTION

[0052] Provided herein are nanoparticles (such as nanoparticles according to Section 5.1) comprising an AAV capsid peptide (such as an AAV capsid peptide according to Section 5.2.1) and / or an transgene product peptide (such as a transgene product peptide according to Section 5.2.2). The present disclosure also provides pharmaceutical compositions comprising the nanoparticles disclosed herein (such as pharmaceutical composition according to Section 5.3). In certain aspects, provided herein are methods of inducing tolerance (e.g., inducing tolerance of CD4+ T cells and / or CD8+ T cells) to a polypeptide encoded by an AAV-vectored transgene in a subject in need thereof by administering the nanoparticles provided herein (such as a method according to Section 5.5). In other aspects, provided herein are methods of inducing tolerance (e.g., inducing tolerance of CD4+ T cells and / or CD8+ T cells) to an AAV particle, specifically to an AAV capsid protein, in a subject in need thereof by administering the nanoparticles provided herein (such as a method according to Section 5.5). Also provided herein are methods of reducing a humoral immune response to a polypeptide encoded by a transgene or to an AAV particle by administering the nanoparticles provided herein (such as a method according to Section 5.5.2). In yet another aspect, provided herein are methods of increasing and / or prolonging expression of a transgene in a subject in need thereof by administering the nanoparticles provided herein (such as a method according to Section 5.5.3).

[0053] As used herein, the term “adeno-associated virus (AAV)” is intended to mean both naturally occurring, including all the different AAV serotypes, as well as non-naturally occurring forms of AAV (e.g., recombinant rAAV, and pseudotypes), and variants thereof.

[0054] As used herein, the term “viral particle” or “AAV particle,” is intended to mean an infectious form of an AAV virus outside a host cell, that contains nucleic acids, such as a transgene for gene therapy flanked by ITRs of an AAV genome, surrounded by a protective coat of protein called a capsid.

[0055] As used herein, the term “transgene product” is intended to mean a polypeptide encoded by a transgene.

[0056] As used herein, the term “about” is intended to mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0057] As used herein, the term “peptide” when used in reference to an AAV capsid peptide (such as an AAV capsid peptide described in Section 5.2.1) or a transgene product peptide (such as a transgene product peptide described in Section 5.2.2) refers to an amino acid sequence derived from the AAV capsid or transgene product, respectively, and does not encompass adjacent amino acid residues, such as an N-terminal linker. Accordingly, by way of example, a nanoparticle comprising a peptide that is 100% identical to a fragment of a polypeptide can also include additional amino acid residues adjacent to the peptide that are not identical to the fragment of the polypeptide, such as a peptide with an N-terminal linker.

[0058] It should be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0059] The practice of embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, and AAV mediated delivery of gene therapy, which are within the skill of those working in the art. Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Glover, ed., DNA Cloning, Volumes I and II (1985); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, N.Y., 2d ed. 1987).

[0060] In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections, or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples. The application contemplates use of any of the applicable components in any combination, whether or not a particular combination is expressly described.5.1 Nanoparticles

[0061] In one aspect, provided herein is a nanoparticle comprising: (a) an amphiphilic polymer (such as an amphiphilic polymer according to Section 5.1.2); and (b) an AAV capsid peptide (such as an AAV capsid peptide according to Section 5.2.1). In certain embodiments, provided herein is a nanoparticle comprising: (a) an amphiphilic polymer (such as an amphiphilic polymer according to Section 5.1.2); and (b) a transgene product peptide (such as a transgene product peptide according to Section 5.2.2). In other embodiments, provided herein is a nanoparticle comprising: (a) an amphiphilic polymer (such as an amphiphilic polymer according to Section 5.1.2); (b) an AAV capsid peptide (such as an AAV capsid peptide according to Section 5.2.1); and (c) a transgene product peptide (such as a transgene product peptide according to Section 5.2.2). In certain embodiments, a nanoparticle provided herein can have the characteristics described in Section 5.1.1.5.1.1 Nanoparticle properties

[0062] In some embodiments, the nanoparticles comprise a solid hydrophobic core which is coated by the micelle. In some embodiments, the nanoparticle provided herein comprises a solid inorganic core. In certain embodiments, the nanoparticle provided herein does not comprise a solid inorganic core. In some embodiments, the nanoparticle provided herein comprises (a) an amphiphilic polymer; and (b) a first lipid comprising a polar head group and a non-polar tail group, such as a nanoparticle as described in International Patent Application Publication No.: WO / 2025 / 149619, which is incorporated herein by reference in its entirety.

[0063] In certain embodiments, the nanoparticle provided herein has a hydrodynamic diameter (z-average) in the range of 10 to 100 nm as determined by Dynamic Light Scattering (DLS). In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 10 to 60 nm as determined by DLS. In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 10 to 50 nm as determined by DLS. In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 10 to 40 nm as determined by DLS. In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 10 to 30 nm as determined by DLS. In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 20 to 30 nm as determined by DLS.

[0064] In certain embodiments, the nanoparticle provided herein has a zeta potential betweenabout -20 and -50 mV. In certain embodiments, the nanoparticle provided herein has a zeta potential between about -25 and -45 mV. In certain embodiments, the nanoparticle provided herein has a zeta potential between about -28 and -42 mV. In certain embodiments, the zeta potential of the nanoparticle is measured at a solution pH of 6 to 7. In certain embodiments, the zeta potential of the nanoparticle is measured using a Malvern Zetasizer Nano ZS instrument.

[0065] Without being bound by theory, a nanoparticle provided herein is capable of being internalized by liver sinusoidal endothelial cells (LSECs) after administration to a subject or in an in vitro model. In some embodiments, the degree or efficiency of internalization of the nanoparticle by LSECs is determined according to the Internalization Assay (see Section 5.6.1). Without wishing to be bound by theory, nanoparticles having a composition according to Section 5.1 such that the hydrodynamic diameter of the nanoparticle is less than about 60 nm are capable of being internalized by LSECs.

[0066] In some embodiments, the nanoparticle is capable of being processed after internalization by LSECs to release one or more of the peptides associated or covalently linked to the nanoparticle. In some embodiments, the nanoparticle is capable of being processed after internalization by LSECs via hydrolysis of the amide bonds covalently linking the N-termini of one or more peptides to the nanoparticle. In some embodiments, the nanoparticle is capable of being processed after internalization by LSECs via hydrolysis of the ester or amide bonds covalently linking the C-termini of one or more peptides to the nanoparticle.

[0067] In some embodiments, the nanoparticle is capable of suppressing a specific immune response. In some embodiments, the nanoparticle is capable of suppressing a specific immune response to a peptide described in Section 5.2.1 or Section 5.2.2, or to a protein comprising the amino acid sequence of a peptide described in Section 5.2.1 or Section 5.2.2.5.1.2 Amphiphilic Polymer

[0068] The present disclosure relates, in part, to nanoparticles comprising an amphiphilic polymer. In certain embodiments, the amphiphilic polymer comprises: (a) a hydrophobic region; and (b) a hydrophilic region. In certain embodiments, the hydrophobic region comprises a hydrocarbyl chain comprising 4 to 30, or preferably 7 to 19, carbon atoms. In certain embodiments, the hydrophilic region of the amphiphilic polymer comprises a protonatable or deprotonatable moiety (e.g. -OH or -COOH) such that the amphiphilic polymer is capable ofhaving a net charge (e.g. net negative charge) in solution. One of skill in the art will recognize that the degree of protonation or deprotonation of the moiety and, thus, the net charge of the amphiphilic polymer is an equilibrium process that depends on the p / <aof the moiety and the pH of the solution.

[0069] In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is about 20,000 g / mol or less, about 10,000 g / mol or less, or about 6,000 g / mol or less. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is between about 1,000 g / mol and about 20,000 g / mol, between about 2,500 g / mol and about 20,000 g / mol, between about 1,000 g / mol and about 10,000 g / mol, between about 2,500 g / mol and about 10,000 g / mol, between about 1,000 g / mol and about 6,000 g / mol, between about 2,500 g / mol and about 6,000 g / mol, between about 1,000 g / mol and about 4,000 g / mol, or between about 2,500 g / mol and about 4,000 g / mol. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is between about 1,000 g / mol and about 6,000 g / mol. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is between about 2,500 g / mol and about 6,000 g / mol. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is between about 2,500 g / mol and about 4,000 g / mol.

[0070] In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is determined using gel permeation chromatography (GPC). In some embodiments, polystyrene is used as a calibration standard. In some embodiments the number average molecular weight (Mn) of the amphiphilic polymer is determined using a PL-gel mixed D column at a temperature of 40°C, a mobile phase consisting of tetrahydrofuran / acetic acid 90 / 10% (v / v), a flow rate of 1.0 ml / min, in combination with a refractive index detector at a temperature of 35 °C and polystyrene as calibration standard. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is determined using GPC and the following measurement conditions:

[0071] In some embodiments, the amphiphilic polymer is a copolymer. In some embodiments, the amphiphilic polymer is an alternating copolymer. Without wishing to be bound by theory, an alternating copolymer is a copolymer comprising two species of monomeric units in alternating sequence. In some embodiments, the amphiphilic polymer is a copolymer of maleic anhydride and at least one alkene. In some embodiments, the amphiphilic polymer is an alternating copolymer of maleic anhydride and at least one alkene. In some embodiments, the at least one alkene is selected from the group consisting of 1 -decene, 1 -undecene, 1 -dodecene, 1- tridecene, 1 -tetradecene, 1 -pentadecene, 1 -hexadecene, 1 -heptadecene, 1 -octadecene, 1- nonadecene and 1-eicosene. In some embodiments, the alkene is 1-octadecene. In some embodiments, the amphiphilic polymer is a copolymer of maleic anhydride and 1-octadecene. In some embodiments, the amphiphilic polymer is an alternating copolymer of maleic anhydride and 1-octadecene.

[0072] In some embodiments, the amphiphilic polymer comprises a hydrophilic poly-maleic anhydride backbone and hydrophobic hydrocarbyl side chains. In some embodiments, the side chain is a linear alkyl chain. In some embodiments, the side chain comprises 5 to 31 carbon atoms. In some embodiments, the side chain comprises 8 to 20 carbon atoms. In some embodiments, the side chain comprises 10 to 18 carbon atoms. In some embodiments, the sidechain comprises 16 carbon atoms.

[0073] In some embodiments, the amphiphilic polymer comprises the following building block:wherein R is a hydrocarbyl group or a substituted hydrocarbyl group. In some embodiments, R is a linear, unsubstituted hydrocarbyl group. In some embodiments, R is a C4 to C30 alkyl group. In some embodiments, R is a linear, unsubstituted C4 to C30 alkyl group. In some embodiments, R is a C7 to C19 alkyl group. In some embodiments, R is a linear, unsubstituted C7 to C19 alkyl group. In some embodiments, R is a C9 to C17 alkyl group. In some embodiments, R is a linear, unsubstituted C9 to C17 alkyl group. In some embodiments, R is a C15 alkyl group. In some embodiments, R is a C15 linear, unsubstituted alkyl group. In some embodiments, the amphiphilic polymer comprises repeating units of the building block. In some embodiments, the amphiphilic polymer comprises n repeating units of the building block, wherein n a number between about 6 and about 11. In some embodiments, the amphiphilic polymer consists of repeating units of the building block. In some embodiments, the amphiphilic polymer consists of n repeating units of the building block, wherein n a number between about 6 and about 11.

[0074] In some embodiments, the amphiphilic polymer is selected from the group consisting of poly(maleic acid-cZ / - 1 -octadecene), poly(maleic acid-cZ / - 1 -tetradecene) and poly(maleic acid- alt-1 -dodecene). In some embodiments, the amphiphilic polymer is poly(maleic acid-alt- 1 - octadecene). In some embodiments, the amphiphilic polymer is poly(maleic acid- 1 -octadecene) and the number average molecular weight of the polymer is from about 1 ,000 to 6,000 g / mol.

[0075] In certain embodiments, the nanoparticle provided herein comprises the amphiphilic polymer in an amount of about 50-85% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises the amphiphilic polymer in an amount of about 55-65% by weight of the total weight of the nanoparticle. In certainembodiments, the nanoparticle provided herein comprises the amphiphilic polymer in an amount of about 65-75% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises the amphiphilic polymer in an amount of about 75-85% by weight of the total weight of the nanoparticle.

[0076] Nanoparticles can be prepared by any method known in the art. Specifically, nanoparticles as described above can be prepared using a poly(maleic acid-alt- 1 -octadecene), a poly(maleic acid-alt- 1 -tetradecene) or a poly(maleic acid-alt- 1 -dodecene) polymer. Non-limiting methods for preparing nanoparticles include those described in International Patent Application Publication Nos.: WO 2013 / 072051 and WO 2021 / 165227, each of which is incorporated herein by reference in its entirety. In certain embodiments, the nanoparticles are prepared according to the methods described in the Examples, infra. It should be understood that these methods are merely exemplary and that the nanoparticles provided herein can be prepared according to other methods.5.2 Peptides

[0077] The present disclosure relates, in part, to nanoparticles comprising a peptide (such as an AAV capsid peptide according to Section 5.2.1 and / or a transgene product peptide according to Section 5.2.2). In certain embodiments, the peptide is covalently linked to a component of the nanoparticle such that the peptide remains on the outside of the nanoparticle. In some embodiments, peptide is associated with the outside of the nanoparticle. In some embodiments, the peptide is non-covalently associated with the outside of the nanoparticle. In some embodiments, the peptide is non-covalently associated with the amphiphilic polymer. In some embodiments, the peptide is associated with the amphiphilic polymer by a linker. In some embodiments, the peptide is covalently associated with the outside of the nanoparticle. In some embodiments, the peptide is covalently linked to the amphiphilic polymer. For example, in some embodiments, the peptide can be covalently linked to amphiphilic polymer via formation of an amide bond between the N-terminus of the peptide and a carboxylate group of the amphiphilic polymer. Known methods of covalently coupling peptides include carbodiimide or succinimide coupling. In some embodiments, the peptide is covalently linked using l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) chemistry or any other peptide coupling reagents. See, for example, Jaradat et al., Advances in solid-phase peptide synthesis in aqueous media (ASPPS) ((Critical Review) Green Chem. , 2022, 24, 6360-6372); Albericio et al., Choosing theRight Coupling Reagent for Peptides: A Twenty-Five-Year Journey, Org. Process Res.Dev. 2018, 22, 7, 760-772.

[0078] In certain embodiments, the nanoparticle provided herein comprises a peptide (such as an AAV capsid peptide according to Section 5.2.1 and / or a transgene product peptide according to Section 5.2.2) in an amount of about 5-15% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises the peptide in an amount of about 8-12% by weight of the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises the peptide in an amount of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% by weight of the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises the peptide in an amount of about 8%, about 9%, about 10%, about 11%, or about 12% by weight of the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises the peptide in an amount of about 8% by weight of the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises the peptide in an amount of about 9% by weight of the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises the peptide in an amount of about 10% by weight of the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises the peptide in an amount of about 11% by weight of the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises the peptide in an amount of about 12% by weight of the total weight of the nanoparticle.

[0079] In certain embodiments, the nanoparticle provided herein comprises a peptide (such as an AAV capsid peptide according to Section 5.2.1 and / or a transgene product peptide according to Section 5.2.2) in an amount of about 1-3 mol% of all components of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises the peptide in an amount of about 1.5-2.5 mol% of all components of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises the peptide in an amount of about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5 mol% of all components of the nanoparticle. In some embodiments, the nanoparticle comprises the peptide in an amount of about 2 mol% of all components of the nanoparticle.

[0080] In certain embodiments, the nanoparticle provided herein comprises a peptide (such as an AAV capsid peptide according to Section 5.2.1 and / or a transgene product peptide according to Section 5.2.2) and the amphiphilic polymer in a ratio of between about 0.03:1 (w / w) and 0.25:1 (w / w) of peptide to amphiphilic polymer. In certain embodiments, the nanoparticle provided herein comprises the peptide and the amphiphilic polymer in a ratio of between about 0.05:1 (w / w) and 0.20:1 (w / w) of peptide to amphiphilic polymer.

[0081] In certain embodiments, the nanoparticle provided herein comprises a peptide (such as an AAV capsid peptide according to Section 5.2.1 and / or a transgene product peptide according to Section 5.2.2) and the amphiphilic polymer in a ratio of between about 0.03:1 (mol / mol) and 0.25:1 (mol / mol) of peptide to amphiphilic polymer. In certain embodiments, the nanoparticle provided herein comprises the peptide and the amphiphilic polymer in a ratio of between about 0.05:1 (mol / mol) and 0.20:1 (mol / mol) of peptide to amphiphilic polymer.

[0082] In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 50-85% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 5-15% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 55-65% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 5-15% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 55-65% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 8-12% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 65-75% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 5-15% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 65-75% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 8-12% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 75-85% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 5-15% by weight of the total weight of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) theamphiphilic polymer in an amount of about 75-85% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 8-12% by weight of the total weight of the nanoparticle.

[0083] In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 50-85% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 1-3 mol% of all components of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 55-65% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 1-3 mol% of all components of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 55-65% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 1.5-2.5 mol% of all components of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 65-75% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 1-3 mol% of all components of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 65-75% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 1.5-2.5 mol% of all components of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 75-85% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 1-3 mol% of all components of the nanoparticle. In certain embodiments, the nanoparticle provided herein comprises: (i) the amphiphilic polymer in an amount of about 75-85% by weight of the total weight of the nanoparticle; and (ii) the peptide in an amount of about 1.5-2.5 mol% of all components of the nanoparticle.

[0084] In some embodiments, the peptide (such as an AAV capsid peptide according to Section 5.2.1 or a transgene product peptide according to Section 5.2.2) is 4 to 50 amino acids long. In some embodiments, the peptide is 5 to 50 amino acids long. In some embodiments, the peptide is 6 to 50 amino acids long. In some embodiments, the peptide is 7 to 50 amino acids long. In some embodiments, the peptide is 8 to 50 amino acids long. In some embodiments, the peptide is 8 to 40 amino acids long. In some embodiments, the peptide is 8 to 30 amino acidslong. In some embodiments, the peptide is 8 to 20 amino acids long. In some embodiments, the peptide is 8 to 15 amino acids long. In some embodiments, the peptide is 8 to 14 amino acids long. In some embodiments, the peptide is 8 to 13 amino acids long. In some embodiments, the peptide is 8 to 12 amino acids long. In some embodiments, the peptide is 8 to 11 amino acids long. In some embodiments, the peptide is 8 to 10 amino acids long. In some embodiments, the peptide is 8 to 9 amino acids long. In some embodiments, the peptide is 8 to 40 amino acids long. In some embodiments, the peptide is 10 to 50 amino acids long. In some embodiments, the peptide is 10 to 40 amino acids long. In some embodiments, the peptide is 10 to 30 amino acids long. In some embodiments, the peptide is 10 to 20 amino acids long. In some embodiments, the peptide is 12 to 30 amino acids long. In some embodiments, the peptide is 12 to 25 amino acids long. In some embodiments, the peptide is 12 to 20 amino acids long. In some embodiments, the peptide is 12 to 19 amino acids long. In some embodiments, the peptide is 12 to 18 amino acids long. In some embodiments, the peptide is 12 to 17 amino acids long. In some embodiments, the peptide is 12 to 16 amino acids long. In some embodiments, the peptide is 12 to 15 amino acids long. In some embodiments, the peptide is 13 to 25 amino acids long. In some embodiments, the peptide is 13 to 20 amino acids long. In some embodiments, the peptide is 13 to 19 amino acids long. In some embodiments, the peptide is 13 to 18 amino acids long. In some embodiments, the peptide is 13 to 17 amino acids long. In some embodiments, the peptide is 13 to 16 amino acids long. In some embodiments, the peptide is 14 to 25 amino acids long. In some embodiments, the peptide is 14 to 20 amino acids long. In some embodiments, the peptide is 14 to 19 amino acids long. In some embodiments, the peptide is 14 to 18 amino acids long. In some embodiments, the peptide is 14 to 17 amino acids long. In some embodiments, the peptide is 14 to 16 amino acids long. In some embodiments, the peptide is 12 to 15 amino acids long. In some embodiments, the peptide is 25 to 50 amino acids long. In some embodiments, the peptide is 30 to 50 amino acids long. In some embodiments, the peptide is 40 to 50 amino acids long. In some embodiments, the peptide is 5 amino acids long. In some embodiments, the peptide is 6 amino acids long. In some embodiments, the peptide is 7 amino acids long. In some embodiments, the peptide is 8 amino acids long. In some embodiments, the peptide is 9 amino acids long. In some embodiments, the peptide is 10 amino acids long. In some embodiments, the peptide is 11 amino acids long. In some embodiments, the peptide is 12 amino acids long. In some embodiments, the peptide is 13 amino acids long. In some embodiments, the peptide is 14 amino acids long. In someembodiments, the peptide is 15 amino acids long. In some embodiments, the peptide is 16 amino acids long. In some embodiments, the peptide is 17 amino acids long. In some embodiments, the peptide is 18 amino acids long. In some embodiments, the peptide is 19 amino acids long. In some embodiments, the peptide is 20 amino acids long. In some embodiments, the peptide is 21 amino acids long. In some embodiments, the peptide is 22 amino acids long. In some embodiments, the peptide is 23 amino acids long. In some embodiments, the peptide is 24 amino acids long. In some embodiments, the peptide is 25 amino acids long.

[0085] In some embodiments, the peptide (such as an AAV capsid peptide according to Section 5.2.1 or a transgene product peptide according to Section 5.2.2) comprises an MHC-I epitope. In some embodiments, the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises two or more different peptides linked to each other as a fusion polypeptide. In certain embodiments, the peptide comprises two or more different peptides comprising MHC-I epitopes linked to each other as a fusion polypeptide. In certain embodiments, the peptide comprises two or more different peptides comprising MHC-II epitopes linked to each other as a fusion polypeptide. In certain embodiments, the peptide comprises two or more different peptides comprising one or more MHC-I epitopes linked to one or more MHC- II epitopes as a fusion polypeptide.

[0086] In some embodiments, the peptide (such as an AAV capsid peptide according to Section 5.2.1 or a transgene product peptide according to Section 5.2.2) is synthesized, recombinantly expressed, or isolated or modified from natural sources.

[0087] In some embodiments, the composition of the nanoparticle is determined by standard analytical means known in the art, such as, for example, Nuclear Magnetic Resonance (NMR) or Mass Spectrometry, with or without complete or partial decomposition of the nanoparticle and separation of the individual components of the nanoparticle by chromatographic or other means. For example, the amount of peptides associated with and / or covalently linked to the nanoparticle may be determined following cleavage of the peptides from the nanoparticle, for example by hydrolysis of amide and / or ester linkages at the N- or C-terminus of peptide, respectively. In some embodiments, the composition of the nanoparticle corresponds to the average composition of a population of nanoparticles.5.2.1 AAV capsid peptides

[0088] AAV particles can serve as vectors for delivery of transgenes for gene therapy. However, host immune responses against the AAV capsid and the transgene product may limit the use and ability to re-administer AAV-vectored transgenes. As provided herein, a nanoparticle (such as a nanoparticle described in Section 5.1) comprising an AAV capsid peptide that is derived from an AAV capsid polypeptide can induce tolerance and reduce host immune responses against the AAV particle and transgene.

[0089] In one aspect, provided herein is a nanoparticle comprising an AAV capsid peptide comprising an amino acid sequence at least 90% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 91 % identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 92% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 93% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 94% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 95% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 96% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 97% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 98% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 99% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 50 amino acids long. In specific embodiments, an AAV capsid peptide comprising an amino acid sequence less than 100% identical to a fragment of anAAV capsid polypeptide does not increase a cellular and / or a humoral immune response in a subject.

[0090] In certain embodiments, provided herein is a nanoparticle comprising an AAV capsid peptide comprising an amino acid sequence at least 90% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 91% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 92% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 93% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 94% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 95% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 96% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 97 % identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 98% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 99% identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence identical to a fragment of an AAV capsid polypeptide, and the peptide is 8 to 11 amino acids long.

[0091] In certain embodiments, provided herein is a nanoparticle comprising an AAV capsid peptide comprising an amino acid sequence at least 90% identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 91 % identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 92% identical to a fragmentof an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 93% identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 94% identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 95% identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 96% identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 97% identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 98% identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 99% identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long. In some embodiments, the AAV capsid peptide comprises an amino acid sequence identical to a fragment of an AAV capsid polypeptide, and the peptide is 13 to 25 amino acids long.

[0092] In some embodiments, the present disclosure provides a AAV capsid peptide comprising an amino acid sequence at least 90% identical to a fragment of an AAV capsid. In some embodiments, the AAV capsid is from or derived from an AAV capsid of an AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype rhlO (AAV rhlO), a synthetic AAV serotype (e.g., AAV-DJ), an AAV serotype variant, combinations, engineered, or modified versions thereof. In some embodiments, an AAV capsid is from or derived from an AAV ancestral serotype. In some embodiments, an AAV capsid is an ancestral (Anc) AAV capsid. An Anc capsid is created from a construct sequence that is constructed using evolutionary probabilities and evolutionary modeling to determine a probable ancestral sequence. In some embodiments, an AAV capsid has been modified in a manner known in the art (see e.g., Biining and Srivastava, Capsid modifications for targeting and improving the efficacy of AAV vectors,Mol Ther Methods Clin Dev. 2019). In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV1 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV2 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV3 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV4 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV5 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV 6 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV7 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV8 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV9 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an AAV10 capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of a synthetic AAV capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of an ancestral AAV capsid. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90% identical to a fragment of a modified AAV capsid. Exemplary amino acid sequences of AAV capsids are provided in Table 1.

[0093] Table 1: Exemplary AAV capsid amino acid sequences

[0094] In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a linear, contiguous fragment of the amino acid sequence of an adeno- associated viral (AAV) capsid polypeptide. For example, non-limiting examples include an AAV capsid peptide comprising amino acids 372-379 of SEQ ID NO:1, amino acids 376-383 of SEQ ID NO:8, amino acids 414-421 of SEQ ID NO:8, amino acids 416-430 of SEQ ID NO:3, amino acids 516- 530 of SEQ ID NO: 8, or amino acids 461-475 of SEQ ID NO: 8. In other embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a non-linear fragment of an adeno- associated viral (AAV) capsid polypeptide that mimics the 3D structure of the AAV capsid. In some embodiments, the AAV capsid peptide comprises an MHC-I epitope. In some embodiments, the AAV capsid peptide comprises an MHC-II epitope. In some embodiments, the AAV capsid peptide comprises two or more different peptides linked to each other as a fusion polypeptide. In certain embodiments, the AAV capsid peptide comprises two or more different peptides comprising MHC-I epitopes linked to each other as a fusion polypeptide. In certain embodiments, the AAV capsid peptide comprises two or more different peptides comprising MHC-II epitopes linked to each other as a fusion polypeptide. In certain embodiments, the AAV capsid peptide comprises two or more different peptides comprising one or more MHC-I epitopes linked to one or more MHC-II epitopes as a fusion polypeptide.

[0095] In specific embodiments, the AAV capsid peptide comprises an amino acid sequence provided in Table 2. In some embodiments, the AAV capsid peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10-SEQ ID NO: 15. In some embodiments, the AAV capsid peptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:10-SEQ ID NO: 14. In some embodiments, the AAV capsid peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10- SEQ ID NO: 12. In some embodiments, the AAV capsid peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13-SEQ ID NO:15. In some embodiments, the AAV capsid peptide comprises an amino acid sequence selected from the SEQ ID NO: 13 or SEQ ID NO: 14. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the AAV capsid peptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:15. In specific embodiments, the AAV capsid peptide comprises SEQ ID NO:10. In specific embodiments, the AAV capsid peptide comprises SEQ ID NO: 11. In specific embodiments, the AAV capsid peptide comprises SEQ ID NO: 12. In specific embodiments, the AAV capsid peptide comprises SEQ ID NO: 13. In specific embodiments, the AAV capsid peptide comprises SEQ ID NO: 14. In specific embodiments, the AAV capsid peptide comprises SEQ ID NO: 15. In specific embodiments, the AAV capsid peptide consists of SEQ ID NO: 10. In specific embodiments, the AAV capsid peptide consists of SEQ ID NO:11. In specific embodiments, the AAV capsid peptide consists of SEQ ID NO: 12. In specific embodiments, the AAV capsid peptide consists of SEQ ID NO: 13. In specific embodiments, the AAV capsid peptide consists of SEQ ID NO: 14. In specific embodiments, the AAV capsid peptide consists of SEQ ID NO: 15.

[0096] Table 2: Exemplary AAV Capsid Peptide Sequences

[0097] In some embodiments, the nanoparticle comprises a linker at the N-terminus of the AAV capsid peptide. In specific embodiments, the nanoparticle comprises a linker at the N- terminus of a AAV capsid peptide provided in Table 2. In some embodiments, the linker comprises one arginine (R) residue. In some embodiments, the linker comprises two arginine residues (RR). In some embodiments, the linker comprises more than two arginine residues. In specific embodiments, the AAV capsid peptide with a linker comprises an amino acid sequence provided in Table 3. In some embodiments, the AAV capsid peptide with a linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:48-SEQ ID NO:57. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:48. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:49. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 50. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:51. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 52. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:53. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 54. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:55. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 56. In specific embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 57. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:48. In specific embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:49. In specific embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:50. In specific embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:51. In specific embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:52. In specific embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:53. In specificembodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:54. In specific embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:55. In specific embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:56. In specific embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:57.

[0098] Table 3: Exemplary AAV Capsid Peptides with a Linker

[0099] In some embodiments, the nanoparticle comprises more than one AAV capsid peptide. In some embodiments, the nanoparticle comprises only one type of AAV capsid peptide, e.g. peptides having the same amino acid sequence. In some embodiments, the nanoparticle comprises only one type of AAV capsid peptide, e.g. AAV capsid peptides having the same amino acid sequence. In some embodiments, only one type of AAV capsid peptide, e.g. AAV capsid peptides having the same amino acid sequence, is associated with the nanoparticle. In some embodiments, only one type of AAV capsid peptide, e.g. AAV capsid peptides having the same amino acid sequence, is covalently linked to the nanoparticle.

[0100] In other embodiments, the nanoparticle comprises more than one AAV capsid peptide, e.g. AAV capsid peptides having different amino acid sequences. In some embodiments, the nanoparticle comprises two or more different types of peptides. In some embodiments, the two or more different types of AAV capsid peptides, e.g. peptides having different amino acid sequences, are associated with or covalently linked to the nanoparticle. In some embodiments, the nanoparticle comprises three or more different types of AAV capsid peptides. In some embodiments, the three or more different types of AAV capsid peptides, e.g. peptides having different amino acid sequences, are associated with or covalently linked to the nanoparticle. Insome embodiments, the nanoparticle comprises four or more different types of AAV capsid peptides. In some embodiments, the four or more different types of AAV capsid peptides, e.g. peptides having different amino acid sequences, are associated with or covalently linked to the nanoparticle. In some embodiments, the nanoparticle comprises five or more different types of AAV capsid peptides. In some embodiments, the five or more different types of AAV capsid peptides, e.g. peptides having different amino acid sequences, are associated with or covalently linked to the nanoparticle. In some embodiments, the nanoparticle comprises one or more AAV capsid peptides (such as an AAV capsid peptide described in Section 5.2.1), and one or more transgene product peptides (such as a transgene product peptide described in Section 5.2.2). In some embodiments, the nanoparticle comprises one or more AAV capsid peptides (such as an AAV capsid peptide described in Section 5.2.1), and one or more transgene product peptides (such as a transgene product peptide described in Section 5.2.2) that are associated with and / or covalently linked to the nanoparticle.5.2.2 Transgene product peptides

[0101] The present disclosure relates, in part, to the transfer of foreign genetic material (e.g., a transgene) using an adeno-associated viral (AAV) vector. By way of example, upon delivery to a subject, a transgene can be transcribed and translated into a transgene product (i.e., a polypeptide encoded by a transgene). The introduction of new genetic materials into a subject can alter gene or protein expression. In certain aspects, the transgene can provide a one-time curative treatment, including for many diseases that currently have no cure. In certain embodiments, a transgene can encode: a protein whose gene is mutated or deficient in the subject to be treated or an antibody that affects protein activity in the subject to be treated.

[0102] In one aspect, provided herein is a nanoparticle comprising a transgene product peptide that comprises an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by a transgene, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 91% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 92% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence atleast about 93% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 94% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 95% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 96% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 97% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 98% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 99% identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence identical to a fragment of a transgene product, and the peptide is 8 to 50 amino acids long. In specific embodiments, a transgene product peptide comprising an amino acid sequence less than 100% identical to a fragment of a transgene product does not increase a cellular and / or a humoral immune response in a subject.

[0103] In one aspect, provided herein is a nanoparticle comprising a transgene product peptide that comprises an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by a transgene, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 91% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 92% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 93% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 94% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an aminoacid sequence at least about 95% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 96% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 97% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 98% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 99% identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence identical to a fragment of a transgene product, and the peptide is 8 to 11 amino acids long.

[0104] In one aspect, provided herein is a nanoparticle comprising a transgene product peptide that comprises an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by a transgene, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 91% identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 92% identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 93% identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 94% identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 95% identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 96% identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 97% identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 98% identical to a fragment of atransgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 99% identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long. In some embodiments, the transgene product peptide comprises an amino acid sequence identical to a fragment of a transgene product, and the peptide is 13 to 25 amino acids long.

[0105] In some embodiments, the transgene encodes for a functioning version of a protein of interest. In certain embodiments, the transgene does not encode for an autoantigen. In certain embodiments, the transgene encodes for a protein that elicits an iatrogenic response when the transgene or transgene product is administered two or more times to a subject in the absence of the nanoparticle comprising the transgene product peptide.

[0106] An exemplary transgene suitable for use with the present disclosure is the gene for clotting Factor VIII (termed 8). Factor VIII (Accession Number: KAI4001687.1), also known as antihemophilic factor, is a protein that helps blood clot properly and prevents excessive bleeding. It is a co-factor in the blood coagulation cascade, and is produced primarily by cells in the liver. Hemophilia A patients have an inherited gene mutation that impairs their ability to produce normal levels of Factor VIII. However, it should be understood that Factor VIII is exemplary and that other transgenes can similarly be used.

[0107] In some embodiments, provided herein is a nanoparticle comprising a transgene product peptide that comprises an amino acid sequence at least about 90% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 91% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 92% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 93% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 94% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 95% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 96% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 97% identical to a fragment of FactorVIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 98% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 99% identical to a fragment of Factor VIII. In some embodiments, the transgene product peptide comprises an amino acid sequence identical to a fragment of Factor VIII.

[0108] Table 4: Exemplary Factor VIII amino acid sequence

[0109] In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a linear, contiguous fragment of a transgene product. For example, non-limiting examples include a transgene product peptide comprising amino acids 43-62 of SEQ ID NO:16, or amino acids 1906-1914 of SEQ ID NO:16. In other embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a non-linear fragment of a transgene product that mimics the 3D structure of the transgene product. In some embodiments, the transgene product peptide comprises an MHC-I epitope. In some embodiments, the transgene product peptide comprises an MHC-II epitope. In some embodiments, the transgene product peptide comprises an MHC-I epitope and an MHC-II epitope. In some embodiments, the transgene product peptide comprises two or more different peptides linked to each other as a fusion polypeptide. In certain embodiments, the transgene product peptide comprises two or more different peptides comprising MHC-I epitopes linked to each other as a fusion polypeptide. In certain embodiments, the transgene product peptide comprises two or more different peptides comprising MHC-II epitopes linked to each other as a fusion polypeptide. In certainembodiments, the transgene product peptide comprises two or more different peptides comprising one or more MHC-I epitopes linked to one or more MHC-II epitopes as a fusion polypeptide.

[0110] In specific embodiments, the transgene product peptide comprises an amino acid sequence provided in Table 5. In some embodiments, the transgene product peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:17-SEQ ID NO:47. In some embodiments, the transgene product peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:17-SEQ ID NO:25. In some embodiments, the transgene product peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:26-SEQ ID NO:47. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 17. In some embodiments, the transgene product peptide comprises about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 18. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 19. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 20. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:21. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:22. In some embodiments, the transgene product peptide comprises an amino acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:23. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:24. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:25. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:26. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 27. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:28. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:29. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:30. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:31. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:32. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:33. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:34. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 35. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:36. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:37. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:38. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:39. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:40. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:41. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 42. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:43. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:44. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:45. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:46. In some embodiments, the transgene product peptide comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 47. In some embodiments, the transgene product peptide is modified by a linker at its N-terminus.

[0111] In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO: 17. In specific embodiments, the transgene product peptide comprisesan amino acid sequence of SEQ ID NO: 18. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO: 19. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO: 20. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO: 21. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:22. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:23. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:24. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO: 25. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:26. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:27. In specific embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:28. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:29. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:30. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO: 31. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:32. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:33. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:34. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:35. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:36. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:37. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO: 38. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:39. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:40. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:41. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:42. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO: 43. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ IDNO:44. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:45. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:46. In some embodiments, the transgene product peptide comprises an amino acid sequence of SEQ ID NO:47. In some embodiments, the transgene product peptide is modified by a linker at its N-terminus.

[0112] In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO: 17. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO: 18. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO: 19. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:20. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO: 21. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:22. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:23. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:24. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:25. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:26. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:27. In specific embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:28. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:29. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:30. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:31. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:32. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:33. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:34. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO: 35. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:36. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:37. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ IDNO: 38. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:39. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:40. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO: 41. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:42. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:43. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:44. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:45. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO:46. In some embodiments, the transgene product peptide consists of an amino acid sequence of SEQ ID NO: 47.

[0113] Table 5: Exemplary Transgene Product Peptides

[0114] In some embodiments, the nanoparticle comprises a linker at the N-terminus of the transgene product peptide. In specific embodiments, the nanoparticle comprises a linker at the N- terminus of a transgene product peptide provided in Table 5. In some embodiments, the linker comprises one arginine (R) residue. In some embodiments, the linker comprises two arginine residues (RR). In some embodiments, the linker comprises more than two arginine residues. In specific embodiments, the transgene product peptide with a linker comprises an amino acid sequence provided in Table 6. In some embodiments, the transgene product peptide with a linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:58-SEQ ID NO:88. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:58. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:59. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 60. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:61. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 62. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:63. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 64. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:65. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 66. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 67. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 68. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 69. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:70. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:71. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:72. In someembodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:73. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:74. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:75. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:76. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:77. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:78. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:79. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 80. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 81. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 82. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:83. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 84. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO:85. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 86. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 87. In some embodiments, the AAV capsid peptide with a linker comprises SEQ ID NO: 88. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:58. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:59. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:60. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:61. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:62. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:63. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:64. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:65. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:66. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:67. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:68. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:69. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:70. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:71. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:72. In someembodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:73. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:74. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:75. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:76. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:77. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:78. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:79. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO: 80. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:81. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO: 82. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO: 83. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO: 84. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO: 85. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO: 86. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO:87. In some embodiments, the AAV capsid peptide with a linker consists of SEQ ID NO: 88.

[0115] Table 6: Exemplary Transgene Product Peptides with a Linker

[0116] In some embodiments, the nanoparticle comprises only one type of transgene product peptide, e.g. peptides having the same amino acid sequence. In some embodiments, the nanoparticle comprises only one type of transgene product peptide, e.g. transgene product peptides having the same amino acid sequence. In some embodiments, only one type of transgene product peptide, e.g. transgene product peptides having the same amino acid sequence, is associated with the nanoparticle. In some embodiments, only one type of transgene product peptide, e.g. transgene product peptides having the same amino acid sequence, is covalently linked to the nanoparticle.

[0117] In other embodiments, the nanoparticle comprises more than one transgene product peptide, e.g. transgene product peptides having different amino acid sequences. In some embodiments, the nanoparticle comprises two or more different types of peptides. In some embodiments, the two or more different types of transgene product peptides, e.g. peptides having different amino acid sequences, are associated with or covalently linked to the nanoparticle. In some embodiments, the nanoparticle comprises three or more different types of transgene product peptides. In some embodiments, the three or more different types of transgene product peptides, e.g. peptides having different amino acid sequences, are associated with or covalently linked to the nanoparticle. In some embodiments, the nanoparticle comprises four or moredifferent types of transgene product peptides. In some embodiments, the four or more different types of transgene product peptides, e.g. peptides having different amino acid sequences, are associated with or covalently linked to the nanoparticle. In some embodiments, the nanoparticle comprises five or more different types of transgene product peptides. In some embodiments, the five or more different types of transgene product peptides, e.g. peptides having different amino acid sequences, are associated with or covalently linked to the nanoparticle. In some embodiments, the nanoparticle comprises one or more AAV capsid peptides (such as an AAV capsid peptide described in Section 5.2.1), and one or more transgene product peptides (such as a transgene product peptide described in Section 5.2.2). In some embodiments, the nanoparticle comprises one or more AAV capsid peptides (such as an AAV capsid peptide described in Section 5.2.1), and one or more transgene product peptides (such as a transgene product peptide described in Section 5.2.2) that are associated with and / or covalently linked to the nanoparticle.5.3 Pharmaceutical Composition

[0118] Also provided herein are pharmaceutical compositions comprising one or more nanoparticles according to Section 5.1, and a pharmaceutically acceptable carrier.

[0119] In certain embodiments, the pharmaceutical composition comprises the nanoparticles in a concentration below 100 pM. In some embodiments, the pharmaceutical composition comprises the nanoparticles in a concentration of between about 0.5 to 80 pM. In some embodiments, the pharmaceutical composition comprises the nanoparticles in a concentration of between about 1 to 50 pM.

[0120] In certain embodiments, the pharmaceutical composition comprises a homogenous composition of nanoparticles comprising the same peptide ((such as an AAV capsid peptide described in Section 5.2.1 or a transgene product peptide described in Section 5.2.2). In other embodiments, the pharmaceutical composition comprises more than one type of nanoparticle, wherein the different types of nanoparticles are associated with or covalently linked to different peptides. Without wishing to be bound by theory, using a mixture of nanoparticles can induce broader immune tolerance against the AAV capsid and / or transgene product at the same time. In some embodiments, the peptides are derived from the AAV capsid, such as an AAV capsid peptide described in Section 5.2.1. In some embodiments, the peptides are derived from the transgene product, such as a transgene product peptide described in Section 5.2.2. In otherembodiments, the peptides are derived from (a) the AAV capsid, such as an AAV capsid peptide described in Section 5.2.1, and (b) the transgene product, such as a transgene product peptide described in Section 5.2.2. In some embodiments, the pharmaceutical composition comprises the different types of nanoparticles in equimolar concentrations.

[0121] In certain embodiments, the pharmaceutical composition comprises an AAV capsid peptide (such as an AAV capsid peptide described in Section 5.2.1) in a concentration between about 0.01 and 2 mM. In some embodiments, the pharmaceutical composition comprises an AAV capsid peptide (such as an AAV capsid peptide described in Section 5.2.1) in a concentration between about 0.1 and 1 mM. In some embodiments, the pharmaceutical composition comprises an AAV capsid peptide (such as an AAV capsid peptide described in Section 5.2.1) in a concentration between about 0.45 mM to 1 mM.

[0122] In certain embodiments, the pharmaceutical composition comprises a transgene product peptide (such as a transgene product peptide described in Section 5.2.2) in a concentration between about 0.01 and 2 mM. In some embodiments, the pharmaceutical composition comprises a transgene product peptide (such as a transgene product peptide described in Section 5.2.2) in a concentration between about 0.1 and 1 mM. In some embodiments, the pharmaceutical composition comprises a transgene product peptide (such as a transgene product peptide described in Section 5.2.2) in a concentration between about 0.45 mM to 1 mM.

[0123] In certain embodiments, the pharmaceutical composition comprises less than 10%, less than 5%, or less than 2% free amphiphilic polymer, i.e. amphiphilic polymer not incorporated into a nanoparticle.

[0124] In some embodiments, the pharmaceutical composition further comprises an excipient and / or diluent. In some embodiments, the diluent is water or an aqueous solution, e.g., a buffer such as Phosphate buffered saline (PBS), Ringer solution, TRIS buffer or sodium chloride solution. In some embodiments, the pharmaceutical composition comprises the nanoparticles in a solution comprising D-mannitol, TRIS and / or L-lactic acid.5.4 Kits and Medical Systems

[0125] In one aspect, the disclosure provides a kit comprising the nanoparticles (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) or the pharmaceutical composition of the disclosure (such as a pharmaceutical composition according to Section 5.3) and instructions for use. In certain embodiments, the kit comprises the nanoparticles (such as a nanoparticle according to Section 5.1) or the pharmaceutical composition of the disclosure (such as a pharmaceutical composition according to Section 5.3), an AAV particle comprising a transgene, and instructions for use.

[0126] In another aspect, the disclosure provides a medical system comprising an AAV particle comprising a transgene, and the nanoparticles (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) or the pharmaceutical composition of the disclosure (such as a pharmaceutical composition according to Section 5.3). As used herein, the term “medical system” is intended to describe the combination of the AAV particle and the nanoparticle without any requirement for the components to be simultaneously adjacent in the same composition. In some embodiments, the medical system comprises the sequential combination of the AAV particle and the nanoparticle provided herein. In some embodiments, the medical system comprises the simultaneous combination of the AAV particle and the nanoparticle provided herein.5.5 Method of treatment

[0127] In one aspect, provided herein is a method of treating a subject in need of an AAV particle comprising a transgene. In certain embodiments, the method comprises administering a nanoparticle (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) to the subject. In specific embodiments, the method comprises intravenously administering a nanoparticle (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) to the subject. In some embodiments, the method further comprises administering an AAV particle to the subject, wherein the AAV particle comprises anAAV capsid, and a transgene. In some embodiments, a nanoparticle provided herein (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) is administered at least once prior to administration of the AAV particle. In some embodiments, the nanoparticle is administered at least twice prior to administration of the AAV particle. In certain embodiments, the nanoparticle is administered one day prior to administration of the AAV particle. In specific embodiments, the nanoparticle is administered five days prior and one day prior to administration of the AAV particle. In some embodiments, the nanoparticle is administered at least once after administration of the AAV particle. In some embodiments, the nanoparticle is administered at least twice after administration of the AAV particle. In certain embodiments, the nanoparticle is administered at least once prior to administration of the AAV particle, and at least once after administration of the AAV particle. In certain embodiments, the nanoparticle is administered at least twice prior to administration of the AAV particle, and at least once after administration of the AAV particle. In certain embodiments, the nanoparticle is administered at least twice prior to administration of the AAV particle, and at least twice after administration of the AAV particle.

[0128] In certain aspects, the route of administration of an AAV particle provided herein is oral, intrathecal, intraperitoneal, subcutaneous, pulmonary, nasal, transdermal, or ocular. In some embodiments, the route of administration of the AAV particle is intravenous or intramuscular. In some embodiments, the route of administration of the nanoparticle provided herein (such as a nanoparticle according to Section 5.1) is intravenous. In some embodiments, the route of administration of the nanoparticle provided herein (such as a nanoparticle according to Section 5.1) is oral, intrathecal, intramuscular, intraperitoneal, subcutaneous, pulmonary, nasal, transdermal, or ocular. In some embodiments, the route of administration of the AAV particle and the nanoparticle is the same (e.g., the nanoparticle and the AAV particle are both administered intravenously). In some embodiments, the route of administration of the AAV particle and the nanoparticle is different (e.g., the nanoparticle is administered intravenously and the AAV particle is administered intramuscularly or intrathecally.)5.5.1 Methods of inducing tolerance

[0129] As provided herein, the nanoparticles of the present disclosure (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) reduce or prevent the immunogenicity of an AAV particle that comprises an AAV capsid and a transgene. In some embodiments, administration of the nanoparticles provided herein before administration of an AAV particle that comprises an AAV capsid and a transgene reduces or prevents an immune response, relative to administration of the AAV particle alone without pre-administration of the nanoparticle.

[0130] In some embodiments, administering the nanoparticles provided herein (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2), before administration of an AAV particle that comprises an AAV capsid and a transgene, reduces or prevents a cellular immune response, relative to administration of the AAV particle without the nanoparticle. In some embodiments, the nanoparticle comprises a peptide (such as an AAV capsid peptide described in Section 5.2.1) and the cellular immune response against the AAV capsid is reduced by about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%. In some embodiments, the nanoparticle comprises a peptide (such as an AAV capsid peptide described in Section 5.2.1) and the cellular immune response against the transgene is reduced by about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%. In some embodiments, the nanoparticle comprises a peptide (a transgene product peptide described in Section 5.2.2) and the cellular immune response against the AAV capsid is reduced by about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%. In some embodiments, the nanoparticle comprises a peptide (a transgene product peptide described in Section 5.2.2) and the cellular immune response against the transgene is reduced by about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%. In some embodiments, the cellular immune response is measured by detection of IFNy ELISPOT.

[0131] In one aspect, provided herein is a method of inducing tolerance to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, and the method comprises administering to the subject a nanoparticle provided herein(such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2). In some embodiments, provided herein is a method of inducing tolerance to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the method comprises administering a nanoparticle provided herein (such as a nanoparticle according to Section 5.1).

[0132] As provided herein, the nanoparticle of the present disclosure (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) can induce tolerance of CD4+ T and / or CD8+ T cells to an AAV particle comprising the peptide. In one aspect, provided herein is a method of inducing tolerance of CD4+ T cells to the AAV particle comprising an AAV capsid and a transgene, wherein the method comprises administering a nanoparticle (such as a nanoparticle according to Section 5.1) comprising an AAV capsid peptide (such as an AAV capsid peptide described in Section 5.2.1). In another aspect, provided herein is a method of inducing tolerance of CD8+ T cells to the AAV particle comprising an AAV capsid and a transgene, wherein the method comprises administering a nanoparticle (such as a nanoparticle according to Section 5.1) comprising an AAV capsid peptide (such as an AAV capsid peptide described in Section 5.2.1). In a further aspect, provided herein is a method of inducing tolerance of CD4+ T cells to the AAV particle comprising an AAV capsid and a transgene, wherein the method comprises administering a nanoparticle (such as a nanoparticle according to Section 5.1) comprising a transgene product peptide (such as a transgene product peptide described in Section 5.2.2)). In another aspect, provided herein is a method of inducing tolerance of CD8+ T cells to the AAV particle comprising an AAV capsid and a transgene, wherein the method comprises administering a nanoparticle (such as a nanoparticle according to Section 5.1) comprising a transgene product peptide (such as a transgene product peptide described in Section 5.2.2). In some embodiments, tolerance is maintained for at least 30 days after administration of the AAV particle. In some embodiments, tolerance is maintained for at least 40 days after administration of the AAV particle. In some embodiments, tolerance is maintained for at least 50 days after administration of the AAV particle. In some embodiments, tolerance is maintained for at least 60 days after administration of the AAV particle. In someembodiments, tolerance is determined by IFNy ELISPOT. In specific embodiments, the method further comprises administering the AAV vector to the subject.5.5.2 Methods of reducing a humoral immune response

[0133] As provided herein, the nanoparticle of the present disclosure (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) can reduce a humoral immune response, relative to a method that does not include administering the nanoparticles provided herein.

[0134] In some embodiments, administering the nanoparticles provided herein (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2), before administration of an AAV particle that comprises an AAV capsid and a transgene, reduces or prevents a humoral immune response, relative to administration of the AAV particle without the nanoparticle. In some embodiments, the nanoparticle comprises a peptide (such as an AAV capsid peptide described in Section 5.2.1) and the humoral immune response against the AAV capsid is reduced by about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%. In some embodiments, the nanoparticle comprises a peptide (such as an AAV capsid peptide described in Section 5.2.1) and the humoral immune response against the transgene is reduced by about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%, relative to a method that does not include administering the nanoparticles provided herein. In some embodiments, the nanoparticle comprises a peptide (a transgene product peptide described in Section 5.2.2) and the humoral immune response against the AAV capsid is reduced by about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%. In some embodiments, the nanoparticle comprises a peptide (a transgene product peptide described in Section 5.2.2) and the humoral immune response against the transgene is reduced by about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%, relative to a method that does not include administering the nanoparticles provided herein.

[0135] In some embodiments, provided herein is a method of reducing a humoral immune response to an AAV particle in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle (such as a nanoparticle according to Section 5.1)comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2); and (b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and a transgene. In some embodiments, the method further comprises administering an inhibitor of mTOR (mammalian target of rapamycin). In specific embodiments, the inhibitor of mTOR is Temsirolimus. In some embodiments, Temsirolimus is administered in an amount sufficient to reduce a humoral immune response to an AAV particle in a subject in need thereof, relative to administration of the AAV particle without Temsirolimus. In some embodiments, Temsirolimus is administered in an amount sufficient to increase Treg survival and / or functionality. In specific embodiments, Temsirolimus is administered at a dose of about 0.4 mg / kg. In some embodiments, Temsirolimus is administered at a dose less than 2 mg / kg. In some embodiments, Temsirolimus is administered at a dose about 0.2 mg / kg to about 2 mg / kg. In some embodiments, Temsirolimus is administered at a dose about 0.2 mg / kg to about 1.5 mg / kg. In some embodiments, Temsirolimus is administered at a dose about 0.4 mg / kg to about 0.9 mg / kg. In certain embodiments, the inhibitor of mTOR is not rapamycin.5.5.3 Increasing Transgene Expression

[0136] In one aspect, provided herein is a method of increasing and / or prolonging expression of a transgene in a subject in need thereof, wherein the method comprises: (a) administering a nanoparticle to the subject; and (b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene, wherein the nanoparticle comprises: (i) an amphiphilic polymer; and (ii) a peptide comprising an amino acid sequence at least 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the expression is increased and / or prolonged relative to a method comprising step (b), but not step (a). In some embodiments, expression of the transgene is increased relative to a method comprising step (b), but not step (a). In specific embodiments, the transgene is increased by about 2-fold, about 3- fold, about 4-fold, about 5-fold, or about 10-fold, relative to a method comprising step (b), but not step (a). In specific embodiments, the expression of the transgene is prolonged for more than 30 days, more than 40 days, more than 50 days, or more than 60 days after administering the AAV particle.5.5.4 Cross-tolerization

[0137] The present disclosure is based, in part, on the observation that the nanoparticles provided herein (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) can induce cross-tolerization. In some embodiments, the nanoparticles comprise a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) comprising a MHC-I epitope and cross-tolerize against CD4+ T cells. In some embodiments, the nanoparticles comprise a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) comprising a MHC-II epitope and cross-tolerize against CD8+ T cells. In some embodiments, the nanoparticles comprise an AAV capsid peptide (such as an AAV capsid peptide described in Section 5.2.1) and cross-tolerize against the transgene product. In some embodiments, the nanoparticles comprise a transgene product peptide (such as a transgene product peptide described in Section 5.2.2) and cross-tolerize against the AAV capsid peptide. In specific embodiments, the nanoparticles provided herein (such as a nanoparticle according to Section 5.1) comprising a peptide (such as an AAV capsid peptide described in Section 5.2.1 and / or a transgene product peptide described in Section 5.2.2) do not result in tolerization against an unrelated antigen.5.6 Assays5.6.1 Assays for Characterization of Nanoparticles

[0138] Characterization of the nanoparticles provided herein can be performed by any suitable method known in the art.Nanoparticle size

[0139] Characterization of nanoparticle size and distribution can be performed by, for example, dynamic light scattering (DLS). In certain embodiments, the hydrodynamic diameter (z-average) and poly dispersity index is determined by using a Malvern Zetasizer Nano ZS or equivalent in unimodal mode. In certain embodiments, these measurements are performed on nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L-lactic acid. In other embodiments, these measurements are performed particles dispersed in an appropriate buffer anddesired pH.

[0140] In certain embodiments, the determination of the hydrodynamic diameter and the polydispersity index is carried out using electrophoretic light scattering analysis methods, such as a Malvern Zetasizer. In some embodiments, the method for determining the hydrodynamic diameter and the polydispersity index is carried out using electrophoretic light scattering, disposable polystyrene cuvettes, Zetasizer Software 7.12, and milli-Q water. The nanosphere size standards of 20 nm and 100 nm (NIST certified or equivalent) are diluted in an aqueous 0.9% sodium chloride solution and the test samples are diluted in water. All aqueous reagents are filtered through 0.22 pm membrane prior to use. In specific embodiments, the method for determining the hydrodynamic diameter and the poly dispersity index is carried out using electrophoretic light scattering in combination with the following analysis conditions:

[0141] Overview of the analysis conditions:

[0142] In certain embodiments, the nanoparticle size is based on mean diameter (Z- Average, nm by intensity), which is a parameter also known in DLS as the cumulants mean and Polydispersity index (PDI), which is used as a measure of the size distribution.Surface Charge Assay

[0143] In certain embodiments, the surface charge of the particles is analyzed by measuringthe zeta potential at pH 6 to 7 (pH during measurement) using a Malvern Zetasizer Nano ZS instrument. In certain embodiments, these measurements are performed on nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L- lactic acid.Polymer Content Assay

[0144] In certain embodiments, the total polymer content of the nanoparticles is determined by gel permeation chromatography (GPC). In certain embodiments, for measuring the total polymer content, the peptides are hydrolyzed, and the particles are destroyed (e.g. using a 6 M HC1 solution). In certain embodiments, the polymer is then extracted (e.g. with ethyl acetate) after addition of EDTA. After evaporation of solvent, the residue is re-dissolved (e.g. in THF / acetic acid mixture) and the polymer content is determined by GPC.

[0145] In certain embodiments, the determination of total polymer content of the nanoparticles is carried out using the following reagents and reference standards water (HPLC grade), acetonitrile (HPLC grade), tetrahydrofuran with BHT (THF-HPLC grade), acetic acid 100% (analytical grade), hydrochloric acid 37% (analytical grade), ethylenediaminetetraacetic acid disodium salt dihydrate (analytical grade), ethyl acetate (analytical grade), sodium hydroxide (analytical grade) and poly (maleic acid-alt- 1 -octadecene) as reference material.

[0146] In certain embodiments, the chromatographic conditions for the determination of total polymer content are:Peptide Content Assay

[0147] Sample preparation: Defined sample volumes of the liquid samples were put into hydrolysis vials and were dried under vacuum. 6N HC1 was added to the residues, the vials were closed under vacuum (< lOmbar) and were hydrolysed for 24 - 60 hours at 110°C. The HC1 was evaporated, and the samples were solved in sample dilution buffer. If the amount of the measured amino acids is too high, the solution must be accordingly diluted.

[0148] Measurement: The amino acid analysis was done with an Amino Acid Analyzer, LC 3000 (Eppendorf-Biotronik), using cation exchange technique with a five-step gradient and postcolumn derivatisation with ninhydrin and photometric detection at 440nm / 570nm.

[0149] Eluent: sodium acetate buffer (4-step gradient).

[0150] Column: cation exchange column, particle size 4pm, 125 x 4 mm ID.

[0151] Injection: 20pl (sample loop).

[0152] Derivatisation: post-column reactor, 125°C, ninhydrin as reagent.

[0153] Detection: 2-channel photometric detection (570nm / 440nm) The 440nm wavelength is especially for the determination of proline and hydroxy-proline.

[0154] Calibration standard: Sigma Standard for protein hydrolysates, A2908 Amino acids added: cysteine, allo-isoleucine, kynurenine (Deg. Prod. Trp) concentration: 200nmol / ml.Storage Stability Assay

[0155] In certain embodiments, the stability of the nanoparticles is determined following storage of the nanoparticles for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, or at least 12 days at 25 °C in a buffer at a given pH. In certain embodiments, the buffer and pH conditions are: (1) 25 mM sodium acetate buffer at pH about 5; (2) 25 mM MES buffer at pH about 6; (3) O.lx PBS buffer at pH about 7; (4) 25 mM HEPES buffer at pH about 8; or (5) 25 mM SBB buffer at pH about 9. Changes in the physical and / or biological properties of the nanoparticles after storage are then determined according to another assay described in this section, for example the Nanoparticle Size and Distribution Assay inSection 5.6.1.Stress Test Assay

[0156] After the production of the nanoparticles have been purified via TFF to remove the organic solvent present in the sample. This is an essential step since a high organic solvent percentage negatively affects the stability of the particles.

[0157] From the purified sample 1 mL has been taken per each stress test condition mentioned in the table below.

[0158] In total there have been four stress test conditions selected: Vortexing, Sonification, and Heating the samples to 37°C & 70°C. Per conditions there have been two timepoints selected. For example, the timepoints for heating the sample have been selected according to our standard coupling conditions which consist out of a 15 min incubation with EDC and a 2.5 h coupling with the corresponding peptide. The second timepoints always refer to the total overall time.

[0159] After each timepoint 100 pL of the sample has been taken and diluted with 900 pL of 0.1 x PBS for a DLS measurement. The result of the DLS measurement was used to evaluate the stability of the sample (size distribution, z-average, PDI, monodisperse / polydisperse).Internalization Assay

[0160] The Internalization Assay is designed to semi-quantitatively determine the biological activity of nanoparticles described herein that are functionalized by surface expression of a peptide, which represents the active moiety of the nanoparticle. The Internalization Assay allows for assessment of the activity of nanoparticles.

[0161] Without wishing to be bound by theory, T cells require two signals to get activated - an antigen-specific signal transmitted via the T cell receptor (TCR) after binding to its natural ligand, a peptide presented by the Major histocompatibility complex (MHC), and a costimulatory signal provided by the interaction with the antigen presenting cell (APC).

[0162] In this potency assay, the 2D2 mouse strain is used as a model system. These mice have a transgenic TCR which is specific for myelin oligodendrocyte glycoprotein (MOG). 2D2 CD4 T cells specifically recognize MOG35-55 peptide, which is presented on MHCII on APCs. The assay is appropriate to test the in vitro potency of nanoparticle batches that are functionalized with the MOG35-55 peptide.

[0163] Nanoparticles are incubated for three days at 37°C with cells isolated from spleen and lymph nodes of 2D2 mice. The cell mix contains MOG35-55-specific CD4 T cells and APCs, such as dendritic cells that take up nanoparticles and present the nanoparticle-derived MOG35- 55 peptide on MHCII to MOG35-55-specific CD4 T cells. The nanoparticle-induced T cell activation is then measured by assessing the IFNy release into the supernatant via enzyme-linked immunosorbent assay.

[0164] Without wishing to be bound by theory, presentation of nanoparticle-derived peptide on MHC molecules and subsequent recognition of said peptide by T cells is a pre-requisite for tolerance and later clinical effectiveness.5.6.2 Assays for Measuring Gene and Protein Expression

[0165] Any assay known in the art for measuring gene expression can be used to measure expression of a transgene. Non-limiting examples include measuring expression of the transgene’s RNA transcript by polymerase chain reaction (PCR), reverse transcriptase PCR (RT- PCR), real-time quantitative reverse transcription PCR (qRT-PCR), serial analysis of gene expression (SAGE), RNA Seq, northern blotting, microarray, in situ hybridization, or a combination thereof. Other conventional methods can also be employed as suitable. In certain embodiments, any assay known in the art for measuring protein expression can be used. Nonlimiting examples include Western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, HPLC, flow cytometry, fluorescence-activated cell sorting (FACS), liquid chromatography-mass spectrometry (LC / MS), immunoelectrophoresis, translation complex profile sequencing (TCP-seq), protein microarray, protein chip, capture arrays, reverse phaseprotein microarray (RPPA), two-dimensional gel electrophoresis or (2D-PAGE), functional protein microarrays, electrospray ionization (ESI), matrix-assisted laser desorption / ionization (MALDI), or a combination thereof. Other conventional methods can also be employed as suitable.5.6.3 Assays for Measuring Immune Response

[0166] Any assay known in the art for measuring humoral and / or cellular immune responses can be used. Non-limiting examples include enzyme-linked immunosorbent spot (ELIS pot) and / or FluoroSpot assays to measure antigen-specific T cells; detection of neutrophils and / or macrophages (e.g., flow cytometry); cytokine / chemokine detection (e.g., ELISA Multi-plexing, or flow cytometry); cell proliferation (e.g., beta counter, or flow cytometry); T-cell-dependent antibody response - TDAR (e.g., ELISA); in vivo functional assay to characterize immune response to an immunogen; immunophenotyping (e.g., flow cytometry; T-cell cytotoxicity); extended histopathological examination, such as by examination of lymphoid tissues and organs; CD8+ T-cell response model (adenovirus-based vaccine), or a combination thereof. Other conventional methods can also be employed as suitable.6. EMBODIMENTS

[0167] The present disclosure provides the following non-limiting embodiments:

[0168] 1. A nanoparticle comprising:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a transgene product, wherein the peptide is 8 to 50 amino acids long.

[0169] 2. The nanoparticle of embodiment 1 , wherein the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the transgene product.

[0170] 3. The nanoparticle of embodiment 1 , wherein the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the transgene product.

[0171] 4. The nanoparticle of embodiment 1 , wherein the peptide comprises an amino acid sequence 100% identical to the fragment of the transgene product.

[0172] 5. The nanoparticle of any one of embodiments 1 to 4, wherein the transgene product is suitable for use in a gene therapy.

[0173] 6. The nanoparticle of any one of embodiments 1 to 4, wherein the transgene product is encoded by human F8.

[0174] 7. The nanoparticle of any one of embodiments 1 to 4, wherein the transgene product is not an autoantigen.

[0175] 8. A nanoparticle comprising:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of an adeno-associated viral (AAV) capsid polypeptide, wherein the peptide is 8 to 50 amino acids long.

[0176] 9. The nanoparticle of embodiment 8, wherein the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid polypeptide.

[0177] 10. The nanoparticle of embodiment 8, wherein the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid polypeptide.

[0178] 11. The nanoparticle of embodiment 8, wherein the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid polypeptide.

[0179] 12. The nanoparticle of any one of embodiments 1 to 11, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV rhlO, a synthetic AAV, combinations, engineered, and modified versions thereof.

[0180] 13. The nanoparticle of embodiment 12, wherein the AAV capsid is AAV1 or AAV8.

[0181] 14. The nanoparticle of any one of embodiments 1 to 13, wherein the peptide is 8 to11 amino acids long.

[0182] 15. The nanoparticle of any one of embodiments 1 to 13, wherein the peptide is 13 to25 amino acids long.

[0183] 16. The nanoparticle of any one of embodiments 1 to 14, wherein the peptide comprises an MHC-I epitope.

[0184] 17. The nanoparticle of any one of embodiments 1 to 13 or 15, wherein the peptide comprises an MHC-II epitope.

[0185] 18. The nanoparticle of embodiment 1, wherein the peptide comprises the amino acid sequence of SEQ ID NOs: 17-47.

[0186] 19. The nanoparticle of embodiment 8, wherein the peptide comprises the amino acid sequence of SEQ ID NOs: 10-15.

[0187] 20. The nanoparticle of any one of embodiments 1 to 19, wherein the nanoparticle comprises a linker at the N-terminus of the peptide.

[0188] 21. The nanoparticle of any one of embodiments 1 to 20, wherein the nanoparticle has a hydrodynamic diameter (z-average) in the range of 10 to 100 nm, as determined by Dynamic Light Scattering (DLS).

[0189] 22. The nanoparticle of any one of embodiments 1 to 21, wherein the nanoparticle has a zeta potential between about -20 and -50 mV, as measured at pH 6 to pH 7.

[0190] 23. The nanoparticle of any one of embodiments 1 to 22, wherein the nanoparticle provided herein has a hydrodynamic diameter (z-average) between 10 and 100 nm, as measured by DLS.

[0191] 24. A pharmaceutical composition comprising the nanoparticle of any one of embodiments 1 to 23, and a pharmaceutically acceptable carrier.

[0192] 25. A kit comprising: the nanoparticle of any one of embodiments 1 to 23, and anAAV particle comprising the AAV capsid and / or the transgene.

[0193] 26. A method of inducing tolerance to a polypeptide encoded by an AAV-vectored transgene in a subject in need thereof, wherein the method comprises administering the nanoparticle of any one of embodiments 1 to 23 to the subject.

[0194] 27. A method of inducing tolerance of CD4+ T cells to a polypeptide encoded by anAAV-vectored transgene in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope.

[0195] 28. A method of inducing tolerance of CD4+ T cells to a polypeptide encoded by anAAV-vectored transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0196] 29. A method of inducing tolerance of CD8+ T cells to a polypeptide encoded by anAAV-vectored transgene in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope.

[0197] 30. A method of inducing tolerance of CD8+ T cells to a polypeptide encoded by anAAV-vectored transgene in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene,wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0198] 31. The method of any one of embodiments 27 to 30, wherein the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene.

[0199] 32. The method of any one of embodiments 27 to 30, wherein the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene.

[0200] 33. The method of any one of embodiments 27 to 30, wherein the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene.

[0201] 34. The method of any one of embodiments 27 to 30, wherein the transgene is humanF8.

[0202] 35. A method of inducing tolerance of CD4+ T cells to a polypeptide encoded by anA AV- vectored transgene in a subject in need thereof, wherein the AAV vector comprises an AAV capsid, and wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0203] 36. A method of inducing tolerance of CD4+ T cells to a polypeptide encoded by anA AV- vectored transgene in a subject in need thereof, wherein the AAV vector comprises an AAV capsid, and wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope.

[0204] 37. A method of inducing tolerance of CD8+ T cells to a polypeptide encoded by anA AV- vectored transgene in a subject in need thereof, wherein the AAV vector comprises an AAV capsid, and wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0205] 38. A method of inducing tolerance of CD8+ T cells to a polypeptide encoded by anA AV- vectored transgene in a subject in need thereof, wherein the AAV vector comprises an AAV capsid, and wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope.

[0206] 39. The method of any one of embodiments 35 to 38, wherein the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid.

[0207] 40. The method of any one of embodiments 35 to 38, wherein the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid.

[0208] 41. The method of any one of embodiments 35 to 38, wherein the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid.

[0209] 42. The method of any one of embodiments 35 to 41, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV rhlO, a synthetic AAV, combinations, engineered, and modified versions thereof.

[0210] 43. The method of embodiment 42, wherein the AAV capsid is AAV1 or AAV8.

[0211] 44. The method of any one of embodiments 35 to 38, wherein the peptide comprises the amino acid sequence of SEQ ID NOs: SEQ ID NOs: 10-15.

[0212] 45. A method of inducing tolerance to an AAV particle in a subject in need thereof, wherein the method comprises administering to the subject the nanoparticle of any one of embodiments 1 to 23.

[0213] 46. A method of inducing tolerance of CD4+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope.

[0214] 47. A method of inducing tolerance of CD4+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, whereinthe method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0215] 48. A method of inducing tolerance of CD8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope.

[0216] 49. A method of inducing tolerance of CD8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0217] 50. The method of any one of embodiments 46 to 49, wherein the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid.

[0218] 51. The method of any one of embodiments 46 to 49, wherein the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid.

[0219] 52. The method of any one of embodiments 46 to 49, wherein the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid.

[0220] 53. The method of any one of embodiments 46 to 49, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV rhlO, a synthetic AAV, combinations, engineered, and modified versions thereof.

[0221] 54. The method of embodiment 53, wherein the AAV capsid is AAV1 or AAV8.

[0222] 55. The method of any one of embodiments 46 to 49, wherein the peptide comprises the amino acid sequence of SEQ ID NOs:10-18.

[0223] 56. A method of inducing tolerance of CD4+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope.

[0224] 57. A method of inducing tolerance of CD4+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene,wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0225] 58. A method of inducing tolerance of CD8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I epitope.

[0226] 59. A method of inducing tolerance of CD8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-II epitope.

[0227] 60. The method of any one of embodiments 56 to 59, wherein the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene.

[0228] 61. The method of any one of embodiments 56 to 59, wherein the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene.

[0229] 62. The method of any one of embodiments 56 to 59, wherein the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene.

[0230] 63. The method of any one of embodiments 56 to 62, wherein the transgene is humanF8.

[0231] 64. The method of any one of embodiments 26 to 63, wherein tolerance is maintained for at least 30 days after administration of the AAV particle.

[0232] 65. The method of any one of embodiments 26 to 63, wherein tolerance is maintained for at least 50 days after administration of the AAV particle.

[0233] 66. The method of any one of embodiments 26 to 63, wherein tolerance is maintained for at least 60 days after administration of the AAV particle.

[0234] 67. The method of any one of embodiments 26 to 66, wherein tolerance is determined by IFN^ ELISPOT.

[0235] 68. The method of any one of embodiments 26 to 67, further comprises administering the AAV vector to the subject.

[0236] 69. A method of reducing a humoral immune response to a polypeptide encoded by a transgene in a subject in need thereof, wherein the method comprises:(a) administering a nanoparticle to the subject; and(b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene, wherein the nanoparticle comprises:(i) an amphiphilic polymer; and(ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the humoral immune response is reduced relative to a method comprising step (b) but not step (a).

[0237] 70. A method of reducing a humoral immune response to an AAV particle in a subject in need thereof, wherein the method comprises:(a) administering a nanoparticle to the subject; and(b) administering the AAV particle to the subject, wherein the AAV particle comprises an AAV capsid, wherein the nanoparticle comprises:(i) an amphiphilic polymer; and(ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, wherein the humoral immune response is reduced relative to a method comprising step (b) but not step (a).

[0238] 71. The method of embodiment 69 or 70, wherein the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the AAV capsid polypeptide.

[0239] 72. The method of embodiment 69 or 70, wherein the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the AAV capsid polypeptide.

[0240] 73. The method of embodiment 69 or 70, wherein the peptide comprises an amino acid sequence 100% identical to the fragment of the AAV capsid polypeptide.

[0241] 74. The method of any one of embodiments 69 to 73, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, a synthetic AAV, combinations, engineered, and modified versions thereof.

[0242] 75. The method of embodiment 74, wherein the AAV capsid is AAV1 or AAV8.

[0243] 76. A method of reducing a humoral immune response to a polypeptide encoded by a transgene in a subject in need thereof, wherein the method comprises:(a) administering a nanoparticle to the subject; and(b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene,wherein the nanoparticle comprises:(i) an amphiphilic polymer; and(ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the humoral immune response is reduced relative to a method comprising step (b) but not step (a).

[0244] 77. A method of reducing a humoral immune response to an AAV particle in a subject in need thereof, wherein the method comprises:(a) administering a nanoparticle to the subject; and(b) administering the AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the nanoparticle comprises:(i) an amphiphilic polymer; and(ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the humoral immune response is reduced relative to a method comprising step (b) but not step (a).

[0245] 78. The method of embodiment 76 or 77, wherein the peptide comprises an amino acid sequence at least about 95% identical to the fragment of the polypeptide encoded by the transgene.

[0246] 79. The method of embodiment 76 or 77, wherein the peptide comprises an amino acid sequence at least about 99% identical to the fragment of the polypeptide encoded by the transgene.

[0247] 80. The method of embodiment 76 or 77, wherein the peptide comprises an amino acid sequence 100% identical to the fragment of the polypeptide encoded by the transgene.

[0248] 81. The method of any one of embodiments 76 to 80, wherein the transgene is humanF8.

[0249] 82. The method of embodiment 69 or 76, wherein the humoral immune response is measured by detecting absolute units of an anti-transgene IgG titer.

[0250] 83. The method of embodiment 70 or 77, wherein the humoral immune response is measured by detecting absolute units of an anti-AAV capsid IgG titer.

[0251] 84. The method of any one of embodiments 69 to 83, wherein the humoral immune response is reduced by more than about 60%, relative to a method comprising step (b) but not step (a).

[0252] 85. The method of any one of embodiments 69 to 83, wherein the humoral immune response is reduced by more than about 70%, relative to a method comprising step (b) but not step (a).

[0253] 86. The method of any one of embodiments 69 to 83, wherein the humoral immune response is reduced by more than about 80%, relative to a method comprising step (b) but not step (a).

[0254] 87. The method of any one of embodiments 69 to 83, wherein the humoral immune response is reduced by more than about 90%, relative to a method comprising step (b) but not step (a).

[0255] 88. The method of any one of embodiments 69 to 87, wherein the method further comprises administering Temsirolimus.

[0256] 89. A method of increasing and / or prolonging expression of a transgene in a subject in need thereof, wherein the method comprises:(a) administering a nanoparticle to the subject; and(b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene, wherein the nanoparticle comprises:(i) an amphiphilic polymer; and(ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene,wherein the expression is increased and / or prolonged relative to a method comprising step (b), but not step (a).

[0257] 90. The method of embodiment 89, wherein expression of the transgene is increased relative to a method comprising step (b), but not step (a).

[0258] 91. The method of embodiment 89, wherein the expression of the transgene is increased by about 2-fold, relative to a method comprising step (b), but not step (a).

[0259] 92. The method of embodiment 89, wherein the expression of the transgene is increased by about 4-fold, relative to a method comprising step (b), but not step (a).

[0260] 93. The method of embodiment 89, wherein the expression of the transgene is increased by more than 5-fold, relative to a method comprising step (b), but not step (a).

[0261] 94. The method of any one of embodiments 89 to 93, wherein the expression of the transgene is prolonged for more than 30 days after administering the AAV particle.

[0262] 95. The method of any one of embodiments 89 to 93, wherein the expression of the transgene is prolonged for more than 40 days after administering the AAV particle.

[0263] 96. The method of any one of embodiments 89 to 93, wherein the expression of the transgene is prolonged for more than 50 days after administering the AAV particle.

[0264] 97. The method of any one of embodiments 89 to 93, wherein the expression of the transgene is prolonged for more than 60 days after administering the AAV particle.

[0265] 98. The method of any one of embodiments 26 to 97, wherein administering the nanoparticle particle is performed intravenous.

[0266] 99. The method of any one of embodiments 26 to 98, wherein administering the AAV particle is performed intravenous or intramuscular.

[0267] 100. The method of any one of embodiments 68 to 99, wherein the nanoparticle is administered at least once prior to administration of the AAV particle.

[0268] 101. The method of embodiment 100, wherein the nanoparticle is administered one day prior to administration of the AAV particle.

[0269] 102. The method of any one of embodiments 68 to 99, wherein the nanoparticle is administered at least twice prior to administration of the AAV particle.

[0270] 103. The method of embodiment 102, wherein the nanoparticle is administered five days prior and one day prior to administration of the AAV particle.

[0271] 104. The method of any one of embodiments 68 to 103, wherein the nanoparticle is administered at least once after administration of the AAV particle.

[0272] 105. The method of any one of embodiments 68 to 103, wherein the nanoparticle is administered at least twice after administration of the AAV particle.

[0273] 106. Use of the nanoparticle of any one of embodiments 1 to 23 in the manufacture of a medicament for reducing an immune response to the AAV capsid and / or the transgene.

[0274] 107. A nanoparticle of any one of embodiments 1 to 23 or the pharmaceutical composition of embodiment 24 for use in reducing an immune response to the AAV capsid and / or the transgene.7. EXAMPLES

[0275] The examples in this section are offered by way of illustration, and not by way of limitation. The following examples are presented as exemplary embodiments of the invention. They should not be construed as limiting the scope of the invention.Example 1: Ova-A Vl tolerization by nanoparticles administration

[0276] This example demonstrates the therapeutic potential of nanoparticles-induced tolerization in AAV-gene therapy settings. Nanoparticles combined with Ovalbumin (Ova) peptides were used to evaluate their tolerizing effect in mice transduced with AAVl-sOva vector. Ova-specific CD8 and CD4 T cells and anti-Ova IgG antibodies abundance were evaluated, as well as persistence of Ova transgene expression. Multiple injection regimen and concentration of the nanoparticles were tested to determine optimal conditions for further exploration.

[0277] Briefly, the Ovalbumin transgene was cloned into AAV 1 serotype adenovirus vector. Nanoparticles comprising MHC-I Ova257-264 peptide (Ova-I Nanoparticles), MHC-II Ova323-339 peptide (Ova- II Nanoparticles), or a combination of both (Ova-l / Ova-II Nanoparticles). Micewere injected with AAVl-sOva (AVV vector coding for a secreted form of Ovalbumin) at day 0 intramuscularly (i.m.), and two or three times with Ova-I Nanoparticles, Ova-II Nanoparticles, or Ova-I / Ova-II Nanoparticles at days -1, 7 and / or 13 intravenously (i.v.). The nanoparticles contain between 7.5 and 30 nmol / 150 pl injection volume.

[0278] For evaluating the dose response of Ova Nanoparticles, concentrations ranging from 2 to 28 nmol / mouse were used. Tolerizing effects of nanoparticles loaded with Ova-I, Ova-II or both peptides versus controls were evaluated by flow cytometry to determine the frequency of Ova-specific CD8+and CD4+T cells, by measuring anti-Ova IgG antibody titers, and by qRT- PCR to determine Ova mRNA expression. Frequency of Ova-specific CD8+T cells in the blood was determined using H-2Kb / Ova-I dextramers staining (gated Ova-specific CD8+T cells).

[0279] In the first study, nanoparticles were injected twice (days -1 and 7) i.v. and AAV1- sOva was injected i.m. at day 0. In the group treated with Ova-I nanoparticles, a significant inhibition of both Ova-specific CD8+T cells as well as anti-Ova IgG antibodies was observed. Reemergence of immune responses was evidenced at the late timepoints of the study suggesting partial loss of tolerance, but immune responses remained overall lower compared to control groups. However, nanoparticles coupled with Ova II peptide alone did not result in these tolerization effects. The inhibition of immune responses in the groups that have received Ova-I nanoparticles correlated with a better persistence of Ova expression in transduced muscles, as measured by the abundance of Ova mRNA 90 days after AAVl-sOva injection.

[0280] For the second study based on the same settings and groups, a third injection of nanoparticles was added (days -1, 7 and 13) with the aim to increase tolerization effects and to prevent later reemergence of immune responses. The efficiency of Ova-I nanoparticles to inhibit Ova-specific CD8+T responses was confirmed in the early timepoints but did not persist at late timepoints, suggesting that addition of the third administration of nanoparticles at day 13 was not enough to prevent late reemergence of the immune response. Inhibition of immune responses was not evidenced in the groups injected with Ova-II nanoparticles.

[0281] The next study aimed to establish a dose response using Ova-I nanoparticles (from 2 to 28 nmol / mouse) in the same settings as the first study. All the doses tested inhibited Ova- specific CD8+T cells at the early timepoints of the kinetic (at days 14 and 21), but only the highest dose (28 nmol / mouse) significantly inhibited Ova-specific CD8+T cells at the lastanalyzed timepoint (day 57). Amelioration of the persistence of transgene expression was noticeable with the two highest doses (14 and 28 nmol / mouse).

[0282] In the group treated with Ova-I nanoparticles, the significant inhibition of both Ova- specific CD8+T cells as well as anti-Ova IgG antibodies suggest a direct tolerization towards the Ova-specific CD8+T cells and cross-tolerization mechanisms towards the Ova-specific CD4+T cell compartment, thereby inhibiting CD4+T cell dependent B cell responses, resulting in lower anti-Ova IgG levels. Immune response reemerged in the later timepoints, suggesting partial loss of tolerance which was not improved by an additional nanoparticles administration. The dose response study revealed that only the highest dose (28 nmol / mouse) significantly inhibited Ova- specific CD8+ T cells until the last analyzed timepoint (day 57) and amelioration of the persistence of transgene expression was noticeable with the two highest doses (14 and 28 nmol / mouse).

[0283] On the other hand, nanoparticles coupled with Ova-II peptide alone did not result in these tolerization effects. Quality controls (QC) of Ova-II nanoparticles could not rule out inefficient coupling and / or nanoparticles aggregation and suggested that lower peptide dose (7 nmol vs 14 nmol) may explain poor efficiency of Ova-II nanoparticles to inhibit immune responses.

[0284] Overall, these results demonstrated that Ova-I nanoparticles administration at doses greater than or equal to 14 nmol / mouse could efficiently inhibit cellular and humoral immune responses against Ovalbumin transgene. Furthermore, in each study, the immune response inhibition correlated with a better persistence of Ova expression, suggesting therapeutic potential of nanoparticles-induced tolerization in the context of AAV-gene therapy.EXAMPLE 2: Ova-AAV8 tolerization by nanoparticles administration

[0285] This example demonstrates that nanoparticles also inhibit cellular immune response in the context of muscular AAV8 gene therapy. Specifically, the following study aimed to evaluate another AAV serotype (different than AAV1). As one of the vectors of choice used in clinical trials in liver gene therapy, AAV8 was selected. The same strategy as in Example 1 were used to evaluate tolerization. Additionally, CD8 and CD4 T cells were tested for Ova-specific immune response and Ova-specific CD8 functionality was evaluated. Other objectives of thefollowing studies were to extend and maintain tolerance in the late phase of the kinetic by increasing the number nanoparticles injections, and to increase Ova-I nanoparticles efficiency with the co-administration of other nanoparticles loaded with MHC-II restricted Ova- II peptide, and / or MHC-I and MHC-II restricted AAV8 peptides.

[0286] Briefly, Ovalbumin trans gene was cloned into AAV8 serotype adenovirus vector. Nanoparticles were combined with MHC-I Ova257-264 peptide (Ova-I nanoparticles), MHC-II Ova323-339 peptide (Ova-II nanoparticles), a combination of both (Ova-I / Ova-II nanoparticles), MHC-I Ova257-264 / MHC-I AAV epitope ML8 (MIPQYGYL, (SEQ ID NO:10)) / MHC-I AAV epitope PL8 (PQYGYLTL (SEQ ID NO: 11)) peptides (Ova-I / PL8 / ML8 nanoparticles), or a combination of Ova-I, Ova-II, PL8 and MHC-II AAV epitope RK15 (RNSLANPGIAMATHK (SEQ ID NO: 13)) peptides (Ova-I / Ova-II / PL8 / RK15 nanoparticles) in comparison to nanoparticles without peptides (TP).

[0287] Mice were injected with AAV8-sOva (AAV vector coding for a secreted form of Ovalbumin) at day 0 intramuscularly (i.m.), and two or five times with Ova-I nanoparticles, Ova- I / Ova-II nanoparticles, Ova-I / PL8 / ML8 nanoparticles, or Ova-I / PL8 / RK15 nanoparticles at days -1 and 7 or at days -1, 3, 7, 12 and 15 intravenously (i.v.) at a concentration of 14 and / or 28 nmole / mouse. Tolerizing effects of nanoparticles was evaluated as in Example 1. Additionally, CD8 and CD4 T cells were evaluated for Ova-specific immunity upon stimulation with MHC-I and MHC-II restricted Ova-epitopes and for AAV -specific immunity upon stimulation with MHC-I and MHC-II restricted AAV-epitope using ELISpot assays. Phenotypic evaluation of Ova-specific CD8 T cells was performed by interrogating their expression of functional and regulatory markers by flow cytometry (PD1, CXC3CR, KLRG-1, CD45RB, CD73, and FR4).

[0288] The frequency of Ova-specific CD8+T cells in the blood was determined using H- 2Kb / Ova-I dextramers staining (gated Ova-specific CD8+T cells).

[0289] Splenocytes were collected at 67 p.i. and analyzed by ex vivo enzyme-linked immunospot (ELISpot) assays for their capability to secrete IFNy after restimulation with three MHC-I-restricted epitopes (ML8: MIPQYGYL (SEQ ID NO: 10); PL8: PQYGYLTL (SEQ ID NO:11); FV8-8: FTYTFEDV (SEQ ID NO:12)), three MHC-II-restricted epitopes (EL15: EDVPFHSSYAHSQSL (SEQ ID NO:15); RK15: RNSLANPGIAMATHK (SEQ ID NO:13); QN15: QTLGFSQGGPNTMAN (SEQ ID NO: 14)), and Ova-I peptide.

[0290] In the first study, the AAV8-sOva vector was administered according to the regimen described in Example 1 for AAVl-sOva. Ova-I nanoparticles doses of 28 or 14 nmol / mice was administrated twice (days -1 and 7). Results showed that i.m. injection of AAV8-sOva was able to induce a robust immune response against Ova. Groups treated with Ova-I nanoparticles displayed significantly lower cellular Ova-specific CD8+T cells immune responses, at the early time-points. As in EXAMPLE 1, reemergence of the immune responses was noticeable in the late phase of the kinetic. This was associated in the end of the study, at day 62 post AAV8-sOva injection, to a non-significant tendency in the group treated with 14 nmol / mouse Ova-I nanoparticles to improve transgene persistence in transduced muscles.

[0291] In parallel, this study also allowed to evaluate novel A A V8 -derived-epitopes that were predicted in silico. Three MHC-I-restricted epitopes (ML8, PL8 and FV8-8) and three MHC-II-restricted epitopes (EL15, RK15, and QN15) were evaluated using ELISpot assays. The analysis revealed that PL8 and RK15 were appropriate to detect AAV8-specific cellular CD8+and CD4+T cells immune responses. Analysis of the anti-AAV8 immune responses in the different groups demonstrated that Ova-I nanoparticles treatment significantly inhibited anti- AAV8 cellular immune response.

[0292] In the next study, groups of 4 mice were treated with Ova-I nanoparticles, Ova-I / Ova- II nanoparticles, Ova-PPL8 / ML8 nanoparticles, or with Ova-I / Ova-II / PL8 / RK 15 nanoparticles, respectively, and immune responses against Ova and AAV8 capsid were followed upon i.m. injection of AAV8-sOVA. Remarkably, Ova-specific CD8+T cell responses were significantly inhibited in all the timepoints of the kinetic, including in the late phase suggesting that tolerance can be extended by multiple nanoparticles injections (FIG. 1, FIG. 2A-D).

[0293] Phenotypic evaluation of the few detectable Ova-specific CD8+T cells demonstrated their lower functionality (as assessed by lower expression of KLRG-1 and CX3CR1), their lower affinity to cognate antigen (as assessed by higher expression of CD45RB), and their expression of regulatory markers (e.g., CD73 and FR4 expression) suggesting their conversion to anergic or regulatory cells (FIG. 2E-I, FIG. 2K-M), and their inability to secrete IFNy upon re-stimulation in vitro in ELISpots assays (FIG. 2J).

[0294] Anti-Ova IgG responses were inhibited up to 38 days post AAV8-sOva injection despite the continuous endogenous production of secreted immunogenic Ova protein in theserum of these animals. Importantly, nanoparticles treatment also significantly inhibited CD4+and CD8+T cell responses directed against AAV8 capsid (FIG. 6A-B).

[0295] Analysis of transgene expression by qRT-PCR in transduced muscles indeed confirmed better transgene expression in groups treated with Ova-I / Ova-II nanoparticles (FIG. 4A, FIG. 4C).

[0296] Overall, these results confirmed the efficacy of Ova-I nanoparticles to inhibit cellular immune response in the context of muscular AAV8 gene therapy, extending the results obtained with AAV1 vector from Example 1, and identified immune-dominant AAV8-derived epitopes that could be coupled to nanoparticles in future studies (ML8 and RK15).

[0297] Taken together, these data demonstrate the capacity of multiple nanoparticles treatment to improve tolerization over a longer time period in the context of AAV-mediated gene transfer despite the use of a very immunogenic Ova-antigen in this exemplary model.

[0298] Furthermore, analysis of the anti-AAV8 immune responses in the different groups demonstrated that Ova-I nanoparticles treatment significantly inhibited anti-AAV8 cellular immune response suggesting the possibility to cross-tolerize towards unrelated-epitopes presented by the same cells (e.g., transduced cells display Ova-derived epitopes as well as AAV8-derived epitopes).EXAMPLE 3: Early timepoint kinetics of Ova- V8 tolerization by nanoparticles administration

[0299] This example elucidates the immunomodulatory mechanisms underlying the significant tolerization effects induced by nanoparticles. In the following studies, the objective was to assess potential immunomodulatory effects at an early phase of the study, focusing on T cell changes that might be detected at the early priming phase and / or tolerization events induced by nanoparticle treatment (up to day 12 post AAV8-CMV-sOva injection). Further study also included later timepoints for mechanistic analysis, i.e.. monitoring the immune responses at days 8, 12, 16 and 21 p.i. to better cover the emergence and evolution of the immune response beyond day 12.

[0300] Briefly, mice were injected with AAV8-CMV-sOva at day 0 intramuscularly (i.m.), and three times with Ova-I / Ova-II nanoparticles at days -5, -1 and 5 intravenously (i.v.) at aconcentration of 14 or 28 nmol / mouse. Tolerizing effects of Ova-l / Ova-II nanoparticles versus controls were evaluated at days 3, 6, 9 and 12 or at days 8, 12, 16 and 21 p.i. using the same method as described in the previous Examples.

[0301] In the first study, Ova-I- / Ova-II-nanoparticles and empty nanoparticles were i.v. injected at days -5, -1 and 5, and liver and spleen from mice were collected at different early timepoints (i.e., 3, 6, 9 and 12 days post i.v. AAV8-CMV-sOva injection). FACS-Analysis and ELISA results show that both CD8+T cellular and also humoral immune responses against Ova transgene were first detectable at day 12 post- AAV injection, not at earlier timepoints. While Ova-specific CD8+T cells were clearly visible in the liver of the control group receiving empty nanoparticles at day 12, these cells were barely detectable in the spleen. Ova-specific CD8+T cells in the liver were markedly reduced in the group that had received Ova-L / Ova-II- nanoparticles in comparison to the group treated with empty nanoparticles (FIG. 3A), but did not reach statistical significance.

[0302] The next study was designed in a similar way but included later timepoints for mechanistic analysis (days 8, 12, 16 and 21 p.i.). In line with the first study, in the empty nanoparticles control group Ova-specific CD8+T cells were first detected in the liver at day 12 post-AAV injection and their frequency increased rapidly to reach almost 60% of the CD8+cells. In contrast, throughout the study until day 21 the frequency of these cells in the liver remained modest and significantly lower in the mice treated with Ova-I / Ova-II-nanoparticles (FIG 3B).

[0303] In the spleen, the emergence of Ova-specific CD8+T cells in the empty nanoparticles control group also started from day 12, and further increased overtime to reach 5 % of the CD8+pool at day 21. In contrast, Ova-specific CD8+T cells were barely detectable in the spleen of mice treated with Ova-I / Ova-II-nanoparticles (FIG. 3C).

[0304] Anti-Ova IgG antibodies were also first detected at day 12 post-AAV injection. The titers remained lower in the mice treated with Ova-EOva-II-nanoparticles compared with those treated with empty nanoparticle, albeit not statistically significant.

[0305] Summarizing these results, i.v. injection of AAV8-CMV-sOva induced a prominent Ova-specific CD8+T cell response in the liver of control mice but not in the liver of nanoparticle-tolerized mice suggesting an active control of the activation, proliferation and / or recruitment of the specific cytotoxic CD8+T cells in the vicinity of the target organ. Tolerizationwas also demonstrable in the peripheral circulation as assessed by the very low frequency of Ova-specific CD8+T cells in the spleen of these mice throughout the study course. The lack of statistical significance in some of the timepoints is believed to be related to the low number of animals per group (n=4). These data again suggest that tolerization of transgene-specific CD8+T cells is actively induced in the liver over a period of at least 21 days and may possibly rely on the induction of cellular deletion, anergy or locally induced regulatory CD4+T cells. At the early timepoints, the nanoparticle’s tolerizing effects on Ova-specific CD8+T cells were overall less noticeable and not significantly different from the control mice, which suggests that clonal deletion of antigen-specific CD8+T cells by nanoparticles application may be one mechanism of tolerization.EXAMPLE 4: Tolerization upon liver-specific Ova-AAV8 transduction

[0306] This example demonstrates that immune responses can be difficult to detect when using a liver-specific promoter at low subthreshold vector doses. Liver gene transfer, when using a liver-specific promoter, is well documented to be tolerogenic, and generally, low doses of vector induce immune responses, whereas higher doses induce tolerance. Therefore, in the following studies, liver gene transfer using AAV8 vectors was evaluated when using Ova expression under the control of either ubiquitous or liver-specific promoters.

[0307] AAV8 vectors were generated containing either the CMV ubiquitous promoter or the liver restricted ApoE / hAAT promoter (AAV8-ApoE / hAAT-sOVA, AAV8-ApoE / hAAT- cytoOVA, AAV8-ApoE / hAAT-cytoOVA-2WlS and AAV8-CMV-sOVA). AAV8-CMV-sOva was used at a dose of 1010vg / mouse to elicit expression of secreted Ova under an ubiquitous promoter, while AAV8-ApoE / hAAT-cOva at a dose of 109vg / mouse to elicit expression of cytoplasmic Ova in hepatocytes. In both studies, groups of mice were treated with Ova-POva-II nanoparticles, PL8 / ML8 nanoparticles, or with Ova-I / Ova-II / PL8 / RK15-coupled nanoparticles, and compared to the control group treated with empty nanoparticles. Mice were treated with nanoparticles five times (days -1, 3, 7, 12 and 25) and the selected AAV8 vectors were injected intravenously at day 0. Tolerizing effects of nanoparticles versus controls were evaluated using the same method as described in previous Examples, infra.

[0308] In mice receiving AAV8-CMV-sOva at a dose of 1010vg / mouse, treatments with Ova-I / Ova-II nanoparticles and with Ova-I / Ova-II / PL8 / RK15 nanoparticles were able tosignificantly inhibit CD8+T cell-mediated immune responses against Ova as well as CD4+T cells against AAV8 capsid. As in the previous examples, we observed the reappearance of Ova- specific T cells in tolerized groups at later timepoints that displayed an anergic / regulatory phenotype (PD-lhlgh, CD73+FR4+) (FIG. 2K-M). Anti-Ova IgG humoral immune responses remained low at this dose of vector and were not found to differ significantly between groups. In mice receiving AAV8-ApoE / hAAT-cOva at a dose of 109vg / mouse, we did not observe any detectable immune responses in treated and control groups from blood and spleen samples.

[0309] These results highlighted challenges to inducing immune responses when using liver- restricted promoter at low sub threshold vector doses.EXAMPLE 5: Adoptive T cells transfer

[0310] This example demonstrates that the nanoparticles not only have a direct tolerization effect but also a bystander tolerization potential. Briefly, to further evaluate the mechanisms involved in nanoparticle-induced and sustained tolerization, two adoptive transfer studies aimed to specifically track Ova-antigen-specific T effector cells during tolerization. In the following studies, the transgenic T cells were characterized by the congenic marker CD45.1, which allows to track their cell fate and to discriminate them from the endogenous CD45.2 cells of the recipient mice.

[0311] In this study, mice were i.v. injected with either a mixture of Ova-L / Ova-II- coupled nanoparticles or empty control nanoparticles at days -5, -1 and 5, 10 and 18, and liver and spleen were collected at different timepoints post i.v. injection of AAV8-CMV-sOva performed at day 0. To monitor the fate of the CD4+and CD8+antigen- specific T cells during the tolerization protocol in vivo, 5xl04Ova-specific CD4+(OT-II) and 5xl04CD8+(OT-I) T cells were transferred at day -6. Liver and spleen were collected and the immune responses were monitored using flow cytometry analysis at different timepoints (at days 6, 8, 12, 16 and 20) using the same methods described in previous Examples, infra.

[0312] Transferred OT-I cells could be detected in the liver of the animal starting from day 12 post AAV injection. In the control group treated with empty nanoparticles, the frequency of these OT-I TCR transgenic cells increased dramatically to reach about 20 % of the CD8+repertoire by day 20. In contrast, in the tolerized group treated with Ova-I / Ova-II-nanoparticles,these cells were markedly suppressed and remained at a significantly lower frequency (at least 10-times lower than the control group) (FIG. 3D). The overall frequency of transgenic OT-II cells in the liver remained very low in all groups, in agreement with the lower affinity of their TCR to their cognate MHC-class II restricted antigen. Of note, these cells were not detected in the liver, nor in the spleen of the animals. Importantly, the evaluation of the frequency of all Ova-specific CD8+cells (i.e., combining adoptively transferred OT-I TCR-transgenic as well as endogenously expanded Ova-specific CD8+T cells) revealed a significant reduction of these cells in the group treated with Ova-I- / Ova-II-nanoparticle compared to empty nanoparticle from day 16 onwards until the end of the study (FIG. 3E).

[0313] Although the frequency of total Tregs was not upregulated in the Ova-l / Ova-II- nanoparticle group compared to the empty nanoparticle group, the frequency of activated CD25+Tregs and the expression of Ki67 on Tregs (as a marker indicating proliferation) were both significantly higher in the group treated with Ova-I / Ova-II-nanoparticle. This suggests a stronger activation of the CD4+endogenous Treg cells in the tolerized group. This effect was observed at day 16 post AAV-inj ection, but was no longer visible at day 20 (FIG. 5D-E).

[0314] Considering the significant tolerization effects on adoptively transferred OT-I cells on day 16 and 20, we aimed to further assess whether those effects might be detectable already at earlier timepoints. The next study was performed under the same condition, however, following i.v. AAV8-CMV-sOva injection at day 0, liver and spleen were collected and the immune responses were monitored at different timepoints (at days -4, 0, 2 and 6).

[0315] The results showed that early timepoints (day -4 until day 6) after transfer of OT-I and OT-II TCR-transgenic cells and following three administrations of nanoparticles, a very low frequency of transferred cells could be detected and no clear indication of deletion of transferred cells in the tolerized group could be observed in the liver or in the spleen. A small increase of total Treg population in the liver of nanoparticle-treated mice was detected at day 2 (i.e., 7 days after first nanoparticle application). Additionally, a significant increase of PD-1 and TIM-3 double positive population among Ova-specific CD8+T cells was observed in the liver of nanoparticle-treated animals at day 6 (i.e., 12 days after the first nanoparticle application), suggesting that nanoparticle treatment correlates with the occurrence of immunoregulatory markers known to be associated with anergic phenotypes.

[0316] The results of these adoptive transfer studies showed successful monitoring of the fate of the CD4+and CD8+antigen- specific T cells in tolerized mice. In the tolerized group, OT-I TCR-transgenic CD8+cells were markedly suppressed and remained at a significantly lower frequency compared to endogenous T cells. Overall, a similar inhibition of CD8 T cells activation was observed as described in the Example, infra. Furthermore, these results have also indicated a stronger activation of the CD4+endogenous Treg cells in the tolerized group and enhanced occurrence of immunoregulatory markers known to be associated with anergic phenotypes in CD8 T cells, supporting the observation that nanoparticles not only have a direct tolerization effect but also a bystander tolerization potential.EXAMPLE 6: AAV vector redosing

[0317] This example demonstrates that nanoparticles allow for AAV redosing. Briefly, the following studies were designed to evaluate efficacy of nanoparticles to allow vector redosing, which remains an unmet challenge in AAV-gene therapy.

[0318] In a first study, AAV8-ApoE / hAAT-cOva was injected first i.v. at a dose of 1010vg / mouse to elicit antibody formation, followed 21 days later with the i.m. administration of 3xl010vg / mouse of AAV8-CMV-sOva. This study was designed to evaluate the potential of nanoparticles to tolerize against the anti-capsid antibody response. In a second study, AAV8- CMV-sOva was injected first i.v. at a dose of 1010vg / mouse to elicit antibody formation as well as cellular response against transgene and against AAV8-capsid. This was followed 21 days later with the i.m. administration of 3xl010vg / mouse of the same AAV8-CMV-sOva vector. This study was designed to evaluate the potential of nanoparticles to tolerize against anti-capsid antibody as well as cellular response against transgene and AAV8-capsid. In both studies, groups of mice were treated five times with Ova-I / Ova-II nanoparticles, PL8 / ML8 nanoparticles, or with Ova-I / Ova-II / PL8 / RK15 nanoparticles and compared to the control group treated with empty nanoparticles. Tolerizing effects of nanoparticles versus controls were evaluated using the same methods as described in the Examples, infra.

[0319] No significant reduction of the level of anti-capsid IgG was observed in either study. However, the mice receiving two doses of the AAV8-CMV-sOva vector exhibited a significant reduction of cellular immune T cells responses (i.e. CD8+ T cell responses and notably also of CD4+ T cell responses) directed against AAV capsid epitopes and Ova-transgene epitopes in theELISpot from splenocytes collected at day 60 (40 days after the second AAV transduction), in the groups treated with Ova-l / Ova-II nanoparticles, PL8 / ML8 nanoparticles, and with Ova- I / Ova-Il / PL8 / RK15 nanoparticles (FIG. 6).

[0320] Although no significant reduction of anti-capsid IgG was observed, the significant reduction of cellular immune T cells responses against Ova and AAV capsid was shown in the group treated with Ova-I / Ova-II nanoparticles (transgene epitopes only), the group treated with PL8 / ML8 nanoparticles (viral epitopes only), as well as in the group treated with Ova-I / Ova- I1 / PL8 / RK15 nanoparticles (transgene and viral epitopes), demonstrating the promising potential for efficacious tolerization and cross-tolerization upon nanoparticle treatment that render AAV redosing possible.EXAMPLE 7: Ova-I and Oval / II-nanoparticle-mediated tolerization on CD4 T cells

[0321] This Example demonstrates that administration of nanoparticles coupled to Ova-I and mixtures of Ova-I and Ova-II resulted in a strong tolerization effect on both humoral and cellular responses against the transgene Ova, thereby resulting in a significantly increased persistence of transgene expression.

[0322] Briefly, four groups of mice (n=8) received five applications of either Ova-I nanoparticles, or Ova-II nanoparticles alone, or a mixture of Ova-I- / Ova-II nanoparticles. In the control group empty nanoparticles were injected at days -5, -1, 5, 10, and 20. All groups were injected at day 0 with IxlO10vg / mouse of AAV8-CMV-sOva using the i.v. route. Tolerizing effects of nanoparticles versus controls were evaluated using the same methods as described in previous Examples, infra.

[0323] Results showed a nearly complete inhibition of CD8+T cellular responses in the groups treated with nanoparticles coupled to Ova-I peptide or treated with nanoparticles coupled to Ova-I and Ova-II peptides, when evaluated at day 15, day 29, and day 62 using flow cytometry. Also, a significant inhibition of antigen-specific CD8+and CD4+T cell responses was demonstrated at the end of the study at day 62 when using peptide restimulation in vitro in ELISpots assays. The control group with empty nanoparticles displayed prominent levels of CD8+and CD4+cellular immune responses against Ova, while the control group receiving PBS instead of AAV vectors did not show any cellular response. Interestingly, beside tolerance effects against the transgenic protein, Ova-I nanoparticles and Ova-I / Ova-II nanoparticles alsoshowed a strong inhibition of AAV-specific T cell responses (tolerance against the vector). This effect is consistent with the results observed in other Examples, infra.

[0324] In summary, after transduction of a liver-targeting AAV8-Ova-vector a strong tolerization effect on both humoral and cellular responses against the transgene Ova resulting in a significantly increased persistence of transgene expression can be demonstrated and confirmed when using nanoparticles coupled to Ova-I and nanoparticle-mixtures of Ova-I and Ova- II.

[0325] Interestingly, beside tolerance effects against the transgenic protein, Ova-I nanoparticles and Ova-I / Ova-II nanoparticles also show a strong inhibition of AAV-specific T cell responses (tolerance against the vector). As the nanoparticles are not coupled to AAV- specific peptides, but contain Ova-specific peptides, this effect could only be explained by a strong bystander tolerization (or cross-tolerization). This may reflect the induction of a tolerogenic environment protecting the cells expressing the transgenic Ova-protein and displaying at the same time capsid-derived peptides (for instance through the induction of regulatory T cells, Tregs, recognizing Ova and / or Capsid derived peptides). In addition, this may also result from the lower release of capsid proteins in the extracellular milieu (due to the absence / tolerization of cytotoxic T cells) resulting in absence / lower presentation of capsid derived antigens by antigen presenting cells. This important result highlights and strengthens the notion that nanoparticle-mediated tolerization against the transgene-derived epitope can be propagated towards capsid antigens delivered by the same viral vector.EXAMPLE 8: Effect of Ova -AAV tolerization on anti-tumor immunization

[0326] This example demonstrates the antigen-specificity of the tolerance induced by nanoparticles treatment, and further demonstrate that nanoparticle-induced tolerization towards the target antigen does not hinder the immune responses directed against another third-party antigen.

[0327] Briefly, the efficacy of anti-tumor vaccination in mice otherwise tolerized to Ova- AAV was evaluated in a model in which mice were vaccinated in order to develop immunity against the Trp2 antigen expressed by the B16 melanoma cell line (Mansour et al., 2007). The B16F10 melanoma cell line was used for xenograft studies. For anti-tumor vaccination protocol mice received i.m. injections (gastrocnemius muscles) of 200 pg of delta-Trp2 peptide (derived from Trp2180-188 peptide, harboring a single mutation improving its affinity for H-2Kb MHC)together with 100 pg of poly(I:C), a TLR3 ligand as an adjuvant. Study was then conducted with groups of mice that were tolerized against Ova using Ova-I / Ova-II nanoparticles (days -5, -1, 5, 9 and 16), and i.v. injected with our AAV8-CMV-sOva vector (1010vg / mouse) at day 0. Some groups were also vaccinated against the Trp2-tumor antigen at days 7 and 14. Next, 5xl05B16F10 melanoma cancer cells were inoculated s.c. at day 21 to monitor tumor development and survival in the different groups (FIG. 8A). Successful Trp2 immunization was evaluated by flow cytometry measuring the frequency of Trp2-specific CD8+ T cells. Tolerizing effects of nanoparticles versus controls were evaluated using the same methods as described in previous Example, infra.

[0328] Analysis of immune responses by flow cytometry confirmed that the protocol of vaccination induced Trp2-specific CD8+T cells (FIG. 8B-D) and that tolerance towards Ova remained robust despite injections of vaccine / adjuvant in these mice (FIG. 8E-F). Monitoring of animals’ survival following tumor development confirmed a better survival in vaccinated mice, irrespective of their treatment with Ova-I / Ova-II nanoparticles, thereby demonstrating efficient third-party antigen vaccination in Ova-I / Ova-II nanoparticle-tolerized mice. However, a slightly lower number of Trp2-specific cells in mice tolerized with Ova-I / Ova-II nanoparticles was observed when compared with the mice injected with empty nanoparticles. The next study followed the same design with only minor technical improvements (i.e., lowering the volume used during the i.m. vaccination to avoid potential leakage of the injected Trp2 -peptides from the muscle and their capture by the liver, and using a B16 cancer cell line less aggressive than the B16F10).

[0329] The results this study confirmed and further improved what was observed in the previous one. Indeed, data showed efficient anti-tumor vaccination irrespective of treatment with Ova-I / Ova-II nanoparticles, comparable numbers of Trp2-specific T cells in mice tolerized with nanoparticles Ova-I / Ova-II compared with the mice injected with empty nanoparticles, improved mice survival, but also better control of tumor growth in the vaccinated mice (FIG. 8G-H).

[0330] These studies demonstrated antigen-specificity of the tolerance induction by nanoparticle-treatment and confirmed that nanoparticle-induced tolerization toward the target antigen (i.e., Ova in this model) does not hamper vaccination nor response against another third- party unrelated antigen (i.e., Trp2 tumor antigen in this model). Indeed, in the presence of Ova-directed immune tolerance, the animals being vaccinated against a tumor antigen at the same time could significantly control tumor growth. This result represents an important finding that further highlights the safety of nanoparticle-tolerization that is not expected to induce a general state of immunosuppression, but only a selective antigen-specific tolerization, exclusively directed against the unwanted immune response.EXAMPLE 9: Induced Tolerization of Humoral Responses by TPC and a combination of TPC and Temsirolimus

[0331] This example demonstrates that nanoparticles provided herein as well as coapplication of the nanoparticles provided herein and temsirolimus induce tolerization of humoral responses. Previous studies suggested that the humoral response characterized by formation of anti-Ova IgG and anti-AAV IgG was only partially inhibited by TPC treatment in the applied model and not at all evaluated timepoints. Therefore, TPC treatment was combined with a small molecule drug, Temsirolimus, to reinforce tolerization and to prevent antigen specific IgG response more significantly than TPC alone.

[0332] Temsirolimus is an analog of Rapamycin that has been reported to prevent humoral immune responses and to preserve regulatory T cells (Tregs). Briefly, Ova-I / Ova-II TPCs were repeatedly administered i.v. at days -5, -1, 5, 10 and 20 and combined with the application of Temsirolimus (0.4 mg / kg), injected i.p. every other day from days -2 to day 20, in comparison to groups tolerized with TPCs, but without co-application of Temsirolimus.

[0333] The results showed that in the group treated with Ova-l / Ova-II TPCs without Temsirolimus co-application, a significant inhibition of anti-Ova IgG response until day 40 was observed, which vanished over time. In contrast, co-application of Ova-I / Ova-II TPCs and Temsirolimus (at low dose of 0.4 mg / kg) significantly prevented both, the generation of anti- AAV IgG and anti-Ova IgG until the last evaluated timepoint at day 106 (FIG. 9A-FIG. 9D). Interestingly, at this late timepoint the cellular immune responses against Ova transgene and AAV capsid, measured by peptide restimulation and ELISpot assays were not inhibited by Temsirolimus without TPCs, and only the groups that were treated with the combination therapy (i.e., mixed Ova-l / Ova-II TPCs together with low dose Temsirolimus administered during the first 20 days of the protocol) demonstrated a significant inhibition of both the cellular immuneresponses (mediated mainly by mixed Oval / Ova-II TPCs) and the IgG antibody responses (greatly improved by Temsirolimus short-term treatment).

[0334] Hence, this example demonstrates that the strong efficacy of TPC treatment to inhibit both types of immune responses can be further complemented and leveraged by low dose and short-term treatment with the already approved and clinically used drug Temsirolimus. This important result further expands the potential portfolio of clinical situations where TPCs can be applied.EXAMPLE 10: FVIII-AAV8 tolerization by nanoparticles administration

[0335] This example demonstrates the therapeutic potential of nanoparticles-induced tolerization against AAV8-transduced FVIII peptide (F8).

[0336] Briefly, the same evaluation of immune response and tolerization were used as in the previous Examples, infra. Nanoparticles were combined with F8 I + F8 II CD8 peptides (Fl .2 and F2.1), F8 I + F8 II + F8 III CD4 peptides (Fl.3 and F2.2), or F8 I + F8 II CD8 and F8 I / II / III CD4 peptides (Fl.4 and F2.3 = 5 nanoparticle). Note: Empty nanoparticles control = Fl.l and F2.4.

[0337] F8 knock-out mice (n=10 / timepoint / group) were injected with AAV8-CMV-FVIII (3 x 1011vg / mouse) at day 0 i.m.. Nanoparticles coupled with peptides derived from F8 were repeatedly administered i.v. at days -5, -1, 5, 10 and 20. Tolerizing effects of nanoparticles versus controls were evaluated using the same methods as described in previous Examples, infra.

[0338] In the first study, tolerization was evaluated at days 14 and 21 post AAV8-CMV- FVIII injection. Results showed that i.m. injection of AAV8-CMV-FVIII was able to induce a robust immune response against F8. Groups treated with F8 nanoparticles displayed significantly lower cellular F8-specific CD8+and CD4+T cells immune responses, at the early time-points, particularly in the group that received the mixture of five nanoparticle treatment as shown by ELISpot assays (FIG. 10A-FIG. 10C). Additionally, Anti-F8 IgG responses were inhibited until the last timepoint of this study (day 21-23) (FIG. 10D).

[0339] In parallel, this study also allowed to evaluate different F8 -derived-epitopes for their immunogenicity in the F8-knockout animal model. Seven MHC-I-restricted epitopes (CD8 Al-1, CD8 Al-2, CD8 A2-1, CD8 A3-1, CD8 A3-2, CD8 Cl, and CD8 C2) and seven MHC-II-restricted epitopes (CD4 Al-1, CD4 Al-2, CD4 A2, CD4 A3, CD4 Cl, CD4 Al-3, and CD4 C2) were evaluated using ELISpot assays. The analysis revealed that CD8 Cl and CD8 C2 were appropriate to detect F8-specific cellular CD8+ T cells immune response and that CD4 Al-1, CD4 Cl, and CD4 C2 were appropriate to detect F8-specific cellular CD4+ T cells immune response, which were used in the first study described above.

[0340] The next studies followed the same study design as the first, but aimed at evaluating tolerization at later timepoints, with monitoring at days 14, 21, 28, 35, 42, 49, and 56 post AAV8-CMV-FVIII injection. Similar results were observed, with a persistence of F8-specific immune response inhibition all the way to day 56, at least for the group treated with the mixture of five nanoparticle-peptides (F2.3) (FIG. 11A-C). Additionally, Anti-F8 IgG responses were inhibited at each timepoint tested (days 14, 28, 42, and 56), at least in for the group treated with the mixture of five nanoparticle-peptides (F2.3) (FIG. 12A-E).

[0341] In the previous studies in an AAV-Ova gene therapy setting, nanoparticle treatment had shown the potential to efficiently tolerize cellular responses by preventing the emergence and / or effector functions of antigen-specific CD8+as well as CD4+T cells. The results of the studies presented in Example 10 demonstrate that such tolerization is possible to obtain against different transgenes. The results with AAV-FVIII transduction showed nanoparticle-induced tolerization against F8 transgene.EXAMPLE 11: Preparation of Precursor Nanoparticles

[0342] Superparamagnetic iron oxide crystalline cores (SPIONs) and low molecular weight poly(maleic acid-alt- 1 -octadecene) (LM-PMAcOD) can be prepared as described in International Patent Application Publication No.: WO 2021 / 165227, which is incorporated herein by reference in its entirety.

[0343] An exemplary procedure for the synthesis of precursor nanoparticles is as follows: 100 mg LM-PMAcOD are dissolved in 4 mL chloroform in a 100 mL round bottomed flask. The mixture is heated until the polymer is fully dissolved. 3.3 mL of the oleate-SPION solution as described in WO 2021 / 165227 is added to the mixture and subsequently evaporated at <10 mbar for 15 minutes at 40°C on the rotavap with 280 RPM. Then, 10 mL 5 mM NaOH is added to the mixture and stirred on the rotavap for 15 minutes at 50°C until all black solids are dissolved. The solution is diluted 8 times using 70 mL 25 mM NaOH to dissolve the entirepolymer. The obtained solution is stirred on the rotavap for 15 minutes, resulting in a brown solution.

[0344] The product is filtered over a 0.45 pm and a 0.2 pm PES filter. Afterwards, the probe is purified by tangential flow filtration (TFF).EXAMPLE 12: Peptides and Peptide Coupling

[0345] An exemplary procedure for the synthesis of peptides is as follows: The synthesis of the peptides is accomplished via Fmoc chemistry from the C to N direction using solid phase peptide synthesis (SPPS). The alpha amino group of each amino acid is protected with a fluoren- 9-ylmethoxycarbonyl (Fmoc) group, while side chain functional groups are also blocked with various appropriate protective groups.

[0346] In general, the SPPS consists of repeated cycles of N-terminal deprotection followed by coupling reactions. The first Fmoc protected amino acid is coupled to the resin. Afterwards, the amine group is deprotected with a mixture of piperidine in dimethylformamide (DMF) , and then coupled with the free acid of the second Fmoc-protected amino acid. The cycle is repeated until the desired sequence is obtained. The resin is washed between each step. The completion of each coupling reaction is monitored by a qualitative ninhydrin test. In the last step of the synthesis, the crude peptide-resin is successively washed with DMF and methanol, and dried. Then, the protective groups are removed from the peptide and the peptide is cleaved from the resin using trifluoroacetic acid (TFA). The obtained crude peptide is isolated by ether precipitation from the cleavage mixture.

[0347] Further, the peptide is purified through preparative HPLC to reach purity requirements, and the counter ion TFA is replaced with chloride by using an appropriate solventbuffer system. Finally, the purified peptide is lyophilized.

[0348] The peptides can have an amino acid at the N-terminus and a free acid (HC1 salt) at the C-terminus.

[0349] Characterization of the free peptides (starting materials) is performed by LC-MS. The molecular weight of the peptides is measured by multimode electrospray atmospheric pressure chemical ionization mass spectrometry.

[0350] An exemplary procedure for the coupling of peptides to precursor nanoparticles is as follows: The peptides are coupled to the surface of the precursor nanoparticles obtained in Example 11 using l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) acid / sodium tetraborate decahydrate (SBB) chemistry in boric buffer.

[0351] EDC in SBB buffer is added to the precursor nanoparticles obtained in Example 11. After 15 minutes at RT, the peptides are added and the reaction mixture is stirred for 2 hours and 15 minutes at RT. The resulting nanoparticle solution is filtered and purified by tangential flow filtration (TFF) purification.

[0352] The nanoparticles are characterized using a variety of analytical methods. Characterization of the iron oxide core is performed using TEM and SAXS on the nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L- lactic acid. Characterization of particle size and distribution is performed by dynamic light scattering (DLS).

[0353] The hydrodynamic diameter (z-average) and polydispersity index are determined using a Malvern Zetasizer Nano ZS or equivalent in unimodal mode. These measurements are performed on the nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L- lactic acid.

[0354] The surface charge of the nanoparticles is analyzed by measuring the zeta potential at pH 6 to 7 (pH during measurement) using a Malvern Zetasizer Nano ZS instrument. These measurements are performed on the nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L-lactic acid.

[0355] The total polymer content is determined using GPC. The peptides are hydrolyzed, and the particles destroyed in 6 M HC1. The PMAcOD is extracted with ethyl acetate after addition of EDTA. After evaporation of sol vent, the residue is re-dissolved in a THF / acetic acid mixture before analysis.

[0356] The spectroscopic properties of the nanoparticles are determined by Fourier- transform infrared spectroscopy (FTIR).* * *

[0357] The embodiments described above are intended to be merely exemplary, and those skilled in the art will recognize, or will be able to ascertain using no more than routineexperimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the invention and are encompassed by the appended claims.

Claims

WHAT IS CLAIMED:

1. A nanoparticle comprising:(a) an amphiphilic polymer; and(b) a peptide comprising:(i) an amino acid sequence at least about 90% identical to a fragment of a transgene product; or(ii) an amino acid sequence at least about 90% identical to a fragment of an adeno-associated viral (AAV) capsid polypeptide, wherein the peptide is 8 to 50 amino acids long.

2. The nanoparticle of claim 1, wherein the transgene product is suitable for use in a gene therapy.

3. The nanoparticle of claim 1, wherein the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV rhlO, a synthetic AAV, combinations, engineered, or modified versions thereof.

4. The nanoparticle of any one of claims 1 to 3, wherein the peptide comprises an MHC-I epitope.

5. The nanoparticle of any one of claims 1 to 4, wherein the peptide comprises an MHC-II epitope.

6. The nanoparticle of claim 1 , wherein the peptide comprises the amino acid sequence of SEQ ID NOs: 17-47.

7. The nanoparticle of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NOs: 10-15.

8. The nanoparticle of any one of claims 1 to 7, wherein the nanoparticle comprises a linker at the N-terminus of the peptide.

9. A pharmaceutical composition comprising the nanoparticle of any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

10. A method of inducing tolerance of CD4+ T cells and / or CD8+ T cells to a polypeptide encoded by an A AV- vectored transgene in a subject in need thereof, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising:(i) an amino acid sequence at least about 90% identical to a fragment of a transgene product; or(ii) an amino acid sequence at least about 90% identical to a fragment of an AAV capsid polypeptide, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I or an MHC-II epitope.

11. A method of inducing tolerance of CD4+ T cells and / or CD8+ T cells to an AAV particle in a subject in need thereof, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the method comprises administering to the subject a nanoparticle, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising:(i) an amino acid sequence at least about 90% identical to a fragment of a transgene product; or(ii) an amino acid sequence at least about 90% identical to a fragment of an AAV capsid polypeptide, wherein the peptide is 8 to 50 amino acids long, and wherein the peptide comprises an MHC-I or an MHC-II epitope.

12. A method of reducing a humoral immune response to a polypeptide encoded by a transgene in a subject in need thereof, wherein the method comprises:(A) administering a nanoparticle to the subject; and(B) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising:(i) an amino acid sequence at least about 90% identical to a fragment of the AAV capsid, or(ii) an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the humoral immune response is reduced relative to a method comprising step (B) but not step (A).

13. A method of reducing a humoral immune response to an AAV particle in a subject in need thereof, wherein the method comprises:(A) administering a nanoparticle to the subject; and(B) administering the AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and a transgene, wherein the nanoparticle comprises:(a) an amphiphilic polymer; and(b) a peptide comprising:(i) an amino acid sequence at least about 90% identical to a fragment of the AAV capsid,(ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the humoral immune response is reduced relative to a method comprising step (B) but not step (A).

14. A method of increasing and / or prolonging expression of a transgene in a subject in need thereof, wherein the method comprises:(a) administering a nanoparticle to the subject; andIll(b) administering an AAV particle to the subject, wherein the AAV particle comprises an AAV capsid and the transgene, wherein the nanoparticle comprises:(i) an amphiphilic polymer; and (ii) a peptide comprising an amino acid sequence at least about 90% identical to a fragment of a polypeptide encoded by the transgene, wherein the expression is increased and / or prolonged relative to a method comprising step (b), but not step (a).

15. A nanoparticle of any one of claims 1-8 or the pharmaceutical composition of claim 9 for use in reducing an immune response to the AAV capsid and / or the transgene.

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