Use of IRAK4 modulators for gene therapy

Abstract

Provided herein are methods of enhancing gene therapy in an individual by administering an IRAK modulator (e.g., an IRAK-4 degrading agent) with a gene therapy to inhibit innate immunity to the gene therapy. In some embodiments, the gene therapy uses an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, a herpes simplex virus (HSV) vector, or lipid nanoparticles. Also provided herein are methods for selecting an individual for treatment with an IRAK modulator in combination with a gene therapeutic agent.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 330,239, filed on April 12, 2022, which is incorporated by reference in its entirety. Reference to an electronic sequence listing

[0002] The contents of the electronic Sequence Listing (159792018040SEQLIST.xml; size: 1,987 bytes; and creation date: April 12, 2023) are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to methods of enhancing gene therapy in an individual by administering an IRAK modulator with the gene therapy to suppress innate immunity to the gene therapy. In some aspects, the present invention provides methods for selecting an individual for treatment with an IRAK modulator in combination with a gene therapy agent. Background Art

[0004] The success of gene therapy for the treatment of rare genetic diseases relies heavily on adeno-associated virus (AAV) viral vectors, which provide many attractive features, including tissue-specific tropism, transduction of quiescent cells, and maintenance of modified gene expression. However, the immune response to AAV vectors poses a major challenge to successful clinical transformation. Capsid, viral genome, and transgene trigger immune responses involving activation of both innate and adaptive immunity of the immune system. The innate immune system activated by the TLR pathway subsequently triggers an adaptive immune response in B and T cells of people exposed to pathogens (Iawaski, A and Medzhitov, R, Nat Immunol [Natural Immunology] 2004 5 (10): 987-985). Based on several mouse studies, it is shown that the endosomal DNA sensor TLR9 that recognizes CpG-rich hypomethylated DNA plays an important role in the genome recognition of AAV vectors. The signal transduction cascade triggered by TLR9 activates the adaptive immune response, which ultimately leads to the removal of transgenic-induced cells by T cell-mediated cytotoxicity. Mice lacking the TLR9 sensor show longer maintenance of transgene expression and weakened immune responses (Ashley, SN et al., Cell Immunol [Cellular Immunology] December 2019; 346: 103997; and Faust SM et al., J Clin Invest [Clinical Research Journal] 2013 123 (7): 2994-3001). Consistent with this, data from hemophilia clinical trials revealed that a reduction in the total number of CpG bases reduces the need for pharmacological immunosuppressants and shows that hemophilia patients have reduced cytotoxic T lymphocyte responses, while patients receiving vectors containing more CpG bases in the transgene have a much greater need for immunosuppressants (Wright, JFMol. Ther. [Molecular Therapy] 2020 28 (3): 701-703). Although widely acting immunosuppressants have successfully improved AAV delivery in clinical trials, they still lead to loss of transgene expression and are at risk of side effects and opportunistic infections. Developing vectors without CpG bases is a challenge because non-codon optimized vectors (i.e., vectors without CpG or vectors with low CpG content) show poor transgene expression (Wright, JF Mol. Ther. [Molecular Therapy] 2020 28(3): 701-703). Therefore, what is needed is a different class of immunomodulators that show enhanced specificity and low side effects.

[0005] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the invention

[0006] In some aspects, the present invention provides a method for delivering nucleic acid to a cell of an individual, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for treating an individual in need with a gene therapy agent, the method comprising a) administering an IRAK regulator to the individual, and b) administering the gene therapy agent to the individual. In some aspects, the present invention provides a method for improving gene therapy for an individual, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for suppressing an individual's immune response to a gene therapy agent, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual.

[0007] In some embodiments, the IRAK modulator modulates the activity or expression of an IRAK protein kinase. In some embodiments, the IRAK protein kinase is an IRAK-1 protein kinase, an IRAK-2 protein kinase, an IRAK-3 protein kinase, or an IRAK-4 protein kinase. In some embodiments, the IRAK modulator modulates the activity or expression of an IRAK-4 protein kinase. In some embodiments, the IRAK modulator is an IRAK degrader, an IRAK inhibitor, or an agent that imparts loss of IRAK function. In some embodiments, the IRAK modulator is a small molecule.

[0008] In some embodiments, the IRAK modulator comprises a compound of formula [I]: or a pharmaceutically acceptable salt thereof, wherein each variable is as defined and described herein. In some embodiments, the IRAK modulator comprises any one of the compounds of Formula [II]-[V]: or a pharmaceutically acceptable salt thereof. In some embodiments, the IRAK modulator is a compound of formula [II], PROTAC IRAK-4 degrader 1, or PF 06650833. In some embodiments, the IRAK modulator is CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme, or deoxyribozyme (DNAzyme). In some embodiments, the IRAK modulator blocks TLR9 function.

[0009] In some embodiments, the gene therapy agent comprises a viral vector. In some embodiments, the viral vector is an AAV particle. In some embodiments, the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human Bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof. In some embodiments, the AAV capsid comprises a tyrosine mutation, a heparin binding mutation or an HBKO mutation. In some embodiments, the AAV virus particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR or AAV12 ITR. In some embodiments, the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0010] In some embodiments, the viral vector is an adenoviral particle. In some embodiments, the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4,, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3, or a functional variant thereof.

[0011] In some embodiments, the viral vector is a lentiviral particle. In some embodiments, the lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

[0012] In some embodiments, the viral vector is a herpes simplex virus (HSV) particle. In some embodiments, the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

[0013] In some embodiments, the gene therapy agent comprises lipid nanoparticles.

[0014] In some embodiments, the gene therapy agent comprises a nucleic acid encoding a heterologous transgene. In some embodiments, the heterologous transgene is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0015] In some embodiments, the IRAK modulator is administered prior to, concurrently with, or after administration of the gene therapy agent. In some embodiments, the individual suffers from a disease or disorder suitable for treatment by gene therapy. In some embodiments, the disease or disorder is a monogenic disease or disorder.

[0016] In some embodiments, the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically. In some embodiments, the IRAK modulator is administered orally, intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

[0017] In some aspects, the present invention provides a method for delivering a gene therapy agent to cells of an individual, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the gene therapy agent identifies an individual with innate immunity to the gene therapy agent, c) administering an IRAK modulator to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b). In some aspects, the present invention provides a method for treating an individual in need thereof with a gene therapy agent, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the gene therapy agent identifies an individual with innate immunity to the gene therapy agent, c) administering an IRAK modulator to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b). In some aspects, the present invention provides a method for selecting an individual for treatment with a gene therapy agent and an IRAK modulator, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the expression of one or more cytokines of the innate immune cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent identifies the individual for treatment with the gene therapy agent and the IRAK modulator, c) selecting the individual identified in step b) for treatment with the gene therapy agent and the IRAK modulator. In some embodiments, the method further comprises the steps of: d) administering an IRAK modulator to the individual identified in step b), and e) administering the gene therapy agent to the individual identified in step b).

[0018] In some embodiments, the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell. In some embodiments, the innate immune cells are isolated from peripheral blood mononuclear cells from the individual. In some embodiments, the innate immune cell is a dendritic cell. In some embodiments, the dendritic cell is derived from a monocyte of the individual.

[0019] In some embodiments, the method of the present invention further comprises isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent. In some embodiments, the monocytes are CD14+ monocytes. In some embodiments, the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes. In some embodiments, the innate immune cells are re-plated before incubating with the gene therapy agent of step c). In some embodiments, the innate immune cells are re-plated in a microwell dish.

[0020] In some embodiments, the gene therapy agent is a viral vector, and wherein the innate immune cells are coupled to the viral vector at a rate of about 1×10 3 About 1×10 5 or about 1×10 4 In some embodiments, the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL. In some embodiments, the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

[0021] In some embodiments, the cytokine signature includes the increased expression of one or more of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the increased expression of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the increased expression of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the increased expression of IL6, TNFα and IL-1β. In some embodiments, the expression of these cytokines in the cytokine signature increases compared to a suitable control. In some embodiments, the suitable control is the expression of these cytokines in the cytokine signature of innate immune cells not incubated with the gene therapy agent, or wherein the suitable control is the expression of these cytokines in the cytokine signature of innate immune cells before incubation with the gene therapy agent.

[0022] In some aspects, the present invention provides the use of a composition in the manufacture of a medicament for delivering a nucleic acid to a cell of an individual in need, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator. In some aspects, the present invention provides the use of a composition in the manufacture of a medicament for delivering a nucleic acid to a cell of an individual in need, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent. In some aspects, the present invention provides the use of a composition in the manufacture of a medicament for treating an individual in need of gene therapy, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator. In some aspects, the present invention provides the use of a composition in the manufacture of a medicament for treating an individual in need of gene therapy, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent. In some aspects, the present invention provides the use of a composition in the manufacture of a medicament for regulating an immune response of an individual in need of gene therapy to gene therapy, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator. In some aspects, the present invention provides a composition for use in the manufacture of a medicament for regulating an individual's immune response to gene therapy, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent. In some embodiments, the gene therapy agent is an AAV particle, an adenovirus particle, a lentivirus particle, an HSV particle, or a lipid nanoparticle. In some embodiments, the IRAK modulator is an IRAK-4 degrader.

[0023] In some aspects, the present invention provides a composition comprising a gene therapy agent for use in delivering nucleic acids to cells of individuals in need, wherein the gene therapy agent is used in combination with an IRAK modulator. In some aspects, the present invention provides a composition comprising an IRAK modulator for use in delivering nucleic acids to cells of individuals in need, wherein the IRAK modulator is used in combination with a gene therapy agent. In some aspects, the present invention provides a composition comprising a gene therapy agent for use in treating individuals in need of gene therapy, wherein the gene therapy agent is used in combination with an IRAK modulator. In some aspects, the present invention provides a composition comprising an IRAK modulator for use in treating individuals in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent. In some aspects, the present invention provides a composition comprising an IRAK modulator for use in treating individuals in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent. In some aspects, the present invention provides a composition comprising an IRAK modulator for regulating an immune response of an individual in need of gene therapy to gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent. In some aspects, the present invention provides a composition comprising an IRAK modulator for suppressing an immune response of an individual in need of gene therapy to gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent. In some embodiments, the gene therapy agent is an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle. In some embodiments, the IRAK modulator is an IRAK-4 degrader.

[0024] In some embodiments, the present invention provides kits for use in any of the methods or uses described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The experimental scheme for evaluating the efficacy of IRAK regulators (e.g., IRAK4 degraders and IRAK4 inhibitors) is shown. Peripheral blood mononuclear cells (PBMCs) are isolated from enriched leukocyte apheresis products (leukopak). CD14+ monocytes are purified from (PBMC), a differentiation factor mixture is added to these monocytes to allow differentiation into dendritic cells, and maturation factors are added to obtain mature dendritic cells. Mature dendritic cells are pretreated with IRAK regulators (such as zimlovisertib and PROTAC IRAK4 degraders-1) for 18 hours and then infected with AAV particles. Twenty-four hours after AAV infection, culture supernatants are collected for downstream analysis (such as cytokine release and cytotoxicity). These cells are used to measure the level of IRAK4 inhibition or degradation. In different settings, IRAK4 regulators zimlovisertib IRAK4 and PROTAC IRAK4 degraders-1 are added simultaneously with AAV (referred to as co-treatment), and culture supernatants are collected for downstream analysis (such as cytokine release and cytotoxicity).

[0026] Figure 2A-2B It is shown that pretreatment with two different IRAK modulators (IRAK inhibitor and IRAK4 degrader) resulted in inhibition of cytokine release. Figure 2A It is shown that pretreatment of human monocytic dendritic cells with the IRAK4 inhibitor zimlovisertib resulted in inhibition of cytokine release. Figure 2B It is shown that pre-treatment of human monocytic dendritic cells with the IRAK4 degrader PROTAC IRAK4 degrader-1 resulted in inhibition of cytokine release.

[0027] Figure 3A-3B It is shown that co-treatment with two different IRAK modulators (IRAK inhibitor and IRAK4 degrader) resulted in inhibition of cytokine release. Figure 3A It is shown that pretreatment of human monocytic dendritic cells with the IRAK4 inhibitor zimlovisertib resulted in inhibition of cytokine release. Figure 3B It is shown that co-treatment of human monocytic dendritic cells with PROTAC IRAK4 degrader-1 resulted in inhibition of cytokine release.

[0028] Figure 4A-4B It is shown that treatment with two different IRAK modulators (IRAK inhibitor and IRAK4 degrader) does not induce cytotoxicity in primary human monocytic dendritic cells. Figure 4A Shown are toxicity results following administration of the IRAK4 inhibitor zimlovisertib with AAV. Figure 4BToxicity results following administration of PROTAC IRAK4 Degrader-1 with AAV are shown.

[0029] Figure 5A-Figure 5B It is shown that treatment with an IRAK4 inhibitor blocks phosphorylation of the IRAK4 kinase target protein NFkB, and that an IRAK4 degrader causes degradation of the IRAK4 protein in primary human monocytic dendritic cells. Figure 5A Shown that treatment of human monocytic dendritic cells with the IRAK4 inhibitor zimlovisertib blocks IRAK4 kinase activity induced by LPS. Figure 5B PROTAC IRAK4 Degrader-1AAV is shown to reduce IRAK4 levels in AAV.SAN024 infected cells.

[0030] Figure 6 Shown is a schematic diagram of an in vivo mouse experiment designed to show the effects of IRAK4 inhibition on CD8 T cells and memory T cells after AAV injection.

[0031] Figure 7A-7B It is shown that treatment with an IRAK4 inhibitor reduces transgenic lacZ-specific CD8 T cells and effector memory T cells in PBMCs after AAV injection in mice. Fig. 7A Shown is a decrease in lacZ-specific CD8 T cells in mice treated with the IRAK4 inhibitor zimlovisertib. Figure 7B Shown is a reduction in effector memory cells in mice treated with the IRAK4 inhibitor zimlovisertib.

[0032] Figure 8A-8C Shown that treatment with an IRAK4 inhibitor reduced IFNg-producing CD8 T cells after AAV injection in mice. Fig. 8A Shown is the production of interferon gamma (a measure of activated CD8 T cells) following isolation of splenocytes from treated mice and subsequent stimulation with AAV peptide pools. Figure 8B Shown is the production of interferon gamma following isolation of splenocytes from treated mice and subsequent stimulation with the immunodominant peptide. Figure 8C Shown is the production of interferon gamma after isolation of splenocytes from treated mice and subsequent stimulation with the LacZ immunodominant peptide.

[0033] Figure 9A-9D Treatment with the IRAK4 degrader PROTAC IRAK4-1 is shown to reduce IRAK4 expression in immune cells. Fig.9A Treatment with IRAK degraders is shown to reduce PBMCs. Fig. 9B Treatment with an IRAK degrader is shown to reduce T cells. Fig. 9CTreatment with an IRAK degrader is shown to reduce B cells. Fig.9D Treatment with an IRAK degrader is shown to reduce monocytes.

[0034] Figure 10A-10B It is shown that pre-treatment with the IRAK4 degrader of formula (II) (KT-474) reduced capsid transgene-specific CD8 T cells and interferon-γ-producing immune cells after AAV injection in mice. Fig. 10A Shown is a decrease in lacZ-specific CD8 T cells in PBMCs. Fig. 10B Shown is a decrease in lacZ-specific CD8 T cells in the spleen.

[0035] Fig.11 In vivo mouse experiments with oral treatment of IRAK4 modulators administered in combination with AAV are shown. The IRAK4 modulators can be administered prior to or concurrently with AAV. DETAILED DESCRIPTION

[0036] In some aspects, the present invention provides a method for delivering nucleic acid to an individual's cells, the method comprising a) administering an IRAK modulator (e.g., an IRAK-4 degrader) to the individual, and b) administering a gene therapy agent comprising nucleic acid. As used herein, a "gene therapy agent" can be a therapeutic component and / or a vector component of a gene therapy to be administered to an individual, for example, nucleic acid (end-closed DNA), a viral vector (e.g., an AAV vector, an adenoviral vector, a lentiviral vector, an HSV vector), a lipid nanoparticle, all or part of an antibody (e.g., a nanobody, an Fc region, etc.). In some aspects, the present invention provides a method for treating an individual in need with a composition comprising a gene therapy agent, the method comprising a) administering an IRAK modulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for improving gene therapy for an individual, the method comprising a) administering an IRAK modulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for regulating an immune response to a gene therapy agent, the method comprising a) administering an IRAK modulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for suppressing an immune response to a gene therapy agent, the method comprising a) administering an IRAK modulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for inducing tolerance to a gene therapy agent, the method comprising a) administering an IRAK modulator to the individual, and b) administering a gene therapy agent to the individual.

[0037] In some aspects, the present invention provides a method for delivering nucleic acid to cells of an individual, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles or lipid nanoparticles), b) analyzing the expression of one or more cytokines of these innate immune cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent identifies an individual with immunity (e.g., innate immunity, adaptive immunity) to the gene therapy agent, c) administering an IRAK modulator (e.g., an IRAK-4 degrader) to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b). In some embodiments, the innate immune cells are dendritic cells, monocytes, macrophages or natural killer (NK) cells. In some embodiments, the method further comprises the steps of isolating innate immune cells from the individual and then incubating these innate immune cells with the gene therapy agent. In some embodiments, the method further comprises the steps of isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent. As used herein, the term "derive" dendritic cells includes differentiation of cells (e.g., monocytes) to produce dendritic cells.

[0038] In some aspects, the present invention provides a method for treating an individual in need thereof, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles), b) analyzing the expression of one or more cytokines of these innate immune cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent identifies an individual with immunity (e.g., innate immunity, adaptive immunity) to the gene therapy agent, c) administering an IRAK modulator (e.g., an IRAK-4 degrader) to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b). In some embodiments, the innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells. In some embodiments, the method further comprises the steps of isolating innate immune cells from the individual and then incubating these innate immune cells with the gene therapy agent. In some embodiments, the method further comprises the steps of isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0039] In some aspects, the present invention provides a method for selecting an individual for treatment with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) and an IRAK modulator (e.g., an IRAK-4 degrader), the method comprising a) incubating innate immune cells with the gene therapy agent, b) analyzing the expression of one or more cytokines of the innate immune cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent identifies the individual treated with the gene therapy agent and the IRAK modulator, c) selecting the individual identified in step b) for treatment with the gene therapy agent and the IRAK modulator. In some embodiments, the innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells. In some embodiments, the method further comprises the steps of isolating innate immune cells from the individual and then incubating the innate immune cells with the gene therapy agent. In some embodiments, the method further comprises the steps of isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent. In some embodiments, the method further comprises the steps of: d) administering an IRAK modulator to the individual identified in step b), and e) administering the gene therapy agent to the individual identified in step b). General Technology

[0040] The techniques and procedures described or referenced herein are generally well known to those skilled in the art and are routinely employed using conventional methods, such as the widely used methods described in the following references: Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications [Cultivation of Animal Cells: A Handbook of Basic Techniques and Specialized Applications] (RI Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis [Oligonucleotide Synthesis] (M. J. Gait, ed., 1984); Methods in Molecular Biology [Molecular Biology Methods], Humana Press [Humana Press]; Cell Biology: A Laboratory Notebook [Cell Biology: Laboratory Notebook] (J. E. Cellis, ed., Academic Press [Academic Press], 1998); Introduction to Cell and Tissue Culture [Introduction to Cell and Tissue Culture] (J. P. Heathen and P. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures [Cell and Tissue Culture: Laboratory Procedures] (A. Doyle, J. B. Griffiths and D. G. Smith, ed., Plenum Press, 1998);Newell, ed., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 1993-8); Sons [John Wiley & Sons, 2002); Immunobiology [Immunobiology] (CA Janeway et al., 2004); Antibodies [Antibodies] (P. Finch, 1997); Antibodies: A Practical Approach [Antibodies: A Practical Approach] (D. Catty. ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach [Monoclonal Antibodies: A Practical Approach] (P. Shepherd and C. Dean eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual [Using Antibodies: A Laboratory Manual] (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies [Antibodies] (M. Zanetti and JD Capra eds., Harwood Academic Press, 1996); Publishers [Harwood Academic Publishers, 1995]; and Cancer: Principles and Practice of Oncology [Cancer: Principles and Practice of Oncology] (VT DeVita et al., eds., JB Lippincott Company [JBLippincott & Co., 2011). definition

[0041] As used herein, the term "IRAK degrader" is a heterobifunctional compound that binds to and / or inhibits (completely or partially) both IRAK kinase and E3 ligase with measurable affinity, resulting in ubiquitination and subsequent degradation of IRAK kinase. In certain embodiments, the degrader has a DC of less than about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. 50 As used herein, the term "monovalent" refers to a degrader compound that does not contain an additional E3 ligase binding moiety.

[0042] As used herein, the term "inhibitor" with respect to IRAK is a compound that binds to and / or inhibits (completely or partially) the IRAK kinase with measurable affinity. In certain embodiments, the inhibitor has an IC of less than about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. 50 and / or binding constant.

[0043] As used herein, the term "modulator" with respect to IRAK is a compound that stimulates, delays, inhibits and / or suppresses (completely or partially) the activity of IRAK kinase.

[0044] As used herein, the term "gene therapy" refers to a therapy that modifies the expression of nucleic acids (e.g., genes, mRNA, etc.) in the cells of an individual to change the biological properties of the cells. In some instances, gene therapy includes delivering exogenous nucleic acids to be expressed in the cells of an individual. In some instances, gene therapy changes (e.g., degrades, inhibits, enhances) the expression of endogenous genes in the cells of an individual. In some instances, gene therapy is an in vivo therapy. In some instances, gene therapy is an ex vivo therapy (e.g., cell therapy).

[0045] As used herein, the term "gene therapy agent" refers to a nucleic acid (e.g., an expression construct, miRNA, antisense, shRNA, siRNA) or a combination of a nucleic acid and an agent that is used to deliver a nucleic acid to an individual or cell to modify or manipulate the expression of one or more nucleic acids (e.g., genes, mRNA) in an individual or cell to change the biological properties of a living cell. Examples of gene therapy agents include, but are not limited to, viral vectors (e.g., adeno-associated viruses, adenoviruses, lentiviruses, herpes simplex viruses, baculoviruses), bacterial vectors, and non-viral vectors (e.g., lipid nanoparticles encapsulating therapeutic nucleic acids or encapsulating plasmid DNA (e.g., end-blocked DNA) containing therapeutic nucleic acids and / or encoding therapeutic polypeptides).

[0046] As used herein, "vector" refers to a recombinant plasmid or virus comprising a nucleic acid to be delivered into a host cell in vitro or in vivo.

[0047] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, the term includes but is not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of the nucleic acid can include sugar and phosphate groups (as can be found in RNA or DNA generally), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the nucleic acid can include a polymer of synthetic subunits such as aminophosphorylates, therefore oligodeoxynucleoside aminophosphorylates (P-NH2) or mixed aminophosphorylates-phosphodiester oligomers. In addition, double-stranded nucleic acids can be obtained from chemically synthesized single-stranded polynucleotide products in the following manner: by synthesizing complementary chains and annealing the chains under appropriate conditions, or by using DNA polymerase to synthesize complementary chains from scratch with appropriate primers.

[0048] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. This definition encompasses both full-length proteins and fragments thereof. The term also includes post-translational modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications (such as deletions, additions and substitutions, which are generally conservative in nature) relative to the native sequence, as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host producing the protein or due to errors in PCR amplification.

[0049] "Recombinant viral vector" refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (ie, nucleic acid sequences of non-viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one, for example two, inverted terminal repeats (ITRs).

[0050] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences from non-AAV sources), which are flanked by at least one, for example, two AAV inverted terminal repeats (ITRs). When present in a host cell that has been infected with a suitable helper virus (or is expressing a suitable helper function) and is expressing AAVrep and cap gene products (i.e., AAV Rep and Cap proteins), such rAAV vectors can be replicated and packaged into infectious viral particles. When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or another vector such as a plasmid for cloning or transfection), the rAAV vector can be referred to as a "front vector", which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. The rAAV vector can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles (particularly AAV particles) in an embodiment. rAAV vectors can be packaged into AAV viral capsids to produce "recombinant adeno-associated virus particles (rAAV particles)".

[0051] "rAAV virus" or "rAAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0052] "Recombinant adenoviral vector" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-adenoviral origin), which are flanked by at least one adenoviral inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeats (ITR). When present in a host cell expressing essential adenoviral genes (e.g., E1 gene, E2 gene, E4 gene, etc.) deleted from the recombinant viral genome, such recombinant viral vectors can be replicated and packaged into infectious viral particles. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid for cloning or transfection), the recombinant viral vector can be referred to as a "pre-vector", which can be "rescued" by replication and encapsidation in the presence of an adenoviral packaging function. The recombinant viral vector can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles (e.g., adenoviral particles). The recombinant viral vector can be packaged into an adenoviral capsid to produce a "recombinant adenoviral particle".

[0053] "Recombinant lentiviral vector" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-lentiviral origin) flanked by at least one lentiviral terminal repeat (LTR). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral terminal repeats (LTR). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with appropriate helper functions. Recombinant lentiviral vectors can be packaged into lentiviral capsids to produce "recombinant lentiviral particles".

[0054] "Recombinant herpes simplex virus vector (recombinant HSV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences not derived from HSV), which are flanked by HSV terminal repeats. When present in a host cell that has been infected with a suitable helper function, such a recombinant viral vector can be replicated and packaged into infectious viral particles. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid for cloning or transfection), the recombinant viral vector can be referred to as a "provector", which can be "rescued" by replication and encapsidation in the presence of an HSV packaging function. The recombinant viral vector can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles (e.g., HSV particles). The recombinant viral vector can be packaged into an HSV capsid to produce a "recombinant herpes simplex virus particle".

[0055] As used herein, "solid lipid nanoparticles" (SLN, sLNP) or "lipid nanoparticles" (LNP) refer to nanoparticles composed of lipids. In some instances, there is only one phospholipid layer, and the majority of the interior of the particle is composed of lipophilic substances. Payloads (such as nucleic acids) can be embedded inside. In some instances, the lipid nanoparticle is a liposome comprising a lipid bilayer.

[0056] As used herein, the term "improvement" when it comes to gene therapy can refer to the act of strengthening, improving, prolonging or otherwise increasing the expression of the therapeutic gene payload of the gene therapy agent. In some embodiments, the improved gene therapy is such a gene therapy, wherein the expression of the therapeutic gene payload of the gene therapy agent administered with the IRAK modulator is increased by greater than about 10%, 25%, 50%, 75% or 100% compared to the gene therapy not administered with the IRAK modulator. In some embodiments, the improved gene therapy is such a gene therapy, wherein the expression time of the therapeutic gene payload of the gene therapy agent administered with the IRAK modulator is extended by greater than about 10%, 25%, 50%, 75% or 100% compared to the gene therapy not administered with the IRAK modulator. In some instances, the gene therapy is improved by reducing the immune response (e.g., innate immune response) to the gene therapy agent. In some embodiments, the improved gene therapy is a gene therapy wherein the immune response to the gene therapy administered with the IRAK modulator is reduced by greater than about any of 10%, 25%, 50%, 75%, or 100% compared to the gene therapy administered without the IRAK modulator. In some embodiments, the reduction in immune response to the gene therapy agent is measured as a reduction in cytokine signature after exposure of the gene therapy agent to immune cells in the presence of the IRAK modulator compared to exposure of the gene therapy agent to immune cells in the absence of the IRAK modulator.

[0057] As used herein, the term "modulate" in reference to gene therapy can refer to an action that changes, alters, modulates, improves, or otherwise modifies the presence or activity of a gene therapy agent. For example, modulating an immune response to a gene therapy agent can refer to any action that results in a change, alteration, modification, improvement, or otherwise modifies the immune response to a gene therapy agent (e.g., reducing, delaying, and / or eliminating an immune response (e.g., an innate immune response) to a gene therapy agent).

[0058] As used herein, the term "cytokine signature" when referring to an immune response (e.g., an innate immune response) to a gene therapy agent refers to the expression of an alteration (e.g., increase, decrease) of one or more cytokines after exposing innate immune cells to a gene therapy agent. In some instances, the cytokines of the cytokine signature are specific to a TLR pathway (e.g., a TLR2, TLR3, TLR4, or TLR9 pathway).

[0059] Innate immune cells are leukocytes that mediate innate immunity, and include basophils, dendritic cells, eosinophils, Langerhans cells, mast cells, monocytes and macrophages, neutrophils and NK cells. Different AAV capsids can enter these innate immune cells with different efficiencies (commonly referred to as transduction efficiencies). Some serotypes (such as AAV1) are effective in transducing certain immune cells like monocytes, while other AAVs (such as AAV6) are effective in transducing cells like dendritic cells (Grimm, D et al., J. Virol. [Journal of Virology], 2008, 82 (12): 5887-5911). AAV after cell entry can cause an immune response. The extent of this immune response depends on the AAV serotype and cell type. Once AAV transduces host immune cells, they can engage immune receptors such as TLR (e.g., TLR9). Several studies using mouse models have revealed that TLR9 is a key DNA sensor that contributes to AAV immunogenicity (Zhu, J et al., J Clin Invest. [Clinical Research Journal] 2009; 119 (8): 2388-2398; Ashley SN et al., Cell. Immunol. [Cellular Immunology] 2019, 346: 103997). Once these TLRs are activated by the virus, they secrete cytokines that establish an antiviral state within the infected cell and alert neighboring cells. (Carty, M and Bowie, AG, Clin Exp Immunol, 2010, 161(3):397-406; Lester, SN and Li, K, J Mol Biol. 2014; 426(6):1246-1264; Fitzgerald, KA and Kagan, JC, Cell, 2020180(6):1044-1066).

[0060] These cytokines are also responsible for activating the adaptive immune system, which includes B cells and T cells that produce antibodies and cytotoxicity to kill virus-infected cells, respectively. As used herein, the upregulation or downregulation of certain subsets of cytokines is referred to as a "cytokine signature". These cytokine signatures containing three or more cytokines can be used as predictive markers for disease and therapy success. Examples of cytokine signatures can be found in Zuniga, J et al., Int. J. Infect. Diseases [International Journal of Infectious Diseases], 2020, 94: 4-11, Bergamaschi, C et al., Cell Reports [Cell Report], 2021, 36: 109504; Del Valle, DM et al., Nat. Med. [Natural Medicine] 2020, 26: 1636-1643.

[0061] "Heterologous" means an entity that is genotypically different from the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a nucleic acid introduced into a different cell type by genetic engineering techniques is a heterologous nucleic acid (and may encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0062] The term "transgenic" refers to a nucleic acid that is introduced into a cell and can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In some aspects, it imparts desired properties to the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic outcome. On the other hand, it can be transcribed into a molecule that mediates RNA interference, such as siRNA.

[0063] The terms "genomic particles (gp)", "genomic equivalents" or "genomic copies" as used in relation to viral titer refer to the number of virions containing the recombinant AAV DNA genome, without regard to infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by procedures such as those described in the Examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther. [Human Gene Therapy], 10: 1031-1039; Veldwijk et al. (2002) Mol. Ther. [Molecular Therapy], 6: 272-278.

[0064] The terms "infectious unit (iu)", "infectious particles" or "replication units" as used in reference to viral titers refer to the number of infectious and replicative recombinant AAV vector particles measured by the infectious center assay (also called the replication center assay), as described, for example, in McLaughlin et al. (1988) J. Virol., 62: 1963-1973.

[0065] The term "transducing unit (tu)" as used in reference to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured in a functional assay such as described in the Examples herein or, for example, in Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al. (1996) J. Virol., 70: 520-532 (LFU assay).

[0066] "Inverted terminal repeat" or "ITR" sequence is a term well known in the art and refers to relatively short sequences in opposite orientation found at the ends of the viral genome.

[0067] The "AAV inverted terminal repeat (ITR)" sequence is a term well known in the art and is a sequence of approximately 145 nucleotides present at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can exist in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (designated as region A, region A', region B, region B', region C, region C', and region D) that allow intrastrand base pairing to occur within this portion of the ITR.

[0068] "Terminal dissociation sequence" or "trs" is a sequence in the D region of AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. Mutant terminal dissociation sequences are resistant to cleavage by the AAV rep protein. "AAV helper function" refers to a function that allows AAV to be replicated and packaged by a host cell. AAV helper function can be provided in any of a variety of forms, including but not limited to helper viruses or helper viral genes that assist in AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0069] "AAV helper function" refers to a function that allows AAV to be replicated and packaged by a host cell. AAV helper function can be provided in any of a variety of forms, including but not limited to a helper virus or helper virus gene that assists in AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0070] The "helper virus" of AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by host cells. Many such helper viruses have been identified, including adenovirus, herpes virus, pox virus (such as vaccinia) and baculovirus. Adenovirus covers many different subgroups, but the 5-type adenovirus (Ad5) of subgroup C is the most commonly used. Many adenoviruses in human, non-human mammal and avian sources are known and can be obtained from depository institutions such as ATCC. The viruses of the herpes family that can also be obtained from depository institutions such as ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and pseudorabies virus (PRV). The baculovirus that can be obtained from depository institutions includes Autographa californica nuclear polyhedrosis virus.

[0071] "Percentage (%) of sequence identity" with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues or nucleotides in a reference polypeptide or nucleic acid sequence after aligning the sequences and introducing spaces (if necessary) to obtain the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology [Molecular Biology Experiment Manual] (Ausubel et al., 1987), Supplement 30, Section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Potential alignment programs are ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequence. For the purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, and or to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, and or to a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as identical matches by a sequence alignment program in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. For the purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, and or for a given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C having or comprising a certain % nucleic acid sequence identity to, and or for a given nucleic acid sequence D) is calculated as follows: 100 multiplied by a fraction W / Z, where W is the number of nucleotides that are scored as identical matches by a sequence alignment program in the program's alignment of C and D, and where Z is the total number of nucleotides in D. It should be understood that when the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not be equal to the % nucleic acid sequence identity of D to C.

[0072] An "effective amount" of an agent is an amount effective to achieve the desired therapeutic outcome at the dosage and time period necessary. For example, an effective amount of a gene therapy agent is an amount effective to achieve the desired gene therapy outcome at the dosage and time period necessary. In another example, an effective amount of an IRAK modulator can be an amount effective to achieve the desired outcome of improved gene therapy at the dosage and time period necessary.

[0073] A "therapeutically effective amount" of a substance / molecule of the invention (e.g., a gene therapy agent and / or an IRAK modulator) can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects.

[0074] The term "appropriate control" when referring to a cytokine signature is the expression of cytokines in a cytokine signature from innate immune cells that have not been incubated with the gene therapy agent, or the expression of cytokines in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

[0075] Administration "in combination with" with respect to a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator) includes simultaneous (concurrent), consecutive, or sequential administration of the gene therapy agent and the innate immune response modulator (e.g., an IRAK modulator) in any order.

[0076] The term "concurrently" is used herein to refer to the administration of a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator) wherein at least a portion of the administration overlaps in time. Thus, concurrent administration includes dosing regimens when administration of a gene therapy agent or an innate immune response modulator (e.g., an IRAK modulator) continues after administration of the other agent / modulator has ceased.

[0077] As used herein, "in combination with" means administering another therapeutic modality in addition to one therapeutic modality. Thus, "in combination with" means administering another therapeutic modality (gene therapy agent or innate immune response modulator (e.g., IRAK modulator)) before, during, or after one therapeutic modality is administered to an individual.

[0078] An "isolated" molecule (eg, nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0079] References herein to "about" a value or parameter include (and describe) embodiments involving that value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X".

[0080] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0081] It should be understood that the aspects and embodiments of the present invention described herein include "comprising the aspects and embodiments," "consisting of," and / or "consisting essentially of," the aspects and embodiments. Treatment

[0082] In some aspects, the present invention provides a method for delivering nucleic acid to a cell of an individual, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for treating an individual in need, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for improving gene therapy for an individual, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for regulating an immune response to a gene therapy agent, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for suppressing an immune response to a gene therapy agent, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual. In some aspects, the present invention provides a method for inducing tolerance to a gene therapy agent, the method comprising a) administering an IRAK regulator to the individual, and b) administering a gene therapy agent to the individual. In some embodiments, the IRAK modulator modulates the activity of IRAK protein kinase. In some embodiments, the IRAK modulator modulates the activity of IRAK-4 protein kinase. In some embodiments, the gene therapy agent is a viral gene therapy agent (e.g., a viral vector) or a non-viral gene therapy agent (e.g., a lipid nanoparticle comprising a non-viral gene therapy agent). In some embodiments, the gene therapy agent is an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, or a herpes simplex virus (HSV) vector.

[0083] In some embodiments of the present invention, a gene therapy agent (e.g., AAV particles, adenovirus particles, lentivirus particles, HSV particles, or lipid nanoparticles) can be administered to a specific target tissue, or it can be administered systemically. In some embodiments, an effective amount of a gene therapy agent can be administered to a subject. In some embodiments, an effective amount of a gene therapy agent can be administered parenterally. Parenteral administration routes can include but are not limited to intravenous, intraperitoneal, intraosseous, intraarterial, intracerebral, intramuscular, intrathecal, subcutaneous, intraventricular, intrahepatic, etc. In some embodiments, an effective amount of a gene therapy agent can be administered by a route of administration. In some embodiments, an effective amount of a gene therapy agent can be administered by a combination of routes of administration or multiple (e.g., two, three, etc.) routes of administration. In some embodiments, an effective amount of a gene therapy agent is administered to one site. In other embodiments, an effective amount of a gene therapy agent can be administered to more than one site.

[0084] An effective amount of a gene therapy agent (e.g., AAV particles, adenovirus particles, lentiviral particles, HSV particles, or lipid nanoparticles) is administered according to the treatment goal. For example, in the case where a low percentage of transduction or transfection can achieve the desired therapeutic effect, the treatment goal is usually to meet or exceed this transduction or transfection level. In some cases, this transduction or transfection level can be achieved by transducing or transfecting only about 1% to 5% of the target cells of the desired tissue type, at least about 20% of the cells of the desired tissue type in some embodiments, at least about 50% in some embodiments, at least about 80% in some embodiments, at least about 95% in some embodiments, and at least about 99% of the cells of the desired tissue type in some embodiments. The gene therapy agent can be administered during the same procedure or spaced apart for several days, weeks, months or years by one or more administrations. One or more of any administration routes described herein can be used. In some embodiments, a variety of gene therapy agents can be used to treat humans; for example, AAV vectors and lentiviral vectors.

[0085] Methods for identifying cells transduced or transfected with a gene therapy agent are known in the art; for example, cells transduced or transfected with a gene therapy agent can be detected using immunohistochemistry or using a marker such as enhanced green fluorescent protein.

[0086] In some embodiments, an effective amount of a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) is administered to more than one site simultaneously or sequentially. In other embodiments, an effective amount of a gene therapy agent is administered to a single site more than once (e.g., repeatedly). In some embodiments, multiple injections of a gene therapy agent are spaced no more than one hour, two hours, three hours, four hours, five hours, six hours, nine hours, twelve hours, or 24 hours apart.

[0087] In some embodiments, the methods include administering an effective amount of a pharmaceutical composition comprising a gene therapy agent to treat an individual in need of gene therapy treatment. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 12 , 6×10 12 ,7×10 12 , 8×10 12 ,9×10 12 , 10×10 12 , 11×10 12 , 15×10 12 , 20×10 12 , 25×10 12 ,30×10 12 or 50×10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 12 Up to 6×10 12 , 6×10 12 Up to 7×10 12 ,7×10 12 Up to 8×10 12 , 8×10 12 Up to 9×10 12 ,9×10 12 Up to 10×10 12 , 10×10 12 Up to 11×10 12 , 11×10 12 Up to 15×10 12 , 15×10 12 Up to 20×10 12 , 20×10 12 Up to 25×10 12 , 25×10 12 Up to 30×10 12 ,30×10 12 Up to 50×10 12 or 50×10 12 Up to 100×10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 12 Up to 10×10 12 , 10×10 12 Up to 25×10 12 or 25×10 12 Up to 50×10 12In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 9 , 6×10 9 ,7×10 9 , 8×10 9 ,9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 ,30×10 9 or 50×10 9 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 9 Up to 6×10 9 , 6×10 9 Up to 7×10 9 ,7×10 9 Up to 8×10 9 , 8×10 9 Up to 9×10 9 ,9×10 9 Up to 10×10 9 , 10×10 9 Up to 11×10 9 , 11×10 9 Up to 15×10 9 , 15×10 9 Up to 20×10 9 , 20×10 9 Up to 25×10 9 , 25×10 9 Up to 30×10 9 ,30×10 9 Up to 50×10 9 or 50×10 9 Up to 100×10 9 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 9 Up to 10×10 9 , 10×10 9 Up to 15×10 9 , 15×10 9 Up to 25×10 9 or 25×10 9 Up to 50×10 9In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 10 , 6×10 10 ,7×10 10 , 8×10 10 ,9×10 10 , 10×10 10 , 11×10 10 , 15×10 10 , 20×10 10 , 25×10 10 ,30×10 10 , 40×10 10 or 50×10 10 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 10 Up to 6×10 10 , 6×10 10 Up to 7×10 10 ,7×10 10 Up to 8×10 10 , 8×10 10 Up to 9×10 10 ,9×10 10 Up to 10×10 10 , 10×10 10 Up to 11×10 10 , 11×10 10 Up to 15×10 10 , 15×10 10 Up to 20×10 10 , 20×10 10 Up to 25×10 10 , 25×10 10 Up to 30×10 10 ,30×10 10 Up to 40×10 10 , 40×10 10 Up to 50×10 10 or 50×10 10 Up to 100×10 10 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 10 Up to 10×10 10 , 10×10 10 Up to 15×10 10 , 15×10 10 Up to 25×10 10 or 25×10 10 Up to 50×1010 Any of 10 infectious units / mL.

[0088] In some embodiments, the dose of the gene therapy agent (e.g., AAV particle, adenoviral particle, lentiviral particle, HSV particle, or lipid nanoparticle) administered to an individual is at least about 1×10 8 About 6×10 13 In some embodiments, the dose of the gene therapy agent administered to an individual is about 1×10 8 About 6×10 13 Any of genomic copies / kg body weight.

[0089] In some embodiments, the total amount of gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) administered to an individual is at least about 1×10 9 About 1×10 14 In some embodiments, the total amount of gene therapy agent administered to an individual is about 1×10 9 About 1×10 14 Any of the genome copies.

[0090] Compositions of the invention comprising gene therapy (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles or lipid nanoparticles) can be used alone or in combination with one or more additional therapeutic agents in addition to IRAK modulators. The interval between sequential administrations can be calculated as at least (or alternatively, less than) minutes, hours or days.

[0091] In some embodiments of the present invention, the composition comprising an IRAK modulator (e.g., an IRAK-4 degrader) can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the composition is administered orally, intraperitoneally, or intravenously. The sterile injectable form of the composition of the present invention can be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in l,3-butanediol. Among acceptable vehicles and solvents, water, Ringer's solution, and isotonic sodium chloride solution can be used. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0092] The amount of an IRAK modulator (e.g., IRAK-4 degrader) that can be combined with a carrier material to produce a single dosage form of a composition (e.g., a pharmaceutical composition) will vary depending on the individual and the particular mode of administration. In some embodiments, a composition comprising an IRAK modulator is formulated so that a dose of an IRAK modulator between about 0.01 mg / kg to about 100 mg / kg body weight is administered to an individual. In some embodiments, an IRAK modulator is administered at about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 75 mg / kg, about 0.01 mg / kg to about 500 mg / kg, about 0.01 mg / kg to about 25 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 1.0 mg / kg, about 1.0 mg / kg to about 100 mg / kg, about 1.0 mg / kg to about 75 mg / kg, about 1.0 mg / kg to about 50 mg / kg, about 1.0 mg / kg to about 25 mg / kg, about 1.0 mg / kg to about 50 mg / kg. The invention can be administered orally or parenterally at a dosage level of any of 0 mg / kg to about 10 mg / kg, about 1.0 mg / kg to about 5 mg / kg, about 10 mg / kg to about 100 mg / kg, about 10 mg / kg to about 75 mg / kg, about 10 mg / kg to about 50 mg / kg, about 10 mg / kg to about 25 mg / kg, about 25 mg / kg to about 100 mg / kg, about 25 mg / kg to about 75 mg / kg, about 25 mg / kg to about 50 mg / kg, about 50 mg / kg to about 100 mg / kg, about 50 mg / kg to about 75 mg / kg, or about 75 mg / kg to about 100 mg / kg of subject body weight. In some embodiments, an IRAK modulator is administered orally or parenterally at a dosage level of greater than about any of 0.01 mg / kg, 1.0 mg / kg, 5 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg body weight, 200 mg / kg body weight, 300 mg / kg body weight, 400 mg / kg body weight, or 500 mg / kg body weight of the individual.

[0093] Liquid dosage forms for oral administration of IRAK modulators (e.g., IRAK-4 degraders) include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art (e.g., such as water or other solvents), solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatics.

[0094] Injectable preparations, such as sterile injectable aqueous or oily suspensions or IRAK modulators (e.g., IRAK-4 degradation agents) can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents, for example as solutions in 1,3-butanediol. Among acceptable vehicles and solvents, water, Ringer's solution, USP and isotonic sodium chloride solution can be used. In addition, sterile fixed oils are routinely used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used to prepare injectables.

[0095] Injectable formulations or IRAK modulators (e.g., IRAK-4 degraders) can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0096] Solid dosage forms for oral administration of an IRAK modulator (eg, an IRAK-4 degrader) include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier (such as sodium citrate or dicalcium phosphate) and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarder such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as, for example, cetyl alcohol and glyceryl monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain a buffer.

[0097] Solid compositions of similar types can also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose (lactose or milk sugar) and high molecular weight polyethylene glycol. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings known in the field of pharmaceutical formulation. They can optionally contain opacifiers and can also have a composition that releases one or more active ingredients only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of similar types can also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose (lactose or milk sugar) and high molecular weight polyethylene glycol.

[0098] IRAK modulators (e.g., IRAK-4 degraders) may also be in the form of microencapsulation with one or more excipients as described above. Solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings, controlled release coatings, and other coatings well known in the art of pharmaceutical formulation. In such solid dosage forms, the active compound may be mixed with at least one inert diluent (such as sucrose, lactose, or starch). Such dosage forms may also contain (as normal practice) additional substances in addition to inert diluents (e.g., tableting lubricants) and other tableting aids (such as magnesium stearate and microcrystalline cellulose). In the case of capsules, tablets, and pills, the dosage form may also include a buffer. They may optionally contain an opacifier, and may also have a composition that releases only or preferentially one or more active ingredients in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0099] In some embodiments, an IRAK modulator (e.g., an IRAK-4 degrader) is administered prior to administration of a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle). In some embodiments, an IRAK modulator is administered to an individual about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, one week, or more than one week prior to administration of a gene therapy agent. In some embodiments, an IRAK modulator is administered to an individual less than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, or one week prior to administration of a gene therapy agent. In some embodiments, an IRAK modulator and a gene therapy agent are administered at about the same time (e.g., within about an hour). In some embodiments, an IRAK modulator is administered after administration of a gene therapy agent. In some embodiments, the IRAK modulator is administered to an individual at about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, one week, or more than one week after the administration of the gene therapy agent. In some embodiments, the IRAK modulator is administered to an individual less than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, or one week after the administration of the gene therapy agent.

[0100] In some embodiments, a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) is used in combination with an IRAK modulator (e.g., an IRAK-4 degrader) to treat a disease or disorder suitable for treatment by gene therapy. In some embodiments, the disease or disorder is a monogenic disease or disorder.

[0101] In some embodiments, a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) is used in combination with an IRAK modulator (e.g., an IRAK-4 degrader) to treat CNS disorders. Non-limiting CNS disorders include stroke, Huntington's disease, epilepsy, Parkinson's disease, Lou Gehrig's disease (also known as amyotrophic lateral sclerosis), Alzheimer's disease, corticobasal degeneration or CBD, corticobasal ganglionic degeneration or CBGD, frontotemporal dementia or FTD, progressive supranuclear palsy or PSP, multiple system atrophy or MSA, brain cancer, and lysosomal storage disease (LSD). Other non-limiting examples of disorders of the present invention that can be treated by gene therapy in combination with IRAK modulators include traumatic brain injury, enzyme dysfunction disorders, mental disorders (including post-traumatic stress syndrome), neurodegenerative diseases, and cognitive disorders (including dementia, autism, and depression), and enzyme dysfunction includes, but is not limited to, leukodystrophy (including Canavan disease).

[0102] In some embodiments, a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) is used in combination with an IRAK modulator (e.g., an IRAK-4 degrader) to treat lysosomal storage diseases. As is generally known in the art, lysosomal storage diseases are rare inherited metabolic disorders characterized by lysosomal functional defects. Such disorders are usually caused by a lack of enzymes required for the metabolism of appropriate mucopolysaccharides, glycoproteins, and / or lipids, resulting in pathological accumulation of lysosomal stored cellular materials. Non-limiting examples of lysosomal storage diseases of the invention that can be treated by the therapeutic polypeptides or therapeutic nucleic acids of the invention include Gaucher disease type 2 or 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, mannosidosis, beta mannosidosis, metachromatic leukodystrophy, mucolipidosis type II / III, Niemann-Pick disease type A, Niemann-Pick disease type C, Pompe disease, Sandhoff disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Schendler disease, Slater disease, Tay-Sachs disease, and Wolman disease.

[0103] In some embodiments, a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) is used in combination with an IRAK modulator (e.g., an IRAK-4 degrader) to treat hemophilia A, hemophilia B, age-related macular degeneration, diabetic retinopathy, glaucoma, muscular dystrophy, X-linked myotubular myopathy, spinal muscular atrophy, Leber's congenital amaurosis, choroideremia, Leber's hereditary optic neuropathy, ornithine transcarbamylase (OTC) deficiency, citrullinemia type 1, phenylketonuria (PKU), adrenoleukodystrophy, sickle cell disease, muscular dystrophy, or beta thalassemia.

[0104] In some aspects, the present invention provides a composition for use in the manufacture of a medicament for delivering nucleic acid to a cell of an individual in need, wherein the composition comprises a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles), and wherein the composition is formulated to be used in combination with an IRAK modulator (e.g., an IRAK-4 degrader). In some aspects, the present invention provides a composition for use in the manufacture of a medicament for delivering nucleic acid to a cell of an individual in need, wherein the composition comprises an IRAK modulator (e.g., an IRAK-4 degrader), and wherein the composition is formulated to be used in combination with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles).

[0105] In some aspects, the present invention provides a composition for use in the manufacture of a medicament for treating an individual in need of gene therapy, wherein the composition comprises a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles), and wherein the composition is formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrader). In some aspects, the present invention provides a composition for use in the manufacture of a medicament for treating an individual in need of gene therapy, wherein the composition comprises an IRAK modulator (e.g., an IRAK-4 degrader), and wherein the composition is formulated for use in combination with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles).

[0106] In some aspects, the present invention provides a composition for use in the manufacture of a medicament for regulating an immune response of an individual to gene therapy in need thereof, wherein the composition comprises a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles), and wherein the composition is formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrader). In some aspects, the present invention provides a composition for use in the manufacture of a medicament for regulating an immune response of an individual to gene therapy, wherein the composition comprises an IRAK modulator (e.g., an IRAK-4 degrader), and wherein the composition is formulated for use in combination with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles).

[0107] In some aspects, the present invention provides a composition for use in the manufacture of a medicament for suppressing an immune response of an individual to gene therapy in need thereof, wherein the composition comprises a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles), and wherein the composition is formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrader). In some aspects, the present invention provides a composition for use in the manufacture of a medicament for suppressing an immune response of an individual to gene therapy, wherein the composition comprises an IRAK modulator (e.g., an IRAK-4 degrader), and wherein the composition is formulated for use in combination with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles).

[0108] In some aspects, the present invention provides a composition for use in the manufacture of a medicament for improving gene therapy in an individual in need of gene therapy, wherein the composition comprises a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles), and wherein the composition is formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrader). In some aspects, the present invention provides a composition for use in the manufacture of a medicament for improving gene therapy in an individual, wherein the composition comprises an IRAK modulator (e.g., an IRAK-4 degrader), and wherein the composition is formulated for use in combination with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles).

[0109] In some aspects, the present invention provides a composition for use in the manufacture of a medicament for inducing tolerance to gene therapy in an individual in need of gene therapy, wherein the composition comprises a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles), and wherein the composition is formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrader). In some aspects, the present invention provides a composition for use in the manufacture of a medicament for inducing tolerance to gene therapy in an individual, wherein the composition comprises an IRAK modulator (e.g., an IRAK-4 degrader), and wherein the composition is formulated for use in combination with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles).

[0110] In some aspects, the invention provides a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) for use in delivering nucleic acids to cells of individuals in need, wherein the gene therapy agent is used in combination with an IRAK modulator (e.g., an IRAK-4 degrader). In some aspects, the invention provides an IRAK modulator (e.g., an IRAK-4 degrader) for use in delivering nucleic acids to cells of individuals in need, wherein the IRAK modulator is used in combination with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles).

[0111] In some aspects, the present invention provides a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) for use in treating an individual in need of gene therapy, wherein the gene therapy agent is used in combination with an IRAK modulator (e.g., an IRAK-4 degrader). In some aspects, the present invention provides an IRAK modulator (e.g., an IRAK-4 degrader) for use in treating an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles).

[0112] In some aspects, the present invention provides an IRAK modulator (e.g., an IRAK-4 degrader) for modulating an immune response to gene therapy in an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle).

[0113] In some aspects, the present invention provides an IRAK modulator (e.g., an IRAK-4 degrader) for use in suppressing an immune response to gene therapy in an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle). IRAK modulators

[0114] In some aspects, the present invention provides methods for improving gene therapy by suppressing immune responses (e.g., innate immune responses, adaptive immune responses) to gene therapy agents using IRAK modulators and gene therapy agents. IRAK plays a central role in the protective response to pathogens introduced into the human body by inducing acute inflammation and subsequently inducing additional adaptive immune responses. IRAK is an essential component of the interleukin-1 receptor signaling pathway and some Toll-like receptor signaling pathways. Toll-like receptors (TLRs) detect microorganisms by recognizing specific pathogen-associated molecular patterns (PAMPs), and IL-1R family members respond to interleukin-1 (IL-1) family cytokines. These receptors initiate intracellular signaling cascades through adapter proteins (mainly MyD88).

[0115] The IRAK family consists of IRAK-1, IRAK-2 and IRAK-4 (which are expressed in a variety of human immune cell types), and IRAK-M (also known as IRAK-3) (their expression is mainly limited to monocytes and macrophages). All four IRAK family proteins contain an N-terminal death domain (DD), a ProST domain, and a centrally located kinase domain. IRAK-1, IRAK-2, and IRAK-M also contain a C-terminal domain. DD serves as a platform that allows protein-protein interactions with other DD-containing proteins, the most important of which is the adapter protein myeloid differentiation factor 88 (MyD88). The inventors assume that blocking IRAK function will result in specific blocking of the TLR9 pathway, thereby resulting in specific immunomodulation. In some embodiments, an IRAK regulator is used to block the TLR9 pathway. In some embodiments, the IRAK regulator blocks TLR9 function. In some embodiments, the IRAK regulator is an IRAK inhibitor and / or an IRAK degrader.

[0116] In some embodiments, the IRAK degrader is a bifunctional compound that functions to recruit IRAK kinase to an E3 ubiquitin ligase for degradation. In some embodiments, the IRAK degrader is a modulator of targeted ubiquitination of IRAK kinase.

[0117] Protein degraders are bifunctional compounds comprising the following three components: E3 ubiquitin ligase ligands, linkers, and ligands of target proteins. They induce the formation of ternary complexes by binding both E3 ligases and target proteins simultaneously. The formation of ternary complexes effectively recruits E3 ligases to polyubiquitinate the target of interest, thereby inducing subsequent degradation by proteasomes. Degraders are attractive tools that can be used to induce selective protein knockdown in a reversible and adjustable manner. In some embodiments, IRAK degraders are proteolysis targeting chimerics (PROTAC).

[0118] In some embodiments, the IRAK degrader is an IRAK-1 degrader, an IRAK-2 degrader, an IRAK-M degrader (or an IRAK-3 degrader), or an IRAK-4 degrader.

[0119] Suitable IRAK4 degrader compounds for use in the methods of the invention are described in patent applications WO 2019 / 133531, WO 2020 / 113233, WO 2020 / 264490, WO 2021 / 127283 or WO 2021 / 011868.

[0120] In some embodiments, the IRAK-4 degrading agent comprises a compound of formula [I] described in U.S. Pat. No. 11,117,889: or a pharmaceutically acceptable salt thereof, wherein: X 1 is a divalent moiety selected from the group consisting of a covalent bond, -CH2-, -C(O)-, -C(S)-, and R 1a is hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O)2R, -N(R)2, -Si(R)3, or an optionally substituted C l-4 aliphatic; Each R 2a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Ring A ais a bicyclic or tricyclic ring selected from: Ring B a is a fused ring selected from the following: a 6-membered aryl group containing 0-2 nitrogen atoms, a 5- to 7-membered partially saturated carbocyclic group, a 5- to 7-membered partially saturated heterocyclic group having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur, or a 5-membered heteroaryl group having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur; R 3a is selected from hydrogen, halogen, -OR, -N(R)2 or -SR; Each R 4a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 5a It is hydrogen, C l-4 Aliphatic or -CN; Each R 6a is independently an optionally substituted group selected from the following: 1-6 aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring A is a 4-10 membered saturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring C is phenyl or a 5-10 membered monocyclic or bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen and sulfur; L 2 and L 3 Each of the 1-3 A divalent straight or branched saturated or unsaturated hydrocarbon chain, wherein 1 to 3 methylene units of the chain are independently and optionally replaced by: -O-, -C(O)-, -C(S)-, -C(R)2-, -CH(R)-, -C(F)2-, -N(R)-, -S-, -S(O)2-, or -CR=CR-; Each R 1 are independently hydrogen, R 5, Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CFR2, -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR or -C(O)NR2; Each R is independently hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or: two R groups on the same atom are optionally taken together with their intervening atoms to form an optionally substituted 4-11 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic monocyclic, bicyclic, bridged bicyclic, spirocyclic, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur in addition to the atoms to which they are attached; Each R 2 are independently hydrogen, R 5 , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CF2(R), -CF3, -CR2(OR), -C R2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 4 Selected from hydrogen, or an optionally substituted group selected from: C 1-6 aliphatic or 4-11 membered saturated or partially unsaturated monocyclic, bicyclic, bridged bicyclic or spirocyclic carbocyclic ring or heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring D is phenyl, a 4-10 membered saturated or partially unsaturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Each R 3 are independently hydrogen, R 5 、Halogen、-CN、-NO2、-OR、-SR、-NR 2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2) 2 , -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)O R, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Each R 5 is independently an optionally substituted group selected from the following: C1-6 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; n is 0, 1, or 2; Each m is independently 0, 1, 2, 3 or 4; and p is 0, 1, 2, 3 or 4.

[0121] In some embodiments, the IRAK-4 degrader comprises 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]hept-5-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-((4-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1Hbenzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)piperidin-1-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide and pharmaceutically acceptable salts thereof as described in WO 2021 / 247899. In some embodiments, the IRAK-4 degrader comprises a crystalline form of 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]hept-5-yl-N-(3-(difluoromethyl)-1-((1r,4R)-4-((4-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1Hbenzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)piperidin-1-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide and pharmaceutically acceptable salts thereof.

[0122] In some embodiments, the IRAK-4 degrader comprises a compound of formula [II] or a deuterated form of a compound of formula [II] or a pharmaceutically acceptable salt thereof (described in WO 2021 / 247897).

[0123] The compound of formula [II] is 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]hept-5-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-((4-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1Hbenzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)piperidin-1-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0124] In some embodiments, the IRAK-4 degrader comprises a compound of formula [III] or [IV] or a pharmaceutically acceptable salt thereof (described in ACS Med. Chem. Lett. 2019, 10, 7, 1081-1085). as well as

[0125] In some embodiments, the IRAK-4 degrading agent is 1-(((2S,3S,4S)-3-ethyl-4-fluoro-5-oxopyrrolidin-2-yl)methoxy)-4-(3-(9-(5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamoyl)pyrimidin-2-yl)-3,9-diazaspiro[5.5]undec-3-yl)prop-1-yn-1-yl)-7-methoxyisoquinoline-6-carboxamide.

[0126] In some embodiments, the IRAK-4 degrading agent comprises an IRAK-4 degrading agent described in WO 2019 / 160915, WO 2020 / 006265, or WO 2021 / 168197.

[0127] In some embodiments, the IRAK-4 inhibitor is Zimlovisertib (which is a compound of formula [V]) or a pharmaceutically acceptable salt thereof. In some embodiments, the IRAK-4 degrader is 1-[[(2S,3S,4S)-3-ethyl-4-fluoro-5-oxo-2-pyrrolidinyl]methoxy]-7-methoxy-6-isoquinolinecarboxamide or a pharmaceutically acceptable salt thereof. In some embodiments, the IRAK-4 degrader is a compound with CAS number 1817626-54-2.

[0128] The compound of formula [V] is 1-[[(2S,3S,4S)-3-ethyl-4-fluoro-5-oxo-2-pyrrolidinyl]methoxy]-7-methoxy-6-isoquinolinecarboxamide, which is also known as Zimlovisertib and has a CAS number of 1817626-54-2.

[0129] In some embodiments, the IRAK-4 degrader is PROTAC IRAK4 degrader 1 (MedChem Express, catalog number HY-129966). In some embodiments, the IRAK-4 degrader comprises a compound of formula [VI] or a pharmaceutically acceptable salt thereof. In some embodiments, the IRAK-4 degrader is a compound with CAS number 2360533-90-8.

[0130] The compound of formula [VI] is PROTAC IRAK4 Degrader 1 (Medicinal Chemistry Expression, Inc., catalog number HY-129966) and has a CAS number of 2360533-90-8.

[0131] In some embodiments, the IRAK modulator is an anti-IRAK antibody (e.g., all or a functional portion (i.e., the portion of the anti-IRAK4 antibody that retains its binding to IRAK4 and inhibits IRAK4 activity) of an anti-IRAK4 antibody. In some embodiments, the IRAK modulator is an anti-IRAK antibody described in WO 01 / 051641 or WO 2008 / 091535.

[0132] In some embodiments, the IRAK modulator is an IRAK inhibitor (e.g., an IRAK4 inhibitor). In some embodiments, the IRAK modulator is an IRAK-4 inhibitor described in WO 2017 / 004133, WO 2018 / 081294, WO 2017 / 009806, WO 2015 / 164374, WO 2019 / 192962, WO 2016 / 053769, WO 2017 / 024589, or WO 2016 / 144847.

[0133] In some embodiments, the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or deoxyribozyme targeting IRAK (e.g., IRAK-4). In some embodiments, the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or deoxyribozyme that blocks a TLR pathway. In some embodiments, the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or deoxyribozyme that blocks TLR function. Gene therapy agents

[0134] In some aspects, the present invention provides methods for improving gene therapy by suppressing the innate immune response to gene therapy agents using IRAK modulators and gene therapy agents. In some embodiments, the gene therapy agent is a viral particle or a lipid nanoparticle. In some embodiments, the gene therapy agent is an adeno-associated virus (AAV) particle, an adenovirus particle, a lentiviral particle, or a herpes simplex virus (HAV) particle. In some embodiments, the gene therapy agent is a lipid nanoparticle or a liposome. In some embodiments, the immune response to the gene therapy agent is an immune response to viral particles (e.g., viral capsid proteins, viral envelopes, etc.). In some embodiments, the immune response to the gene therapy agent is an immune response to LNP (e.g., one or more lipids for producing LNP). In some embodiments, the immune response to the gene therapy agent is an immune response to a gene therapy payload, for example, a nucleic acid encoding a therapeutic transgene (a viral genome, a plasmid, end-blocked DNA, mRNA, antisense nucleic acid, siRNA, shRNA, etc.). In some embodiments, the immune response to the gene therapy agent is an immune response to a transgenic product (e.g., a therapeutic polypeptide or a therapeutic nucleic acid). AAV

[0135] In some embodiments, the present invention provides methods for improving gene therapy using IRAK modulators and AAV particles. In AAV particles for gene therapy, a recombinant AAV (rAAV) genome encoding a heterologous nucleic acid (e.g., a therapeutic transgene) is encapsidated in an AAV capsid. In some embodiments, the viral genome comprises one or more of a heterologous nucleic acid and / or the following components operably connected in the transcription direction: a control sequence (including a transcription start sequence and a termination sequence) to form an expression cassette.

[0136] In some embodiments, the rAAV genome comprises one or more AAV inverted terminal repeat (ITR) sequences (typically two AAV ITR sequences). For example, the expression cassette can be flanked at least one functional AAV ITR sequence at the 5' and 3' ends. "Functional AAV ITR sequence" means that the ITR sequence functions as intended for rescue, replication and packaging of AAV virions. See Davidson et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 2000, 97 (7) 3428-32; Passini et al., J. Virol. [Journal of Virology], 2003, 77 (12): 7034-40; and Pechan et al., Gene Ther. [Gene Therapy], 2009, 16: 10-16, all of which are incorporated herein by reference in their entirety. For the purpose of implementing some aspects of the present invention, the recombinant viral genome comprises at least all AAV sequences necessary for encapsidation into an AAV capsid and the physical structure for infection by AAV particles. The AAV ITRs for use in the vectors of the invention need not have wild-type nucleotide sequences (e.g., as described in Gene Ther., 1994, 5:793-801), and may be altered by insertion, deletion, or substitution of nucleotides, or the AAV ITRs may be derived from any of several AAV serotypes. More than 40 AAV serotypes are currently known, and new serotypes and variants of existing serotypes are continually being identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is contemplated as being within the scope of the invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype including but not limited to the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, goat AAV, bovine AAV or mouse AAV ITR, etc.In some embodiments, an AAV nucleic acid (e.g., a rAAV vector) comprises one or more (e.g., in some aspects, two) of the following ITRs: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, goat AAV, bovine AAV, or mouse AAV ITRs, etc. In some embodiments, an AAV particle comprises an AAV vector encoding a heterologous transgene flanked by one or more AAV ITRs.

[0137] In some embodiments, the AAV particle comprises a capsid protein selected from the group consisting of: AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human Bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof. "Functional variant" of AAV capsid means that the variant capsid is able to package the AAV genome to produce infectious AAV virions. In another embodiment, the rAAV particle comprises a capsid protein of an AAV serotype from clade AF.

[0138] In some aspects, the present invention provides AAV particles comprising a recombinant self-complementary genome (e.g., a self-complementary or self-complementary AAV vector). rAAV virus particles with self-complementary vector genomes and methods of using self-complementary AAV genomes are described in U.S. Patent Nos. 6,596,535; 7,125,717; 7,465,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111, each of which is incorporated herein by reference in its entirety. AAV particles comprising a self-complementary genome will rapidly form double-stranded DNA molecules by virtue of their partially complementary sequences (e.g., complementary coding strands and non-coding strands of heterologous nucleic acids). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding the complement of the nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.

[0139] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are connected by a mutated ITR (e.g., the right ITR). In some embodiments, the ITR comprises a polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 1). The mutated ITR comprises a deletion of the D region containing the terminal dissociation sequence. Therefore, when the rAAV genome is replicated, the rep protein will not cut the viral genome at the mutated ITR, and therefore, the recombinant viral genome comprising the following items in 5' to 3' order will be packaged in the viral capsid: AAV ITR, a first heterologous polynucleotide sequence comprising a regulatory sequence, a mutated AAV ITR, a second heterologous polynucleotide in the opposite orientation to the first heterologous polynucleotide, and a third AAV ITR.

[0140] Different AAV serotypes are used to optimize transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). AAV can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes. For example, an AAV particle can contain one or more ITRs and capsids derived from the same AAV serotype, or an AAV particle can contain one or more ITRs derived from an AAV serotype different from the capsid of the AAV particle.

[0141] In some embodiments, the AAV capsid comprises a mutation, for example, the capsid comprises a mutant capsid protein. In some embodiments, the mutation is a tyrosine mutation or a heparin binding mutation. In some embodiments, the mutant capsid protein retains the ability to form an AAV capsid. In some embodiments, the AAV particles comprise AAV2 or AAV5 tyrosine mutant capsids (see, e.g., Zhong L. et al., (2008) Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States] 105 (22): 7827-7832), such as mutations in Y444 or Y730 (numbered according to AAV2). In another embodiment, the AAV particles comprise capsid proteins of AAV serotypes from clade AF (Gao et al., J. Virol. [Journal of Virology] 2004, 78 (12): 6381).

[0142] A variety of methods are known in the art for producing AAV particles for gene therapy, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids (Urabe, M. et al., (2002) Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet. 20(R1):R2-R6). AAV production cultures for producing AAV particles all require; 1) suitable host cells, 2) suitable helper virus functions, 3) AAV rep and cap genes and gene products; 4) nucleic acids flanked by at least one AAV ITR sequence (such as therapeutic nucleic acids); and 5) suitable culture media and culture media components that support AAV production. In some embodiments, suitable host cells are primate host cells. In some embodiments, suitable host cells are cell lines of human origin, such as HeLa, A549, 293 or Perc.6 cells. In some embodiments, suitable helper virus functions are provided by wild-type or mutant adenoviruses (such as temperature-sensitive adenoviruses), herpes viruses (HSV), baculoviruses, or plasmid constructs that provide helper functions. In some embodiments, AAV rep and cap gene products can be from any AAV serotype. Generally, but not necessarily, the AAV rep gene product has the same serotype as the ITR of the rAAV genome, as long as the rep gene product can play a role in replicating and packaging the rAAV genome. Suitable culture media known in the art can be used to produce AAV particles. In some embodiments, the AAV helper function is provided by adenovirus or HSV. In some embodiments, the AAV helper functions are provided by a baculovirus and the host cell is an insect cell (eg, a Spodoptera frugiperda (Sf9) cell).

[0143] One method for producing AAV particles is a triple transfection method. In short, a plasmid containing a rep gene and a capsid gene can be transfected (e.g., using a calcium phosphate method) into a cell line (e.g., HEK-293 cells) together with a helper adenovirus plasmid, and the virus can be collected and optionally purified. Therefore, in some embodiments, AAV particles are produced by triple transfection of a nucleic acid encoding an AAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding an AAV helper virus function into a host cell, wherein the nucleic acid is transfected into the host cell to produce a host cell capable of producing AAV particles.

[0144] In some embodiments, AAV particles can be produced by a production cell line method (see Martin et al., (2013) Human Gene Therapy Methods [Human Gene Therapy Methods] 24: 253-269; U.S. Patent Pre-Grant Publication No. US2004 / 0224411; and Liu, XL et al. (1999) Gene Ther. [Gene Therapy] 6: 293-299). Briefly, a cell line (e.g., HeLa, 293, A549 or Perc.6 cell line) can be stably transfected with a plasmid containing a rep gene, a capsid gene, and a vector genome comprising a promoter-heterologous nucleic acid sequence. The cell line can be screened to select a leading clone for AAV production, which can then be amplified into a production bioreactor and infected with a helper virus (e.g., adenovirus or HSV) to initiate AAV production. The virus can then be harvested, the adenovirus can be inactivated (e.g., by heating) and / or removed, and the AAV particles can be purified. Thus, in some embodiments, AAV particles are produced by a producer cell line comprising one or more of: nucleic acid encoding the rAAV genome, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

[0145] In some embodiments, nucleic acids encoding AAV rep and cap genes and / or AAV viral genomes are stably maintained in production cell lines. In some embodiments, nucleic acids encoding AAV rep and cap genes and / or rAAV genomes are introduced into cell lines on one or more plasmids to produce production cell lines. In some embodiments, AAV rep, AAV cap and AAV genomes are introduced into cells on the same plasmid. In other embodiments, AAV rep, AAV cap and rAAV genomes are introduced into cells on different plasmids. In some embodiments, cell lines stably transfected with plasmids maintain plasmids in multiple passages (e.g., 5, 10, 20, 30, 40, 50 or more than 50 cell passages) of cell lines. For example, one or more plasmids can be replicated as cells replicate, or one or more plasmids can be integrated into the cell genome. A variety of sequences that enable plasmids to replicate autonomously in cells (e.g., human cells) have been identified (see, e.g., Krysan, PJ et al. (1989) Mol. Cell Biol. [Molecular Cell Biology] 9: 1026-1033). In certain embodiments, one or more plasmids may contain a selective marker (e.g., an antibiotic resistance marker) that allows the cells maintaining the plasmid to be selected. Selective markers commonly used in mammalian cells include, but are not limited to, blasticidin, G418, hygromycin B, bleomycin, puromycin and derivatives thereof. Methods for introducing nucleic acid into cells are known in the art, and include, but are not limited to, viral transduction, cationic transfection (e.g., using cationic polymers such as DEAE-dextran or cationic lipids such as lipofectamine), calcium phosphate transfection, microinjection, particle bombardment, electroporation and nanoparticle transfection (for more details, see, for example, Kim, TK and Eberwine, JH (2010) Anal. Bioanal. Chem. [Analysis and Bioanalytical Chemistry] 397: 3173-3178).

[0146] In some embodiments, the production cell line is derived from a primate cell line (e.g., a non-human primate cell line, such as a Vero or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the production cell line is derived from HeLa, 293, A549, or (Crucell) cells. For example, the cell line is HeLa, 293, A549 or (Crusell) cell line or its derivatives.

[0147] In some embodiments, the production cell line is suitable for growth in suspension. As known in the art, anchorage-dependent cells typically cannot grow in suspension without a matrix (such as microcarrier beads). Adapting the cell line to growth in suspension can include, for example, growing the cell line in a rotary culture with a stirring paddle, using a culture medium lacking calcium and magnesium ions to prevent agglomeration (and optionally a defoaming agent), using a culture vessel coated with a siliconized compound, and selecting cells in the culture (rather than in a large mass or on the side of the vessel) at each passage.

[0148] The AAV particles of the invention can be harvested from AAV production cultures by lysing the host cells of the production culture or by harvesting spent culture medium from the production culture, provided that the cells are cultured under conditions known in the art to cause release of AAV particles from intact cells into the culture medium, as described more fully in U.S. Pat. No. 6,566, 118. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0149] In another embodiment, the AAV particles are purified. As used herein, the term "purified" includes the following preparations of AAV particles, which lack at least some other components that may also be present in the natural presence of the AAV particles or the place where they were originally prepared. Thus, for example, the separated AAV particles can be prepared by enriching them from a source mixture (such as a culture lysate or a production culture supernatant) using purification techniques. Enrichment can be measured in a variety of ways, such as by the ratio of DNase-resistant particles (DRP) or genome copies (gc) present in the solution, or by infectivity, or enrichment can be measured relative to a second potential interfering substance present in the source mixture, the second potential interfering substance such as a contaminant, including a production culture contaminant or a process contaminant, including a helper virus, a culture medium component, etc.

[0150] In some embodiments, the AAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtering through a series of depth filters, including, for example, DOHC grade Millipore Millistak + HC Pod filter, A1HC grade Millipore Millistak + HC Pod filter, and 0.2 μm Filter Opticap XL10 Millipore Express SHC hydrophilic membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtering through any cellulose acetate filter of 0.2 μm or greater pore size known in the art.

[0151] In some embodiments, The AAV production culture harvest is further processed to digest any high molecular weight DNA present in the production culture. Digestion was performed under standard conditions known in the art, including, for example, 1-2.5 units / ml of The concentration of 50% HCl is 0.1% HCl and the ... temperature ranges from ambient temperature to 37°C for a period of time ranging from 30 minutes to several hours.

[0152] AAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating AAV particles; AAV capture by apatite chromatography; heat inactivation of helper virus; AAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and AAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. In some embodiments, the method includes all steps in the order described below. Methods for purifying AAV particles can be found in, for example, Xiao et al., (1998) Journal of Virology 72: 2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO 2010 / 148143. Adenovirus

[0153] In some embodiments, the present invention provides methods for improving gene therapy using IRAK modulators and adenoviral particles. Adenoviral vectors for gene therapy are typically adenoviral particles having a recombinant adenoviral (rAd) genome that contains one or more heterologous sequences (i.e., nucleic acid sequences of non-adenoviral origin) between two adenoviral ITRs encapsidated into an adenoviral capsid. In some embodiments, the heterologous sequence encodes a therapeutic transgene. In some embodiments, the rAd genome lacks one or more E1 genes or contains defective copies of one or more E1 genes, which renders the adenovirus replication-defective. Adenoviruses are large (approximately ) includes a linear double-stranded DNA genome within a non-enveloped icosahedral capsid. Adenoviruses have large genomes that can incorporate heterologous sequences of more than 30 kb (e.g., replacing E1 and / or E3 regions), making them uniquely suited for use with larger heterologous genes. They are also known to infect dividing and non-dividing cells and do not naturally integrate into the host genome (although hybrid variants may have this ability). In some embodiments, the adenoviral vector may be a first-generation adenoviral vector with a heterologous sequence replacing E1. In some embodiments, the adenoviral vector may be a second-generation adenoviral vector with additional mutations or deletions in E2A, E2B, and / or E4. In some embodiments, the adenoviral vector may be a third-generation or internally destroyed (gutted) adenoviral vector that lacks all viral encoding genes (retaining only ITRs and packaging signals) and requires a trans-acting helper adenovirus for replication and packaging. Adenoviral particles have been studied for use as vectors for transient transfection of mammalian cells and as gene therapy vectors. For further description, see, e.g., Danthinne, X. and Imperiale, MJ (2000) Gene Ther. 7:1707-14; and Tatsis, N. and Ertl, HC (2004) Mol. Ther. 10:616-29.

[0154] In some embodiments, the adenoviral particles comprise a rAd genome containing a therapeutic transgene. The use of any adenoviral serotype is considered to be within the scope of the present invention. In some embodiments, the adenoviral particles are derived from adenoviral serotypes including but not limited to AdHu2, AdHu3, AdHu4, AdHu5, AdHu7, AdHu11, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad 3 type, canine Ad 2 type, sheep Ad and porcine Ad 3 type. The adenoviral particles also comprise capsid proteins. In some embodiments, the adenoviral particles include one or more foreign viral capsid proteins. Such combinations can be referred to as pseudotyped adenoviral particles. In some embodiments, the foreign viral capsid proteins used in the pseudotyped adenoviral particles are derived from foreign viruses or derived from another adenoviral serotype. In some embodiments, the foreign viral capsid proteins are derived from (including but not limited to) reovirus type 3. Examples of vector and capsid protein combinations used in pseudotyped adenoviral particles can be found in the following references (Tatsis, N. et al. (2004) Mol. Ther. [Molecular Therapy] 10 (4): 616-629; and Ahi, Y. et al. (2011) Curr. Gene Ther. [Current Gene Therapy] 11 (4): 307-320). Different adenoviral serotypes can be used to optimize transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). Tissues or cells targeted by specific adenoviral serotypes include, but are not limited to, lung (e.g., HuAd3), spleen and liver (e.g., HuAd37), smooth muscle, synoviocytes, dendritic cells, cardiovascular cells, tumor cell lines (e.g., HuAd11), and dendritic cells (e.g., HuAd5, HuAd30, or HuAd35 pseudotyped with reovirus type 3). For further description, see Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4):307-320; Kay, M. et al. (2001) Nat. Med. 7(1):33-40; and Tatsis, N. et al. (2004) Mol. Ther. 10(4):616-629.

[0155] Many methods for producing adenoviral particles are known in the art. For example, for an internally destroyed adenoviral vector, the adenoviral vector genome and the helper adenoviral genome can be transfected into a packaging cell line (e.g., a 293 cell line). In some embodiments, the helper adenoviral genome can contain recombination sites flanking its packaging signal, and the two genomes can be transfected into a packaging cell line expressing a recombinase (e.g., a Cre / loxP system can be used), so that the adenoviral vector of interest is more efficiently packaged than the helper adenovirus (see, e.g., Alba, R. et al. (2005) Gene Ther. [Gene Therapy] 12 Supplement 1: S18-27). Adenoviral vectors can be harvested and purified using standard methods (such as those described herein). Lentivirus

[0156] In some embodiments, the present invention provides a method for improving gene therapy using an IRAK regulator and a lentiviral particle. The lentiviral vector for gene therapy is typically a lentiviral particle with a recombinant lentiviral genome, which contains one or more heterologous sequences (i.e., nucleic acid sequences from non-lentiviral sources) between two long terminal repeats (LTR). In some embodiments, the heterologous sequence encodes a therapeutic transgene. A lentivirus is a positive ssRNA retrovirus with a genome of about 10kb. The lentivirus is integrated into the genome of a dividing and non-dividing cell. For example, lentiviral particles can be produced by transfecting multiple plasmids (typically lentiviral genomes and genes required for replication and / or packaging are separated to prevent viral replication) into a packaging cell line, which packages the modified lentiviral genome into lentiviral particles. In some embodiments, lentiviral particles can refer to first-generation vectors lacking envelope proteins. In some embodiments, lentiviral particles can refer to second-generation vectors lacking all genes except gag / pol and tat / rev regions. In some embodiments, the lentiviral particle may refer to a third generation vector that contains only endogenous rev, gag, and pol genes and has a chimeric LTR for transduction without a tat gene (see Dull, T. et al. (1998) J. Virol. [Journal of Virology] 72: 8463-71). For further description, see Durand, S. and Cimarelli, A. (2011) Viruses [Virus] 3: 132-59.

[0157] The use of any lentiviral vector is considered to be within the scope of the present invention. In some embodiments, the lentiviral vector is derived from a lentivirus including but not limited to human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), visnavirus (VV) and caprine arthritis encephalitis virus (CAEV). The lentiviral particle also comprises a capsid protein. In some embodiments, the lentiviral particle includes one or more foreign viral capsid proteins. Such combinations can be referred to as pseudotyped lentiviral particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped lentiviral particles is derived from a foreign virus. In some embodiments, the foreign viral capsid protein used in the pseudotyped lentiviral particles is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with ubiquitous cell receptors to provide a wide range of tissue tropism for pseudotyped lentiviral particles. Furthermore, VSV-GP is thought to provide increased stability to pseudotyped lentiviral particles. In other embodiments, the foreign viral capsid protein is derived from, including but not limited to, Chandipura virus, rabies virus, Mokola virus, lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Sindbis virus, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Reston ebola virus, Zaire ebola virus, Marburg virus, Lassa virus, avian leukosis virus (ALV), sheep lung adenomatous retrovirus (JSRV), Moloney murine leukemia virus (MLV), Gibbon ape leukemia virus (GALV), feline endogenous retrovirus (RD114), human T-lymphotropic virus 1 (HTLV-1), human foamy virus, Mae-Visna virus (MVV), SARS-CoV, Sendai virus, respiratory syncytial virus (RSV), human parainfluenza virus type 3, hepatitis C virus (HCV), influenza virus, fowl plague virus (FPV) or Autographa californica nuclear polyhedrosis virus (AcMNPV). Examples of vector and capsid protein combinations used in pseudotyped lentiviral particles can be found, for example, in Cronin, J. et al. (2005). Curr. Gene Ther. [Current Gene Therapy] 5(4): 387-398. Different pseudotyped lentiviral particles can be used to optimize transduction of specific target cells or to target specific cell types within a specific target tissue (e.g., diseased tissue).For example, tissues targeted by specifically pseudotyped lentiviral particles include, without limitation, liver (e.g., pseudotyped with VSV-G, LCMV, RRV or SeV F proteins), lung (e.g., pseudotyped with Ebola, Marburg, SeV F and HN or JSRV proteins), pancreatic islet cells (e.g., pseudotyped with LCMV proteins), central nervous system (e.g., pseudotyped with VSV-G, LCMV, rabies or Merckola proteins), retina (e.g., pseudotyped with VSV-G or Merckola proteins), monocytes or muscle (e.g., pseudotyped with Merckola or Ebola proteins), hematopoietic system (e.g., pseudotyped with RD114 or GALV proteins), or cancer cells (e.g., pseudotyped with GALV or LCMV proteins). For further description, see Cronin, J. et al. (2005). Curr. Gene Ther. 5(4):387-398; and Kay, M. et al. (2001) Nat. Med. 7(1):33-40.

[0158] Many methods for producing lentiviral particles are known in the art. For example, for third-generation lentiviral vectors, a vector containing a recombinant lentiviral genome of interest with gag and pol genes can be co-transfected into a packaging cell line (e.g., 293 cell line) together with a vector containing the rev gene. The recombinant lentiviral genome of interest also contains a chimeric LTR that promotes transcription in the absence of Tat (see Dull, T. et al. (1998) J. Virol. [Journal of Virology] 72: 8463-71). The lentiviral vector can be harvested and purified using the methods described herein (e.g., Segura MM et al., (2013) Expert Opin Biol Ther. [Expert Opinion on Biological Therapy] 13 (7): 987-1011). HSV

[0159] In some embodiments, the present invention provides methods for improving gene therapy using IRAK modulators and HSV particles. HSV vectors for gene therapy are typically HSV particles with a recombinant HSV genome, which contains one or more heterologous sequences (i.e., nucleic acid sequences from non-HSV sources) between two terminal repeats (TRs). In some embodiments, the heterologous sequence encodes a therapeutic transgene. HSV is an enveloped double-stranded DNA virus with an approximately 152kb genome. Advantageously, about half of its genes are non-essential and can be deleted to accommodate heterologous sequences. HSV particles infect non-dividing cells. In addition, they naturally establish latency in neurons, travel by reverse translocation, and can be transferred across synapses, making them conducive to transfection of neurons and / or conducive to gene therapy methods involving the nervous system. In some embodiments, HSV particles can be replication-deficient or replication-type (e.g., capable of performing a single replication cycle by inactivating one or more late genes). For further description, see Manservigi, R. et al. (2010) Open Virol. J. [Open Virology Magazine] 4: 123-56.

[0160] In some embodiments, the HSV particles include a recombinant HSV genome containing a transgene. The use of any HSV vector is considered to be within the scope of the present invention. In some embodiments, the HSV vector is derived from an HSV serotype, including but not limited to HSV-1 and HSV-2. The HSV particles also include capsid proteins. In some embodiments, the HSV particles include one or more foreign virus capsid proteins. Such combinations can be referred to as pseudotyped HSV particles. In some embodiments, the foreign virus capsid proteins used in the pseudotyped HSV particles are derived from foreign viruses or derived from another HSV serotype. In some embodiments, the foreign virus capsid proteins used in the pseudotyped HSV particles are vesicular stomatitis virus glycoproteins (VSV-GP). VSV-GP interacts with ubiquitous cell receptors to provide a wide range of tissue tropism for pseudotyped HSV particles. In addition, VSV-GP is considered to provide higher stability for pseudotyped HSV particles. In other embodiments, foreign virus capsid proteins can be from different HSV serotypes. For example, an HSV-1 vector can contain one or more HSV-2 capsid proteins. Different HSV serotypes can be used to optimize transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). Tissues or cells targeted by specific adenovirus serotypes include, but are not limited to, the central nervous system and neurons (e.g., HSV-1). For further description, see Manservigi, R. et al. (2010) Open Virol J [Open Virology Journal] 4: 123-156; Kay, M. et al. (2001) Nat. Med. [Natural Medicine] 7 (1): 33-40; and Meignier, B. et al. (1987) J. Infect. Dis. [Infectious Diseases Journal] 155 (5): 921-930.

[0161] Many methods for producing HSV particles are known in the art. Standard methods (such as those described herein) can be used to harvest and purify HSV vectors. For example, for replication-defective HSV vectors, the target HSV genome lacking all immediate early (IE) genes can be transfected into a complementary cell line that provides genes (such as ICP4, ICP27, and ICP0) required for virus production (see, for example, Samaniego, LA et al. (1998) J. Virol. [Journal of Virology] 72: 3307-20). The described method (e.g., Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology [Herpes Simplex Virus Methods in Molecular Biology] 1144: 63-79) can be used to harvest and purify HSV vectors. Non-viral gene therapy agents

[0162] In certain embodiments, the invention provides methods for gene therapy using IRAK modulators and non-viral gene transfer methods. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids compounded with delivery systems. For example, carriers can be compounded with lipids (e.g., cationic lipids or neutral lipids), liposomes, polycations, lipid nanoparticles, or agents that enhance cell uptake of nucleic acids. Nucleic acids can be compounded with agents suitable for any delivery method described herein. In certain embodiments, nucleic acid encoding therapeutic transgenes.

[0163] Lipid nanoparticles for gene therapy typically include a vector genome encapsulated in a lipid particle or a vector genome complexed with lipids. In some embodiments, the heterologous sequence encodes a therapeutic transgene. In some embodiments, the vector genome is formulated in a cationic liposome / nucleic acid complex (lipoplex) nanoparticle or liposome. In some embodiments, the cationic liposome / nucleic acid complex nanoparticle formulation for gene therapy agents includes a synthetic cationic lipid (R)-N,N,N-trimethyl-2,3-dioleoyloxy-1-propylammonium chloride (DOTMA) and a phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the DOTMA / DOPE liposome component is optimized for delivery and targeting cells in an individual.

[0164] In some embodiments, a nucleic acid comprising a vector genome is mixed with a pharmaceutical composition comprising one or more cationic lipids, including, for example, (R)-N,N,N-trimethyl-2,3-dioleoyloxy-1-propylammonium chloride (DOTMA) and phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the pharmaceutical composition comprises at least one lipid. In some embodiments, the pharmaceutical composition comprises at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphiphilic molecule (e.g., a molecule comprising at least one hydrophilic and lipophilic portion) is a cationic lipid within the meaning of the present invention. In some embodiments, the positive charge is contributed by at least one cationic lipid, and the negative charge is contributed by the nucleic acid. In some embodiments, the pharmaceutical composition comprises at least one auxiliary lipid. The auxiliary lipid can be a neutral lipid or an anionic lipid. The auxiliary lipid can be a natural lipid (e.g., phospholipid) or an analog of a natural lipid, or a completely synthetic lipid or lipid-like molecule that has no similarity to a natural lipid. In one embodiment, the cationic lipid and / or the auxiliary lipid are lipids that form a bilayer. Examples of helper lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analog or derivative thereof, cholesterol (Chol) or an analog or derivative thereof, and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analog or derivative thereof.

[0165] In some embodiments, the molar ratio of at least one cationic lipid to at least one helper lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1: 1. In some embodiments, at this ratio, the molar amount of the cationic lipid is generated by multiplying the molar amount of the cationic lipid by the number of positive charges in the cationic lipid.

[0166] In some embodiments, the lipid is contained in a vesicle that encapsulates the vector genome. The vesicle can be a multilamellar vesicle, a unilamellar vesicle, or a mixture thereof. The vesicle can be a liposome. Vector genome

[0167] In some embodiments, the invention provides methods of using IRAK modulators with gene therapy agents for delivering a therapeutic transgene to a desired target in an individual. In some embodiments, the gene therapy agent comprises a vector genome for delivering and expressing a therapeutic transgene in a desired target in an individual.

[0168] The present invention contemplates the use of gene therapy agents for the introduction of one or more nucleic acid sequences encoding therapeutic polypeptides and / or nucleic acids for packaging into viral particles (for viral gene therapy agents). The vector genome may include any element that establishes expression of the therapeutic polypeptide and / or nucleic acid, such as a promoter, ITRs of the present disclosure, ribosome binding elements, terminators, enhancers, selectable markers, introns, poly A signals, and / or replication origins.

[0169] In certain embodiments, therapeutic transgenic encoding therapeutic polypeptide.Therapeutic polypeptide can for example provide polypeptide and / or enzymatic activity that does not exist in cell or organism or exists with the level of reduction.Alternately, therapeutic polypeptide can provide polypeptide and / or enzymatic activity that indirectly offsets the imbalance in cell or organism.For example, the therapeutic polypeptide that is used for the obstacle relevant to the accumulation of metabolites caused by metabolic enzyme or activity defect can provide the metabolic enzyme or activity of missing, or it can provide the alternative metabolic enzyme or activity that causes metabolite reduction.Therapeutic polypeptide can also be used for reducing the activity of polypeptide (for example, overexpressed, activated by functional acquisition mutation or its activity is otherwise misregulated) by for example acting as dominant negative polypeptide.

[0170] The vector genome of the present invention can encode polypeptides that are intracellular proteins, anchored in the cell membrane, retained in the cell, or secreted by cells transduced with the vector of the present invention. With respect to polypeptides secreted by cells receiving the vector; these polypeptides can be soluble (i.e., not attached to the cell). For example, soluble polypeptides lack a transmembrane region and are secreted from the cell. Techniques for identifying and removing nucleic acid sequences encoding transmembrane domains are known in the art.

[0171] In some embodiments, the vector genome of the present invention encodes a polypeptide for treating a disease or disorder in an individual. Diseases and disorders treated by the gene therapy agents of the present invention include, but are not limited to, Huntington's disease (HD), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), metachromatic leukodystrophy (MLD), amyotrophic lateral sclerosis (ALS), age-related macular degeneration (AMD), congenital muscular dystrophy (CMD), phenylketonuria (PKU), muscular dystrophy (MD), A1AT deficiency, focal segmental glomerulosclerosis (FSGS), cystinuria, hemophilia A, hemophilia B, Gaucher disease (GBA), Parkinson's disease (PD) and Pompe disease.

[0172] In some embodiments, the therapeutic polypeptide is huntingtin protein (HTT), tau protein, amyloid precursor protein, alpha-synuclein, pseudoarylsulfatase (ARSA), superoxide dismutase 1 (SOD1), phenylalanine hydroxylase (PAH), dystrophin, alpha-1-antitrypsin (A1AT), cysteine ​​transporter, factor VIII (FVIII), factor IX (FIX), acid β-glucosidase, glial-derived growth factor (GDNF), brain-derived growth factor (BDNF), tyrosine hydroxylase (TH), GTP cyclohydrolase (GTPCH) and / or amino acid decarboxylase (AADC) or alpha-glucosidase.

[0173] In some embodiments, heterologous nucleic acid encoding, for example, can be used to replace or knock down one or more defective gene therapeutic nucleic acids. In some embodiments, therapeutic nucleic acids can include but are not limited to DNA, siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozymes or deoxyribozymes. Therefore, therapeutic nucleic acids can encode RNA, when the RNA is transcribed from the nucleic acid of the carrier, the RNA can treat the disorder by interfering with the translation or transcription of the abnormal or excessive protein associated with the obstacle of the present invention. For example, the nucleic acid of the present invention can encode RNA, which treats the disorder by eliminating or reducing the mRNA encoding abnormal and / or excessive protein with high specificity. Therapeutic RNA sequences include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA) and / or ribozymes (such as hammerhead and hairpin ribozymes), which can treat the disorder by eliminating or reducing the mRNA encoding abnormal and / or excessive protein with high specificity.

[0174] In some embodiments, therapeutic polypeptides or therapeutic nucleic acids are used to treat CNS disorders. Without wishing to be bound by theory, it is believed that therapeutic polypeptides or therapeutic nucleic acids can be used to replace mutant genes with wild-type or improved genes, reduce or eliminate the expression and / or activity of polypeptides associated with disorders, or enhance the expression and / or activity of polypeptides to supplement defects associated with disorders (e.g., mutations in genes whose expression shows similar or related activities). Non-limiting examples of disorders of the present invention that can be treated by therapeutic polypeptides or therapeutic nucleic acids of the present invention (exemplary genes that can be targeted or provided are provided in brackets for each disorder) include stroke (e.g., caspase-3, Beclin1, Ask1, PAR1, HIF1α, PUMA, and / or any gene described in Fukuda, AM and Badaut, J. (2013) Genes (Basel) 4: 435-456), Huntington's disease (mutant HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and / or Boison, D. (2 010) Epilepsy [Epilepsia] 51: 1659-1668), Parkinson's disease (alpha-synuclein), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis; SOD1), Alzheimer's disease (tau protein, amyloid precursor protein), corticobasal degeneration or CBD (tau protein), corticobasal ganglionic degeneration or CBGD (tau protein), frontotemporal dementia or FTD (tau protein), progressive supranuclear palsy or PSP (tau protein), multiple system atrophy or MSA (alpha-synuclein), brain cancer (e.g., mutant or overexpressed oncogenes involved in brain cancer), and lysosomal storage diseases (LSD). Disorders of the invention can include those involving large areas of the cortex (e.g., more than one functional area of ​​the cortex, more than one lobe of the cortex, and / or the entire cortex). Other non-limiting examples of disorders of the invention that can be treated by the therapeutic polypeptides or therapeutic nucleic acids of the invention include traumatic brain injury, enzyme dysfunction disorders, psychiatric disorders (including post-traumatic stress syndrome), neurodegenerative diseases, and cognitive disorders (including dementia, autism, and depression). Enzyme dysfunction disorders include, but are not limited to, leukodystrophy (including Canavan's disease) and any of the lysosomal storage diseases described below.

[0175] In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat lysosomal storage diseases. As is generally known in the art, lysosomal storage diseases are rare inherited metabolic disorders characterized by defective lysosomal function. Such disorders are usually caused by a deficiency of enzymes required for proper mucopolysaccharide, glycoprotein and / or lipid metabolism, resulting in pathological accumulation of lysosomal stored cellular material. Non-limiting examples of lysosomal storage diseases of the invention that can be treated by the therapeutic polypeptides or therapeutic nucleic acids of the invention (exemplary genes that can be targeted or provided are provided in parentheses for each disorder) include Gaucher disease type 2 or type 3 (acid β-glucosidase, GBA), GM1 gangliosidosis (β-galactosidase-1, GLB1), Hunter disease (iduronate-2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), alpha mannosidase (alpha mannosidase, such as α-D-mannosidase, MAN2B1), beta mannosidase (β-mannosidase, MANBA), metachromatic leukodystrophy (pseudoarylsulfatase A, ARSA), mucolipidosis type II / III (N-acetylglucosamine-1-phosphotransferase, GNPTAB), Niemann-Pick disease type A ( acid sphingomyelinase, ASM), Niemann-Pick disease type C (Niemann-Pick protein C, NPC1), Pompe disease (acid alpha-1,4-glucosidase, GAA), Sandhoff disease (hexosaminidase beta subunit, HEXB), Sanfilippo disease type A (N-sulfoglucosamine sulfohydrolase, MPS3A), Sanfilippo disease type B (N-alpha-acetylglucosaminidase, NAGLU), Sanfilippo disease type C (heparin acetylcopherol, ACE Enzyme A-CoA: α-glucosaminidase N-acetyltransferase, MPS3C), Sanfilippo type D disease (N-acetylglucosamine-6-sulfatase, GNS), Schendler disease (α-N-acetylgalactosaminidase, NAGA), Slae disease (β-glucuronidase, GUSB), Tay-Sachs disease (hexosaminidase alpha subunit, HEXA), and Wolman disease (lysosomal acid lipase, LIPA).

[0176] In some embodiments, the therapeutic polypeptide encodes factor VIII, factor IX, myotubularin, survival motor neuron protein (SMN), retinoid isomerohydrolase (RPE65), NADH-ubiquinone oxidoreductase chain 4, choroideremia protein (CHM), ornithine transcarbamylase, arginylsuccinate synthetase, β-globin, γ-globin, phenylalanine hydroxylase, adrenoleukodystrophy protein (ALD), dystrophin, truncated dystrophin, anti-VEGF agent or a functional variant thereof.

[0177] In some embodiments, the heterologous nucleic acid is operably linked to a promoter. Exemplary promoters include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter and CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG promoter; Niwa et al., Gene [Gene], 1991, 108 (2): 193-9) and elongation factor 1-alpha promoter (EFl-alpha) promoter (Kim et al., Gene [Gene], 1990, 91 (2): 217-23 and Guo ... Ther. [Gene Therapy], 1996, 3(9):802-10). In some embodiments, the promoter comprises a human β-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β-actin (CBA) promoter. The promoter may be a constitutive promoter, an inducible promoter, or a repressible promoter. In some embodiments, the present invention provides a recombinant vector comprising a nucleic acid encoding a heterologous transgene of the present disclosure operably linked to a CBA promoter. Exemplary promoters and descriptions can be found, for example, in U.S. Pre-Grant Publication 20140335054.

[0178] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 13-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter [Invitrogen].

[0179] Inducible promoters allow for regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors (such as temperature), or the presence of a specific physiological state, such as the acute phase, a specific differentiation state of the cell, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen, Clontech, and Ariad. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 2000). 98 / 10088); ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), tetracycline repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetracycline inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al. =Also, Curr. Opin. Chem. Biol. [Current Opinion in Chemical Biology], 2:512-518 (1998)), the RU486 inducible system (Wang et al., Nat. Biotech. [Nature Biotechnology], 15:239-243 (1997) and Wang et al., Gene Ther. [Gene Therapy], 4:432-441 (1997)) and the rapamycin inducible system (Magari et al., J. Clin. Invest. [Clinical Examination Journal], 100:2865-2872 (1997)). Other types of inducible promoters useful in this context are promoters that are regulated by specific physiological states, such as temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells.

[0180] In another embodiment, a natural promoter or fragment thereof for the transgene will be used. A natural promoter may be used when it is desired that expression of the transgene should mimic natural expression. A natural promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In additional embodiments, other natural expression control elements (such as enhancer elements, polyadenylation sites, or Kozak consensus sequences) may also be used to mimic natural expression.

[0181] In certain embodiments, regulatory sequences confer tissue-specific gene expression capability. In some cases, tissue-specific regulatory sequences are combined with tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art.

[0182] In some embodiments, the vector comprises an intron. For example, in some embodiments, the intron is a chimeric intron derived from chicken β-actin and rabbit β-globin. In some embodiments, the intron is a minute virus of mice (MVM) intron.

[0183] In some embodiments, the vector comprises a polyadenylation (poly A) sequence. Various examples of polyadenylation sequences are known in the art, such as the bovine growth hormone (BGH) poly (A) sequence (see, e.g., Accession No. EF592533), the SV40 polyadenylation sequence, and the HSV TK pA polyadenylation sequence. Methods for selecting patients for treatment with gene therapy agents and IRAK modulators

[0184] In some aspects, the present invention provides a method for delivering nucleic acid to cells of an individual, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles or lipid nanoparticles), b) analyzing the expression of one or more cytokines of these innate immune cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent identifies an individual with innate immunity to the gene therapy agent, c) administering an IRAK modulator (e.g., an IRAK-4 degrader) to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b). In some embodiments, the innate immune cells are dendritic cells, monocytes, macrophages or natural killer (NK) cells. In some embodiments, the method further comprises the steps of isolating innate immune cells from the individual and then incubating these innate immune cells with the gene therapy agent. In some embodiments, the method further comprises the steps of isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0185] In some aspects, the present invention provides a method for treating an individual in need thereof, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles), b) analyzing the expression of one or more cytokines of these innate immune cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent identifies an individual with innate immunity to the gene therapy agent, c) administering an IRAK modulator (e.g., an IRAK-4 degrader) to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b). In some embodiments, the innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells. In some embodiments, the method further comprises the steps of isolating innate immune cells from the individual and then incubating these innate immune cells with the gene therapy agent. In some embodiments, the method further comprises the steps of isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0186] In some aspects, the present invention provides a method for selecting an individual for treatment with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) and an IRAK modulator (e.g., an IRAK-4 degrader), the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the expression of one or more cytokines of the dendritic cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent identifies the individual for treatment with the gene therapy agent and the IRAK modulator, c) selecting the individual identified in step b) for treatment with the gene therapy agent and the IRAK modulator. In some embodiments, the method further comprises the following steps: d) administering an IRAK modulator to the individual identified in step b), and e) administering the gene therapy agent to the individual identified in step b). In some embodiments, the innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells. In some embodiments, the method further comprises the following steps: isolating innate immune cells from the individual and then incubating the innate immune cells with the gene therapy agent. In some embodiments, the method further comprises the steps of isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0187] In some embodiments, these innate immune cells are separated from peripheral blood mononuclear cells from the individual. In some embodiments, the innate immune cells are dendritic cells. In some embodiments, the dendritic cells are derived (e.g., differentiated) from the monocytes of the individual. In some embodiments, these monocytes are separated from peripheral blood mononuclear cells from the individual. In some embodiments, these monocytes are CD14+ monocytes. In some embodiments, these monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from these monocytes. In some embodiments, monocytes are incubated with dendritic cell culture medium for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 days to derive dendritic cells from monocytes. In some embodiments, before incubating with the gene therapy agent of step c), dendritic cells are re-plated. In some embodiments, before incubating with the gene therapy agent, dendritic cells are re-plated in a microwell dish.

[0188] In some embodiments, dendritic cells are administered with a viral gene therapy agent at a concentration of about 1×10 3 About 1×10 5 or about 1×10 4 In some embodiments, dendritic cells are incubated with the gene therapy agent at an MOI of less than about 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 or 5×10 5 The cells were incubated with any MOI.

[0189] In some embodiments, dendritic cells are incubated with a non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL. In some embodiments, dendritic cells are incubated with a non-viral gene therapy agent at a concentration of about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 100 ng / mL, about 100 ng / mL to about 1 μg / mL, about 1 μg / mL to about 10 μg / mL, about 10 μg / mL to about 100 μg / mL, or about 100 μg / mL to about 1 mg / mL.

[0190] In some embodiments, the dendritic cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours. In some embodiments, the dendritic cells are incubated with the gene therapy agent for between about 6 hours and about 48 hours, between about 6 hours and about 36 hours, between about 6 hours and about 24 hours, between about 6 hours and about 18 hours, between about 6 hours and about 12 hours, between about 12 hours and about 48 hours, between about 12 hours and about 36 hours, between about 12 hours and about 24 hours, between about 12 hours and about 18 hours, between about 18 hours and about 48 hours, between about 18 hours and about 36 hours, between about 18 hours and about 24 hours, between about 24 hours and about 48 hours, between about 24 hours and about 36 hours, or between about 36 hours and about 48 hours.

[0191] In some embodiments, by contacting specific immune cells from multiple individuals with a gene therapy agent and determining changes in the expression of one or more cytokines associated with an innate immune response, a cytokine signature in a specific immune cell (e.g., dendritic cells, monocytes, macrophages, NK cells, etc.) is determined for a gene therapy agent, wherein the commonality of the expression change (e.g., increase or decrease in expression) of the one or more cytokines indicates the presence of a cytokine signature. In some embodiments, the cytokines associated with an innate immune response are associated with a toll-like receptor (TLR) pathway (e.g., TLR2, TLR3, TLR4, or TLR9 pathway). In some embodiments, the cytokine signature includes changes in the expression of any one of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cytokines. In some embodiments, a plurality of individuals include any one of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 individuals. In some embodiments, the commonality of expression changes includes similar changes in the expression levels of cytokines in innate immune cells in individuals greater than about 25%, 50%, 75%, or 90% of a plurality of individuals.

[0192] In some embodiments, the cytokine signature includes the expression of one or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of two or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of three or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of four or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of increases in IL6, TNFα and IL-1β.

[0193] In some embodiments, the innate immune cell is a dendritic cell, and the cytokine signature includes the expression of one or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the innate immune cell is a dendritic cell, and the cytokine signature includes the expression of two or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the innate immune cell is a dendritic cell, and the cytokine signature includes the expression of three or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of four or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the innate immune cell is a dendritic cell, and the cytokine signature includes the expression of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of increases in IL6, TNFα and IL-1β.

[0194] In certain embodiments, compared with the expression of the cytokine in a suitable control, the expression of these cytokines in the cytokine signature increases.The example of a suitable control includes the cytokine signature of the innate immune cells not hatched with a gene therapy agent (in the absence of a gene therapy agent), and the expression of cytokines in the cytokine signature of the same or similar innate immune cells before hatching with a gene therapy agent (for example, wherein the cytokine signature includes the expression of one or more of the increase in IL6, TNFα, IL-1β, MCP1 and MIP-1α). In certain embodiments, the cytokine signature includes the expression of two or more of the increase in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In certain embodiments, the cytokine signature includes the expression of three or more of the increase in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In certain embodiments, the cytokine signature includes the expression of four or more of the increase in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, and IL-1β. In some embodiments, any one of an increase in expression of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or more than 100% identifies an individual treated with a gene therapy agent and an IRAK modulator.

[0195] In some embodiments, the expression of cytokines in the cytokine signature of dendritic cells incubated in the absence of a gene therapy agent is compared to the expression of cytokines in the cytokine signature of dendritic cells before incubation with a gene therapy agent, or compared to the expression of cytokines in the cytokine signature of dendritic cells before incubation with a gene therapy agent, the expression of cytokines in the cytokine signature is increased, wherein the cytokine signature includes the expression of one or more of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of two or more of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of three or more of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of four or more of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, and IL-1β. In some embodiments, any of an increase in expression of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or more than 100% identifies an individual treated with a gene therapy agent and an IRAK modulator. Pharmaceutical composition

[0196] In some aspects, the present invention relates to a pharmaceutical composition comprising a gene therapy agent as described herein (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles or lipid nanoparticles) and / or an IRAK modulator (e.g., an IRAK-4 degrader). The pharmaceutical composition may be suitable for any mode of administration described herein or known in the art.

[0197] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that contribute to the administration of pharmacologically effective substances and can be provided as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolving or suspending in liquids before use. For example, an excipient can impart form or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts for changing osmotic pressure molar concentrations, encapsulating agents, pH buffer substances, and buffers. Such excipients include any medicament suitable for direct delivery to the eye, which can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of various TWEEN compounds, and liquids (such as water, saline, glycerol, and ethanol). Pharmaceutically acceptable salts, such as inorganic acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc., can be included therein; and organic acid salts, such as acetates, propionates, malonates, benzoates, etc. A comprehensive discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., New Jersey 1991). In some embodiments, pharmaceutical compositions comprising rAAV particles described herein and a pharmaceutically acceptable carrier are suitable for administration to humans. Such carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 15th ed., pp. 1035-1038 and 1570-1580).

[0198] Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Saline solutions and dextrose, polyethylene glycol (PEG) and glycerol aqueous solutions can also be used as liquid carriers, particularly for injections. The pharmaceutical composition may further comprise additional ingredients, such as preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting agents or clarifying agents, tackifiers, etc. The pharmaceutical compositions described herein can be packaged in single unit doses or in multiple doses. These compositions are typically formulated as sterile and substantially isotonic solutions. Kits and Products

[0199] The gene therapy agent (e.g., AAV particle, adenoviral particle, lentiviral particle, HSV particle or lipid nanoparticle) and / or IRAK modulator (e.g., IRAK-4 degrader) as described herein can be contained in a kit or article of manufacture designed for use in one of the methods of the invention as described herein.

[0200] In some embodiments, the kit or article further includes instructions for administering an IRAK modulator and / or gene therapy agent. The kit or article described herein may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any method described herein. Suitable packaging materials may also be included, and may be any packaging materials known in the art, including, for example, vials (such as sealed vials), vessels, ampoules, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These products may be further sterilized and / or sealed.

[0201] In some embodiments, the kit or article of manufacture further contains one or more of the buffers and / or pharmaceutically acceptable excipients described herein (e.g., as described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991). In some embodiments, the kit or article of manufacture includes one or more pharmaceutically acceptable excipients, carriers, solutions, and / or additional ingredients described herein. The kits or articles of manufacture described herein may be packaged in single unit dose or in multiple dose form. The contents of the kit or article of manufacture are generally formulated sterile and may be lyophilized or provided as a substantially isotonic solution. Exemplary Embodiments

[0202] The present invention includes the following enumerated embodiments.

[0203] 1. A method for delivering a nucleic acid to a cell of an individual, the method comprising a) administering an IRAK modulator to the individual, and b) administering a gene therapy agent to the individual.

[0204] 2. A method for treating an individual in need thereof with a gene therapy agent, the method comprising a) administering an IRAK modulator to the individual, and b) administering the gene therapy agent to the individual.

[0205] 3. A method for improving gene therapy for an individual, the method comprising a) administering an IRAK modulator to the individual, and b) administering the gene therapy agent to the individual.

[0206] 4. A method for suppressing an individual's immune response to a gene therapy agent, the method comprising a) administering an IRAK modulator to the individual, and b) administering the gene therapy agent to the individual.

[0207] 5. The method of any one of embodiments 1-4, wherein the IRAK modulator modulates the activity or expression of IRAK protein kinase.

[0208] 6. The method of embodiment 5, wherein the IRAK protein kinase is IRAK-1 protein kinase, IRAK-2 protein kinase, IRAK-3 protein kinase or IRAK-4 protein kinase.

[0209] 7. The method of any one of embodiments 1-6, wherein the IRAK modulator modulates the activity or expression of IRAK-4 protein kinase.

[0210] 8. The method of any one of embodiments 1-7, wherein the IRAK modulator is an IRAK degrader, an IRAK inhibitor, or an agent that confers loss of IRAK function.

[0211] 9. The method of any one of embodiments 1-8, wherein the IRAK modulator is a small molecule.

[0212] 10. The method of any one of embodiments 1-9, wherein the IRAK modulator comprises a compound of formula [I]: or a pharmaceutically acceptable salt thereof, wherein: X 1 is a divalent moiety selected from a covalent bond, -CH2-, -C(O)-, -C(S)-, and R 1a is hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O)2R, -N(R)2, -Si(R)3, or an optionally substituted C l-4 aliphatic; Each R 2a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Ring A a is a bicyclic or tricyclic ring selected from: Ring B a is a fused ring selected from the following: a 6-membered aryl group containing 0-2 nitrogen atoms, a 5- to 7-membered partially saturated carbocyclic group, a 5- to 7-membered partially saturated heterocyclic group having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur, or a 5-membered heteroaryl group having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur; R 3a is selected from hydrogen, halogen, -OR, -N(R)2 or -SR; Each R 4a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 5a It is hydrogen, C l-4 Aliphatic or -CN; Each R 6a is independently an optionally substituted group selected from the following: 1-6 aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring A is a 4-10 membered saturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring C is phenyl or a 5-10 membered monocyclic or bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen and sulfur; L 2 and L 3 Each of the 1-3 A divalent straight or branched saturated or unsaturated hydrocarbon chain, wherein 1 to 3 methylene units of the chain are independently and optionally replaced by: -O-, -C(O)-, -C(S)-, -C(R)2-, -CH(R)-, -C(F)2-, -N(R)-, -S-, -S(O)2-, or -CR=CR-; Each R 1 are independently hydrogen, R 5, Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CFR2, -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR or -C(O)NR2; Each R is independently hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or: two R groups on the same atom are optionally taken together with their intervening atoms to form an optionally substituted 4-11 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic monocyclic, bicyclic, bridged bicyclic, spirocyclic, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur in addition to the atoms to which they are attached; Each R 2 are independently hydrogen, R 5 , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CF2(R), -CF3, -CR2(OR), -C R2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 4 Selected from hydrogen, or an optionally substituted group selected from: C 1-6 aliphatic or 4-11 membered saturated or partially unsaturated monocyclic, bicyclic, bridged bicyclic or spirocyclic carbocyclic ring or heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring D is phenyl, a 4-10 membered saturated or partially unsaturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Each R 3 are independently hydrogen, R 5 、Halogen、-CN、-NO2、-OR、-SR、-NR 2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2) 2 , -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)O R, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Each R 5 is independently an optionally substituted group selected from the following: C1-6 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; n is 0, 1, or 2; Each m is independently 0, 1, 2, 3 or 4; and p is 0, 1, 2, 3 or 4.

[0213] 11. The method of any one of embodiments 1-9, wherein the IRAK modulator comprises any one of the compounds of formula [II]-[V]: or a pharmaceutically acceptable salt thereof.

[0214] 12. The method of any one of embodiments 1-9, wherein the IRAK modulator is a compound of formula [II], PROTAC IRAK-4 degrader 1, or PF 06650833.

[0215] 13. The method of any one of embodiments 1-9, wherein the IRAK modulator is CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or deoxyribozyme.

[0216] 14. The method of any one of embodiments 1-13, wherein the IRAK modulator blocks TLR9 function.

[0217] 15. The method of any one of embodiments 1-14, wherein the gene therapy agent comprises a viral vector.

[0218] 16. The method of embodiment 15, wherein the viral vector is an AAV particle.

[0219] 17. The method of embodiment 16, wherein the AAV particles comprise AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

[0220] 18. The method of embodiment 17, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

[0221] 19. The method of any one of embodiments 16-18, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, AAVrh8 ITRs, AAV9 ITRs, AAV10 ITRs, AAVrh10 ITRs, AAV11 ITRs, or AAV12 ITRs.

[0222] 20. The method of embodiment 19, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0223] 21. The method of embodiment 19, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0224] 22. The method of embodiment 15, wherein the viral vector is an adenoviral particle.

[0225] 23. The method of embodiment 22, wherein the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4,, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3 or a functional variant thereof.

[0226] 24. The method of embodiment 15, wherein the viral vector is a lentiviral particle.

[0227] 25. The method of embodiment 24, wherein the recombinant lentiviral particles are pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

[0228] 26. The method of embodiment 15, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0229] 27. A method as described in Example 26, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

[0230] 28. The method of any one of embodiments 1-14, wherein the gene therapy agent comprises lipid nanoparticles.

[0231] 29. The method of any one of embodiments 1-28, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0232] 30. The method of embodiment 29, wherein the heterologous transgene is operably linked to a promoter.

[0233] 31. The method of embodiment 30, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0234] 32. The method of any one of embodiments 1-31, wherein the IRAK modulator is administered prior to, simultaneously with, or after administration of the gene therapy agent.

[0235] 33. The method of any one of embodiments 1-32, wherein the individual suffers from a disease or disorder amenable to treatment by gene therapy.

[0236] 34. The method of embodiment 33, wherein the disease or disorder is a monogenic disease or disorder.

[0237] 35. The method of any one of embodiments 1-34, wherein the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

[0238] 36. The method of any one of embodiments 1-35, wherein the IRAK modulator is administered orally, intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

[0239] 37. A method for delivering a gene therapy agent to cells of an individual, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of the cytokine signature following incubation with the gene therapy agent identifies individuals with innate immunity to the gene therapy agent, c) administering an IRAK modulator to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b).

[0240] 38. A method for treating an individual in need thereof with a gene therapy agent, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of the cytokine signature following incubation with the gene therapy agent identifies individuals with innate immunity to the gene therapy agent, c) administering an IRAK modulator to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b).

[0241] 39. A method for selecting an individual for treatment with a gene therapy agent and an IRAK modulator, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of the cytokine signature following incubation with the gene therapy agent identifies the individual treated with the gene therapy agent and the IRAK modulator, c) selecting the individual identified in step b) for treatment with a gene therapy agent and an IRAK modulator.

[0242] 40. The method of embodiment 39, further comprising the following steps d) administering an IRAK modulator to the individual identified in step b), and e) administering the gene therapy agent to the individual identified in step b).

[0243] 41. The method of any one of embodiments 37-40, wherein the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell.

[0244] 42. The method of any one of embodiments 37-41, wherein the innate immune cells are isolated from peripheral blood mononuclear cells from the individual.

[0245] 43. The method of any one of embodiments 38-42, wherein the innate immune cell is a dendritic cell.

[0246] 44. The method of embodiment 43, wherein the dendritic cells are derived from monocytes of the individual.

[0247] 45. The method of embodiment 44, further comprising isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0248] 46. ​​A method as described in Example 44 or 45, wherein the monocytes are CD14+ monocytes.

[0249] 47. The method of any one of embodiments 44-46, wherein the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes.

[0250] 48. The method of any one of embodiments 37-47, wherein the innate immune cells are replated prior to incubation with the gene therapy agent of step c).

[0251] 49. The method of embodiment 48, wherein the innate immune cells are re-plated into microwell dishes.

[0252] 50. The method of any one of embodiments 37-49, wherein the gene therapy agent is a viral vector, and wherein the innate immune cells are coupled to the viral vector at a rate of about 1×10 3 About 1×10 5 or about 1×10 4 MOI was determined and incubated together.

[0253] 51. The method of any one of embodiments 37-49, wherein the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

[0254] 52. The method of any one of embodiments 37-51, wherein the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

[0255] 53. The method of any one of embodiments 37-52, wherein the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α.

[0256] 54. The method of any one of embodiments 37-53, wherein the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α.

[0257] 55. The method of any one of embodiments 37-53, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0258] 56. The method of any one of embodiments 37-55, wherein expression of the cytokines in the cytokine signature is increased compared to a suitable control.

[0259] 57. A method as described in Example 56, wherein the appropriate control is the expression of these cytokines in the cytokine signature of innate immune cells that have not been incubated with the gene therapy agent, or wherein the appropriate control is the expression of these cytokines in the cytokine signature of innate immune cells before incubation with the gene therapy agent.

[0260] 58. The method of any one of embodiments 37-57, wherein the IRAK modulator modulates the activity of IRAK protein kinase.

[0261] 59. The method of embodiment 58, wherein the IRAK protein kinase is IRAK-1 protein kinase, IRAK-2 protein kinase, IRAK-3 protein kinase or IRAK-4 protein kinase.

[0262] 60. The method of any one of embodiments 37-59, wherein the IRAK modulator modulates the activity of IRAK-4 protein kinase.

[0263] 61. The method of any one of embodiments 37-60, wherein the IRAK modulator is an IRAK degrader, an IRAK inhibitor, or an agent that confers loss of IRAK function.

[0264] 62. The method of any one of embodiments 37-61, wherein the IRAK modulator is a small molecule.

[0265] 63. The method of any one of embodiments 37-62, wherein the IRAK modulator comprises a compound of formula [I]: or a pharmaceutically acceptable salt thereof, wherein: X 1 is a divalent moiety selected from a covalent bond, -CH2-, -C(O)-, -C(S)-, and R 1a is hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O)2R, -N(R)2, -Si(R)3, or an optionally substituted C l-4 aliphatic; Each R 2a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Ring A a is a bicyclic or tricyclic ring selected from: Ring B a is a fused ring selected from the following: a 6-membered aryl group containing 0-2 nitrogen atoms, a 5- to 7-membered partially saturated carbocyclic group, a 5- to 7-membered partially saturated heterocyclic group having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur, or a 5-membered heteroaryl group having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur; R 3a is selected from hydrogen, halogen, -OR, -N(R)2 or -SR; Each R 4a are independently hydrogen, R 6a, Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 5a It is hydrogen, C l-4 Aliphatic or -CN; Each R 6a is independently an optionally substituted group selected from the following: 1-6 aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring A is a 4-10 membered saturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring C is phenyl or a 5-10 membered monocyclic or bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen and sulfur; L 2 and L 3 Each of the 1-3 A divalent straight or branched saturated or unsaturated hydrocarbon chain, wherein 1 to 3 methylene units of the chain are independently and optionally replaced by: -O-, -C(O)-, -C(S)-, -C(R)2-, -CH(R)-, -C(F)2-, -N(R)-, -S-, -S(O)2-, or -CR=CR-; Each R 1 are independently hydrogen, R 5 , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CFR2, -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR or -C(O)NR2; Each R is independently hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or: two R groups on the same atom are optionally taken together with their intervening atoms to form an optionally substituted 4-11 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic monocyclic, bicyclic, bridged bicyclic, spirocyclic, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur in addition to the atoms to which they are attached; Each R 2 are independently hydrogen, R 5 , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CF2(R), -CF3, -CR2(OR), -C R2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 4 Selected from hydrogen, or an optionally substituted group selected from: C 1-6 aliphatic or 4-11 membered saturated or partially unsaturated monocyclic, bicyclic, bridged bicyclic or spirocyclic carbocyclic ring or heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring D is phenyl, a 4-10 membered saturated or partially unsaturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Each R 3 are independently hydrogen, R 5 、Halogen、-CN、-NO2、-OR、-SR、-NR 2 , -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2) 2 , -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)O R, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Each R 5is independently an optionally substituted group selected from the following: C1-6 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; n is 0, 1, or 2; Each m is independently 0, 1, 2, 3 or 4; and p is 0, 1, 2, 3 or 4.

[0266] 64. The method of any one of embodiments 37-62, wherein the IRAK modulator comprises any one of the compounds of formula [II]-[V]: or a pharmaceutically acceptable salt thereof.

[0267] 65. The method of any one of embodiments 37-62, wherein the IRAK modulator is a compound of formula [II], PROTAC IRAK-4 degrader 1, or PF 06650833.

[0268] 66. The method of any one of embodiments 37-61, wherein the IRAK modulator is CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or deoxyribozyme.

[0269] 67. The method of any one of embodiments 37-66, wherein the IRAK modulator blocks TLR9 function.

[0270] 68. The method of any one of embodiments 37-67, wherein the gene therapy agent is a viral vector.

[0271] 69. A method as described in Example 68, wherein the viral vector is an AAV particle.

[0272] 70. A method as described in Example 69, wherein the AAV particles comprise AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

[0273] 71. The method of embodiment 70, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

[0274] 72. The method of any one of embodiments 69-71, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR, or AAV12 ITR.

[0275] 73. The method of embodiment 72, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0276] 74. The method of embodiment 72, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0277] 75. The method of Example 68, wherein the viral vector is an adenoviral particle.

[0278] 76. The method of embodiment 75, wherein the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4,, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3 or a functional variant thereof.

[0279] 77. The method of embodiment 68, wherein the viral vector is a lentiviral particle.

[0280] 78. The method of embodiment 77, wherein the recombinant lentiviral particles are pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

[0281] 79. The method of embodiment 68, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0282] 80. A method as described in Example 79, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

[0283] 81. The method of any one of embodiments 37-80, wherein the gene therapy agent is a lipid nanoparticle.

[0284] 82. The method of any one of embodiments 37-81, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0285] 83. The method of embodiment 82, wherein the heterologous transgene is operably linked to a promoter.

[0286] 84. The method of embodiment 83, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0287] 85. The method of any one of embodiments 37-84, wherein the IRAK modulator is administered prior to, simultaneously with, or after administration of the gene therapy agent.

[0288] 86. The method of any one of embodiments 37-85, wherein the individual suffers from a disease or disorder amenable to treatment by gene therapy.

[0289] 87. The method of embodiment 86, wherein the disease or disorder is a monogenic disease or disorder.

[0290] 88. The method of any one of embodiments 37-87, wherein the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

[0291] 89. The method of any one of embodiments 37-88, wherein the IRAK modulator is administered orally, intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

[0292] 90. Use of a composition in the manufacture of a medicament for delivering a nucleic acid to a cell of an individual in need thereof, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator.

[0293] 91. Use of a composition in the manufacture of a medicament for delivering a nucleic acid to a cell of an individual in need thereof, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent.

[0294] 92. Use of a composition in the manufacture of a medicament for treating an individual in need of gene therapy, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator.

[0295] 93. Use of a composition in the manufacture of a medicament for treating an individual in need of gene therapy, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent.

[0296] 94. Use of a composition in the manufacture of a medicament for modulating an immune response to gene therapy in an individual in need of gene therapy, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator.

[0297] 95. Use of a composition in the manufacture of a medicament for modulating an immune response of an individual to a gene therapy, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent.

[0298] 96. The use of any one of embodiments 90-95, wherein the gene therapy agent is an AAV particle, an adenovirus particle, a lentivirus particle, an HSV particle or a lipid nanoparticle.

[0299] 97. The use of any one of embodiments 90-96, wherein the IRAK modulator is an IRAK-4 degrader.

[0300] 98. A composition comprising a gene therapy agent for use in delivering a nucleic acid to a cell of an individual in need thereof, wherein the gene therapy agent is used in combination with an IRAK modulator.

[0301] 99. A composition comprising an IRAK modulator for use in delivering a nucleic acid to a cell of an individual in need thereof, wherein the IRAK modulator is used in combination with a gene therapy agent.

[0302] 100. A composition comprising a gene therapy agent for use in treating an individual in need of gene therapy, wherein the gene therapy agent is used in combination with an IRAK modulator.

[0303] 101. A composition comprising an IRAK modulator for use in treating an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent.

[0304] 102. A composition comprising an IRAK modulator for use in modulating an immune response to gene therapy in an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent.

[0305] 103. A composition comprising an IRAK modulator for use in suppressing an immune response to gene therapy in an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent.

[0306] 104. The composition of any one of embodiments 98-103, wherein the gene therapy agent is an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle.

[0307] 105. The composition of any one of embodiments 98-104, wherein the IRAK modulator is an IRAK-4 degrader.

[0308] 106. A kit for use in the method of any one of embodiments 1-89.

[0309] 107. A kit for use as described in any one of embodiments 90-97.

[0310] 108. A kit comprising the composition of any one of embodiments 98-105. Examples

[0311] The present invention will be more fully understood by reference to the following examples. However, these examples should not be construed as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and the various modifications or changes made according to the examples and embodiments will be conceived by those skilled in the art and should be included in the spirit and scope of the present application and within the scope of the appended claims. Example 1: Ex vivo protocol to evaluate the effects of IRAK4 degraders on human cells treated with AAV.

[0312] This example provides a strategy to examine the effects of IRAK4 inhibition on lymphocytes and dendritic cells exposed to AAV.

[0313] AAV triggers an immune response that involves activation of both the innate and adaptive immune systems. While the adaptive immune response to AAV is relatively well characterized, the innate immune activation caused by AAV is poorly understood.

[0314] IRAK4 is a kinase within the TLR pathway that activates the innate immune response. Although broad-acting immunosuppressants improve AAV delivery, this results in loss of transgene expression, side effects, and risk of opportunistic infections. Therefore, inhibition of IRAk4 could lead to more specific immunomodulation that could benefit AAV therapy.

[0315] In order to prepare peripheral blood mononuclear cells (PBMC), the blood from the enriched leukocyte apheresis product is decanted in a 50mL tube, and DPBS is added with a ratio of 1:1. Blood is pipetted gently in a separate 50mL tube containing 15mL Ficoll (GE17-5442-02), to ensure that blood and Ficoll layers do not mix. Mixture is centrifuged at room temperature for 25 minutes (9 accelerations and no braking) at 2000RPM. The buffy coat containing leukocytes and platelets is collected, transferred to a new tube, and centrifuged for five minutes at 400RCF. Cells are washed three times with the PBS containing 1% FBS or FCS and counted.

[0316] CD14+ monocytes were isolated from PBMCs using CD14 microbeads according to the manufacturer's protocol available online (miltenyibiotec.com / upload / assets / IM0001260.PDF; Miltenyi Biotech, Germany, order number 130-050-201). Briefly, cells were incubated with 20 μL CD14 microbeads / 10 μL of 1% paraformaldehyde at 2°C-8°C. 7Total cells were incubated together for 15 minutes. The cells were applied to the magnetic column and unlabeled cells were allowed to pass through. After the column was washed three times, the column was taken out of the magnetic separator, placed on a collection tube, and the magnetically labeled cells were flushed out by pushing the plunger into the column.

[0317] Using ImmunoCult-ACF dendritic cell culture medium, differentiation supplement and maturation supplement (cdn.stemcell.com / media / Files / pis / DX20521-PIS_1_2_0.pdf?_ga=2.81451927.1035383195.16421057001174975582.1603298321; Stem Cell Technologies, catalog numbers 10986, 10988 and 10989), monocytes were differentiated into dendritic cells according to the manufacturer's protocol available online. Briefly, purified monocytes were added to dendritic cell culture medium containing differentiation supplements and incubated at 37°C for three days. On day 3, the culture medium was changed and the cells were incubated for another two days. Maturation supplements were added on day 5 at a 1 to 100 dilution (e.g., 50 μL supplements / 5 mL culture).

[0318] The differentiated cells were harvested on the seventh day. T cells were stimulated by adding 4 mL of ImmunoCult XF T cell expansion medium (Stem Cell Technology, Cat. No. 10981) to thawed PBMCs. The cells were centrifuged at 400 g for 5 minutes, the medium was aspirated, and the cells were allowed to stand overnight in 10 mL of fresh expansion medium.

[0319] After collection and counting, the stationary cells were centrifuged at 400g for five minutes and resuspended. Cell stimulation was performed in a 96-well plate in T cell complete medium. The final concentration of cytokines in the medium was 100IU IL-2, 5ng / mL IL-7 and 25ng / mL IL-15. AAV stimulation was performed with 1e5 MOI, wherein 10μg PHA-p was used as a positive control. Half depletion was performed every three days. Ten days after thawing PBMC, cells were stimulated again with AAV in common T cell medium. Culture medium was collected after 24 hours of stimulation.

[0320] rAAV vectors (e.g., AAV1, AAV2, and / or AAVrh32.33) were produced using a standard triple transfection method (Sena-Esteves and Gao, Cold Spring Harb Protoc; doi: 10.1101 / pdb.top095513, 2020). The virus was purified by cesium chloride ultracentrifugation and titrated using both silver staining and quantitative polymerase chain reaction (qPCR).

[0321] Lymphocytes and dendritic cells are incubated with AAV vectors and pretreated, posttreated or co-treated with IRAK4 degraders. Pretreated and posttreated with IRAK4 degraders 3, 6, 12, 24 or 48 hours before or after AAV incubation. Cells are incubated with AAV at 1e5 MOI for 6, 12, 24, 48 or 72 hours. Cells are treated with PROTAC IRAK4 degraders-1 (Medicinal Chemical Expression, Catalog No. HY-129966), PZ0327 (Sigma-Aldrich, Catalog No. PF06650833) or compounds of formula [II] (Kymera Therapeutics) in a dose range of 1nM to 1M. LPS (300 ng / mL for 24 hours, Sigma L2630100MG) and R848 (1 μg / mL for 24 hours, Invitrogen tlrl-r948-5) were used as controls. Additional test groups included treatment with AAV alone, IRAK4 inhibitor alone, or IRAK4 inhibitor with LPS or R848.

[0322] To determine the extent to which the TLR9 pathway is inhibited in cells treated with IRAK4 degraders, cell culture media were collected and analyzed using Meso Scale Discovery (MSD) assay to determine the level of cytokine release. V-PLEX human biomarker 54-Plex kit was used and assayed according to the manufacturer's protocol (Meso Scale Discovery, Rockville, Maryland). Example 2: In vivo analysis of IRAK4 degraders in mice treated with AAV or LNP

[0323] This example provides an in vivo strategy to examine whether AAV immunogenicity is suppressed when animals are treated with an IRAK4 degrader.

[0324] All animal housing, maintenance, handling, and experiments were performed according to the Institutional Animal Care and Use Committee (IACUC) guidelines.

[0325] Animals received AAV or LNP injections and were pre-treated, post-treated, or co-treated with an IRAK4 inhibitor. Pre-treatment with an IRAK4 degrader was performed 6 hours, overnight, 24 hours, or 48 hours before AAV or LNP injection, or post-treatment with an IRAK4 degrader was performed 6 hours, overnight, 24 hours, or 48 hours after AAV or LNP injection. AAV was injected intramuscularly at 5x10 11 vg / kg dose, and monitoring 7, 14, 21, 28, 35, 42 and 63 days. LNP injection is carried out intravenously, subcutaneously or intraperitoneally at a dose of 1mg / kg, 10mg / kg or 100mg / kg. In addition, animals are pretreated, post-treated or co-treated with 1mg / kg, 10mg / kg or 100mg / kg PROTAC IRAK4 degrader-1 (Medicinal Chemistry Expression, Catalog No. HY-129966), PZ0327 (Sigma-Aldrich, Catalog No. PF06650833) or a compound of formula [II] (Chemray Therapeutics) administered subcutaneously, intraperitoneally or orally. Additional test groups include treatment with AAV alone, treatment with IRAK4 inhibitors alone, or treatment with IRAK4 inhibitors together with LPS or R848.

[0326] Animals were sacrificed 7, 14, 21, 28, 35, 42 or 63 days after treatment, and muscle, liver, bone marrow, spleen, blood and thymus were isolated. Tissue samples were immediately frozen on dry ice. DNA and RNA samples were extracted, and vector genome copies and transgene expression were quantified using real-time quantitative PCR (qPCR).

[0327] Tissue inflammation in treated animals was determined using hematoxylin and eosin (H&E) staining. Formalin-fixed and paraffin-embedded muscle and liver tissues were stained according to methods well known in the art. (Gernoux, G et al., Mol Ther [Molecular Therapy], 2020, 28(3): 747-757).

[0328] According to methods well known in the art (Gernoux, G et al., Mol Ther [Molecular Therapy], 2020, 28 (3): 747-757), frozen sections of muscle, liver and spleen were subjected to immunohistochemistry. The samples were collected on slides, air-dried, and fixed with 3% paraformaldehyde. Activated immune cells (such as T cells (using anti-CD3, CD4 and MHC II) and macrophages (using anti-F4 / 80 and MHC II)) and AAV transgene expression in the samples were analyzed. Nuclear and cytoplasmic β-gal were evaluated using standard X-gal protocols.

[0329] To analyze cytokine levels in treated animals, serum from these subjects was collected and used for the MSD assay. The assay was performed using the V-PLEX Human Biomarker 54-Plex Kit and according to the manufacturer's protocol (Mercedes-Benz, Rockville, MD).

[0330] To measure IFN-γ secretion in treated animals, ELISpot assays were performed on spleen cells isolated after injection (Gernoux, G et al., Mol Ther [Molecular Therapy], 2020, 28 (3): 747-757). The isolated cells were stimulated in vitro for 48 hours using AAV. The number of spots was determined using iSpot ELISpot Reader ELR068IFL (AID) and analyzed using AID ELISpot Reader software v.6.0. The response was considered positive when the spot-forming units exceeded 50 / 1e6 cells and were at least three times that of the control condition. Unstimulated cells were used as negative controls, and CEFT and CD3 / CD28 stimulation were used as positive controls.

[0331] Single cell suspensions were obtained from spleen and bone marrow samples. Cells were incubated in CD16 / 32 (Fc Block; BD Biosciences) and stained with antibodies. Cells were collected on LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0332] To analyze cytokine expression in treated animals, isolated splenocytes were stained for IFN-γ, TNF-α, IL-2, IL-4, and IL-6 using intracellular cytokine staining. Splenocytes were prepared as described in Mays, J Immunol [Journal of Immunology] 2009 182 (1) 6051-6060. Splenocytes were plated in T cell assay medium supplemented with 1 μg / mL brefeldin A (GolgiPlug, BD Pharmaceuticals (BD Pharmigen)) and 20ng / mL mouse IL-2 (BD Pharmaceuticals). Prior to staining, cells were stimulated with AAV for 5 hours at 37°C in 10% CO2 in the presence or absence of IRAK4 degraders. After stimulation, cells were washed, stained with antibodies, and examined using flow cytometry. Samples were collected on LSR II and analyzed using FlowJo software.

[0333] According to the protocol described in Mays, J Immunol [Journal of Immunology] 2009 182 (1) 6051-6060, the T cell response of animals treated with AAV and IRAK4 degradation agents was determined by MHC class I tetramer staining using PE-conjugated MHC class I H2-Kb-ICPMYARV tetramer complex (Beckman Coulter). At different times after treatment with AAV and IRAK4 degradation agents, heparinized whole blood cells isolated by retroorbital bleeding were subjected to tetramer staining. Cells were co-stained with PE-conjugated tetramers and FITC-conjugated anti-CD8α (Ly-2) antibodies (BD Pharmaceuticals) at room temperature for 30 minutes and fixed with iTAg MHC tetramer lysis solution supplemented with fixation solution (Beckman Coulter) for 15 minutes at room temperature. Cells were washed three times in PBS and resuspended in 0.01% BD CytoFix (BD Biosciences). Samples were collected on the LSR II and analyzed using FlowJo software. Example 3: Ex vivo analysis of the effects of IRAK4 degraders on human cells treated with AAV Materials and methods

[0334] Preparation of peripheral blood mononuclear cells.Blood from enriched leukocyte apheresis products (Stem Cell Technology Company) from different donors is decanted into 50mL tubes, and Dulbecco's phosphate buffered saline (DPBS) is added with a ratio of 1:1.Blood is gently pipetted into a separate 50mL tube containing 15mL Ficoll (GE17-5442-02) with the mixture of DBPS, to ensure that blood and Ficoll do not mix.The mixture is centrifuged at room temperature with 2000RPM for 25 minutes (9 accelerations and no braking).Collect the buffy coat containing peripheral blood mononuclear cells (PBMC), transfer it to a new tube, and centrifuge for five minutes at 400RCF.PBMC is washed three times and counted with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FBS) or fetal calf serum (FCS).

[0335] Isolation of monocytes. CD14+ monocytes were isolated from PBMCs using CD14 microbeads according to the manufacturer's protocol (Miltenyi Biotec, Germany, order number 130-050-201, protocol available online at www.miltenyibiotec.com / upload / assets / IM0001260.PDF). Briefly, PBMCs were incubated with 20 μL CD14 microbeads / 10 μL at 2°C-8°C. 7 Total cells were incubated together for 15 minutes. PBMCs were applied to a magnetic column (Miltenyi Biotec; World Wide Web miltenyibiotec.com / US-en / products / ls-columns.html#130-042-401), and unlabeled cells were allowed to pass through. After the column was washed three times, the column was taken out from a magnetic separator (Miltenyi Biotec; World Wide Web miltenyibiotec.com / US-en / products / quadromacs-separator-and-starting-kits.html#130-091-051), placed on a collection tube, and magnetically labeled CD14+ monocytes were flushed out by pushing the plunger into the column.

[0336] Differentiation of monocytes. Using ImmunoCult-ACF dendritic cell culture medium, differentiation supplements and maturation supplements (Stem Cell Technologies, Catalog Nos. 10986, 10988 and 10989; World Wide Web cdn.stemcell.com / media / Files / pis / DX20521-PIS_1_2_0.pdf?_ga=2.81451927.1035383195.1642105700-1174975582.1603298321), CD14+ monocytes were differentiated into dendritic cells according to the manufacturer's protocol available online. In brief, purified CD14+ monocytes were added to dendritic cell culture medium containing differentiation supplements and incubated at 37°C for three days. On the 3rd day, the culture medium was replaced with fresh dendritic cell culture medium containing differentiation supplements, and the cells were incubated for another two days. On day 5, maturation supplement was added to the cells at a 1 in 100 dilution (eg, 50 μL supplement / 5 mL culture). Differentiated dendritic cells were harvested on day 7.

[0337] rAAV production and titration. rAAV vectors (AAV.SAN024 and AAV.SAN029) were produced using a standard triple transfection method (Sena-Esteves and Gao, Cold Spring Harb Protoc [Cold Spring Harb Protocol]; doi: 10.1101 / pdb.top095513, 2020). All serotypes tested encode the same GFP transgene. The virus was purified by cesium chloride ultracentrifugation and titrated using both silver staining and quantitative polymerase chain reaction (qPCR).

[0338] IRAK4 targeted drugs and AAV treatment. Dendritic cells were plated in 96-well plates at 200,000 cells / well. Each treatment was performed in triplicate. The cells were treated with zimlovisertib IRAK4 inhibitor (alias: PF-06650833) (Catalog No.: HY-19836 (Medicinal Chemical Expression)) and PROTAC IRAK4 degrader-1 (Catalog No.: HY-129966 (Medicinal Chemical Expression)) at 16nm, 80nm, 400nm and 2000nm in an incubator at 37°C, 5% CO2 for 18h. After pretreatment with drugs for 18h, AAV vectors were used at 1e5 MOI. The cells were incubated in an incubator at 37°C, 5% CO2 for 24 hours. After 24 hours, the plates were centrifuged and the culture supernatant was collected. Through the MSD (World Wide Web) provided by DC3 Therapeutics https: / / www.dc3therapeutics.com / services mesoscale.com / products / v-plex-proinflammatory-panel-1-human-kit-k15049d / ) analysis of clarified culture medium. An outline of the experiments for PBMC isolation, monocyte purification, and dendritic cell differentiation is presented in Figure 1 This figure also shows how dendritic cells were treated with different drugs and AAV. In some experiments, lipopolysaccharide LPS (300 ng / mL) (Sigma Aldrich Fine Chemicals Biosciences L2630100MG) was used as a positive control, which is known to activate IRAK 4 and induce phosphorylation of IRAK4 target proteins.

[0339] LDH release assay. Cytotoxicity was measured by the release of lactate dehydrogenase (LDH) in the culture supernatant. LDH was quantified using a Promega kit (Cat. No. G1780) following the manufacturer's protocol. Briefly, after experimental treatment, supernatant samples were transferred to a 96-well plate and an equal volume of CytoTox Reagents were added to each well and incubated for 30 minutes. Stop solution was added and the absorbance signal was measured at 490 nm in a plate reader. https: / / www.promega.com / products / cell-health-assays / cell-viability-and- cytotoxi city-assays / cytotox-96-non_radioactive-cytotoxicity-assay / ? catNum= G1780#protocols

[0340] AlphaLISA assay. This assay was performed to measure the level of IRAK4 protein in cells. The protocol was followed as recommended by the manufacturer (Perkin Elmer (Catalog No. AL3117C)). Briefly, cells were lysed and 5 μL of lysate was added to anti-IRAK4 acceptor beads and incubated at 23°C for 30 min, followed by addition of biotinylated anti-IRAK4 antibody and incubation at 23°C for 60 min. Finally, SA donor beads were added and the plate was read using EnVision-Alpha Reader (615 nm) after 30 min. https: / / resources.perkinelmer.com / lab-solutions / resources / docs / MAN_AlphaLIS A_IRAK4_AL3117.pdf? _gl=1*7jzf5d*_ga* MzQ4MTQ1NzA3LjE2NDY2NzY5MjI.*_ga_W34ZJ1Z1Q1*MTY3NzE5MjAyOS4zLjEuMTY3NzE5MjA2 Mi4yN y4wLjA.&_ga=2.178521252.1409175695.1677192029-348145707. 1646676922. Phosphorylation levels of NFkB were measured using the same protocol using the AlphaLISA SureFire Ultra p-NFκB (Ser536) Assay Kit - High Capacity Catalog No. ALSU-PNFKB-A-HV. result Pretreatment with IRAK4 modulators results in inhibition of cytokine release

[0341] Human monocytic dendritic cells were pretreated with different doses of drugs (16nM, 80nM, 400nM and 2000nM) as indicated for 18h, then the same cells were infected with AAV.SAN024 at an MOI of 1e5 for 24h, and the culture supernatants were analyzed for cytokine release. Treatment with AAV induced secretion of IL1b cytokine by dendritic cells, and this secretion was inhibited by all doses of the IRAK4 inhibitor zimlovisertib ( Figure 2A ) and IRAK4 degrader PROTAC IRAK4 degrader-1 ( Figure 2B ) were blocked. Each point on the bar graph represents one donor. Experiments were performed on three donors and statistical significance was determined using one-way ANOVA. Cytokine secretion is expressed as a percentage of all donors. Cytokine blocking of other cytokines such as IL6 and TNFa was also observed. Co-treatment with IRAK4 modulators results in inhibition of cytokine release

[0342] Human monocytic dendritic cells were co-treated with drugs at different doses (16nM, 80nM, 400nM and 2000nM) as indicated, and the same cells were simultaneously infected with AAV.SAN024 at an MOI of 1e5 for 24h, and the culture supernatants were analyzed for cytokine release. Treatment with AAV induced secretion of IL1b cytokine by dendritic cells, and this secretion was inhibited by all doses of the IRAK4 inhibitor zimlovisertib ( Figure 3A ) and IRAK4 degrader PROTAC IRAK4 degrader-1 ( Figure 3B ) are blocked. Each point on the bar graph represents the technical replicate of a representative donor. Experiments were performed on three donors, and statistical significance was determined using one-way analysis of variance. Cytokine secretion is expressed as a percentage of all donors. Cytokine blocking of other cytokines (such as IL6 and TNFa) was also observed. IRAK4 modulators do not induce cytotoxicity in primary human monocytic dendritic cells

[0343] Human monocytic dendritic cells were treated with different doses (16 nM, 80 nM, 400 nM and 2000 nM) of the IRAK4 inhibitor zimlovisertib ( Figure 4A ) or IRAK degrader PROTAC IRAK4 degrader-1 ( Figure 4B) and the same cells were infected with AAV.SAN024 at an MOI of 1e5 for 24 h. The culture supernatant was analyzed for LDH release. No significant differences were observed between the control (untreated / uninfected cells) and the treated cells and cells infected with AAV.SAN024, indicating that both IRAK4 modulators were not cytotoxic. Treatment with IRAK4 inhibitors blocks phosphorylation of IRAK4 kinase target proteins, and IRAK4 degraders induce degradation of IRAK4 protein in primary human monocytic dendritic cells

[0344] Human monocytic dendritic cells were treated with different doses of drugs as indicated (16nM, 80nM, 400nM and 2000nM), and the same cells were treated with LPS 300ng / ml. LPS induces IRAK4 kinase activity and allows phosphorylation of IRAK4 target proteins such as NFkB. Treatment with the IRAK4 kinase inhibitor zimlovisertib at 400nM and 2000nM ( Figure 5A ) blocks the phosphorylation of NFkB.

[0345] To determine the activity of PROTAC degraders, human monocytic dendritic cells were treated with different doses of drug as indicated (16nM, 80nM, 400nM and 2000nM), and the same cells were infected with AAV.SAN024 at an MOI of 1e5 for 24h. Cell lysates were analyzed for IRAK4 degradation. No significant differences were observed between control (untreated / uninfected) cells and treated cells and cells infected with AAV.SAN024, but treatment of AAV.SAN024-infected cells with all doses of degraders resulted in a decrease in IRAK4 levels ( Figure 5B ). Example 4: Analysis of the Effects of IRAK4 Modulators on Immune Responses Induced by AAV in an In Vivo Mouse Model Materials and methods

[0346] In vivo mouse experiments. C57BL / 6J mice were purchased from Jackson Laboratories. Male mice aged 6-8 weeks were divided into 3 groups - PBS group, AAV group and AAV+IRAK4 group, all groups had 10 mice each. Zimlovisertib IRAK4 inhibitor (alias: PF-06650833) (Catalog No.: HY-19836 (Medicinal Chemical Expression)) was administered at a dose of 30 mg / kg with mouse food (Catalog No. 2016, Teklad Global 16% Protein Rodent Diet) https: / / www.inotivco.com / rodent-natural-ingredient-2016-diets) were mixed. The AAV+IRAK4 group was fed food and Zimlovisertib IRAK4 inhibitor 15 days before AAV injection, while the PBS group and AAV group were fed mouse food (Catalog No. 2016, Teklad Global 16% Protein Rodent Diet https: / / www.inotivco.com / rodent-natural-ingredient-2016-diets Mice in the PBS group were intramuscularly injected with 100ul PBS, and mice in the AAV group were intramuscularly injected with 1e11 vg (100ul volume) into each quadriceps muscle of both legs.

[0347] The PBS group, AAV group, and AAV+IRAK4 degrader PROTAC IRAK4 degrader-1 (Catalog number: HY-129966 (Medicinal Chemistry Expression)) with n=6 mice in each group (mixed with food at 100 mg / kg and fed to mice 15 days before AAV injection) followed the same experimental design. Two different IRAK4 PROTAC degraders, namely the commercially available PROTAC IRAK4 degrader-1 (Catalog number: HY-129966 (Medicinal Chemistry Expression)) and KT-474, were mixed with food at a dose of 100 mg / kg and fed 15 days before AAV administration. The AAV capsid used in the study had a LacZ transgene. All animal experiments were performed at Sanofi in Framingham in accordance with the Institutional Animal Care and Use Committee. Submandibular blood was drawn weekly, and autopsies were performed three weeks after AAV injection.

[0348] PBMC separation and surface staining.120ul blood is collected in a tube coated with K2EDTA.Blood is mixed with DBPS in a 1:1 ratio, and pipetted into a separate tube containing Ficoll (GE17-5442-02) to ensure that blood and Ficoll do not mix.The mixture is centrifuged at room temperature for 25 minutes (9 accelerations and no brakes) at 2000RPM.The buffy coat containing peripheral blood mononuclear cells (PBMC) is collected, transferred to a new tube, and centrifuged for five minutes at 400RCF.PBMC is washed three times and counted with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FBS) or fetal calf serum (FCS).The PBMC cells so obtained are stained with different antibodies to quantitatively determine the percentage of different immune cell subsets in peripheral blood.

[0349] PBMC CD8 TEM and lacZ tetramer staining. PBMC is separated from about 120 μl of mouse blood, and is transferred to 96-well U-shaped bottom plates. PBMC is washed once with 200 μl FACS buffer by centrifugation at 2,000 rpm for 5 minutes, and PBMC is stained for 30 minutes at 4 ° C with a flow antibody mixture (1:100 anti-CD4 PE-Cy7, 1:50 anti-CD8a FITC, 1:100 anti-CD62L APC, 1:100 anti-CD44 Pacific Blue, 1:100 live / dead cell staining kit and 1:20 H-2Kb β-galactosidase tetramer). After incubation, cells are washed 2 times with FACS buffer, and then fixed with 100 μl BD Cytofix / Cytoperm fixation / permeabilization solution kit at 4 ° C for 15 minutes. Cells were washed twice with FACS buffer and samples were run on a flow cytometer (Novocyte Penteon Flow Cytometer System 5 Laser, Agilent Technology).

[0350] Intracellular cytokine staining. Splenocytes were harvested from mouse spleens and 2 million cells were seeded in 96-well U-bottom plates. The cells were centrifuged at 2,000 rpm for 5 minutes and then resuspended in 100 μl of RPMI containing 10% FBS and 1X 2-mercaptoethanol. AAVrh32.33 overlapping peptides, AAVrh32.33 LacZ immunodominant peptides, and LacZ immunodominant peptides were prepared at a concentration of 10 μg / ml in RPMI1640 containing 10% FBS, 1X 2-mercaptoethanol, and 1:500 GolgiStop. For positive controls, 0.10 μg / ml PMA and 2 μg / ml ionomycin solutions were prepared in RPIM1640 containing 10% FBS, 1X 2-mercaptoethanol, and 1:500 GolgiStop. Then, 100 μl of the prepared solution was added to the cells so that the final concentration was 5 μg / ml (or 0.05 μg / ml PMA and 1 μg / ml ionomycin). The cells were incubated overnight in a 37°C, 5% CO2 incubator to stimulate the cells. The next day, the cells were centrifuged at 2,000 rpm for 5 minutes and then washed once with FACS buffer. The cells were stained with an antibody mixture (1:100 anti-CD4 FITC, 1:100 anti-CD8a PerCP-Cy5.5, 1:100 anti-CD44 Pacific Blue, 1:100 live / dead cell staining kit) at 4°C for 30 minutes. Then, the cells were washed twice with FACS buffer. After washing, the cells were permeabilized with 100 μl BD Cytofix / Cytoperm fixation / permeabilization solution kit at 4°C for 20 minutes. Cells were washed twice with 1X PermWash and then stained with an antibody cocktail (1:100 anti-IFNγAPC, 1:100 anti-IL2PE, 1:100 anti-TNFαPE-Cy7) for 30 minutes at 4° C. Cells were washed twice with FACS buffer and samples were run on a flow cytometer.

[0351] Table 1 shows a list of suppliers of various reagents used in the assay described in this example. Table 1: List of reagents and suppliers result Treatment with IRAK4 inhibitor reduced transgenic LacZ-specific CD8 T cells and effector memory T cells in PBMCs derived from peripheral blood 14 days after AAV injection, and reduced IFNg-producing CD8 T cells

[0352] In vivo mouse experiments were designed to show the effects of IRAK4 inhibition on CD8 T cells and memory T cells after AAV injection ( Figure 6 ). Figure 6 The mice depicted in Figure 4 show the AAV+IRAK4 group, in which the IRAK4 inhibitor zimlovisertib was mixed with the diet and fed to the mice 15 days before the AAV injection, and then blood was collected for PBMC analysis on day 14. The mice were necropsied on day 21 to harvest the spleen. The PBS-only group and the AAV-only group were fed regular diet, and blood was drawn on day 14, and necropsies were performed on day 21.

[0353] Mice were bled on day 14 after AAV injection and PBMCs were isolated. PBMCs were stained with different antibodies to quantify LacZ tetramer-positive CD8 T cells ( Fig. 7A ) and CD44+CD62L+ effector memory cells ( Figure 7B ). These cell populations were upregulated after AAV administration compared to PBS controls. Fig. 7A and Figure 7B As shown, a significant reduction in CD8 T cells and effector cells was observed in mice receiving the IRAK4 inhibitor. N = 10 mice per group, and one-way ANOVA was performed to determine statistical significance.

[0354] On day 21, mice were euthanized and splenocytes were harvested. Splenocytes were incubated with AAV peptide libraries ( Fig. 8A ) or AAV immunodominant peptide ( Figure 8B ) to account for the activation of capsid-specific CD8 T cells. Similarly, cells were stimulated with LacZ immunodominant peptide ( Figure 8C ) were treated to quantify transgene-specific activated CD8 T cells. Activated CD8 T cells produced the cytokine interferon gamma, which was quantified by flow cytometry. In all cases, these cell populations were upregulated after AAV administration and then significantly reduced after IRAK4 inhibitor treatment compared to PBS controls. N = 10 mice per group, and one-way ANOVA was performed to determine statistical significance. Treatment with the IRAK4 degrader PROTAC IRAK4 degrader-1 reduces IRAK4 expression in immune cells

[0355] Mice were bled on day 14 and PBMCs were harvested. Different immune cell types in PBMCs were identified by flow cytometry ( Figure 9A-9D ). IRAK4 protein expression was significantly reduced following IRAK4 degrader treatment via the diet in all the different immune cell types evaluated. N=6 mice per group and one-way ANOVA was performed to determine statistical significance. Food treatment with KT-474 reduced transgene-specific CD8 T cells in PBMCs and spleen.

[0356] Mice were bled on day 14 after AAV injection, and PBMCs were isolated. Splenocytes were harvested on day 21 after AAV injection. PBMCs and splenocytes were stained with different antibodies to quantify LacZ tetramer-positive cells. Compared to PBS controls, these cell populations were upregulated after AAV administration and then significantly reduced after KT-474 treatment (Figure 10). Fig. 10A Shown is the reduction of transgenic LacZ-specific CD8 T cells in PBMCs derived from peripheral blood 14 days after AAV injection. Fig. 10B The reduction of transgenic LacZ-specific CD8 T cells in the spleen 21 days after AAV injection is shown, and statistical significance was determined using the ROUT outlier method and one-way ANOVA methods. The ROUT method identified outliers from nonlinear regression. Example 5: General Procedure for Oral Treatment of IRAK Modulators in Combination with AAV

[0357] C57BL / 6J mice were purchased from Jackson Laboratory. Male mice aged 6-8 weeks were divided into 3 groups - PBS group, AAV group and AAV+IRAK4 modulator group, all groups had 10 mice each. IRAK 4 modulators (such as IRAK4 inhibitor Zimlovisertib IRAK4 inhibitor (alias: PF-06650833) (Catalog number: HY-19836 (Medicinal Chemical Expression)), PROTAC IRAK4 degrader-1 (Catalog number HY-129966 (Medicinal Chemical Expression)) and KT-474 were tested via oral administration at a dose of 100 mg / kg twice a day. IRAK4 modulators are being tested with pre-treatment strategies and co-treatment strategies. In the clinic for other diseases ( https: / / www.tandfonline.com / doi / full / 10.1080 / 13543784.2020.1752660 ) is testing IRAK4 modulators and KT-474 https: / / clinicaltrials.gov / ct2 / show / NCT04772885 KT-474 is an oral drug and should therefore be effective in blocking the IRAK4 protein. Chemray Therapeutics has also shown evidence of complete degradation of the IRAK4 protein in human cells and in mouse tissues using KT-474. https: / / www.kymeratx.com / wp-content / uploads / 2021 / 09 / Euro-Prot-Deg-Summit- Sept-21-Final_Anthony-Slavin.pdf A schematic diagram showing the general procedure for oral treatment of IRAK4 modulators in combination with AAV is shown in Fig.11 middle.

Claims

1. A method for delivering a nucleic acid to a cell of an individual in need thereof, the method comprising a) administering an IRAK modulator to the individual, and b) administering the gene therapy agent to the individual.

2. A method for treating an individual in need thereof with a gene therapy agent, the method comprising a) administering an IRAK modulator to the individual, and b) administering the gene therapy agent to the individual.

3. A method for improving gene therapy for an individual, the method comprising a) administering an IRAK modulator to the individual, and b) administering the gene therapy agent to the individual.

4. A method for suppressing an immune response to a gene therapy agent in an individual in need thereof, the method comprising a) administering an IRAK modulator to the individual, and b) administering the gene therapy agent to the individual.

5. The method of any one of claims 1-4, wherein the IRAK modulator modulates the activity or expression of IRAK protein kinase.

6. The method of claim 5, wherein the IRAK protein kinase is IRAK-1 protein kinase, IRAK-2 protein kinase, IRAK-3 protein kinase or IRAK-4 protein kinase.

7. The method of any one of claims 1-6, wherein the IRAK modulator modulates the activity or expression of IRAK-4 protein kinase.

8. The method of any one of claims 1-7, wherein the IRAK modulator is an IRAK degrader, an IRAK inhibitor, or an agent that confers loss of IRAK function.

9. The method of any one of claims 1-8, wherein the IRAK modulator is a small molecule.

10. The method of any one of claims 1-9, wherein the IRAK modulator comprises a compound of formula [I]: or a pharmaceutically acceptable salt thereof, wherein: X 1 is a divalent moiety selected from a covalent bond, -CH2-, -C(O)-, -C(S)-, and R 1a is hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O)2R, -N(R)2, -Si(R)3, or an optionally substituted C l-4 aliphatic; Each R 2a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Ring A a is a bicyclic or tricyclic ring selected from: Ring B a is a fused ring selected from the following: a 6-membered aryl group containing 0-2 nitrogen atoms, a 5- to 7-membered partially saturated carbocyclic group, a 5- to 7-membered partially saturated heterocyclic group having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur, or a 5-membered heteroaryl group having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur; R 3a is selected from hydrogen, halogen, -OR, -N(R)2 or -SR; Each R 4a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 5a It is hydrogen, C l-4 Aliphatic or -CN; Each R 6a is independently an optionally substituted group selected from the following: 1-6 aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring A is a 4-10 membered saturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring C is phenyl or a 5-10 membered monocyclic or bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen and sulfur; L 2 and L 3 Each of the 1-3 A divalent straight or branched saturated or unsaturated hydrocarbon chain, wherein 1 to 3 methylene units of the chain are independently and optionally replaced by: -O-, -C(O)-, -C(S)-, -C(R)2-, -CH(R)-, -C(F)2-, -N(R)-, -S-, -S(O)2-, or -CR=CR-; Each R 1 are independently hydrogen, R 5 , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CFR2, -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR or -C(O)NR2; Each R is independently hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or: two R groups on the same atom are optionally taken together with their intervening atoms to form an optionally substituted 4-11 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic monocyclic, bicyclic, bridged bicyclic, spirocyclic, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur in addition to the atoms to which they are attached; Each R 2 are independently hydrogen, R 5 , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CF2(R), -CF3, -CR2(OR), -C R2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 4 Selected from hydrogen, or an optionally substituted group selected from: C 1-6 aliphatic or 4-11 membered saturated or partially unsaturated monocyclic, bicyclic, bridged bicyclic or spirocyclic carbocyclic ring or heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring D is phenyl, a 4-10 membered saturated or partially unsaturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Each R 3 are independently hydrogen, R 5 、Halogen、-CN、-NO2、-OR、-SR、-NR 2 , -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2) 2 , -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)O R, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Each R 5 is independently an optionally substituted group selected from the following: C1-6 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; n is 0, 1, or 2; Each m is independently 0, 1, 2, 3 or 4; and p is 0, 1, 2, 3 or 4.

11. The method of any one of claims 1-9, wherein the IRAK modulator comprises any one of the compounds of formula [II]-[V]: or a pharmaceutically acceptable salt thereof.

12. The method of any one of claims 1-9, wherein the IRAK modulator is a compound of formula [II], PROTAC IRAK-4 Degrader 1, or PF 06650833.

13. The method of any one of claims 1-9, wherein the IRAK modulator is CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or deoxyribozyme.

14. The method of any one of claims 1-13, wherein the IRAK modulator blocks TLR9 function.

15. The method of any one of claims 1-14, wherein the gene therapy agent comprises a viral vector.

16. The method of claim 15, wherein the viral vector is an AAV particle.

17. The method of claim 16, wherein the AAV particles comprise AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

18. The method of claim 17, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

19. The method of any one of claims 16-18, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, AAVrh8 ITRs, AAV9 ITRs, AAV10 ITRs, AAVrh10 ITRs, AAV11 ITRs, or AAV12 ITRs.

20. The method of claim 19, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

21. The method of claim 19, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

22. The method of claim 15, wherein the viral vector is an adenoviral particle.

23. The method of claim 22, wherein the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4,, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3, or a functional variant thereof.

24. The method of claim 15, wherein the viral vector is a lentiviral particle.

25. The method of claim 24, wherein the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

26. The method of claim 15, wherein the viral vector is a herpes simplex virus (HSV) particle.

27. The method of claim 26, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

28. The method of any one of claims 1-14, wherein the gene therapy agent comprises lipid nanoparticles.

29. The method of any one of claims 1-28, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

30. The method of claim 29, wherein the heterologous transgene is operably linked to a promoter.

31. The method of claim 30, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

32. The method of any one of claims 1-31, wherein the IRAK modulator is administered prior to, simultaneously with, or after administration of the gene therapy agent.

33. The method of any one of claims 1-32, wherein the individual suffers from a disease or disorder amenable to treatment by gene therapy.

34. The method of claim 33, wherein the disease or disorder is a monogenic disease or disorder.

35. The method of any one of claims 1-34, wherein the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

36. The method of any one of claims 1-35, wherein the IRAK modulator is administered orally, intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

37. A method for delivering a gene therapy agent to cells of an individual in need thereof, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of the cytokine signature following incubation with the gene therapy agent identifies individuals with innate immunity to the gene therapy agent, c) administering an IRAK modulator to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b).

38. A method for treating an individual in need thereof with a gene therapy agent, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of the cytokine signature following incubation with the gene therapy agent identifies individuals with innate immunity to the gene therapy agent, c) administering an IRAK modulator to the individual identified in step b), and d) administering the gene therapy agent to the individual identified in step b).

39. A method for selecting an individual for treatment with a gene therapy agent and an IRAK modulator, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of the cytokine signature following incubation with the gene therapy agent identifies the individual treated with the gene therapy agent and the IRAK modulator, c) selecting the individual identified in step b) for treatment with a gene therapy agent and an IRAK modulator.

40. The method of claim 39, further comprising the steps of: d) administering an IRAK modulator to the individual identified in step b), and e) administering the gene therapy agent to the individual identified in step b).

41. The method of any one of claims 37-40, wherein the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell.

42. The method of any one of claims 37-41, wherein the innate immune cells are isolated from peripheral blood mononuclear cells from the individual.

43. The method of any one of claims 38-42, wherein the innate immune cell is a dendritic cell.

44. The method of claim 43, wherein the dendritic cells are derived from monocytes of the individual.

45. The method of claim 44, further comprising isolating mononuclear cells from the individual, and The monocytes are incubated in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then the dendritic cells are incubated with the gene therapy agent.

46. ​​The method of claim 44 or 45, wherein the monocytes are CD14+ monocytes.

47. The method of any one of claims 44-46, wherein the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes.

48. The method of any one of claims 37-47, wherein the innate immune cells are re-plated prior to incubation with the gene therapy agent of step c).

49. The method of claim 48, wherein the innate immune cells are re-plated into microwell dishes.

50. The method of any one of claims 37-49, wherein the gene therapy agent is a viral vector, and wherein the innate immune cells are coupled to the viral vector at a rate of about 1×10 3 About 1×10 5 or about 1×10 4 MOI was determined and incubated together.

51. The method of any one of claims 37-49, wherein the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

52. The method of any one of claims 37-51, wherein the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

53. The method of any one of claims 37-52, wherein the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α.

54. The method of any one of claims 37-53, wherein the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α.

55. The method of any one of claims 37-53, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

56. The method of any one of claims 37-55, wherein expression of the cytokines in the cytokine signature is increased compared to a suitable control.

57. The method of claim 56, wherein the appropriate control is the expression of the cytokines in the cytokine signature of innate immune cells that have not been incubated with the gene therapy agent, or wherein the appropriate control is the expression of the cytokines in the cytokine signature of innate immune cells prior to incubation with the gene therapy agent.

58. The method of any one of claims 37-57, wherein the IRAK modulator modulates the activity of IRAK protein kinase.

59. The method of claim 58, wherein the IRAK protein kinase is IRAK-1 protein kinase, IRAK-2 protein kinase, IRAK-3 protein kinase or IRAK-4 protein kinase.

60. The method of any one of claims 37-59, wherein the IRAK modulator modulates the activity of IRAK-4 protein kinase.

61. The method of any one of claims 37-60, wherein the IRAK modulator is an IRAK degrader, an IRAK inhibitor, or an agent that confers loss of IRAK function.

62. The method of any one of claims 37-61, wherein the IRAK modulator is a small molecule.

63. The method of any one of claims 37-62, wherein the IRAK modulator comprises a compound of formula [I]: or a pharmaceutically acceptable salt thereof, wherein: X 1 is a divalent moiety selected from a covalent bond, -CH2-, -C(O)-, -C(S)-, and R 1a is hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O)2R, -N(R)2, -Si(R)3, or an optionally substituted C l-4 aliphatic; Each R 2a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Ring A a is a bicyclic or tricyclic ring selected from: Ring B a is a fused ring selected from the following: a 6-membered aryl group containing 0-2 nitrogen atoms, a 5- to 7-membered partially saturated carbocyclic group, a 5- to 7-membered partially saturated heterocyclic group having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur, or a 5-membered heteroaryl group having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur; R 3a is selected from hydrogen, halogen, -OR, -N(R)2 or -SR; Each R 4a are independently hydrogen, R 6a , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, - C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 5a It is hydrogen, C l-4 Aliphatic or -CN; Each R 6a is independently an optionally substituted group selected from the following: 1-6 aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring A is a 4-10 membered saturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring C is phenyl or a 5-10 membered monocyclic or bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen and sulfur; L 2 and L 3 Each of the 1-3 A divalent straight or branched saturated or unsaturated hydrocarbon chain, wherein 1 to 3 methylene units of the chain are independently and optionally replaced by: -O-, -C(O)-, -C(S)-, -C(R)2-, -CH(R)-, -C(F)2-, -N(R)-, -S-, -S(O)2-, or -CR=CR-; Each R 1 are independently hydrogen, R 5 , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CFR2, -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR or -C(O)NR2; Each R is independently hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or: two R groups on the same atom are optionally taken together with their intervening atoms to form an optionally substituted 4-11 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic monocyclic, bicyclic, bridged bicyclic, spirocyclic, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur in addition to the atoms to which they are attached; Each R 2 are independently hydrogen, R 5 , Halogen, -CN, -NO2, -OR, -SR, -NR2, -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2)2, -CF2(R), -CF3, -CR2(OR), -C R2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)OR, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; R 4 Selected from hydrogen, or an optionally substituted group selected from: C 1-6 aliphatic or 4-11 membered saturated or partially unsaturated monocyclic, bicyclic, bridged bicyclic or spirocyclic carbocyclic ring or heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring D is phenyl, a 4-10 membered saturated or partially unsaturated monocyclic or bicyclic carbocyclic ring or a heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Each R 3 are independently hydrogen, R 5 、Halogen、-CN、-NO2、-OR、-SR、-NR 2 , -S(O)2R, -S(O)2NR2, -S(O)R, -S(O)(NR)R, -P(O)(OR)2, -P(O)(NR2) 2 , -CF2(R), -CF3, -CR2(OR), -CR2(NR2), -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)O R, -OC(O)R, -OC(O)NR2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR2 or -N(R)S(O)2R; Each R 5 is independently an optionally substituted group selected from the following: C1-6 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring or a heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; n is 0, 1, or 2; Each m is independently 0, 1, 2, 3 or 4; and p is 0, 1, 2, 3 or 4.

64. The method of any one of claims 37-62, wherein the IRAK modulator comprises any one of the compounds of formula [II]-[V]: or a pharmaceutically acceptable salt thereof.

65. The method of any one of claims 37-62, wherein the IRAK modulator is a compound of formula [II], PROTAC IRAK-4 Degrader 1, or PF 06650833.

66. The method of any one of claims 37-61, wherein the IRAK modulator is CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or deoxyribozyme.

67. The method of any one of claims 37-66, wherein the IRAK modulator blocks TLR9 function.

68. The method of any one of claims 37-67, wherein the gene therapy agent is a viral vector.

69. The method of claim 68, wherein the viral vector is an AAV particle.

70. The method of claim 69, wherein the AAV particles comprise AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

71. The method of claim 70, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

72. The method of any one of claims 69-71, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, AAVrh8 ITRs, AAV9 ITRs, AAV10 ITRs, AAVrh10 ITRs, AAV11 ITRs, or AAV12 ITRs.

73. The method of claim 72, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

74. The method of claim 72, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

75. The method of claim 68, wherein the viral vector is an adenoviral particle.

76. The method of claim 75, wherein the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3, or a functional variant thereof.

77. The method of claim 68, wherein the viral vector is a lentiviral particle.

78. The method of claim 77, wherein the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

79. The method of claim 68, wherein the viral vector is a herpes simplex virus (HSV) particle.

80. The method of claim 79, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

81. The method of any one of claims 37-80, wherein the gene therapy agent is a lipid nanoparticle.

82. The method of any one of claims 37-81, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

83. The method of claim 82, wherein the heterologous transgene is operably linked to a promoter.

84. The method of claim 83, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

85. The method of any one of claims 37-84, wherein the IRAK modulator is administered prior to, simultaneously with, or after administration of the gene therapy agent.

86. The method of any one of claims 37-85, wherein the individual suffers from a disease or disorder amenable to treatment by gene therapy.

87. The method of claim 86, wherein the disease or disorder is a monogenic disease or disorder.

88. The method of any one of claims 37-87, wherein the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

89. The method of any one of claims 37-88, wherein the IRAK modulator is administered orally, intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, or intrahepatically.

90. The method of any one of claims 1-89, wherein the IRAK modulator reduces activated T cells.

91. Use of a composition in the manufacture of a medicament for delivering a nucleic acid to a cell of an individual in need thereof, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator.

92. Use of a composition in the manufacture of a medicament for delivering a nucleic acid to a cell of an individual in need thereof, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent.

93. Use of a composition in the manufacture of a medicament for treating an individual in need of gene therapy, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator.

94. Use of a composition in the manufacture of a medicament for treating an individual in need of gene therapy, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent.

95. Use of a composition in the manufacture of a medicament for modulating an immune response to gene therapy in an individual in need of gene therapy, wherein the composition comprises a gene therapy agent, and wherein the composition is formulated for use in combination with an IRAK modulator.

96. Use of a composition in the manufacture of a medicament for modulating an immune response of an individual to a gene therapy, wherein the composition comprises an IRAK modulator, and wherein the composition is formulated for use in combination with a gene therapy agent.

97. The use of any one of claims 91-96, wherein the gene therapy agent is an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle or a lipid nanoparticle.

98. The use of any one of claims 91-97, wherein the IRAK modulator is an IRAK-4 degrader.

99. A composition comprising a gene therapy agent for use in delivering a nucleic acid to a cell of an individual in need thereof, wherein the gene therapy agent is used in combination with an IRAK modulator.

100. A composition comprising an IRAK modulator for use in delivering a nucleic acid to a cell of an individual in need thereof, wherein the IRAK modulator is used in combination with a gene therapy agent.

101. A composition comprising a gene therapy agent for use in treating an individual in need of gene therapy, wherein the gene therapy agent is used in combination with an IRAK modulator.

102. A composition comprising an IRAK modulator for use in treating an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent.

103. A composition comprising an IRAK modulator for use in modulating an immune response to gene therapy in an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent.

104. A composition comprising an IRAK modulator for use in suppressing an immune response to gene therapy in an individual in need of gene therapy, wherein the IRAK modulator is used in combination with a gene therapy agent.

105. The composition of any one of claims 99-104, wherein the gene therapy agent is an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle.

106. The composition of any one of claims 99-105, wherein the IRAK modulator is an IRAK-4 degrader.

107. A kit for use in the method of any one of claims 1-90.

108. A kit for use as described in any one of claims 91-98.

109. A kit comprising the composition of any one of claims 99-106.

Images