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 degrader) in conjunction with the gene therapy to suppress natural immunity to the gene therapy. In some embodiments, the gene therapy uses an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a herpes simplex virus (HSV) vector, or a lipid nanoparticle. Also provided herein are methods of selecting an individual for treatment with an IRAK modulator in combination with a gene therapy agent.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 330,239, filed April 12, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference 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.

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

[0004] Successful gene therapy for the treatment of rare genetic diseases relies heavily on adeno-associated virus (AAV) viral vectors, which offer many attractive features, including tissue-specific tropism, transduction of quiescent cells, and maintenance of modified gene expression. However, immune responses to AAV vectors pose a major challenge to successful clinical translation. The capsid, viral genome, and transgene trigger an immune response, including activation of both innate and adaptive immunity of the immune system. The innate immune system, activated by the TLR pathway, then induces an adaptive immune response in human B and T cells exposed to pathogens (Iawaski, A and Medzhitov, R, Nat Immunol 2004 5(10):987-985). Based on several mouse studies, it has been shown that the endosomal DNA sensor TLR9, which recognizes CpG-rich hypomethylated DNA, plays a key role in the genome recognition of AAV vectors. The signaling cascade triggered by TLR9 activates the adaptive immune response, ultimately resulting in the clearance of transgene-induced cells by T cell-mediated cytotoxicity. Mice lacking the TLR9 sensor show longer maintenance of transgene expression and reduced immune responses (Ashley, SN et al., Cell Immunol 2019 Dec;346:103997 and Faust SM et al., J Clin Invest 2013 123(7):2994-3001). Consistent with this data, a hemophilia clinical trial revealed that a reduction in the total number of CpG bases reduced the need for pharmacological immunosuppressants in hemophilia patients and showed reduced cytotoxic T lymphocyte responses, while patients receiving vectors containing more CpG bases in the transgene required much more immunosuppressant drugs (Wright, JF Mol. Ther. 2020 28(3):701-703). Broad-acting immunosuppressants have been successful in improving AAV delivery in clinical trials but still result in loss of transgene expression and present the risk of side effects and opportunistic infections.The development of vectors without CpG bases is a challenge because non-codon-optimized vectors (i.e., vectors with no or low CpG content) exhibit poor transgene expression (Wright, JF Mol. Ther. 2020 28(3):701-703). Therefore, there is a need for a different class of immunomodulatory agents that exhibit enhanced specificity and fewer side effects.

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

[0006] In some embodiments, the invention provides a method of 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. In some embodiments, the invention provides a method of treating an individual in need of treatment with 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 embodiments, the invention provides a method of improving gene therapy in 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 embodiments, the invention provides a method of suppressing an immune response to a gene therapy agent in an individual, the method comprising a) administering an IRAK modulator 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 results in loss of function of IRAK. In some embodiments, the IRAK modulator is a small molecule.

[0008] In some embodiments, the IRAK modulator has formula [I]: [ka] or a pharma- ceutically acceptable salt thereof, wherein each variable is as defined and described herein. In some embodiments, the IRAK modulator is represented by formulas [II]-[V]: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the IRAK modulator is a compound of formula [II], PROTAC IRAK-4 degrader 1, or PF06650833. In some embodiments, the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme, or 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 particles are selected from the group consisting of 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 In some embodiments, the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation, or a HBKO mutation. In some embodiments, 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. In some embodiments, the one or more ITRs and capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the one or more ITRs and capsid of the AAV particle are derived from different AAV serotypes.

[0010] In some embodiments, the viral vector is an adenovirus particle. In some embodiments, the adenovirus 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 Ad3, canine Ad2, ovine Ad, or porcine Ad3, or a functional variant thereof.

[0011] In some embodiments, the viral vector is a lentiviral particle, hi some embodiments, the lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokara 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 a lipid nanoparticle.

[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 a gene therapy agent. In some embodiments, the individual has 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 invention provides a method of delivering a gene therapy agent to cells of an individual, the method comprising: a) incubating innate immune cells from the individual with a gene therapy agent; b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies an individual having 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 invention provides a method of treating an individual in need of treatment 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, where expression of a cytokine signature after incubation with the gene therapy agent identifies an individual having 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 invention provides methods of 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, where expression of a cytokine signature after incubation with the gene therapy agent identifies the individual for treatment with the gene therapy agent and an IRAK modulator; and c) selecting the individual identified in step b) for treatment with the gene therapy agent and an 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 cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells. In some embodiments, the innate immune cells are isolated 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 from the individual's monocytes.

[0019] In some embodiments, the method further comprises isolating monocytes from the individual and incubating the monocytes in dendritic cell culture medium to induce dendritic cells from the monocytes prior to 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 dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to induce dendritic cells from the monocytes. In some embodiments, the innate immune cells are replated prior to incubation with the gene therapy agent in step c). In some embodiments, the innate immune cells are replated in a microwell dish.

[0020] In some embodiments, the gene therapy agent is a viral vector and the innate immune cells are about 1×10 3 ~Approx. 1×10 5 Or about 1 x 10 4 In some embodiments, the gene therapy agent is a non-viral vector and 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 comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β. In some embodiments, the expression of a cytokine in the cytokine signature is increased compared to a suitable control. In some embodiments, a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells that have not been incubated with a gene therapy agent, or a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells prior to incubation with a gene therapy agent.

[0022] In some aspects, the invention provides for the use of the 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. In some aspects, the invention provides for the use of the 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 an IRAK modulator. In some aspects, the invention provides for the use of the 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 invention provides for the use of the 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 an IRAK modulator. In some aspects, the invention provides for the use of the 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. In some aspects, the invention provides for the use of a composition in the manufacture of a medicament for modulating an immune response to gene therapy in an individual, wherein the composition comprises an IRAK modulator, and 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 adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle. In some embodiments, the IRAK modulator is an IRAK-4 degrader.

[0023] In some embodiments, the present invention provides a composition comprising a gene therapy agent for use in delivering a nucleic acid to a cell of an individual in need thereof, where the gene therapy agent is used in combination with an IRAK modulator. In some embodiments, the present invention provides a composition comprising an IRAK modulator for use in delivering a nucleic acid to a cell of an individual in need thereof, where the IRAK modulator is used in combination with a gene therapy agent. In some embodiments, the present invention provides a composition comprising a gene therapy agent for use in treating an individual in need of gene therapy, where the gene therapy agent is used in combination with an IRAK modulator. In some embodiments, the present invention provides a composition comprising an IRAK modulator for use in treating an individual in need of gene therapy, where the IRAK modulator is used in combination with a gene therapy agent. In some embodiments, the present invention provides a composition comprising an IRAK modulator for modulating an immune response to gene therapy in an individual in need of gene therapy, where the IRAK modulator is used in combination with a gene therapy agent. In some embodiments, the present invention provides a composition comprising an IRAK modulator for suppressing an immune response to gene therapy in an individual in need of gene therapy, where 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 a kit for use in any of the methods or uses described herein. [Brief description of the drawings]

[0025] [Figure 1]The experimental scheme used to evaluate the efficacy of IRAK modulators (e.g., IRAK4 degraders and IRAK4 inhibitors) is shown. Peripheral blood mononuclear cells (PBMCs) were isolated from leukopak. CD14+ monocytes were purified from (PBMCs), and a differentiation factor cocktail was added to the monocytes to allow differentiation into dendritic cells, and maturation factors were added to obtain mature dendritic cells. Mature dendritic cells were pretreated with IRAK modulators such as dimlobicertib and PROTAC IRAK4 degrader-1 for 18 hours and then infected with AAV particles. 24 hours after AAV infection, the medium supernatant was collected for downstream analysis such as cytokine release and cytotoxicity. The cells were used to measure the level of IRAK4 inhibition or degradation. In a different setting, the IRAK4 modulators dimlobicertib IRAK4 and PROTAC IRAK4 degrader-1 are added simultaneously with AAV, called co-treatment, and the medium supernatant is collected for downstream analysis such as cytokine release and cytotoxicity. [Figure 2A-2B] Figure 2 shows that pretreatment with two different IRAK modulators (IRAK inhibitor and IRAK4 degrader) results in attenuation of cytokine release. Figure 2A shows attenuation of cytokine release from pretreatment of human monocytic dendritic cells with the IRAK4 inhibitor dimrobicertib. Figure 2B shows attenuation of cytokine release from pretreatment of human monocytic dendritic cells with the IRAK4 degrader PROTAC IRAK4 degrader-1. [Figure 3A-3B] Figure 3 shows that co-treatment with two different IRAK modulators (IRAK inhibitor and IRAK4 degrader) results in attenuation of cytokine release. Figure 3A shows attenuation of cytokine release from pre-treatment of human monocytic dendritic cells with the IRAK4 inhibitor dimrobicertib. Figure 3B shows attenuation of cytokine release from co-treatment of human monocytic dendritic cells with the PROTAC IRAK4 degrader-1. [Figure 4A-4B]Figure 4 shows that treatment with two different IRAK modulators (IRAK inhibitor and IRAK4 degrader) does not cause cytotoxicity in primary human monocytic dendritic cells. Figure 4A shows toxicity results after administration of the IRAK4 inhibitor dimrobicertib with AAV. Figure 4B shows toxicity results after administration of the PROTAC IRAK4 degrader-1 with AAV. [Figure 5A] Figure 5 shows 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 shows that treatment of human monocytic dendritic cells with the IRAK4 inhibitor dimrobicertib blocks IRAK4 kinase activity induced by LPS. [Figure 5B] Figure 5B shows 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 5B shows that PROTAC IRAK4 degrader-1AAV reduces IRAK4 levels in AAV.SAN024-infected cells. [Figure 6] FIG. 1 shows a schematic of an in vivo mouse experiment designed to show the effect of IRAK4 inhibition on CD8 and memory T cells following AAV injection. [Figure 7A-7B] Figure 7 shows that treatment with an IRAK4 inhibitor reduces transgene lacZ-specific CD8 T cells and effector memory T cells in PBMCs after AAV injection in mice. Figure 7A shows the reduction of LacZ-specific CD8 T cells in mice treated with the IRAK4 inhibitor dimlobicertib. Figure 7B shows the reduction of effector memory cells in mice treated with the IRAK4 inhibitor dimlobicertib. [Figure 8A-8B]Figure 8 shows that treatment with an IRAK4 inhibitor reduces IFNg-producing CD8 T cells in mice after AAV injection. Figure 8A shows interferon gamma production (a measure of activated CD8 T cells) after isolation of splenocytes from treated mice and subsequent stimulation with AAV peptide pools. Figure 8B shows interferon gamma production after isolation of splenocytes from treated mice and subsequent stimulation with immunodominant peptides. [Figure 8C] Figure 8C shows that treatment with an IRAK4 inhibitor reduces IFNg-producing CD8 T cells after AAV injection in mice, and Figure 8D shows the isolation of splenocytes from treated mice and subsequent interferon gamma production after stimulation with a LacZ immunodominant peptide. [Figure 9A-9B] IRAK4 degrader PROTAC IRAK4 degrader-1 treatment reduces IRAK4 expression in immune cells. Figure 9A shows the reduction of PBMCs by treatment with an IRAK degrader. Figure 9B shows the reduction of T cells by treatment with an IRAK degrader. [Fig. 9C-9D] IRAK4 degrader PROTAC IRAK4 degrader-1 treatment reduces IRAK4 expression in immune cells. Figure 9C shows the reduction of B cells by treatment with an IRAK degrader. Figure 9D shows the reduction of monocytes by treatment with an IRAK degrader. [Figure 10A-10B] Figure 10 shows that pretreatment with the IRAK4 degrader of formula (II) (KT-474) reduces capsids, transgene-specific CD8 T cells, and interferon gamma-producing immune cells after AAV injection in mice. Figure 10A shows the reduction of lacZ-specific CD8 T cells in PBMC. Figure 10B shows the reduction of lacZ-specific CD8 T cells in the spleen. [Figure 11] 1 shows an in vivo mouse experiment for oral treatment of an IRAK4 modulator administered in combination with AAV. The IRAK4 modulator can be administered prior to AAV or simultaneously with AAV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] In some aspects, the invention provides methods of delivering a nucleic acid to a cell of an individual, the method comprising: a) administering to the individual an IRAK modulator (e.g., an IRAK-4 degrader); and b) administering a gene therapy agent comprising the nucleic acid. As used herein, a "gene therapy agent" can be a therapeutic component and / or carrier component of a gene therapy agent (e.g., a nucleic acid (e.g., a terminally-closed DNA), a viral vector (e.g., an AAV vector, an adenoviral vector, a lentiviral vector, an HSV vector, etc.), a lipid nanoparticle, all or a portion of an antibody (e.g., a nanobody, an Fc region, etc.) that is administered to an individual. In some aspects, the invention provides methods of treating an individual in need of treatment with a composition comprising a gene therapy agent, the method comprising: a) administering to the individual an IRAK modulator; and b) administering to the individual a gene therapy agent. In some aspects, the invention provides methods of improving gene therapy in an individual, the method comprising: a) administering to the individual an IRAK modulator; and b) administering to the individual a gene therapy agent. In some embodiments, the invention provides methods of modulating an immune response to a gene therapy agent, the method comprising a) administering an IRAK modulator to an individual, and b) administering a gene therapy agent to the individual. In some embodiments, the invention provides methods of suppressing an immune response to a gene therapy agent, the method comprising a) administering an IRAK modulator to an individual, and b) administering a gene therapy agent to the individual. In some embodiments, the invention provides methods of inducing resistance to a gene therapy agent, the method comprising a) administering an IRAK modulator to an individual, and b) administering a gene therapy agent to the individual.

[0027] In some aspects, the invention provides a method of delivering a nucleic acid to cells of an individual, the method comprising: a) incubating innate immune cells from the individual with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles); b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies an individual having 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 isolating the innate immune cells from the individual prior to incubating the innate immune cells with the gene therapy agent. In some embodiments, the method further comprises isolating monocytes from the individual and incubating the monocytes in dendritic cell culture medium to induce dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent. As used herein, the term "inducing" dendritic cells includes differentiation of cells (e.g., monocytes) to produce dendritic cells.

[0028] In some aspects, the invention provides a method of treating an individual in need of treatment, the method comprising: a) incubating innate immune cells from the individual with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles); b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies an individual having 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 isolating the innate immune cells from the individual prior to incubating the innate immune cells with the gene therapy agent. In some embodiments, the method further comprises isolating monocytes from the individual and incubating the monocytes in dendritic cell culture medium to derive dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent.

[0029] In some aspects, the invention provides a method of selecting an individual for treatment with a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and an IRAK modulator (e.g., an IRAK-4 degrading agent), the method comprising: a) incubating innate immune cells with the gene therapy agent; b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies the individual for treatment with the gene therapy agent and the IRAK modulator; and 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 isolating the innate immune cells from the individual prior to incubating the innate immune cells with the gene therapy agent. In some embodiments, the method further comprises isolating monocytes from the individual and incubating the monocytes in dendritic cell culture medium to derive dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent, in some embodiments, the method further comprises d) administering an IRAK modulator to said individual identified in step b), and e) administering said gene therapy agent to said individual identified in step b).

[0030] general technology The techniques and procedures described or referenced herein are generally well understood by those of skill in the art and are described, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., 2004). thed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,2012);Current Protocols in Molecular Biology(F.M.Ausubel,et al.eds.,2003);Methods in Enzymologyシリーズ(Academic Press,Inc.);PCR 2:A Practical Approach(M.J.MacPherson,B.D.Hames and G.R.Taylor eds.,1995);Antibodies,A Laboratory Manual(Harlow and Lane,eds.,1988);Culture of Animal Cells:A Manual of Basic Technique and Specialized Applications(R.I.Freshney,6 thed.,J.Wiley and Sons,2010);Oligonucleotide Synthesis(M.J.Gait,ed.,1984);Methods in Molecular Biology,Humana Press;Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,Academic Press,1998);Introduction to Cell and Tissue Culture(J.P.Mather and P.E.Roberts,Plenum Press,1998);Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,J.Wiley and Sons,1993-8);Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.,1996);Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987);PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(J.E.Coligan et al.,eds.,1991);Short Protocols in Molecular Biology(Ausubel et al.,eds.,J.Wiley and Sons,2002);Immunobiology(C.A.Janeway et al.,2004);Antibodies(P.Finch,1997);Antibodies:A Practical Approach(D.Catty.,ed.,IRL Press,1988-1989);Monoclonal Antibodies:A Practical Approach(P.Shepherd and C.Dean,eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JBLippincott Company, 2011).

[0031] definition As used herein, the term "IRAK degrader" refers to a heterobifunctional compound that binds to and / or inhibits (fully or partially) both IRAK kinase and an E3 ligase with measurable affinity, resulting in ubiquitination and subsequent degradation of the IRAK kinase. In certain embodiments, the degrader has a DC concentration 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 have an attached E3 ligase binding moiety.

[0032] As used herein, the term "inhibitor" with respect to IRAK is a compound that binds to and / or inhibits (fully or partially) IRAK kinase with measurable affinity. In certain embodiments, an 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 constants.

[0033] As used herein, the term "modulator" with respect to IRAK is a compound that stimulates, retards, inhibits and / or suppresses (fully or partially) the activity of IRAK kinase.

[0034] As used herein, the term "gene therapy" refers to a therapy in which the expression of a nucleic acid (e.g., a gene, an mRNA, etc.) in an individual's cells is modified to change the biological properties of the cells. In some examples, gene therapy involves the delivery of an exogenous nucleic acid that is expressed in the individual's cells. In some examples, gene therapy alters (e.g., degrades, inhibits, enhances) the expression of an endogenous gene in the individual's cells. In some examples, gene therapy is an in vivo therapy. In some examples, gene therapy is an ex vivo therapy (e.g., cell therapy).

[0035] As used herein, the term "gene therapy agent" refers to a nucleic acid (e.g., expression construct, miRNA, antisense, shRNA, siRNA) or a nucleic acid in combination with an agent 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, mRNAs) in an individual or cell to alter the biological properties of a living cell. Examples of gene therapy agents include, but are not limited to, viral vectors (e.g., adeno-associated virus, adenovirus, lentivirus, herpes simplex virus, baculovirus), bacterial vectors, and non-viral vectors (e.g., lipid nanoparticles encapsulating a therapeutic nucleic acid or plasmid DNA (e.g., end-closed DNA) that contains a therapeutic nucleic acid and / or encodes a therapeutic polypeptide).

[0036] As used herein, a "vector" refers to a recombinant plasmid or virus containing a nucleic acid that is delivered to a host cell either in vitro or in vivo.

[0037] The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, 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 containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the nucleic acid may contain sugar and phosphate groups (as typically found in RNA or DNA) or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the nucleic acid may comprise a polymer of synthetic subunits such as phosphoramidates, and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. Furthermore, double-stranded nucleic acids can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing the complementary strand and annealing the strands under appropriate conditions or synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0038] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer 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, including, but not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to a protein containing modifications such as deletions, additions and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host producing the protein or by errors due to PCR amplification.

[0039] "Recombinant viral vector" refers to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one, e.g., two, inverted terminal repeats (ITRs).

[0040] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) flanked by at least one, e.g., two, AAV inverted terminal repeats (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that is infected with a suitable helper virus (or expresses suitable helper functions) and expresses the AAVrep and cap gene products (i.e., AAVRep and Cap proteins). When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector, such as a plasmid used for cloning or transfection), the rAAV vector can be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. rAAV vectors can be in a variety of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and in embodiments, encapsulated in viral particles, particularly AAV particles. The rAAV vector can be packaged into an AAV viral capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)."

[0041] "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.

[0042] "Recombinant adenoviral vector" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of adenoviral origin) 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). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell expressing essential adenoviral genes (e.g., E1, E2, E4, etc.) that have been deleted from the recombinant viral genome. When the recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector, such as a plasmid used for cloning or transfection), the recombinant viral vector can be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of adenoviral packaging functions. Recombinant viral vectors can be in a variety of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsulated in viral particles, such as adenoviral particles. The recombinant viral vector can be packaged into an adenovirus viral capsid to produce a "recombinant adenovirus particle."

[0043] "Recombinant lentiviral vector" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of lentiviral origin) flanked by at least one lentiviral long terminal repeat (LTR). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral long terminal repeats (LTR). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell infected with appropriate helper functions. Recombinant lentiviral vectors can be packaged into lentiviral capsids to generate "recombinant lentiviral particles".

[0044] "Recombinant herpes simplex vector (recombinant HSV vector)" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not of HSV origin) flanked by HSV terminal repeat sequences. Such recombinant viral vectors, when present in a host cell infected with appropriate helper functions, can be replicated and packaged into infectious viral particles. When the recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector, such as a plasmid used for cloning or transfection), the recombinant viral vector can be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of HSV packaging functions. Recombinant viral vectors can be in a variety of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsulated in viral particles, e.g., HSV particles. Recombinant viral vectors can be packaged into HSV capsids to generate "recombinant herpes simplex viral particles."

[0045] As used herein, "solid lipid nanoparticles" (SLNS, 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 materials. A payload, such as a nucleic acid, can be embedded within. In some instances, the lipid nanoparticle is a liposome that includes a lipid bilayer.

[0046] As used herein, the term "improving" with respect to gene therapy may refer to the act of boosting, elevating, prolonging or otherwise increasing the expression of a therapeutic gene payload of a gene therapy agent. In some embodiments, an improved gene therapy is one in which the expression of a therapeutic gene payload of a gene therapy agent administered with an IRAK modulator is increased by more than about any of 10%, 25%, 50%, 75% or 100% compared to gene therapy administered without an IRAK modulator. In some embodiments, an improved gene therapy is one in which the expression time of a therapeutic gene payload of a gene therapy agent administered with an IRAK modulator is prolonged by more than about any of 10%, 25%, 50%, 75% or 100% compared to gene therapy administered without an IRAK modulator. In some examples, the gene therapy is improved by reducing the immune response (e.g., innate immune response) to the gene therapy agent. In some embodiments, an improved gene therapy is one in which the immune response to a gene therapy agent administered with an IRAK modulator is reduced by more than about any of 10%, 25%, 50%, 75% or 100% compared to gene therapy administered without the IRAK modulator. In some embodiments, a reduced immune response to a gene therapy agent is measured as a decrease in cytokine signature following exposure of immune cells to the gene therapy agent in the presence of an IRAK modulator compared to exposure of immune cells to the gene therapy agent in the absence of the IRAK modulator.

[0047] As used herein, the term "modulate" when referring to gene therapy can refer to the act of altering, changing, varying, improving, or otherwise modifying the presence or activity of a gene therapy agent. For example, modulating an immune response to a gene therapy agent can refer to any act that results in altering, changing, varying, improving, or otherwise modifying the immune response to the gene therapy agent (e.g., reducing, delaying, and / or eliminating an immune response (e.g., a natural immune response) to the gene therapy agent).

[0048] As used herein, the term "cytokine signature," in reference to an immune response (e.g., innate immune response) to a gene therapy agent, refers to a change (e.g., increase, decrease) in expression of one or more cytokines following exposure of innate immune cells to a gene therapy agent. In some examples, the cytokines of the cytokine signature are specific to a TLR pathway (e.g., TLR2, TLR3, TLR4, or TLR9 pathway).

[0049] Innate immune cells are white blood cells that mediate natural 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, often referred to as transduction efficiencies. Some serotypes, such as AAV1, are efficient at transducing certain immune cells, such as monocytes, while other AAVs, such as AAV6, are efficient at transducing cells, such as dendritic cells (Grimm, D et al., J. Virol., 2008, 82(12):5887-5911). AAV can induce an immune response upon entering a cell. The strength of this immune response depends on the AAV serotype and cell type. Once AAV transduces host immune cells, they can associate with immune receptors, such as TLRs (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.2009;119(8):2388-2398;Ashley SN et al.,Cell.Immunol.2019,346:103997). When 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,2020 180(6):1044-1066).

[0050] These cytokines also play a role in activating the adaptive immune system, including B cells and T cells, which produce antibodies and generate cytotoxicity to kill virus-infected cells, respectively. As used herein, the upregulation or downregulation of a particular subset of cytokines is referred to as a "cytokine signature." These cytokine signatures, which include three or more cytokines, can be used as predictive markers of disease and treatment success. Examples of cytokine signatures can be found in Zuniga, J et al., Int. J. Infect. Diseases, 2020, 94:4-11; Bergamaschi, C et al., Cell Reports, 2021, 36:109504; Del Valle, DM et al., Nat. Med. 2020, 26:1636-1643.

[0051] "Heterologous" means derived from a genotypically different entity than 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 (which, when expressed, can encode a heterologous polypeptide). 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.

[0052] The term "transgene" refers to a nucleic acid that can be introduced into a cell, transcribed into RNA, and optionally translated and / or expressed under appropriate conditions. In an aspect, it confers a desired property to the cell into which it is introduced, or otherwise results in a desired therapeutic or diagnostic outcome. In another aspect, it can be transcribed into a molecule that mediates RNA interference, such as an siRNA.

[0053] The terms "genomic particles (gp)," "genomic equivalents," or "genomic copies" as used with respect to viral titer refer to the number of virions containing a recombinant AAV DNA genome, regardless of infectiousness or functionality. The number of genome particles in a particular vector preparation can be measured by procedures such as those described in the Examples herein or, for example, Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0054] The terms "infectious unit (iu)," "infectious particle," or "replication unit," when used in reference to viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay (also known as the replication center assay), e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0055] The term "transducing unit (tu)" when used with respect 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 the Examples herein or in a functional assay such as that described in, for example, Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0056] "Inverted terminal repeat" or "ITR" sequences are a term well understood in the art and refer to relatively short sequences found at the ends of inverted viral genomes.

[0057] "AAV inverted terminal repeat (ITR)" sequences, a term well understood in the art, are sequences of approximately 145 nucleotides present at both ends of a naturally occurring single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be in either of two different orientations, resulting in heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides also contain several short self-complementary regions (A, A', B, B', C, C', and D regions), allowing intrastrand base pairing within this portion of the ITR.

[0058] "Terminal resolution sequence" or "TRS" is the sequence of the D region of the AAV ITR that is cleaved by AAVrep protein during viral DNA replication. The mutated terminal resolution sequence is resistant to cleavage by AAVrep protein. "AAV helper function" refers to the function that allows AAV to replicate and be packaged by a host cell. AAV helper function can be provided in any of several forms, including, but not limited to, helper virus or helper virus genes that facilitate AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0059] "AAV helper functions" refers to functions that allow AAV to replicate and be packaged by a host cell. AAV helper functions can be provided in any of several forms, including, but not limited to, helper viruses or helper virus genes that facilitate AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0060] AAV "helper virus" refers to a virus that allows AAV (which is a defective parvovirus) to replicate and be packaged by a host cell. Many such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses such as vaccinia virus, and baculoviruses. Adenoviruses encompass several different subgroups, with adenovirus type 5 (Ad5) of subgroup C being the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as ATCC. Viruses of the herpes family that are also available from depositories such as ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.

[0061] "Percent 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 the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be accomplished in a variety of ways within the skill of the art, such as using publicly available computer software programs, including BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, as described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, Section 7.7.18, Table 7.7.1. A possible alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For purposes herein, the amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having a certain amino acid sequence identity percentage to a given amino acid sequence B) is calculated as follows: fraction X / Y×100, where X is the number of amino acid residues scored as identical matches by a sequence alignment program in the alignment of A and B in that program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity percentage of A to B is not equal to the amino acid sequence identity percentage of B to A.For purposes herein, the nucleic acid sequence identity of a given nucleic acid sequence C to a given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C having a particular % nucleic acid sequence identity to a given nucleic acid sequence D) is calculated as follows: fraction W / Z x 100, where W is the number of nucleotides scored as identical matches by a sequence alignment program in that program's alignment of C and D, and Z is the total number of nucleotides in D. It will be understood that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, then the % nucleic acid sequence identity of C to D will not be equal to the % nucleic acid sequence identity of D to C.

[0062] An "effective amount" of an agent refers to an amount effective at the dosage and for the period of time necessary to achieve a desired therapeutic effect. For example, an "effective amount" of a gene therapy agent refers to an amount effective at the dosage and for the period of time necessary to achieve a desired gene therapy effect. In another example, an effective amount of an IRAK modulator can refer to an amount effective at the dosage and for the period of time necessary to achieve a desired result of improved gene therapy.

[0063] A "therapeutically effective amount" of a substance / molecule (e.g., a gene therapy agent and / or an IRAK modulator) of the invention can vary depending on 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.

[0064] The term "suitable control", as it refers to a cytokine signature, is the expression of a cytokine in a cytokine signature from innate immune cells that have not been incubated with a gene therapy agent, or the expression of a cytokine in a cytokine signature from innate immune cells prior to incubation with a gene therapy agent.

[0065] Administration "in combination with," as it relates to a gene therapy agent and a modulator of an innate immune response (e.g., an IRAK modulator), includes simultaneous (concurrent), sequential or sequential administration in any order of the gene therapy agent and the modulator of an innate immune response (e.g., an IRAK modulator).

[0066] The term "concurrently" is used herein to refer to administration of a gene therapy agent and a modulator of an innate immune response (e.g., an IRAK modulator) where at least a portion of the administration overlaps in time. Thus, concurrent administration includes dosing regimens where administration of a gene therapy agent or a modulator of an innate immune response (e.g., an IRAK modulator) continues after administration of the other agent / modulator is discontinued.

[0067] As used herein, "in combination with" refers to the administration of one therapy in addition to another. Thus, "in combination with" refers to the administration of one therapeutic modality (such as a gene therapy agent or a modulator of the innate immune response (e.g., an IRAK modulator)) before, during, or after the administration of another therapeutic modality to an individual.

[0068] An "isolated" molecule (eg, a nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0069] Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter per se. For example, reference to "about X" includes the description of "X."

[0070] As used herein, the singular articles "a," "an," and "the" include plural references unless otherwise indicated.

[0071] It will be understood that aspects and embodiments of the invention described herein include those that "comprise", "consist" and / or "consist essentially of".

[0072] Treatment method In some embodiments, the invention provides a method of 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. In some embodiments, the invention provides a method of treating an individual in need of treatment, the method comprising a) administering an IRAK modulator to the individual, and b) administering a gene therapy agent to the individual. In some embodiments, the invention provides a method of improving gene therapy in 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 embodiments, the invention provides a method of modulating 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 embodiments, the invention provides a method of 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 invention provides methods of inducing resistance to a gene therapy agent, the method comprising: a) administering an IRAK modulator to an individual; and b) administering a gene therapy agent to the individual. In some embodiments, the IRAK modulator modulates the activity of an IRAK protein kinase. In some embodiments, the IRAK modulator modulates the activity of an 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 adenovirus vector, a lentivirus vector, or a herpes simplex virus (HSV) vector.

[0073] In some embodiments of the invention, the gene therapy agent (e.g., AAV particles, adenovirus particles, lentivirus particles, HSV particles, or lipid nanoparticles) may be administered to a specific tissue of interest or may be administered systemically. In some embodiments, an effective amount of the gene therapy agent may be administered to the subject. In some embodiments, the effective amount of the gene therapy agent may be administered parenterally. Parenteral administration routes include, but are not limited to, intravenous, intraperitoneal, intraosseous, intraarterial, intracerebral, intramuscular, intrathecal, subcutaneous, intraventricular, intrahepatic, and the like. In some embodiments, the effective amount of the gene therapy agent may be administered by one administration route. In some embodiments, the effective amount of the gene therapy agent may be administered by a combination of multiple administration routes (e.g., two, three, etc.). In some embodiments, the effective amount of the gene therapy agent is administered at one location. In other embodiments, the effective amount of the gene therapy agent may be administered at multiple locations.

[0074] An effective amount of a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) is administered depending on the goal of the treatment. For example, if a low percentage of transduction or transfection can achieve the desired therapeutic effect, the goal of the treatment is usually to meet or exceed this level of transduction or transfection. In some cases, this level of transduction or transfection can be achieved by transduction or transfection of only about 1-5% of the target cells of the desired tissue type, in some embodiments 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 at least about 99% of the cells of the desired tissue type. The gene therapy agent may be administered in one or more doses during the same procedure or spaced apart by days, weeks, months, or years. Any one or more of the routes of administration described herein may be used. In some embodiments, multiple gene therapy agents, e.g., AAV vectors and lentiviral vectors, may be used to treat a human.

[0075] Methods for identifying cells that have been transduced or transfected with a gene therapy agent are known in the art. For example, immunohistochemistry or markers such as enhanced green fluorescent protein can be used to detect cells that have been transduced or transfected with a gene therapy agent.

[0076] 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 simultaneously or sequentially at multiple locations. In other embodiments, an effective amount of a gene therapy agent is administered multiple times (e.g., repeatedly) at a single location. In some embodiments, multiple injections of gene therapy agent are administered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, or 24 hours apart.

[0077] In some embodiments, the method includes administering an effective amount of a pharmaceutical composition comprising a gene therapy agent to treat an individual in need of gene therapy. 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 x 10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 12 ~6×10 12 , 6×10 12 ~7×10 12 , 7×10 12 ~8×10 12 , 8×10 12 ~9×10 12 , 9×1012 ~10×10 12 , 10×10 12 ~11×10 12 , 11×10 12 ~15×10 12 , 15×10 12 ~20×10 12 , 20×10 12 ~25×10 12 , 25×10 12 ~30×10 12 , 30×10 12 ~50×10 12 Or 50 x 10 12 ~100×10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 12 ~10×10 12 , 10×10 12 ~25×10 12 Or 25 x 10 12 ~50×10 12 In 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 x 10 9 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 9 ~6×10 9 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9 ~9×10 9 , 9×10 9 ~10×10 9, 10×10 9 ~11×10 9 , 11×10 9 ~15×10 9 , 15×10 9 ~20×10 9 , 20×10 9 ~25×10 9 , 25×10 9 ~30×10 9 , 30×10 9 ~50×10 9 Or 50 x 10 9 ~100×10 9 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9 ~25×10 9 Or 25 x 10 9 ~50×10 9 In 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 x 10 10 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 infectious units / mL. 10 ~6×10 10 , 6×10 10 ~7×10 10 , 7×10 10 ~8×10 10 , 8×10 10 ~9×10 10, 9×10 10 ~10×10 10 , 10×10 10 ~11×10 10 , 11×10 10 ~15×10 10 , 15×10 10 ~20×10 10 , 20×10 10 ~25×10 10 , 25×10 10 ~30×10 10 , 30×10 10 ~40×10 10 , 40×10 10 ~50×10 10 Or 50 x 10 10 ~100×10 10 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 infectious units / mL. 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 Or 25 x 10 10 ~50×10 10 infectious units / mL.

[0078] In some embodiments, the dose of 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 ~Approx. 6×10 13 In some embodiments, the dose of the gene therapy agent administered to an individual is about 1×10 genome copies / kg body weight. 8 ~Approx. 6×10 13 genome copies / kg body weight.

[0079] In some embodiments, the total amount of gene therapy agent (e.g., AAV particle, adenovirus particle, lentivirus particle, HSV particle, or lipid nanoparticle) administered to an individual is at least about 1×10 9 ~Approx. 1×10 14In some embodiments, the total amount of gene therapy administered to an individual is about 1×10 9 ~Approx. 1×10 14 Either of the genome copies.

[0080] The compositions of the invention comprising a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) can be used alone or in combination with one or more additional therapeutic agents in addition to an IRAK modulator. The interval between sequential administrations can be at least minutes, hours, or days (or alternatively less).

[0081] In some embodiments of the present invention, compositions containing an IRAK modulator (e.g., an IRAK-4 degrading agent) may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol solutions. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media.

[0082] The amount of IRAK modulator (e.g., an IRAK-4 degrading agent) that can be combined with carrier materials to produce a composition (e.g., a pharmaceutical composition) in a unitary dosage form will vary depending on the individual and the particular mode of administration. In some embodiments, compositions comprising an IRAK modulator are formulated so that a dosage of about 0.01 mg / kg to about 100 mg / kg body weight of the IRAK modulator is administered to an individual. In some embodiments, the IRAK modulator is administered to an individual at a dose of 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 The compound is orally or parenterally administered at any of the following dosage levels: about 0.0 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 body weight. In some embodiments, the IRAK modulator is administered orally or parenterally at a dosage level 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 per individual.

[0083] Liquid dosage forms for oral administration of IRAK modulators (e.g., IRAK-4 degraders) include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and aromatic agents.

[0084] Injectable preparations, for example, sterile injectable aqueous or oily suspensions or IRAK modulators (e.g., IRAK-4 decomposition agents) can be formulated according to known techniques using appropriate dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol solutions. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0085] The injectable formulations or IRAK modulators (e.g., IRAK-4 degrading agents) can be sterilized, for example, by filtration through a bacterial-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.

[0086] 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, pharma- ceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol, glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0087] Solid compositions of a similar type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can optionally contain opacifying agents and can also be of a composition that releases the active ingredient 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 a similar type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0088] The IRAK modulator (e.g., IRAK-4 degrader) may also be in microencapsulated form with one or more excipients as described above. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings and other coatings well known in the pharmaceutical formulation art. 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 additional substances other than inert diluents, as is customary, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents and may also be of a composition that releases the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

[0089] In some embodiments, the IRAK modulator (e.g., an IRAK-4 degrading agent) 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, the IRAK modulator is administered to the 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, 1 week, or more than 1 week prior to administration of the gene therapy agent. In some embodiments, the IRAK modulator is administered to the 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, or less than 1 week prior to administration of the gene therapy agent. In some embodiments, the IRAK modulator and the gene therapy agent are administered at about the same time (e.g., within about an hour). In some embodiments, the IRAK modulator is administered after administration of the gene therapy agent. In some embodiments, the IRAK modulator is administered to the individual either 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, 1 week, or more than 1 week after administration of the gene therapy agent. In some embodiments, the IRAK modulator is administered to the individual either 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 less than 1 week after administration of the gene therapy agent.

[0090] 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 a disease or disorder amenable to treatment by gene therapy. In some embodiments, the disease or disorder is a monogenic disease or disorder.

[0091] In some embodiments, gene therapy agents (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) are used in combination with IRAK modulators (e.g., IRAK-4 degraders) to treat CNS disorders, including but not limited to 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, corticoganglionic degeneration or CBGD, frontotemporal dementia or FTD, progressive supranuclear palsy or PSP, multiple system atrophy or MSA, brain cancer, and lysosomal storage diseases (LSD). Other non-limiting examples of disorders of the present invention that may be treated by gene therapy in combination with IRAK modulators include traumatic brain injury, enzyme dysfunction disorders, psychiatric disorders (including post-traumatic stress syndrome), neurodegenerative diseases and cognitive disorders (including dementia, autism and depression), enzymatic dysfunction disorders including, but not limited to, leukodystrophies (including Canavan disease).

[0092] In some embodiments, gene therapy agents (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles) are used in combination with IRAK modulators (e.g., IRAK-4 degraders) 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 often caused by deficiencies in enzymes required for proper mucopolysaccharide, glycoprotein, and / or lipid metabolism, resulting in the pathological accumulation of lysosomal stored cellular material. Non-limiting examples of lysosomal storage diseases of the invention that can be treated by a therapeutic polypeptide or therapeutic nucleic acid of the invention include Gaucher disease type 2 or 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, mannosidosis, beta-mannosidosis, metachromatic leukodystrophy, mucolipidosis II / III, Niemann-Pick A disease, Niemann-Pick C disease, Pompe disease, Sandhoff disease, Sanfilippo A disease, Sanfilippo B disease, Sanfilippo C disease, Sanfilippo D disease, Schindler disease, Sly disease, Tay-Sachs disease, and Wolman disease.

[0093] 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 tubular myopathy, spinal muscular atrophy, Leber congenital amaurosis, choroideremia, Leber congenital optic neuropathy, ornithine transcarbamylase (OTC) deficiency, citrullinemia type 1, phenylketonuria (PKU), adrenoleukodystrophy, sickle cell syndrome, muscular dystrophy, or beta thalassemia.

[0094] In some aspects, the invention provides a composition for use in the manufacture of a medicament for delivering a nucleic acid to a cell of an individual in need thereof, the composition comprising a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrading agent). In some aspects, the invention provides a composition for use in the manufacture of a medicament for delivering a nucleic acid to a cell of an individual in need thereof, the composition comprising an IRAK modulator (e.g., an IRAK-4 degrading agent) and formulated for use 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).

[0095] In some aspects, the invention provides a composition for use in the manufacture of a medicament for treating an individual in need of gene therapy, the composition comprising a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrading agent). In some aspects, the invention provides a composition for use in the manufacture of a medicament for treating an individual in need of gene therapy, the composition comprising an IRAK modulator (e.g., an IRAK-4 degrading agent) and formulated for use 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).

[0096] In some aspects, the invention provides a composition for use in the manufacture of a medicament for modulating an immune response to gene therapy in an individual in need of gene therapy, the composition comprising a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrading agent). In some aspects, the invention provides a composition for use in the manufacture of a medicament for modulating an immune response to gene therapy in an individual, the composition comprising an IRAK modulator (e.g., an IRAK-4 degrading agent) and formulated for use 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).

[0097] In some aspects, the invention provides a composition for use in the manufacture of a medicament for suppressing an immune response to gene therapy in an individual in need of gene therapy, the composition comprising a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrading agent). In some aspects, the invention provides a composition for use in the manufacture of a medicament for suppressing an immune response to gene therapy in an individual, the composition comprising an IRAK modulator (e.g., an IRAK-4 degrading agent) and formulated for use 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).

[0098] In some aspects, the invention provides a composition for use in the manufacture of a medicament for improving gene therapy in an individual in need thereof, the composition comprising a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrading agent). In some aspects, the invention provides a composition for use in the manufacture of a medicament for improving gene therapy in an individual, the composition comprising an IRAK modulator (e.g., an IRAK-4 degrading agent) and formulated for use 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).

[0099] In some aspects, the invention provides a composition for use in the manufacture of a medicament for inducing resistance to gene therapy in an individual in need of gene therapy, the composition comprising a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and formulated for use in combination with an IRAK modulator (e.g., an IRAK-4 degrading agent). In some aspects, the invention provides a composition for use in the manufacture of a medicament for inducing resistance to gene therapy in an individual, the composition comprising an IRAK modulator (e.g., an IRAK-4 degrading agent) and formulated for use 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).

[0100] In some aspects, the invention provides a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) for use in delivering a nucleic acid to a cell of an individual in need thereof, the gene therapy agent being used in combination with an IRAK modulator (e.g., an IRAK-4 degrading agent). In some aspects, the invention provides an IRAK modulator (e.g., an IRAK-4 degrading agent) for use in delivering a nucleic acid to a cell of an individual in need thereof, the gene therapy agent being used in combination with an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle.

[0101] In some aspects, the invention provides a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) for use in treating an individual in need of gene therapy, the gene therapy agent being used in combination with an IRAK modulator (e.g., an IRAK-4 degrading agent). In some aspects, the invention provides an IRAK modulator (e.g., an IRAK-4 degrading agent) for use in treating an individual in need of gene therapy, the gene therapy agent being used in combination with an IRAK modulator (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle).

[0102] In some aspects, the present invention provides IRAK modulators (e.g., IRAK-4 degraders) for modulating an immune response to gene therapy in an individual in need of gene therapy, the IRAK modulator being 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).

[0103] In some aspects, the present invention provides IRAK modulators (e.g., IRAK-4 degraders) for suppressing an immune response to gene therapy in an individual in need of gene therapy, the IRAK modulator being 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).

[0104] IRAK Modulator In some aspects, the present invention provides methods of using IRAK modulators with gene therapy agents for improved gene therapy by inhibiting immune responses (e.g., innate immune responses, adaptive immune responses) to gene therapy agents. IRAK plays a central role in defense responses against pathogens introduced into the human body by inducing acute inflammation and subsequently triggering further adaptive immune responses. IRAK is an essential component of the interleukin-1 receptor signaling pathway and several 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 via adaptor proteins, primarily MyD88.

[0105] The IRAK family is composed of IRAK-1, IRAK-2 and IRAK-4, which are expressed in various human immune cell types, and IRAK-M (also called IRAK-3), whose expression is primarily restricted 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. The DD serves as a platform that allows protein-protein interactions with other DD-containing proteins, the most important of which is the adaptor protein myeloid differentiation factor 88 (MyD88). The inventors hypothesize that blocking IRAK function will result in specific blocking of the TLR9 pathway, leading to specific immune modulation. In some embodiments, an IRAK modulator is used to block the TLR9 pathway. In some embodiments, the IRAK modulator blocks TLR9 function. In some embodiments, the IRAK modulator is an IRAK inhibitor and / or an IRAK degrader.

[0106] In some embodiments, the IRAK degraders are bifunctional compounds that function to recruit IRAK kinase to an E3 ubiquitin ligase for degradation. In some embodiments, the IRAK degraders are modulators of targeted ubiquitination of IRAK kinase.

[0107] Protein degraders are bifunctional compounds that contain three components: an E3 ubiquitin ligase ligand, a linker and a ligand of a target protein of interest. They induce the formation of a ternary complex by simultaneously binding to both the E3 ligase and the target protein. The ternary complex formation effectively recruits the E3 ligase to polyubiquitinate the target of interest, inducing subsequent degradation by the proteasome. Degraders are attractive tools for use in inducing selective protein knockdown in a reversible and tunable manner. In some embodiments, the IRAK degrader is a protein degradation targeting chimera (PROTAC).

[0108] In some embodiments, the IRAK degrading agent is an IRAK-1 degrading agent, an IRAK-2 degrading agent, an IRAK-M degrading agent (or an IRAK-3 degrading agent), or an IRAK-4 degrading agent.

[0109] Suitable IRAK4 decomposer compounds for use in the methods of the present invention are described in WO 2019 / 133531, WO 2020 / 113233, WO 2020 / 264490, WO 2021 / 127283 or WO 2021 / 011868.

[0110] In some embodiments, the IRAK-4 degrading agent is of formula [I] as described in U.S. Pat. No. 11,117,889. [ka] or a pharma- ceutically acceptable salt thereof, wherein X 1 are covalent bonds, -CH2-, -C(O)-, -C(S)-, and [ka] is a divalent moiety selected from R 1a is hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O)2R, -N(R)2, -Si(R)3 or optionally substituted C l~4 It is 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 teeth, [ka] [ka] [ka] is a bicyclic or tricyclic ring selected from Ring B a is a fused ring selected from a 6-membered aryl containing 0 to 2 nitrogen atoms, a 5- to 7-membered partially saturated carbocyclyl, a 5- to 7-membered partially saturated heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-membered heteroaryl having 1 to 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 is hydrogen, C l~4 aliphatic or -CN; Each R 6a is independently 1~6 an optionally substituted group selected from aliphatic, phenyl, 4- to 7-membered saturated or partially unsaturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Ring A is a 4-10 membered saturated monocyclic or bicyclic carbocyclic or heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring C is a 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 independently represents a covalent bond or C 1~3 a divalent linear or branched saturated or unsaturated hydrocarbon chain, wherein one to three 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 C 1~6 an optionally substituted group selected from aliphatic, phenyl, a 4- to 7-membered saturated or partially unsaturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom optionally together with their intervening atoms form an optionally substituted 4-11 membered saturated or partially unsaturated carbocyclic or heterocyclic monocyclic, bicyclic, bridged bicyclic, spirocyclic, 5- or heteroaryl ring having, in addition to the atom to which they are attached, 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; 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), -CR 2(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 teeth, [ka] , hydrogen or C 1~6 is selected from optionally substituted groups selected from aliphatic or 4-11 membered saturated or partially unsaturated monocyclic, bicyclic, bridged bicyclic or spirocyclic carbocyclic or heterocyclic rings having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring D is a 4- to 10-membered saturated or partially unsaturated monocyclic or bicyclic carbocyclic or heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl ring having 1 to 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)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, Each R 5are independently optionally substituted groups selected from C1-6 aliphatic, phenyl, 3- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic rings having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl rings 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.

[0111] In some embodiments, the IRAK-4 decomposer includes 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-((4-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[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 pharma- ceutically 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]heptan-5-yl-N-(3-(difluoromethyl)-1-((1r,4R)-4-((4-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[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 pharma- ceutically acceptable salts thereof.

[0112] In some embodiments, the IRAK-4 decomposer comprises a compound of formula [II] or a deuterated form of the compound of formula [II] or a pharma- ceutically acceptable salt thereof (as described in WO 2021 / 247897). [ka]

[0113] The compound of formula [II] is 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-((4-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[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.

[0114] In some embodiments, the IRAK-4 degrader comprises a compound of formula [III] or [IV] or a pharma- ceutically acceptable salt thereof (as described in ACS Med. Chem. Lett. 2019, 10, 7, 1081-1085). [ka]

[0115] In some embodiments, the IRAK-4 degrader 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]undecan-3-yl)prop-1-yn-1-yl)-7-methoxyisoquinoline-6-carboxamide.

[0116] In some embodiments, the IRAK-4 degrader comprises an IRAK-4 degrader described in WO 2019 / 160915, WO 2020 / 006265, or WO 2021 / 168197.

[0117] In some embodiments, the IRAK-4 inhibitor is dimlobicertib, a compound of formula [V], or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof. In some embodiments, the IRAK-4 degrader is a compound having the CAS number: 1817626-54-2. [ka]

[0118] 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 dimlobicertib and has the CAS number: 1817626-54-2.

[0119] In some embodiments, the IRAK-4 degrader is PROTAC IRAK4 degrader 1 (Med Chem Express, catalog number HY-129966). In some embodiments, the IRAK-4 degrader comprises a compound of formula [VI] or a pharma- ceutically acceptable salt thereof. In some embodiments, the IRAK-4 degrader has CAS number: 2360533-90-8. [ka]

[0120] The compound of formula [VI] is PROTAC IRAK4 degrader 1 (Med Chem Express, catalog number HY-129966) and has CAS number: 2360533-90-8.

[0121] In some embodiments, the IRAK modulator is an anti-IRAK antibody (e.g., all or a functional portion of an anti-IRAK4 antibody (i.e., a portion of an anti-IRAK4 antibody that retains the ability to bind to IRAK4 and inhibit IRAK4 activity). In some embodiments, the IRAK modulator is an anti-IRAK antibody described in WO 01 / 051641 or WO 2008 / 091535.

[0122] 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.

[0123] In some embodiments, the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme, or DNAzyme that targets IRAK (e.g., IRAK-4). In some embodiments, the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme, or DNAzyme that blocks the TLR pathway. In some embodiments, the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme, or DNAzyme that blocks TLR function.

[0124] Gene Therapy In some aspects, the invention provides methods of using IRAK modulators with gene therapy agents for improved gene therapy by inhibiting the innate immune response to the gene therapy agent. In some embodiments, the gene therapy agent is a viral particle or lipid nanoparticle. In some embodiments, the gene therapy agent is an adeno-associated virus (AAV) particle, an adenovirus particle, a lentivirus particle, or a herpes simplex virus (HAV) particle. In some embodiments, the gene therapy agent is a lipid nanoparticle or liposome. In some embodiments, the immune response to the gene therapy agent is an immune response to a viral particle (e.g., a viral capsid protein, a viral envelope, etc.). In some embodiments, the immune response to the gene therapy agent is an immune response to a LNP (e.g., one or more lipids used to produce the LNP). In some embodiments, the immune response to the gene therapy agent is an immune response to a gene therapy payload, e.g., a nucleic acid (viral genome, a plasmid, a closed-end DNA, an mRNA, an antisense nucleic acid, an siRNA, an shRNA, etc.) encoding a therapeutic transgene. In some embodiments, the immune response to the gene therapy agent is an immune response to a transgene product (eg, a therapeutic polypeptide or therapeutic nucleic acid).

[0125] AAV In some embodiments, the present invention provides a method of using an IRAK modulator with an AAV particle for improved gene therapy. In an AAV particle for gene therapy, a recombinant AAV (rAAV) genome encoding a heterologous nucleic acid (e.g., a therapeutic transgene) is packaged in an AAV capsid. In some embodiments, the viral genome comprises a heterologous nucleic acid operably linked in the direction of transcription and / or a control sequence, including one or more of the following components: transcription start and stop sequences, thereby forming an expression cassette.

[0126] 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 the 5' and 3' ends by at least one functional AAV ITR sequence. By "functional AAV ITR sequence" it is meant that the ITR sequence functions as intended for the rescue, replication and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein by reference in their entirety. To carry out some aspects of the invention, the recombinant viral genome comprises at least all of the sequences of AAV essential for packaging into an AAV capsid and the physical structure for infection by an AAV particle. AAV ITRs for use in the vectors of the invention need not have a wild-type nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801) but may be modified by nucleotide insertion, deletion or substitution, or may be derived from any of several AAV serotypes. Currently, over 40 AAV serotypes are known, and new serotypes and variants of existing serotypes continue to be 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 considered to be within the scope of the present invention.In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, caprine AAV, bovine AAV, or murine AAV ITR. In some embodiments, the AAV nucleic acid (e.g., a rAAV vector) comprises one or more (e.g., in some aspects, two) ITRs, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, caprine AAV, bovine AAV, or murine AAV ITRs. In some embodiments, the AAV particle comprises an AAV vector encoding a heterologous transgene flanked by one or more AAV ITRs.

[0127] In some embodiments, the AAV particles are selected from the group consisting of 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 The capsid protein is selected from 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 virus 1), AAV2HBKO capsid, AAVPHP.B capsid, or AAVPHP.eB capsid, or a functional variant thereof. By "functional variant" of an AAV capsid, it is meant that the variant capsid is capable of packaging an AAV genome to generate an infectious AAV virion. In further embodiments, the AAV particle comprises capsid proteins of an AAV serotype from clades A-F.

[0128] In some aspects, the present invention provides AAV particles comprising recombinant self-complementary genomes (e.g., self-complementary or self-complementary AAV vectors). AAV viral particles with self-complementary vector genomes and methods of using self-complementary rAAV 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 containing self-complementary genomes rapidly form double-stranded DNA molecules with their partially complementary sequences (e.g., complementary coding and non-coding strands of a heterologous nucleic acid). In some embodiments, the vector comprises a first nucleic acid sequence that encodes a heterologous nucleic acid and a second nucleic acid sequence that encodes the complement of the nucleic acid, where the first nucleic acid sequence is capable of intrastrand base pairing with the second nucleic acid sequence over most or all of its length.

[0129] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are linked by a mutated ITR (e.g., a right ITR). In some embodiments, the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 1). The mutated ITR comprises a deletion of the D region, which includes the terminal release sequence. As a result, upon replication of the rAAV genome, the rep protein does not cleave the viral genome at the mutated ITR, and thus a recombinant viral genome is packaged into the viral capsid, which comprises, in 5' to 3' order: the AAV ITR, the first heterologous polynucleotide sequence including the regulatory sequence, the mutated AAV ITR, the second heterologous polynucleotide in the opposite orientation to the first heterologous polynucleotide, and the third AAV ITR.

[0130] Different AAV serotypes are used to optimize the introduction of specific target cells or target specific cell types in specific target tissues (e.g., diseased tissues).AAV particles can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes.For example, AAV particles can contain one or more ITRs and capsid from the same AAV serotype, or AAV particles can contain one or more ITRs from the capsid of AAV particles that are different from the AAV serotype.

[0131] In some embodiments, the AAV capsid comprises a mutation, e.g., 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 maintains the ability to form an AAV capsid. In some embodiments, the AAV particle comprises an AAV2 or AAV5 tyrosine mutant capsid (see, e.g., Zhong L. et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832), such as a mutation at Y444 or Y730 (numbering according to AAV2). In further embodiments, the AAV particle comprises a capsid protein of an AAV serotype from clades A-F (Gao, et al., J. Virol. 2004, 78(12):6381).

[0132] Numerous methods are known in the art for the production of AAV particles for gene therapy, including transfection, production of stable cell lines and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J. E. 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). All AAV production cultures for the production of AAV particles require 1) a suitable host cell, 2) suitable helper virus functions, 3) AAV rep and cap genes and gene products; 4) nucleic acid (such as a therapeutic nucleic acid) flanking at least one AAV ITR sequence, and 5) suitable media and media components to support AAV production. In some embodiments, the suitable host cell is a primate host cell. In some embodiments, the suitable host cell is a human-derived cell line, such as HeLa, A549, 293, or PERC.6 cells. In some embodiments, the suitable helper virus functions are provided by wild-type or mutant adenovirus (such as a temperature-sensitive adenovirus), herpes virus (HSV), baculovirus, or a plasmid construct that provides helper functions. In some embodiments, the AAV rep and cap gene products can be from any AAV serotype. Generally, but not necessarily, the AAV rep gene product is of the same serotype as the ITRs of the rAAV genome, so long as the rep gene product can function to replicate and package the rAAV genome. Suitable media known in the art can be used for production of AAV particles. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, AAV helper functions are provided by baculovirus and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cell).

[0133] One method for producing AAV particles is the triple transfection method. Briefly, a plasmid containing rep and capsid genes can be transfected (e.g., using calcium phosphate method) into a cell line (e.g., HEK-293 cells) together with a helper adenovirus plasmid, and the virus can be harvested and optionally purified. Thus, in some embodiments, AAV particles are produced by triple transfection of nucleic acid encoding an AAV vector, nucleic acid encoding AAVrep and cap, and nucleic acid encoding AAV helper virus function into a host cell, and transfection of nucleic acid into the host cell produces a host cell capable of producing AAV particles.

[0134] In some embodiments, AAV particles can be produced by a producer cell line method (see Martin et al., (2013) Human Gene Therapy Methods 24:253-269; US Patent Publication No. 2004 / 0224411; and Liu, XL et al. (1999) Gene Ther. 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 vector genome including a rep gene, a capsid gene and a promoter heterologous nucleic acid sequence. The cell line can be screened to select a lead clone for AAV production, which can then be expanded 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 heat) and / or removed, and the AAV particles can be purified. Thus, in some embodiments, the AAV particles were produced by a producer cell line that contains one or more of nucleic acids encoding the rAAV genome, nucleic acids encoding AAVrep and CAP, and nucleic acids encoding AAV helper virus functions.

[0135] In some embodiments, the nucleic acid encoding the AAVrep and cap genes and / or the AAV viral genome is stably maintained in the producer cell line. In some embodiments, the nucleic acid encoding the AAVrep and cap genes and / or the rAAV genome is introduced into the cell line on one or more plasmids to generate the producer cell line. In some embodiments, the AAVrep, AAVcap and AAV genome are introduced into the cell on the same plasmid. In other embodiments, the AAVrep, AAVcap and rAAV genome are introduced into the cell on different plasmids. In some embodiments, the cell line stably transfected with the plasmid maintains the plasmid over multiple passages of the cell line (e.g., 5, 10, 20, 30, 40, 50 or more than 50 passages). For example, the plasmid may replicate when the cell replicates, or the plasmid may integrate into the cell genome. Various sequences have been identified that allow plasmids to replicate autonomously in cells (e.g., human cells) (see, e.g., Krysan, PJ et al. (1989) Mol. Cell Biol. 9:1026-1033). In some embodiments, the plasmid may contain a selection marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Commonly used selection markers in mammalian cells include, but are not limited to, blastitidine, G418, hygromycin B, zeocin, puromycin, and derivatives thereof. Methods of introducing nucleic acids 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 further details see, e.g., Kim, T K and Eberwine, J H (2010) Anal. Bioanal. Chem. 397:3173-3178).

[0136] In some embodiments, the producer 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 producer cell line is derived from a HeLa, 293, A549, or PERC.6® (Crucel) cell. For example, the cell line is a HeLa, 293, A549, or PERC.6® (Crucel) cell line, or a derivative thereof, prior to introducing and / or stably maintaining / integrating nucleic acid encoding the AAVrep and cap genes and / or the rAAV genome into the cell line to generate the producer cell line.

[0137] In some embodiments, the production cell line is adapted to grow in suspension. As known in the art, anchorage-dependent cells typically cannot grow in suspension without a substrate such as microcarrier beads. Adapting a cell line to grow in suspension may include, for example, growing the cell line in a spinner culture using a stirring paddle, using a culture medium lacking calcium and magnesium ions (and optionally an antifoam agent) to prevent clumping, using a culture vessel coated with a siliconizing compound, and selecting cells in the culture (not on large clumps or on the sides of the vessel) at each passage.

[0138] The AAV particles of the invention can be recovered from the AAV production culture by lysis of the host cells of the production culture or by recovery of spent medium from the production culture, provided that the cells are cultured under conditions known in the art to release AAV particles from intact cells into the medium, as described in further detail in U.S. Patent No. 6,566,118. Suitable methods of 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.

[0139] In further embodiments, the AAV particles are purified. The term "purified" as used herein includes preparations of AAV particles that lack at least some of the other components that may also be present when the AAV particles are naturally present or initially prepared. Thus, for example, isolated AAV particles can be prepared using purification techniques to enrich them from a source mixture, such as culture lysate or production culture supernatant. Enrichment can be measured in a variety of ways, such as by the proportion of DNase-resistant particles (DRP) or genome copies (GC) present in the solution or infectivity, or it can be measured in relation to a second potentially interfering substance present in the source mixture, such as a contaminant, including a production culture contaminant or an in-process contaminant, such as a helper virus, medium component, etc.

[0140] 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 filtration through a series of depth filters including, for example, a grade DOHC Millipore Millistak+HC Pod Filter, a grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 μm Filter Opticap XL1O 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 filtration through any cellulose acetate filter with a pore size of 0.2 μm or larger known in the art.

[0141] In some embodiments, the AAV production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase® digestion is performed under standard conditions known in the art, e.g., at a final concentration of 1-2.5 units / ml of Benzonase®, at temperatures ranging from ambient to 37° C., for periods ranging from 30 minutes to several hours.

[0142] 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) to concentrate 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), AAV capture by nanofiltration and 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 the steps in the order described below. Methods for purifying AAV particles can be found, for example, in 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.

[0143] Adenovirus In some embodiments, the present invention provides a method of using IRAK modulators with adenoviral particles for improved gene therapy. Adenoviral vectors for gene therapy are typically adenoviral particles with a recombinant adenovirus (RAD) genome that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of adenoviral origin) between two adenoviral ITRs packaged in the adenoviral capsid. In some embodiments, the heterologous sequences encode a therapeutic transgene. In some embodiments, the rAd genome lacks or contains a defective copy of one or more E1 genes, which renders adenoviral replication defective. Adenoviruses contain a linear double-stranded DNA genome within a large (approximately 950 Å) non-enveloped icosahedral capsid. Adenoviruses have a large genome that can incorporate more than 30 kb of heterologous sequences (e.g., in place of the E1 and / or E3 regions), making them particularly suitable for use with larger heterologous genes. They are also known to infect dividing and non-dividing cells and not naturally integrate into the host genome (although hybrid variants may have this ability). In some embodiments, the adenoviral vector can be a first generation adenoviral vector with a heterologous sequence in place of E1. In some embodiments, the adenoviral vector can be a second generation adenoviral vector with additional mutations or deletions in E2A, E2B and / or E4. In some embodiments, the adenoviral vector can be a third generation or gutted adenoviral vector that lacks all viral coding genes, retaining only the ITRs and packaging signal, and requiring a helper adenovirus in trans 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.

[0144] In some embodiments, the adenoviral particle comprises a rAd genome comprising 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 particle is derived from an adenoviral serotype, 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 Ad3, canine Ad2, ovine Ad, and porcine Ad3. The adenoviral particle also comprises a capsid protein. In some embodiments, the adenoviral particle comprises one or more foreign viral capsid proteins. Such a combination may be referred to as a pseudotyped adenoviral particle. In some embodiments, the foreign viral capsid protein used in the pseudotyped adenoviral particles is derived from a foreign virus or another adenovirus serotype. In some embodiments, the foreign viral capsid protein is derived from a virus, 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. 10(4):616-629 and Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4):307-320). Different adenoviral serotypes can be used to optimize transduction of specific target cells or target specific cell types within a specific target tissue (e.g., diseased tissue). Tissues or cells targeted by particular adenovirus serotypes include, but are not limited to, lung (e.g., HuAd3), spleen and liver (e.g., HuAd37), smooth muscle, synovial cells, dendritic cells, cardiovascular cells, tumor cell lines (e.g., HuAd11) and dendritic cells (e.g., HuAd5 pseudotyped with reovirus type 3, HuAd30 or HuAd35).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.

[0145] Numerous methods for the production of adenoviral particles are known in the art. For example, in the case of gutted adenoviral vectors, the adenoviral vector genome and the helper adenoviral genome can be transfected into a packaging cell line (e.g., 293 cell line). In some embodiments, the helper adenoviral genome can contain recombination sites flanking its packaging signal, and both genomes can be transfected into a packaging cell line expressing a recombinase (e.g., the Cre / loxP system can be used), so that the adenoviral vector of interest is packaged more efficiently than the helper adenovirus (see, e.g., Alba, R. et al. (2005) Gene Ther. 12 Suppl 1:S18-27). The adenoviral vector can be recovered and purified using standard methods such as those described herein.

[0146] Lentivirus In some embodiments, the present invention provides a method of using an IRAK modulator with lentiviral particles for improved gene therapy. Lentiviral vectors for gene therapy are typically lentiviral particles with a recombinant lentiviral genome that includes one or more heterologous sequences (i.e., nucleic acid sequences that are not of lentiviral origin) between two long terminal repeats (LTRs). In some embodiments, the heterologous sequence encodes a therapeutic transgene. Lentiviruses are positive-stranded ssRNA retroviruses with a genome of approximately 10 kb. Lentiviruses integrate into the genome of dividing and non-dividing cells. Lentiviral particles can be produced, for example, by transfecting multiple plasmids (typically the lentiviral genome and genes required for replication and / or packaging are separated to prevent viral replication) into a packaging cell line, which results in the modified lentiviral genome being packaged into the lentiviral particle. In some embodiments, lentiviral particles can refer to a first generation vector that lacks envelope proteins. In some embodiments, lentiviral particles can refer to a second generation vector that lacks all genes except the gag / pol and tat / rev regions. In some embodiments, lentiviral particles may refer to third generation vectors that contain only endogenous rev, gag, and pol genes and have chimeric LTRs for transduction without the tat gene (see Dull, T. et al. (1998) J. Virol. 72:8463-71). For further explanation, see Durand, S. and Cimarelli, A. (2011) Viruses 3:132-59.

[0147] The use of any lentiviral vector is considered 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), visna virus (VV) and caprine arthritis encephalitis virus (CAEV). The lentiviral particle also comprises a capsid protein. In some embodiments, the lentiviral particle comprises one or more foreign viral capsid proteins. Such a combination may be referred to as a pseudotyped lentiviral particle. In some embodiments, the foreign viral capsid protein used in the pseudotyped lentiviral particle is derived from a foreign virus. In some embodiments, the foreign viral capsid protein used in the pseudotyped lentiviral particle is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with ubiquitous cellular receptors, providing broad tissue tropism to pseudotyped lentiviral particles, and is thought to provide greater stability to pseudotyped lentiviral particles.In other embodiments, the foreign viral capsid protein is selected from the group consisting of Chandipura virus, Rabies virus, Mokola virus, Lymphocytic choroiditis virus (LCMV), Ross River virus (RRRV), Sindbis virus, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Ebola virus Reston, Ebola virus Zaire, Marburg virus, Lassa virus, Avian leukosis virus (ALV), Jaagsiekte sheep retrovirus (JSRV), Moloney murine leukemia virus (MLV), Gibbon ape leukemia virus (GALV1), Feline endogenous retrovirus (RD114), Human T-lymphotropic virus (HTLV-1), Human foamy virus, Maedi-visna virus (MV), SARS-Cov, Sendai virus, Respiratory syncytial virus (RSV), Human parainfluenza virus type 3, Hepatitis C virus (HCV), Influenza virus, Avian plague virus (FPV), or Autographa The pseudotyped lentiviral particles are derived from viruses including, but not limited to, the 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. 5(4):387-398. Different pseudotyped lentiviral particles can be used to optimize transduction of specific target cells or target specific cell types within a specific target tissue (e.g., diseased tissue). For example, tissues targeted by particular pseudotyped lentiviral particles include, but are not limited to, the liver (e.g., pseudotyped with VSV-G, LCMV, RRV or SEVF proteins), lung (e.g., pseudotyped with Ebola, Marburg, SeVF 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 Mokola proteins), retina (e.g., pseudotyped with VSV-G or Mokola proteins), monocytes or muscle (e.g., pseudotyped with Mokola 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.

[0148] Numerous methods for the production of lentiviral particles are known in the art. For example, in the case of third generation lentiviral vectors, a vector containing gag and pol genes and a recombinant lentiviral genome of interest can be co-transfected into a packaging cell line (e.g., 293 cell line) with a vector containing rev gene. The recombinant lentiviral genome of interest also contains a chimeric LTR that promotes transcription in the absence of Tat (see Dul, T. et al. (1998) J. Virol. 72: 8463-71). Lentiviral vectors can be collected and purified using the methods described herein (Segura MM, et al., (2013) Expert Opin Biol Ther. 13 (7): 987-1011).

[0149] HSV In some embodiments, the present invention provides a method of using IRAK modulators with HSV particles for improved gene therapy. HSV vectors for gene therapy are typically HSV particles with a recombinant HSV genome that includes one or more heterologous sequences (i.e., nucleic acid sequences that are not of HSV origin) between two terminal repeats (TRs). In some embodiments, the heterologous sequence encodes a therapeutic transgene. HSV is an enveloped double-stranded DNA virus with a genome of approximately 152 kb. Advantageously, approximately half of its genes are non-essential and can be deleted to accommodate the heterologous sequence. HSV particles infect non-dividing cells. In addition, they are advantageous for neuronal transfection and / or gene therapy approaches involving the nervous system, since they can naturally establish latency in neurons, travel by retrograde transport, and transfer across synapses. In some embodiments, HSV particles can be replication-defective or replication-competent (e.g., competent for a single replication cycle by inactivation of one or more late genes). For further explanation, see Manservigi, R. et al. (2010) Open Virol. J. 4:123-56.

[0150] In some embodiments, the HSV particle comprises a recombinant HSV genome containing the 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 particle also comprises a capsid protein. In some embodiments, the HSV particle comprises one or more foreign viral capsid proteins. Such combinations may be referred to as pseudotyped HSV particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped HSV particle is derived from a foreign virus or another HSV serotype. In some embodiments, the foreign viral capsid protein used in the pseudotyped HSV particle is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with a ubiquitous cellular receptor, providing broad tissue tropism for the pseudotyped HSV particle. In addition, VSV-GP is believed to provide greater stability to the pseudotyped HSV particle. In other embodiments, the foreign viral capsid protein may be from a different HSV serotype. For example, HSV-1 vectors may contain one or more HSV-2 capsid proteins. Different HSV serotypes can be used to optimize transduction of specific target cells or target specific cell types within a specific target tissue (e.g., diseased tissue). 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 4:123-156, Kay, M. et al. (2001) Nat. Med. 7(1):33-40 and Meignier, B. et al. (1987) J. Infect. Dis. 155(5):921-930.

[0151] Numerous methods for the production of HSV particles are known in the art. HSV vectors can be harvested and purified using standard methods such as those described herein. For example, in the case of replication-defective HSV vectors, the HSV genome of interest, lacking all immediate early (IE) genes, can be transfected into a complementing cell line that provides genes necessary for virus production, such as ICP4, ICP27 and ICP0 (see, for example, Samaniego, LA et al. (1998) J. Virol. 72:3307-20). HSV vectors can be harvested and purified using described methods (e.g., Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144:63-79).

[0152] Non-viral gene therapy In some embodiments, the present invention provides methods of using IRAK modulators with non-viral gene transfer methods for gene therapy. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed to a delivery system. For example, the vector may be complexed with lipids (e.g., cationic or neutral lipids), liposomes, polycations, lipid nanoparticles, or agents that enhance cellular uptake of the nucleic acid. The nucleic acid may be complexed to an agent appropriate for any of the delivery methods described herein. In some embodiments, the nucleic acid encodes a therapeutic transgene.

[0153] Lipid nanoparticles for gene therapy typically contain a vector genome encapsulated in a lipid particle or complexed with a lipid. In some embodiments, the heterologous sequence encodes a therapeutic transgene. In some embodiments, the vector genome is formulated in a lipoplex nanoparticle or liposome. In some embodiments, a lipoplex nanoparticle formulation of a gene therapy agent contains the synthetic cationic lipid (R)-N,N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA) and the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the DOTMA / DOPE liposome components are optimized for cellular delivery and targeting in an individual.

[0154] In some embodiments, the nucleic acid comprising the vector genome is mixed with a pharmaceutical composition comprising one or more cationic lipids, including, for example, (R)-N,N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA) and the 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 may be monocationic or polycationic. Any cationic amphiphilic molecule, for example 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 helper lipid. The helper lipid may be a neutral or anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue 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 helper lipid is a bilayer-forming lipid. Examples of helper lipids include, but are not limited to, 1,2-di-(9Z-octadecynoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or its analogue or derivative, cholesterol (Chol) or its analogue or derivative, and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or its analogue or derivative.

[0155] 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, in this ratio, the molar amount of cationic lipid is obtained by multiplying the molar amount of cationic lipid by the number of positive charges in the cationic lipid.

[0156] In some embodiments, the lipid is included in a vesicle that encapsulates the vector genome. The vesicle may be a multilamellar vesicle, a unilamellar vesicle, or a mixture thereof. The vesicle may be a liposome.

[0157] Vector genome In some embodiments, the present invention provides methods of using an IRAK modulator in conjunction with a gene therapy agent to deliver a therapeutic transgene to a desired target in an individual, hi some embodiments, the gene therapy agent comprises a vector genome for delivery and expression of a therapeutic transgene in a desired target in an individual.

[0158] 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 for establishing expression of the therapeutic polypeptide and / or nucleic acid, such as a promoter, the ITRs of the present disclosure, a ribosome binding element, a terminator, an enhancer, a selection marker, an intron, a polyA signal, and / or an origin of replication.

[0159] In some embodiments, the therapeutic transgene encodes a therapeutic polypeptide. The therapeutic polypeptide may, for example, provide a polypeptide and / or enzyme activity that is absent or present at reduced levels in a cell or organism. Alternatively, the therapeutic polypeptide may provide a polypeptide and / or enzyme activity that indirectly counteracts an imbalance in a cell or organism. For example, a therapeutic polypeptide for a disorder associated with accumulation of a metabolic product caused by a deficiency in a metabolic enzyme or activity may provide a missing metabolic enzyme or activity, or provide an alternative metabolic enzyme or activity that results in a reduction in the metabolic product. Therapeutic polypeptides may also be used to reduce the activity of a polypeptide (e.g., one that is overexpressed, activated by a gain-of-function mutation, or whose activity is otherwise misregulated), for example, by acting as a dominant-negative polypeptide.

[0160] The vector genome of the present invention may encode a polypeptide that is an intracellular protein, anchored in the cell membrane, retained intracellularly, or secreted by a cell transduced with a vector of the present invention. In the case of a polypeptide that is secreted by a cell receiving the vector, the polypeptide may be soluble (i.e., not attached to the cell). For example, a soluble polypeptide lacks a transmembrane region and is secreted from the cell. Techniques for identifying and removing nucleic acid sequences that encode transmembrane domains are known in the art.

[0161] In some embodiments, the vector genome of the present invention encodes a polypeptide used to treat a disease or disorder in an individual. Diseases and disorders treated by the gene therapy agent 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.

[0162] In some embodiments, the therapeutic polypeptide is huntingtin (HTT), TAU, amyloid precursor protein, alpha-synuclein, pseudotype arylsulfatase (ARSA), superoxide dismutase 1 (SOD1), phenylalanine hydroxylase (PAH), dystrophin, alpha-1-antitrypsin (A1AT), cysteine ​​transporter, factor VIII (FVIII), factor IX (FIX), acid beta-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.

[0163] In some embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid that can be used, for example, to replace or knock down one or more defective genes. In some embodiments, the therapeutic nucleic acid can include, but is not limited to, DNA, siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozymes, or DNAzymes. Thus, the therapeutic nucleic acid can encode an RNA that, when transcribed from the nucleic acid of the vector, can treat a disorder by interfering with the translation or transcription of an abnormal or excess protein associated with a disorder of the invention. For example, the nucleic acid of the invention can encode an RNA that treats a disorder by highly specific removal or reduction of mRNA that codes for the abnormal and / or excess protein. Therapeutic RNA sequences include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA), and / or ribozymes (such as hammerhead and hairpin ribozymes) that can treat a disorder by highly specific removal or reduction of mRNA that codes for the abnormal and / or excess protein.

[0164] In some embodiments, a therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS. Without wishing to be bound by theory, a therapeutic polypeptide or therapeutic nucleic acid may be used to replace a mutated gene with a wild-type or improved gene, to reduce or eliminate expression and / or activity of a polypeptide whose gain of function is associated with a disease, or to enhance expression and / or activity of a polypeptide to complement a deficiency associated with a disorder (e.g., a mutation in a gene whose expression exhibits a similar or related activity). Non-limiting examples of disorders that may be treated by the therapeutic polypeptides or therapeutic nucleic acids of the invention (exemplary genes that may be targeted or delivered are provided in parentheses for each disorder) include stroke (e.g., caspase-3, Beclin1, Ask1, PAR1, HIF1α, PUMA, and / or any of the genes described in Fukuda, A and Badaut, J. (2013) Genes (Basel) 4:435-456), Huntington's disease (mutant HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and / or any of the genes described in Boison, D. (2010) Epilepsy), and / or any of the genes described in Boison, D. (2010) Epilepsy. 51:1659-1668), Parkinson's disease (alpha-synuclein), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis; SOD1), Alzheimer's disease (tau, amyloid precursor protein), corticobasal degeneration or CBD (tau), corticoganglionic degeneration or CBGD (tau), frontotemporal dementia or FTD (tau), progressive supranuclear palsy or PSP (tau), multiple system atrophy or MSA (alpha-synuclein), brain cancer (e.g., mutated or overexpressed cancer genes implicated in brain cancer), and lysosomal storage diseases (LSDs). Disorders of the present invention can include disorders involving large areas of the cortex, such as multiple functional areas of the cortex, multiple lobes of the cortex, and / or the entire cortex. Other non-limiting examples of disorders of the invention that may be treated by a therapeutic polypeptide or nucleic acid of the invention include, but are not limited to, traumatic brain injury, enzyme dysfunction disorders, psychiatric disorders (including post-traumatic stress syndrome), neurodegenerative diseases and cognitive disorders (including dementia, autism and depression).Enzymatic dysfunction disorders include, but are not limited to, leukodystrophies (including Canavan disease) and any of the lysosomal storage diseases described below.

[0165] 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 often caused by deficiencies in enzymes required for proper mucopolysaccharide, glycoprotein and / or lipid metabolism, resulting in pathological accumulation of cellular material stored in lysosomes. Non-limiting examples of lysosomal storage diseases that may be treated by the therapeutic polypeptides or nucleic acids of the invention (exemplary genes that may be targeted or delivered are provided in parentheses for each disorder) include Gaucher disease type 2 or 3 (acid beta-glucosidase, GBA), GM1 gangliosidosis (beta-galactosidase-1, GLB1), Hunter disease (iduronate 2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), mannosidosis (mannosidases, e.g., alpha-D-mannosidase, MAN2B1), beta-mannosidosis (beta-mannosidase, MANBA), metachromatic leukodystrophy (pseudotype arylsulfatase A, ARSA), mucolipidosis II / III (N-acetylglucosamine-1-phosphotransferase, GNPTAB), Niemann-Pick A (acid sphingomyelinase, ASM), ), Niemann-Pick C disease (Niemann-Pick C protein, NPC1), Pompe disease (acid alpha-1,4-glucosidase, GAA), Sandhoff disease (hexosaminidase beta subunit, HEXB), Sanfilippo A disease (N-sulfoglucosamine sulfohydrolase, MPS3A), Sanfilippo B disease (N-alpha-acetylglucosaminidase, NAGLU), Sanfilippo C disease (heparin acetyl-CoA: alpha -glucosaminidase N-acetyltransferase, MPS3C), Sanfilippo D disease (N-acetylglucosamine-6-sulfatase, GNS), Schindler disease (alpha-N-acetylgalactosaminidase, NAGA), Sly disease (beta-glucuronidase, GUSB), Tay-Sachs disease (hexosaminidase alpha subunit, HEXA) and Wolman disease (lysosomal acid lipase, LIPA).

[0166] 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 transcarbomylase, arginine succinate synthase, β-globin, γ-globin, phenylalanine hydroxylase, adrenoleukodystrophy protein (ALD), dystrophin, truncated dystrophin, an anti-VEGF agent, or a functional variant thereof.

[0167] In some embodiments, the heterologous nucleic acid is operably linked to a promoter. Exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, the simian virus 40 (SV40) promoter and the CK6 promoter, the transthyretin promoter (TTR), the TK promoter, the tetracycline responsive promoter (TRE), the HBV promoter, the hAAT promoter, the LSP promoter, the chimeric liver specific promoter (LSP), the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirus enhancer / chicken beta actin / rabbit beta-globin promoter (CAG promoter, Niwa et al., Gene, 1991, 108(2):193-9) and the elongation factor 1-alpha promoter (EF1-alpha) promoter (Kimetal., Gene, 1990, 91(2):217-23 and Guoetal., Genether., 1996, 3(9):802-10). In some embodiments, the promoter comprises a cytomegalovirus enhancer linked to a human β-glucuronidase promoter or a chicken β-actin (CBA) promoter. The promoter can be a constitutive, inducible or 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 are described, for example, in US Patent Publication No. 20140335054.

[0168] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter [Invitrogen].

[0169] Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological states (acute phase, specific differentiation states of cells, or replicating cells only). 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 readily selected by one of skill in the art. Examples of inducible promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), Harvey et al., Science, 268:1766-1769 (1995), and the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995)). al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of cells or only replicating cells.

[0170] In another embodiment, the native promoter for the transgene or a fragment thereof is used. The native promoter can be used when it is desired that the expression of the transgene mimics native expression. The native promoter can be used when the expression of the transgene needs to be regulated temporally or developmentally or in a tissue-specific manner or in response to a specific transcriptional stimulus. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences, can also be used to mimic native expression.

[0171] In some embodiments, the regulatory sequences confer tissue-specific gene expression capabilities.

[0172] In some cases, the tissue-specific regulatory sequence binds to a tissue-specific transcription factor that induces transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the vector comprises an intron. For example, in some embodiments, the intron is a chimeric intron derived from chicken beta-actin and rabbit beta-globin. In some embodiments, the intron is a minute virus of mice (MVM) intron.

[0173] In some embodiments, the vector comprises a polyadenylation (polyA) sequence, many examples of which 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.

[0174] Methods for selecting patients for treatment with gene therapy agents and IRAK modulators - Patents.com In some aspects, the invention provides a method of delivering a nucleic acid to cells of an individual, the method comprising: a) incubating innate immune cells from the individual with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles); b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies an individual having 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 isolating the innate immune cells from the individual prior to incubating the innate immune cells with the gene therapy agent. In some embodiments, the method further comprises isolating monocytes from the individual and incubating the monocytes in dendritic cell culture medium, and deriving dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent.

[0175] In some aspects, the invention provides a method of treating an individual in need of treatment, the method comprising: a) incubating innate immune cells from the individual with a gene therapy agent (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, or lipid nanoparticles); b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies an individual having 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 isolating the innate immune cells from the individual prior to incubating the innate immune cells with the gene therapy agent. In some embodiments, the method further comprises isolating monocytes from the individual and incubating the monocytes in dendritic cell culture medium, and deriving dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent.

[0176] In some aspects, the invention provides a method of selecting an individual for treatment with a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and an IRAK modulator (e.g., an IRAK-4 degrading agent), the method comprising: a) incubating innate immune cells from the individual with the gene therapy agent; b) analyzing dendritic cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies the individual for treatment with the gene therapy agent and the IRAK modulator; and 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: 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 isolating the innate immune cells from the individual prior to incubating the innate immune cells with the gene therapy agent. In some embodiments, the method further comprises isolating monocytes from the individual and incubating the monocytes in dendritic cell culture medium to derive dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent.

[0177] In some embodiments, the innate immune cells are isolated 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 monocytes of the individual. In some embodiments, the monocytes are isolated from peripheral blood mononuclear cells from the individual. In some embodiments, the monocytes are CD14+ monocytes. In some embodiments, the monocytes are incubated with dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to induce dendritic cells from the monocytes. In some embodiments, the monocytes are incubated with dendritic cell culture medium for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days or more than 12 days to induce dendritic cells from the monocytes. In some embodiments, the dendritic cells are replated prior to incubation with the gene therapy agent in step c). In some embodiments, the dendritic cells are replated in the microwell dish prior to incubation with the gene therapy agent.

[0178] In some embodiments, the dendritic cells are about 1×10 3 ~Approx. 1×10 5 Or about 1 x 10 4 In some embodiments, dendritic cells are incubated with the viral gene therapy agent at an MOI of about 1 x 10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 Or 5 x 10 5 The cells are incubated with the gene therapy agent at an MOI smaller than either

[0179] In some embodiments, dendritic cells are incubated with the 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 the 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.

[0180] 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 about 6 hours to about 48 hours, about 6 hours to about 36 hours, about 6 hours to about 24 hours, about 6 hours to about 18 hours, about 6 hours to about 12 hours, about 12 hours to about 48 hours, about 12 hours to about 36 hours, about 12 hours to about 24 hours, about 12 hours to about 18 hours, about 18 hours to about 48 hours, about 18 hours to about 36 hours, about 18 hours to about 24 hours, about 24 hours to about 48 hours, about 24 hours to about 36 hours, or about 36 hours to about 48 hours.

[0181] In some embodiments, a cytokine signature is determined for the gene therapy agent in specific immune cells (e.g., dendritic cells, monocytes, macrophages, NK cells, etc.) by contacting specific immune cells from a plurality of individuals with the gene therapy agent and determining changes in expression of one or more cytokines associated with an innate immune response, where a commonality of changes in expression (e.g., increased or decreased expression) in 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 the Toll-like receptor (TLR) pathway (e.g., the TLR2, TLR3, TLR4, or TLR9 pathway). In some embodiments, the cytokine signature comprises changes in expression in more than any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cytokines. In some embodiments, the plurality of individuals includes more than any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 individuals. In some embodiments, the commonality of changes in expression comprises similar changes in expression levels of cytokines in innate immune cells in more than about 25%, 50%, 75%, or 90% of the plurality of individuals.

[0182] In some embodiments, the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0183] In some embodiments, the innate immune cells are dendritic cells and the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the innate immune cells are dendritic cells and the cytokine signature comprises increased expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the innate immune cells are dendritic cells and the cytokine signature comprises increased expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the innate immune cells are dendritic cells and the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0184] In some embodiments, the expression of a cytokine in the cytokine signature is increased compared to the expression of the cytokine in a suitable control. Examples of suitable controls include a cytokine signature from an innate immune cell not incubated (in the absence) with the gene therapy agent and a cytokine signature from the same or similar innate immune cell prior to incubation with the gene therapy agent (e.g., the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α). In some embodiments, the cytokine signature comprises increased expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β. In some embodiments, an increase in expression of any one of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or greater than 100% identifies the individual for treatment with a gene therapy agent and an IRAK modulator.

[0185] In some embodiments, the expression of cytokines in the cytokine signature is increased compared to the expression of cytokines in a cytokine signature from dendritic cells incubated in the absence of the gene therapy agent or compared to the expression of cytokines in a cytokine signature from dendritic cells prior to incubation with the gene therapy agent, and the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β, hi some embodiments, an increase in expression of any one of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or greater than 100% identifies the individual for treatment with a gene therapy agent and an IRAK modulator.

[0186] Pharmaceutical Compositions In some aspects, the invention relates to pharmaceutical compositions comprising a gene therapy agent (e.g., an AAV particle, an adenoviral particle, a lentiviral particle, an HSV particle, or a lipid nanoparticle) and / or an IRAK modulator (e.g., an IRAK-4 degrading agent) described herein. The pharmaceutical composition may be suitable for any mode of administration described herein or known in the art.

[0187] In some embodiments, the pharmaceutical composition includes a pharma- ceutically acceptable excipient. As is well known in the art, a pharma- ceutically acceptable excipient is a relatively inert substance that facilitates administration of a pharma- ceutically effective substance, and can be provided as a liquid solution or suspension, as an emulsion, or as a solid form suitable for dissolving or suspending in a liquid prior to use. For example, an excipient can provide shape or consistency or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts that change osmotic pressure, encapsulating agents, pH buffering substances and buffers. Such excipients include any agent suitable for direct delivery to the eye that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts can be included, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and organic acid salts such as acetate, propionate, malonate, benzoate, and the like. A thorough discussion of pharma- ceutically acceptable excipients is found in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991). In some embodiments, pharmaceutical compositions comprising the rAAV particles described herein and a pharma- ceutically acceptable carrier are suitable for administration to humans. Such carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580).

[0188] Such pharma-ceutically acceptable carriers can be sterile liquids such as water and oils, for example of petroleum, animal, vegetable or synthetic origin, for example peanut oil, soybean oil, mineral oil, etc. Physiological saline and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. The pharmaceutical compositions can further comprise additional components, for example preservatives, buffers, isotonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, viscosity-increasing agents, etc. The pharmaceutical compositions described herein can be packaged in the form of a single unit dose or multiple doses. The compositions are generally formulated as sterile and substantially isotonic solutions.

[0189] Kits and Products The gene therapy agents (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles or lipid nanoparticles) and / or IRAK modulators (e.g., IRAK-4 degrading agents) described herein can be included, for example, within a kit or article of manufacture designed for use in one of the methods of the invention described herein.

[0190] In some embodiments, the kit or article of manufacture further comprises instructions for administration of the IRAK modulator and / or gene therapy agent. The kits or articles of manufacture described herein may further include other items desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for practicing any of the methods described herein. Suitable packaging materials may also be included, which may be any packaging material known in the art, including, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles may be further sterilized and / or sealed.

[0191] In some embodiments, the kit or article of manufacture further contains one or more of the buffers and / or pharma- ceutically 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 pharma- ceutically acceptable excipients, carriers, solutions and / or additional components described herein. The kit or article of manufacture described herein can be packaged in single unit dose or multi-dose form. The contents of the kit or article of manufacture are typically formulated under sterile conditions and can be lyophilized or provided as a substantially isotonic solution.

[0192] Exemplary embodiments The present invention includes the embodiments listed below.

[0193] 1. A method for delivering a nucleic acid to a cell of an individual, comprising: a) administering to an individual an IRAK modulator; b) administering a gene therapy agent to the individual; The method includes:

[0194] 2. A method of treating an individual in need of treatment with a gene therapy agent, comprising: a) administering to an individual an IRAK modulator; b) administering a gene therapy agent to the individual; The method includes:

[0195] 3. A method for improving gene therapy in an individual, comprising: a) administering to an individual an IRAK modulator; b) administering a gene therapy agent to the individual; The method includes:

[0196] 4. A method of suppressing an immune response to a gene therapy agent in an individual, comprising: a) administering to an individual an IRAK modulator; b) administering a gene therapy agent to the individual; The method includes:

[0197] 5. The method of any one of embodiments 1-4, wherein the IRAK modulator modulates the activity or expression of IRAK protein kinase.

[0198] 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.

[0199] 7. The method of any one of embodiments 1-6, wherein the IRAK modulator modulates the activity or expression of IRAK-4 protein kinase.

[0200] 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 results in loss of function of IRAK.

[0201] 9. The method of any one of embodiments 1-8, wherein the IRAK modulator is a small molecule.

[0202] 10. The IRAK modulator has the formula [I]: [ka] (In the formula, X 1 are covalent bonds, -CH2-, -C(O)-, -C(S)-, and [ka] is a divalent moiety selected from R 1a is hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O)2R, -N(R)2, -Si(R)3 or optionally substituted C l~4 It is aliphatic, Each R2a 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 teeth, [ka] [ka] [ka] is a bicyclic or tricyclic ring selected from Ring B a is a fused ring selected from a 6-membered aryl containing 0 to 2 nitrogen atoms, a 5- to 7-membered partially saturated carbocyclyl, a 5- to 7-membered partially saturated heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-membered heteroaryl having 1 to 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 is hydrogen, C l~4 aliphatic or -CN; Each R 6a is independently1~6 an optionally substituted group selected from aliphatic, phenyl, 4- to 7-membered saturated or partially unsaturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Ring A is a 4-10 membered saturated monocyclic or bicyclic carbocyclic or heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring C is a 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 independently represents a covalent bond or C 1~3 a divalent linear or branched saturated or unsaturated hydrocarbon chain, wherein one to three 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 C 1~6 an optionally substituted group selected from aliphatic, phenyl, a 4- to 7-membered saturated or partially unsaturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom optionally together with their intervening atoms form an optionally substituted 4-11 membered saturated or partially unsaturated carbocyclic or heterocyclic monocyclic, bicyclic, bridged bicyclic, spirocyclic, 5- or heteroaryl ring having, in addition to the atom to which they are attached, 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; 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), -CR 2(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 teeth, [ka] , hydrogen or C 1~6 is selected from optionally substituted groups selected from aliphatic or 4-11 membered saturated or partially unsaturated monocyclic, bicyclic, bridged bicyclic or spirocyclic carbocyclic or heterocyclic rings having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring D is a 4- to 10-membered saturated or partially unsaturated monocyclic or bicyclic carbocyclic or heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl ring having 1 to 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)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, Each R 5 are independently optionally substituted groups selected from C1-6 aliphatic, phenyl, 3- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic rings having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl rings 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. or a pharma- ceutically acceptable salt thereof.

[0203] 11. The IRAK modulator is represented by the formula [II] to [V]: [ka] or a pharma- ceutically acceptable salt thereof.

[0204] 12. The method of any one of embodiments 1 to 9, wherein the IRAK modulator is a compound of formula [II], PROTAC IRAK-4 degrader 1 or PF06650833.

[0205] 13. The method of any one of embodiments 1 to 9, wherein the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or DNAzyme.

[0206] 14. The method of any one of embodiments 1-13, wherein the IRAK modulator blocks TLR9 function.

[0207] 15. The method of any one of embodiments 1 to 14, wherein the gene therapy agent comprises a viral vector.

[0208] 16. The method of embodiment 15, wherein the viral vector is an AAV particle.

[0209] 17. AAV particles include 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 17. The method of embodiment 16, comprising an E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a murine AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid, or an AAVPHP.eB capsid, or a functional variant thereof.

[0210] 18. The method of embodiment 17, wherein the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation, or an HBKO mutation.

[0211] 19. The method of any one of embodiments 16 to 18, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), and 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.

[0212] 20. The method of embodiment 19, wherein one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0213] 21. The method of embodiment 19, wherein one or more of the ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0214] 22. The method of embodiment 15, wherein the viral vector is an adenovirus particle.

[0215] 23. The method of embodiment 22, wherein the adenovirus 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.

[0216] 24. The method of embodiment 15, wherein the viral vector is a lentiviral particle.

[0217] 25. The method of embodiment 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, Mokara virus, rabies virus, RD114 or a functional variant thereof.

[0218] 26. The method of embodiment 15, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0219] 27. The method of embodiment 26, wherein the HSV particles are HSV-1 or HSV-2 particles or functional variants thereof.

[0220] 28. The method of any one of embodiments 1-14, wherein the gene therapy agent comprises a lipid nanoparticle.

[0221] 29. The method of any one of embodiments 1 to 28, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0222] 30. The method of embodiment 29, wherein the heterologous transgene is operably linked to a promoter.

[0223] 31. The method of embodiment 30, wherein the promoter is a constitutive promoter, a tissue-specific promoter or an inducible promoter.

[0224] 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.

[0225] 33. The method of any one of embodiments 1-32, wherein the individual has a disease or disorder suitable for treatment by gene therapy.

[0226] 34. The method of embodiment 33, wherein the disease or disorder is a monogenic disease or disorder.

[0227] 35. The method of any one of embodiments 1 to 34, wherein the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously or intrahepatically.

[0228] 36. The method of any one of embodiments 1-35, wherein the IRAK modulator is administered orally, intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously or intrahepatically.

[0229] 37. A method for delivering a gene therapy agent to cells of an individual, comprising: a) incubating innate immune cells from an individual with a gene therapy agent; b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies individuals with innate immunity to the gene therapy agent; c) administering to the individual identified in step b) an IRAK modulator; and d) administering a gene therapy agent to the individual identified in step b); The method includes:

[0230] 38. A method of treating an individual in need of treatment with a gene therapy agent, comprising: a) incubating innate immune cells from an individual with a gene therapy agent; b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent identifies individuals with innate immunity to the gene therapy agent; c) administering to the individual identified in step b) an IRAK modulator; and d) administering a gene therapy agent to the individual identified in step b); The method includes:

[0231] 39. A method for selecting an individual for treatment with a gene therapy agent and an IRAK modulator, comprising: a) incubating innate immune cells from an individual with a gene therapy agent; b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature following incubation with the gene therapy agent identifies the individual for treatment with the gene therapy agent and an IRAK modulator; c) selecting individuals identified in step b) for treatment with gene therapy agents and IRAK modulators. The method includes:

[0232] 40.d) administering to the individual identified in step b) an IRAK modulator; e) administering a gene therapy agent to the individual identified in step b); 40. The method of embodiment 39, further comprising:

[0233] 41. The method according to any one of embodiments 37 to 40, wherein the innate immune cells are dendritic cells, monocytes, macrophages or natural killer (NK) cells.

[0234] 42. The method according to any one of embodiments 37 to 41, wherein the innate immune cells are isolated from peripheral blood mononuclear cells from an individual.

[0235] 43. The method of any one of embodiments 38 to 42, wherein the innate immune cells are dendritic cells.

[0236] 44. The method of embodiment 43, wherein the dendritic cells are derived from the individual's monocytes.

[0237] 45. The method of embodiment 44, further comprising isolating monocytes from the individual and incubating the monocytes in dendritic cell culture medium to derive dendritic cells from the monocytes prior to incubating the dendritic cells with the gene therapy agent.

[0238] 46. ​​The method of embodiment 44 or 45, wherein the monocytes are CD14+ monocytes.

[0239] 47. The method according to any one of embodiments 44 to 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 induce dendritic cells from the monocytes.

[0240] 48. The method according to any one of embodiments 37 to 47, wherein the innate immune cells are replated prior to incubation with the gene therapy agent in step c).

[0241] 49. The method of embodiment 48, wherein the innate immune cells are replated in microwell dishes.

[0242] 50. The gene therapy agent is a viral vector, and innate immune cells are approximately 1 x 10 3 ~Approx. 1×10 5 Or about 1 x 10 4 The method of any one of embodiments 37 to 49, wherein the cells are incubated with the viral vector at an MOI of 100-1500 μg / ml.

[0243] 51. The method of any one of embodiments 37 to 49, wherein the gene therapy agent is a non-viral vector and the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

[0244] 52. The method of any one of embodiments 37 to 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.

[0245] 53. The method of any one of embodiments 37 to 52, wherein the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1 and MIP-1α.

[0246] 54. A method according to any one of embodiments 37 to 53, wherein the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1 and MIP-1α.

[0247] 55. A method according to any one of embodiments 37 to 53, wherein the cytokine signature comprises increased expression of IL6, TNFα and IL-1β.

[0248] 56. A method according to any one of embodiments 37 to 55, wherein expression of a cytokine in the cytokine signature is increased compared to a suitable control.

[0249] 57. The method of embodiment 56, wherein a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells that have not been incubated with the gene therapy agent, or a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

[0250] 58. The method of any one of embodiments 37 to 57, wherein the IRAK modulator modulates the activity of the IRAK protein kinase.

[0251] 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.

[0252] 60. The method of any one of embodiments 37-59, wherein the IRAK modulator modulates the activity of IRAK-4 protein kinase.

[0253] 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 results in loss of function of IRAK.

[0254] 62. The method of any one of embodiments 37-61, wherein the IRAK modulator is a small molecule.

[0255] 63. The IRAK modulator is represented by the formula [I]: [ka] (In the formula, X 1 are covalent bonds, -CH2-, -C(O)-, -C(S)-, and [ka] is a divalent moiety selected from R1a is hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O)2R, -N(R)2, -Si(R)3 or optionally substituted C l~4 It is 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 teeth, [ka] [ka] [ka] is a bicyclic or tricyclic ring selected from Ring B a is a fused ring selected from a 6-membered aryl containing 0 to 2 nitrogen atoms, a 5- to 7-membered partially saturated carbocyclyl, a 5- to 7-membered partially saturated heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-membered heteroaryl having 1 to 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 is hydrogen, C l~4 aliphatic or -CN; Each R 6a is independently 1~6 an optionally substituted group selected from aliphatic, phenyl, 4- to 7-membered saturated or partially unsaturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Ring A is a 4-10 membered saturated monocyclic or bicyclic carbocyclic or heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring C is a 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 independently represents a covalent bond or C 1~3 a divalent linear or branched saturated or unsaturated hydrocarbon chain, wherein one to three 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 C 1~6an optionally substituted group selected from aliphatic, phenyl, a 4- to 7-membered saturated or partially unsaturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups on the same atom optionally together with their intervening atoms form an optionally substituted 4-11 membered saturated or partially unsaturated carbocyclic or heterocyclic monocyclic, bicyclic, bridged bicyclic, spirocyclic, 5- or heteroaryl ring having, in addition to the atom to which they are attached, 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; 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), -CR 2(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 teeth, [ka] , hydrogen or C 1~6 is selected from optionally substituted groups selected from aliphatic or 4-11 membered saturated or partially unsaturated monocyclic, bicyclic, bridged bicyclic or spirocyclic carbocyclic or heterocyclic rings having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Ring D is a 4- to 10-membered saturated or partially unsaturated monocyclic or bicyclic carbocyclic or heterocyclic ring having 1 to 3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R3 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)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, Each R 5 are independently optionally substituted groups selected from C1-6 aliphatic, phenyl, 3- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic rings having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl rings 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. or a pharma- ceutically acceptable salt thereof.

[0256] 64. The IRAK modulator is represented by the formula [II] to [V]: [ka] or a pharma- ceutically acceptable salt thereof.

[0257] 65. The method of any one of embodiments 37 to 62, wherein the IRAK modulator is a compound of formula [II], PROTAC IRAK-4 degrader 1 or PF06650833.

[0258] 66. The method of any one of embodiments 37 to 61, wherein the IRAK modulator is a CRISPR, siRNA, shRNA, miRNA, RNAi, antisense RNA, ribozyme or DNAzyme.

[0259] 67. The method of any one of embodiments 37-66, wherein the IRAK modulator blocks TLR9 function.

[0260] 68. The method of any one of embodiments 37 to 67, wherein the gene therapy agent is a viral vector.

[0261] 69. The method of embodiment 68, wherein the viral vector is an AAV particle.

[0262] 70. AAV particles include 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 70. The method of embodiment 69, comprising an E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a murine AAV capsid, a rAAV2 / HBoV1 (chimeric AAV / human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid, or an AAVPHP.eB capsid, or a functional variant thereof.

[0263] 71. The method of embodiment 70, wherein the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation or an HBKO mutation.

[0264] 72. The method of any one of embodiments 69 to 71, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), and 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.

[0265] 73. The method of embodiment 72, wherein one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0266] 74. The method of embodiment 72, wherein one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0267] 75. The method of embodiment 68, wherein the viral vector is an adenovirus particle.

[0268] 76. The method of embodiment 75, wherein the adenovirus 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.

[0269] 77. The method of embodiment 68, wherein the viral vector is a lentiviral particle.

[0270] 78. The method of embodiment 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, Mokara virus, rabies virus, RD114 or a functional variant thereof.

[0271] 79. The method of embodiment 68, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0272] 80. The method of embodiment 79, wherein the HSV particles are HSV-1 or HSV-2 particles or functional variants thereof.

[0273] 81. The method of any one of embodiments 37 to 80, wherein the gene therapy agent is a lipid nanoparticle.

[0274] 82. The method of any one of embodiments 37 to 81, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0275] 83. The method of embodiment 82, wherein the heterologous transgene is operably linked to a promoter.

[0276] 84. The method of embodiment 83, wherein the promoter is a constitutive promoter, a tissue-specific promoter or an inducible promoter.

[0277] 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.

[0278] 86. The method of any one of embodiments 37-85, wherein the individual has a disease or disorder suitable for treatment by gene therapy.

[0279] 87. The method of embodiment 86, wherein the disease or disorder is a monogenic disease or disorder.

[0280] 88. The method of any one of embodiments 37 to 87, wherein the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously or intrahepatically.

[0281] 89. The method of any one of embodiments 37-88, wherein the IRAK modulator is administered orally, intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously or intrahepatically.

[0282] 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 the composition is formulated for use in combination with an IRAK modulator.

[0283] 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 the composition is formulated for use in combination with a gene therapy agent.

[0284] 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 the composition is formulated for use in combination with an IRAK modulator.

[0285] 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 the composition is formulated for use in combination with a gene therapy agent.

[0286] 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 the composition is formulated for use in combination with an IRAK modulator.

[0287] 95. Use of a composition in the manufacture of a medicament for modulating an immune response to gene therapy in an individual, wherein the composition comprises an IRAK modulator, and the composition is formulated for use in combination with a gene therapy agent.

[0288] 96. The use according to any one of embodiments 90 to 95, wherein the gene therapy agent is an AAV particle, an adenovirus particle, a lentivirus particle, an HSV particle or a lipid nanoparticle.

[0289] 97. The use according to any one of embodiments 90 to 96, wherein the IRAK modulator is an IRAK-4 degrader.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 102. A composition comprising an IRAK modulator 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.

[0295] 103. A composition comprising an IRAK modulator for 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.

[0296] 104. The composition of any one of embodiments 98 to 103, wherein the gene therapy agent is an AAV particle, an adenovirus particle, a lentivirus particle, an HSV particle or a lipid nanoparticle.

[0297] 105. The composition of any one of embodiments 98-104, wherein the IRAK modulator is an IRAK-4 degrading agent.

[0298] 106. A kit for use in the method according to any one of embodiments 1 to 89.

[0299] 107. A kit for use according to any one of embodiments 90 to 97.

[0300] 108. A kit comprising a composition according to any one of embodiments 98 to 105. EXAMPLES

[0301] The present invention will be more fully understood by referring to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and in light thereof, various modifications or changes may be suggested to those skilled in the art, and should be included within the spirit and scope of this application and the scope of the appended claims.

[0302] Example 1: Ex vivo protocol to evaluate the effect of IRAK4 degraders on AAV-treated human cells This example provides a strategy for examining the effects of IRAK4 inhibition on lymphocytes and dendritic cells exposed to AAV.

[0303] AAV induces an immune response that includes activation of both the innate and adaptive immune systems. While the adaptive immune response to AAV is relatively well characterized, innate immune activation by AAV is less understood.

[0304] IRAK4 is a kinase in the TLR pathway that activates innate immune response. Broad acting immunosuppressants improve AAV delivery, but this leads to loss of transgene expression, side effects and the risk of opportunistic infection. Therefore, inhibiting IRAk4 may provide more specific immunomodulation that is favorable for AAV therapy.

[0305] To prepare peripheral blood mononuclear cells (PBMCs), decant the blood from the leukopak into a 50 mL tube and add DPBS in a 1:1 ratio. Gently pipette the blood into a separate 50 mL tube containing 15 mL of Ficoll (GE17-5442-02) to avoid mixing the blood and Ficoll layers. Centrifuge the mixture at 2000 RPM (acceleration 9, no brake) for 25 minutes at room temperature. Collect the buffy coat containing leukocytes and platelets, transfer to a new tube, and centrifuge at 400 RCF for 5 minutes. Wash the cells 3 times with PBS containing 1% FBS or FCS and count.

[0306] CD14+ monocytes are isolated from PBMCs using CD14 microbeads according to the manufacturer's protocol available online (World Wide Web miltenyibiotec.com / upload / assets / IM0001260.PDF; Miltenyi Biotech, Germany, order number 130-050-201). Briefly, cells are cultured at 10 7 Incubate with 20 µL of CD14 microbeads per total cells for 15 min at 2-8 °C. Apply the cells to a magnetic column and allow the unlabeled cells to pass through. After washing the column three times, remove the column from the magnetic separator, place it in a collection tube, and flush the magnetically labeled cells by pushing the plunger into the column.

[0307] Monocytes are differentiated into dendritic cells using ImmunoCult-ACF dendritic cell medium, differentiation supplements and maturation supplements according to the manufacturer's protocol available online (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). Briefly, purified monocytes are added to dendritic cell medium containing differentiation supplements and incubated at 37° C. for 3 days. On day 3, the medium is changed and the cells are incubated for an additional 2 days. Maturation supplements are added on day 5 at a dilution of 1:100 (e.g., 50 μL of supplement per 5 mL of culture).

[0308] Differentiated cells are harvested on day 7. T cells are stimulated by adding 4 mL of ImmunoCult XF T cell growth medium (Stem Cell Technologies, Cat. No. 10981) to the thawed PBMCs. The cells are spun at 400g for 5 minutes, the medium is aspirated, and the cells are left overnight in 10 mL of fresh growth medium.

[0309] After harvesting and counting, resting cells are spun at 400g for 5 minutes and resuspended. Cell stimulation is performed in 96-well plates in T cell complete medium. Final concentrations of cytokines in the medium are 100IU IL-2, 5ng / mL IL-7 and 25ng / mL IL-15. AAV stimulation is performed at 1e5 MOI and 10μg PHA-p is used as a positive control. Half-depletion is performed every 3 days. 10 days after thawing PBMCs, cells are restimulated with AAV in simple T cell medium. Medium is collected after 24 hours of stimulation.

[0310] rAAV vectors (e.g., AAV1, AAV2 and / or AAVrh32.33) are produced using a standard triple transfection method (Sena-Esteves and Gao, Cold Spring Harb Protoc; doi:10.1101 / pdb.top095513, 2020). Virus is purified by cesium chloride ultracentrifugation and titrated using both silver staining and quantitative polymerase chain reaction (qPCR).

[0311] Lymphocytes and dendritic cells are incubated with AAV vectors and pre-treated, post-treated or co-treated with IRAK4 degraders. Pre-treatment and post-treatment with IRAK4 degraders are performed 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 degrader-1 (Med Chem Express, Catalog No. HY-129966), PZ0327 (Sigma-Aldrich, Catalog No. PF06650833) or compound of formula [II] (Kymera Therapeutics) at doses ranging from 1 nM to 1 M. LPS (300ng / mL, 24 hours, Sigma L2630100MG) and R848 (1μg / mL, 24 hours, Invitrogen tlrl-r948-5) are used as controls. Additional test groups include treatment with AAV alone, an IRAK4 inhibitor alone, or an IRAK4 inhibitor with either LPS or R848.

[0312] To determine the extent to which the TLR9 pathway is inhibited in cells treated with IRAK4 degraders, cell media is collected and analyzed using a Meso Scale Discovery (MSD) assay to determine the level of cytokine release. The V-PLEX Human Biomarker 54-Plex Kit is used and the assay is performed according to the manufacturer's protocol (Meso Scale Discovery, Rockville, MD).

[0313] Example 2: In vivo analysis of IRAK4 degraders in mice treated with AAV or LNP This example provides an in vivo strategy to examine whether treating animals with an IRAK4 degrading agent attenuates AAV immunogenicity.

[0314] All animal care, maintenance, treatment, and experiments are conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC).

[0315] Animals receive either AAV or LNP injections and are pre-treated, post-treated or co-treated with IRAK4 inhibitors. Pre- and post-treatment with IRAK4 degraders is performed 6 hours, overnight, 24 or 48 hours before or after AAV or LNP injection. Intramuscular AAV injections are performed using 5×10 11 vg / kg and monitored for 7, 14, 21, 28, 35, 42 and 63 days. LNP injections are performed intravenously, subcutaneously or intraperitoneally at doses of 1 mg / kg, 10 mg / kg or 100 mg / kg. Additionally, animals are pre-treated, post-treated or co-treated with 1 mg / kg, 10 mg / kg or 100 mg / kg of PROTAC IRAK4 degrader-1 (Med Chem Express, Catalog No. HY-129966), PZ0327 (Sigma-Aldrich, Catalog No. PF06650833) or compound of formula [II] (Kymera Therapeutics) administered subcutaneously, intraperitoneally or orally. Additional test groups include treatment with AAV alone, IRAK4 inhibitor alone or IRAK4 inhibitor with either LPS or R848.

[0316] Animals are sacrificed 7, 14, 21, 28, 35, 42 or 63 days after treatment and muscle, liver, bone marrow, spleen, blood and thymus are isolated. Tissue samples are immediately frozen on dry ice. DNA and RNA samples are extracted and vector genome copies and transgene expression are quantified using real-time quantitative PCR (qPCR).

[0317] Tissue inflammation in treated animals is assayed using hematoxylin and eosin (H&E) staining. Staining is performed on formalin-fixed, paraffin-embedded muscle and liver tissues according to methods well known in the art. (Gernoux, G et al., Mol Ther, 2020, 28(3):747-757).

[0318] Immunohistochemistry is performed on frozen sections of muscle, liver and spleen according to methods well known in the art (Gernoux, G et al., Mol Ther, 2020, 28(3):747-757). Samples are harvested on slides, air-dried and fixed with 3% paraformaldehyde. Samples are analyzed for 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. Nuclear and cytoplasmic β-gal are assessed using standard X-gal protocols.

[0319] Serum is collected from these subjects and used in Meso Scale Discovery (MSD) assays to analyze cytokine levels in treated animals. The V-PLEX Human Biomarker 54-Plex Kit is used and the assay is performed according to the manufacturer's protocol (Meso Scale Discovery, Rockville, MD).

[0320] To measure IFN-γ secretion in treated animals, an ELISpot assay is performed on spleen cells isolated after injection (Gernux, G et al., Mol Ther, 2020, 28(3):747-757). Isolated cells are stimulated in vitro for 48 hours using AAV. Spot numbers are determined using an iSpot ELISpot Reader ELR068IFL (AID) and analyzed using AID ELISpot Reader software v.6.0. Responses are considered positive if spot-forming units are greater than 50 per 1e6 cells and at least 3-fold higher than the control condition. Unstimulated cells are used as negative controls, and CEFT and CD3 / CD28 stimulation are used as positive controls.

[0321] Single cell suspensions are obtained from spleen and bone marrow samples. Cells are incubated in CD16 / 32 (Fc Block; BD Biosciences) and stained with antibodies. Cells are acquired on an LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star).

[0322] To analyze cytokine expression in treated animals, isolated splenocytes are stained for IFN-γ, TNF-α, IL-2, IL-4 and IL-6 using intracellular cytokine staining. Splenocytes are prepared as described in Mays, J Immunol 2009 182(1)6051-6060. Splenocytes are seeded in T cell assay medium supplemented with 1 μg / mL Brefeldin A (GolgiPlug, BD Pharmigen) and 20 ng / mL mouse IL-2 (BD Pharmingen). Prior to staining, cells are stimulated with AAV in the presence or absence of IRAK4 degraders for 5 hours at 37° C. in 10% CO2. After stimulation, cells are washed, stained with antibodies and examined using flow cytometry. Samples are acquired on an LSR II and analyzed using FlowJo software.

[0323] T cell responses of animals treated with AAV and IRAK4 degraders are determined by MHC class I tetramer staining using PE-conjugated MHC class I H2-Kb-ICPMYARV tetramer complexes (Beckman Coulter) according to the protocol described in Mays, J Immunol 2009 182(1)6051-6060. Tetramer staining is performed on heparinized whole blood cells isolated by retro-orbital bleeding at various time points after treatment with AAV and IRAK4 degraders. Cells are co-stained with PE-conjugated tetramers and FITC-conjugated anti-CD8α (Ly-2) antibodies (BD Pharmingen) for 30 minutes at room temperature and fixed with iTAg MHC tetramer lysis solution supplemented with fixative solution (Beckman Coulter) for 15 minutes at room temperature. Cells are washed three times with PBS and resuspended in 0.01% BD CytoFix (BD Biosciences). Samples are acquired on an LSR II and analyzed using FlowJo software.

[0324] Example 3: Ex vivo analysis of the effects of IRAK4 degraders on AAV-treated human cells Materials and Methods Preparation of peripheral blood mononuclear cells. Blood from leukopak (Stem cell technologies) from different donors was decanted into 50 mL tubes and Dulbecco's Phosphate Buffered Saline (DPBS) was added in a 1:1 ratio. The blood + DBP mixture was gently transferred with a pipette into a separate 50 mL tube containing 15 mL of Ficoll (GE17-5442-02) to avoid mixing the blood and Ficoll phases. The mixture was centrifuged at 2000 RPM (9 acceleration, no brake) for 25 minutes at room temperature. The buffy coat containing peripheral blood mononuclear cells (PBMCs) was collected, transferred to a new tube and centrifuged at 400 mm RCF for 5 minutes. PBMCs were washed three times with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FBS) or fetal calf serum (FCS) and counted.

[0325] Isolation of monocytes. CD14+ monocytes were isolated from PBMCs using CD14 microbeads according to the manufacturer's protocol (Miltenyi Biotech, Germany, order number 130-050-201, protocol available online, World Wide Web miltenyibiotec.com / upload / assets / IM0001260.PDF). Briefly, PBMCs were cultured at 10 7 The PBMCs were incubated with 20 µL of CD14 microbeads per total cells for 15 min at 2–8 °C. The PBMCs were applied to a magnetic column (Miltenyi; World Wide Web miltenyibiotec.com / US-en / products / ls-columns.html#130-042-401) and unlabeled cells were allowed to pass through. After washing the column three times, the column was removed from the magnetic separator (Miltneyi; World Wide Web miltenyibiotec.com / US-en / products / quadromacs-separator-and-starting-kits.html#130-091-051), placed in a collection tube, and the magnetically labeled CD14+ monocytes were flushed out by pushing the plunger into the column.

[0326] Differentiation of monocytes. CD14+ monocytes are differentiated into dendritic cells using ImmunoCult-ACF dendritic cell medium, differentiation supplements and maturation supplements according to the manufacturer's protocol available online (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=2.81451927.1035383195.16421057001174975582.1603298321). Briefly, purified CD14+ monocytes were added to dendritic cell medium containing differentiation supplements and incubated for 3 days at 37°C. On the third day, the medium was replaced with fresh dendritic cell medium containing differentiation supplements and the cells were incubated for an additional 2 days. Maturation supplement was added to the cells at a dilution of 1:100 (eg, 50 μL of supplement per 5 mL of culture) on day 5. Differentiated dendritic cells were harvested on day 7.

[0327] rAAV Production and Titration. rAAV vectors (AAV.SAN024 and AAV.SAN029) are produced using a standard triple transfection method (Sena-Esteves and Gao, Cold Spring Harb Protoc; doi:10.1101 / pdb.top095513, 2020). All serotypes tested encode the same GFP transgene. Virus was purified by cesium chloride ultracentrifugation and titrated using both silver staining and quantitative polymerase chain reaction (qPCR).

[0328] IRAK4-targeted drug and AAV treatment. Dendritic cells were seeded in 96-well plates at 200,000 cells per well. Each treatment was performed in triplicate. Cells were treated with dimlobicertib IRAK4 inhibitor (also known as PF-06650833 (catalog number: HY-19836 (MedChemExpress)) and PROTAC IRAK4 degrader-1 (catalog number: HY-129966 (MedChemExpress)) at 16 nm, 80 nm, 400 nm and 2000 nm for 18 hours in an incubator at 37°C and 5% CO2. After 18 hours of drug pretreatment, AAV vectors were used at 1e5 MOI. Cells were incubated for 24 hours in an incubator at 37°C and 5% CO2. After 24 hours, plates were centrifuged and media supernatants were collected. The clarified media was analyzed using Meso Scale Discovery MSD (world wide web) performed by DC3 therapeutics. The results were analyzed by immunofluorescence assay (https: / / www.dc3therapeutics.com / services). An experimental overview of PBMC isolation, monocyte purification and dendritic cell differentiation is shown in Figure 1. The figure also shows how dendritic cells were treated with different drugs and AAV. In some experiments, lipopolysaccharide LPS (300ng / mL) (Sigma Aldrich Fine Chemicals Biosciences L2630100MG) was used as a positive control, known to activate IRAK4 and induce phosphorylation of IRAK4 target proteins.

[0329] LDH release assay. Cytotoxicity was measured by the release of lactate dehydrogenase (LDH) in the culture supernatant. LDH was quantified using a Promega kit (catalog number G1780) according to the manufacturer's protocol. Briefly, after experimental treatment, supernatant samples were transferred to a 96-well plate and an equal volume of CytoTox96® reagent was added to each well and incubated for 30 minutes. Stop solution was added and absorbance signal was measured at 490 nm in a plate reader. https: / / www.promega.com / products / cell-health-assays / cell-viability-and-cytotoxicity-assays / cytotox-96-non_radioactive-cytotoxicity-assay / ?catNum=G1780#protocols

[0330] AlphaLISA assay. This assay was performed to measure the levels of IRAK4 protein in cells. The protocol was performed according to the manufacturer's recommendations, Perkin Elmer (catalog number AL3117C). Briefly, cells were lysed and 5 μL of lysate was added to anti-IRAK4 acceptor beads and incubated at 23° C. for 30 minutes, followed by the addition of biotinylated anti-IRAK4 antibody and incubation at 23° C. for 60 minutes. Finally, SA donor beads are added and the plate is read after 30 min using an EnVision-Alpha Reader (615 nm) (https: / / resources.perkinelmer.com / lab-solutions / resources / docs / MAN_AlphaLISA_IRAK4_AL3117.pdf?_gl=1*7jzf5d*_ga*MzQ4MTQ1NzA3LjE2NDY2NzY5MjI.*_ga_W34ZJ1Z1Q1*MTY3NzE5MjAyOS4zLjEuMTY3NzE5MjA2Mi4yNy4wLjA.&_ga=2.178521252.1409175695.1677192029-348145707.1646676922). The same protocol was used to measure phosphorylation levels of NFkB using the AlphaLISA SureFire Ultra p-NFkB(Ser536)Assay Kit-High Volume, catalogue no. ALSU-PNFKB-A-HV.

[0331] result Pretreatment with IRAK4 modulators results in attenuation of cytokine release Human monocytic dendritic cells were pretreated with different doses of drugs as indicated (16nM, 80nM, 400nM and 2000nM) for 18 hours, then the same cells were infected with AAV at an MOI of 1e5. 24 hours of SAN024 and media supernatants were analyzed for cytokine release. Treatment with AAV induces secretion of IL1B cytokine from dendritic cells, which is blocked at all doses by both the IRAK4 inhibitor dimlobicertib (Figure 2A) and the IRAK4 degrader PROTAC IRAK4 degrader-1 (Figure 2B). 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 across all donors. Cytokine blockade was also observed with other cytokines such as IL6 and TNFa.

[0332] Co-treatment with IRAK4 modulators results in attenuation of cytokine release Human monocytic dendritic cells were co-treated with different doses of drugs as indicated (16 nM, 80 nM, 400 nM and 2000 nM), the same cells were simultaneously infected with AAV.SAN024 at an MOI of 1e5 for 24 h, and culture supernatants were analyzed for cytokine release. Treatment with AAV induces secretion of IL1b cytokine from dendritic cells, which is blocked at all doses by both the IRAK4 inhibitor dimlobicertib (Figure 3A) and the IRAK4 degrader PROTAC IRAK4 degrader-1 (Figure 3B). Each point on the bar graph represents a technical replicate of a representative donor. Experiments were performed on three donors and statistical significance was determined using one-way ANOVA. Cytokine secretion is expressed as a percentage across all donors. Cytokine blockade was also observed with other cytokines such as IL6 and TNFa.

[0333] IRAK4 modulators do not cause cytotoxicity in primary human monocytic dendritic cells Human monocytic dendritic cells were treated with different doses of the IRAK4 inhibitor dimlobicertib (Figure 4A) or the IRAK degrader PROTAC IRAK4 degrader-1 (Figure 4B) as indicated (16 nM, 80 nM, 400 nM and 2000 nM) and the same cells were infected with AAV.SAN024 at an MOI of 1e5 for 24 hours. Culture supernatants were analyzed for LDH release. No significant differences were observed between control (untreated / uninfected cells) compared to treated and AAV. SAN024 infected cells indicate that neither IRAK4 modulator had cytotoxicity.

[0334] Treatment with IRAK4 inhibitors blocks phosphorylation of IRAK4 kinase target proteins, and IRAK4 degraders cause degradation of IRAK4 protein in primary human monocytic dendritic cells Human monocytic dendritic cells were treated with different doses of drugs as indicated (16 nM, 80 nM, 400 nM, and 2000 nM), and the same cells were treated with LPS 300 ng / ml. LPS triggers IRAK4 kinase activity, allowing phosphorylation of IRAK4 target proteins such as NFkB. Treatment with the IRAK4 kinase inhibitor dimlobicertib (Figure 5A) blocks phosphorylation of NFkB at 400 nM and 2000 nM.

[0335] To determine the activity of PROTAC degraders, human monocytic dendritic cells were treated with different doses of drugs as indicated (16 nM, 80 nM, 400 nM and 2000 nM) and the same cells were infected with AAV.SAN024 at an MOI of 1e5 for 24 hours. Cell lysates were analyzed for IRAK4 degradation. No significant differences were observed between control (untreated / uninfected) cells compared to treated and AAV. SAN024 infected cells but not AAV. SAN024 infected cells treated with degraders cause a reduction in IRAK4 levels at all doses (Figure 5B).

[0336] Example 4: Analysis of the effects of IRAK4 modulators on AAV-induced immune responses in an in vivo mouse model Materials and Methods In vivo mouse experiments. C57BL / 6J mice were purchased from Jackson Laboratories. Male mice aged 6–8 weeks were divided into three groups (PBS group, AAV group, and AAV+IRAK4 group), with 10 mice in each group. Dimlobicertib IRAK4 inhibitor (also known as PF-06650833 (catalog number: HY-19836 (MedChemExpress)) was mixed with mouse chow (catalog number 2016, Teklad Global 16% Protein Rodent Diets, https: / / www.inotivco.com / rodent-natural-ingredient-2016-diets) at a dose of 30 mg / kg. The AAV+IRAK4 group was fed chow containing dimlobicertib IRAK4 inhibitor 15 days prior to AAV injection, whereas the PBS and AAV groups were fed chow containing dimlobicertib IRAK4 inhibitor 15 days prior to AAV injection. Mice were fed a diet containing 100 μL of PBS (Natural Diets, https: / / www.inotivco.com / rodent-natural-ingredient-2016-diets). Mice in the PBS group were injected intramuscularly with 100 μL of PBS, and those in the AAV group were injected intramuscularly with 1E11 vg per quadriceps in both legs in a volume of 100 μL.

[0337] Following the same experimental design, n=6 mice were used for each group: PBS, AAV, and AAV+IRAK4 degrader PROTAC IRAK4 degrader-1 (Cat. No. HY-129966 (MedChemEsrses)) mixed in the feed at 100 mg / kg and given to mice 15 days before AAV injection. Two different IRAK4 PROTAC degraders (commercially available PROTAC IRAK4 degrader-1, Cat. No. HY-129966 (MedChemEsrses)) and KT-474 were mixed in the feed at a dose of 100 mg / kg and given to mice 15 days before AAV administration. The AAV capsids used in this study carried the LacZ transgene. All animal experiments were performed in accordance with the Institutional Animal Care and Use Committees of Sanofi, Framingham. Submandibular blood sampling was performed weekly, and necropsy was performed 3 weeks after AAV injection.

[0338] PBMC isolation and surface staining. 120ul of blood was collected in a K2EDTA coated tube. The blood was mixed with DBP in a 1:1 ratio and pipetted into a separate tube containing Ficoll (GE17-5442-02) without mixing the blood and Ficoll phases. The mixture was centrifuged at 2000RPM (9 acceleration, no brake) for 25 minutes at room temperature. The buffy coat containing peripheral blood mononuclear cells (PBMC) was collected, transferred to a new tube and centrifuged at 400mmRCF for 5 minutes. The PBMC were washed three times with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FBS) or fetal calf serum (FCS) and counted. The PBMC cells thus obtained were stained with different antibodies to quantify the percentage of different immune cell subsets in the peripheral blood.

[0339] PBMC CD8 TEM and lacZ tetramer staining. PBMCs were isolated from approximately 120 μL of mouse blood and transferred to a 96-well U-bottom plate. PBMCs were washed once with 200 μl of FACS buffer by centrifugation at 2,000 rpm for 5 min and stained with flow antibody cocktail (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) for 30 min at 4°C. After incubation, cells were washed twice with FACS buffer and fixed with 100 μl of BD Cytofix / Cytoperm Fixation / Permeabilization Solution Kit for 15 min at 4°C. The cells were washed twice with FACS buffer, and the samples were analyzed using a flow cytometer (Novocyte Penteon Flow Cytometer Systems 5 Lasers, Agilent Technology).

[0340] Intracellular cytokine staining. Spleen cells were harvested from mouse spleens and 2 million cells were seeded in 96-well U-bottom plates. 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, AAVrh32.33LacZ immunodominant peptides and LacZ immunodominant peptides at 10 μg / ml concentration were prepared in RPMI1640 containing 10% FBS, 1X 2-mercaptoethanol and 1:500 GolgiStop. As a positive control, 0.10 μg / ml PMA and 2 μg / ml ionomycin solutions were prepared in RPMI1640 containing 10% FBS, 1X 2-mercaptoethanol and 1:500 GolgiStop. Then, 100 μL of the prepared solution was added to the cells to obtain a final concentration of 5 μg / ml (or 0.05 μg / ml PMA and 1 μg / ml ionomycin). To stimulate the cells, the cells were incubated overnight at 37° C. in a 5% CO2 incubator. The next day, the cells were centrifuged at 2,000 rpm for 5 min and washed once with FACS buffer. The cells were stained with an antibody cocktail (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) for 30 min at 4° C. Then, the cells were washed twice with FACS buffer. After washing, the cells were permeabilized with 100 μl of BD Cytofix / Cytoperm Fixation / Permeabilization Solution Kit for 20 min at 4° C. Cells were washed twice with 1XPermWash and stained with an antibody cocktail (1:100 anti-IFNγ APC, 1:100 anti-IL2 PE, 1:100 anti-TNFα PE-Cy7) for 30 minutes at 4° C. Cells were washed twice with FACS buffer and samples were analyzed on a flow cytometer.

[0341] Table 1 shows a list of sources of the various reagents used in the assays described in this example.

[0342] [Table 1]

[0343] result Treatment with an IRAK4 inhibitor reduces transgene LacZ-specific CD8 T cells and effector memory T cells and reduces IFNg-producing CD8 T cells in PBMCs derived from peripheral blood 14 days after AAV injection In vivo mouse experiments were designed to show the effect of IRAK4 inhibition on CD8 T cells and memory T cells after AAV injection (Figure 6). Mice shown in Figure 6 represent the AAV+IRAK4 group, where mice were fed with IRAK4 inhibitor dimlobicertib mixed feed 15 days before AAV injection, and then blood was collected for PBMC analysis on day 14. Necropsy of mice was performed on day 21 to harvest spleens. PBS and AAV only groups were fed normal feed, bled on day 14, and necropsied on day 21.

[0344] Mice were bled 14 days after AAV injection and PBMCs were isolated. PBMCs were stained with different antibodies to quantify LacZ tetramer-positive CD8 T cells (Figure 7A) and CD44+CD62L+ effector memory cells (Figure 7B). These cell populations were upregulated upon AAV administration compared to PBS controls. A significant decrease in CD8 T cells and effector cells was observed in mice administered IRAK4 inhibitors, as shown in Figures 7A and 7B, respectively. One-way ANOVA was performed with N=10 mice per group to determine statistical significance.

[0345] Mice were euthanized on day 21 and splenocytes were harvested. Splenocytes were stimulated with either AAV peptide pools (Figure 8A) or AAV immunodominant peptides (Figure 8B) to generate activated capsid-specific CD8 T cells. Similarly, cells were treated with LacZ immunodominant peptides (Figure 8C) 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 are upregulated upon AAV administration and then significantly reduced upon IRAK4 inhibitor treatment compared to PBS controls. One-way ANOVA with N=10 mice per group was performed to determine statistical significance.

[0346] IRAK4 Degrader Treatment with the PROTAC IRAK4 Degrader-1 reduces IRAK4 expression in immune cells Mice were bled on day 14 and PBMCs were harvested. Different immune cell types in PBMCs were identified by flow cytometry (Figures 9A-9D). In all different immune cell types evaluated, IRAK4 protein expression was significantly decreased with dietary IRAK4 degrader treatment. One-way ANOVA was performed with N=6 mice per group to determine statistical significance.

[0347] KT-474 diet treatment reduces transgene-specific CD8 T cells in PBMCs and spleens. Mice were bled 14 days after AAV injection and PBMCs were isolated. Spleen cells were harvested 21 days after AAV injection. PBMCs and spleen cells were stained with different antibodies to quantify LacZ tetramer positive cells. These cell populations were upregulated upon AAV administration and then significantly decreased upon KT-474 treatment compared to PBS control (Figure 10). Figure 10A shows the decrease in transgene LacZ-specific CD8 T cells in PBMCs derived from peripheral blood 14 days after AAV injection. Figure 10B shows the decrease in transgene LacZ-specific CD8 T cells in spleen 21 days after AAV injection. Statistical significance was determined using the ROUT outlier method and one-way ANOVA. The ROUT method identifies outliers from nonlinear regression.

[0348] Example 5: General protocol for oral therapy with IRAK modulators in combination with AAV C57BL / 6J mice are purchased from Jackson Laboratories. Male mice aged 6-8 weeks are divided into three groups (PBS group, AAV group, and AAV+IRAK4 modulator group), with 10 mice in each group. IRAK4 modulators such as Dimrobicertib IRAK4 inhibitor (also known as PF-06650833) (catalog number: HY-19836 (MedChemExpress)), PROTAC IRAK4 degrader-1 (catalog number: HY-129966 (MedChemerses)) and KT-474 have been tested by oral administration route treated twice daily at a dose of 100 mg / kg. IRAK4 modulators have been tested as pretreatment and cotreatment strategies. Oral administration of IRAK4 modulators and KT-474 (https: / / clinicaltrials.gov / ct2 / show / NCT04772885) has been clinically tested for other diseases (https: / / www.tandfonline.com / doi / full / 10.1080 / 13543784.2020.1752660) and should therefore be effective in blocking IRAK4 protein. Kymera therapeutics has also shown evidence that IRAK4 protein is completely degraded using KT-474 in human cells and mouse tissues (https: / / www.kymeratx.com / wp-content / uploads / 2021 / 09 / Euro-Prot-Deg-Summit-Sept-21-Final_Anthony-Slavin.pdf). A schematic showing the general procedure for oral treatment of IRAK4 modulators in combination with AAV is shown in Figure 11.

Claims

1. A pharmaceutical composition comprising a viral vector for delivering nucleic acid to the cells of an individual that requires it, the pharmaceutical composition being administered in combination with an IRAK-4 degrading agent or an IRAK-4 inhibitor.

2. A pharmaceutical composition comprising a viral vector for treating individuals in need of treatment, which is administered in combination with an IRAK-4 degrading agent or an IRAK-4 inhibitor.

3. A pharmaceutical composition comprising a viral vector for improving gene therapy in an individual, which is administered in combination with an IRAK-4 degrading agent or an IRAK-4 inhibitor.

4. A pharmaceutical composition comprising a viral vector for suppressing the immune response to a viral vector in an individual requiring such suppression, the pharmaceutical composition being administered in combination with an IRAK-4 degrading agent or an IRAK-4 inhibitor.

5. The IRAK-4 degrading agent or IRAK-4 inhibitor is defined by formula [I]: 【Chemistry 1】 (In the formula, X １ は, shared combination, -CH ２ -, -C(O)-, -C(S)-, and び 【Chemistry 2】 The bivalent part selected from, R １ａ These are hydrogen, halogen, -CN, -OR, -SR, -S(O)R, -S(O) ２ R, -N(R) ２ , -Si(R) ３ or C replaced by optional selection ｌ～４ It is aliphatic, Each R ２ａ is, independently, hydrogen, R ６ａ , halogen, -CN, -NO ２ , -OR, -SR, -NR ２ , -S(O) ２ R, -S(O) ２ NR ２ , -S(O)R, -C(O)R, -C(O)OR, -C(O)NR ２ , -C(O)N(R)OR, -OC(O)R, -OC(O)NR ２ , -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR ２ or -N(R)S(O)2R, and Ring A ａ teeth, 【Transformation 3】 【Chemistry 4】 【Transformation 5】 A biring or triring ring selected from the following: Ring B ａ This is a condensed ring selected from a 6-membered aryl containing 0 to 2 nitrogen atoms, a 5-7 membered partially saturated carbocyclyl, a 5-7 membered partially saturated heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-membered heteroaryl having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R ３ａ These are hydrogen, halogen, -OR, and -N(R). ２ Or selected from -SR, Each R ４ａ Independently, hydrogen, R ６ａ , halogen, -CN, -NO ２ , -OR, -SR, -NR ２ , -S(O) ２ R, -S(O) ２ NR ２ , -S(O)R, -C(O)R, -C(O)OR, -C(O)NR ２ , -C(O)N(R)OR, -OC(O)R, -OC(O)NR ２ , -N(R)C(O)OR, N(R)C(O)R, -N(R)C(O)NR ２ or -N(R)S(O) ２ It is R, R ５ａ is hydrogen, C ｌ～４ It is aliphatic or -CN, Each R ６ａ Independently, C １～６ The group is optionally substituted, selected from a 4-7 member saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from aliphatic, phenyl, nitrogen, oxygen, and sulfur, and a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Ring A is a 4-10 member saturated monocyclic or bicyclic carbocyclic or heterocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Ring C is a 5-10 membered monocyclic or bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from phenyl or nitrogen, oxygen, and sulfur, L ２ and L ３ Each of them is independently covalently bonded or C １～３ A divalent linear, branched saturated, or unsaturated hydrocarbon chain, wherein 1 to 3 methylene units of the chain are independently and arbitrarily selected as -O-, -C(O)-, -C(S)-, -C(R)2-, -CH(R)-, -C(F) ２ -, -N(R)-, -S-, -S(O) ２ It is substituted with - or -CR=CR-, Each R １ Independently, hydrogen, R ５ , halogen, -CN, -NO ２ , -OR, -SR, -NR ２ , -S(O) ２ R, -S(O) ２ NR ２ , -S(O)R, -S(O)(NR)R, -P(O)(OR) ２ , -P(O)(NR ２ ) ２ , -CFR ２ , -CF ２ (R), -CF ３ , -CR ２ (OR), -CR ２ (NR ２ ), -C(O)R, -C(O)OR, or -C(O)NR ２ And, Each R is independently either hydrogen or C １～６ A optionally substituted group selected from aliphatic, phenyl, nitrogen, oxygen, and sulfur-independently-selected 4-7 member saturated or partially unsaturated heterocycles having 1-2 heteroatoms, and 5-6 member heteroaryl rings having 1-4 heteroatoms, and independently-selected nitrogen, oxygen, and sulfur, or Two R groups on the same atom, optionally together with their intervening atoms, have optionally substituted 4-11 member saturated or partially unsaturated carbon atoms having 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, in addition to the atom to which they are bonded. Forming a cyclic or heterocyclic monocyclic, bicyclic, bridging bicyclic, spirocyclic, or heteroaryl ring, Each R ２ Independently, hydrogen, R ５ , halogen, -CN, -NO ２ , -OR, -SR, -NR ２ , -S(O) ２ R, -S(O) ２ NR ２ , -S(O)R, -S(O)(NR)R, -P(O)(OR) ２ , -P(O)(NR ２ ) ２ , -CF ２ (R), -CF ３ , -CR ２ (OR), -CR ２ (NR ２ ), -C(O)R, -C(O)OR, -C(O)NR ２ , -C(O)N(R)OR, -OC(O)R, -OC(O)NR ２ , -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR ２ or -N(R)S(O) ２ It is R, ４ teeth, 【Transformation 6】 , hydrogen or C １～６ Selected from optionally substituted groups, which are selected from 4-11 member saturated or partially unsaturated monocyclic, bicyclic, bridging bicyclic, or spirocyclic carbocyclic or heterocyclic rings having 1-3 heteroatoms independently selected from aliphatic, nitrogen, oxygen, and sulfur, Ring D is a 4-10 member saturated or partially unsaturated monocyclic or bicyclic carbocyclic or heterocyclic ring having 1-3 heteroatoms independently selected from phenyl, nitrogen, oxygen, and sulfur, or a 5-6 member heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Each R ３ is, independently, hydrogen, R ５ , halogen, -CN, -NO ２ , -OR, -SR, -NR ２ , -S(O) ２ R, -S(O) ２ NR ２ , -S(O)R, -S(O)(NR)R, -P(O)(OR) ２ , -P(O)(NR ２ ) ２ , -CF ２ (R), -CF ３ , -CR ２ (OR), -CR ２ (NR ２ ), -C(O)R, -C(O)OR, -C(O)NR ２ , -C(O)N(R)OR, -OC(O)R, -OC(O)NR ２ , -N(R)C(O)OR, -N(R)C(O)R, -N(R)C(O)NR ２ or -N(R)S(O) ２ R; and each R ５ is, independently, an optionally substituted group selected from C1-6 aliphatic, phenyl, a 3-7 member saturated or partially unsaturated carbocyclic or heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5-6 member 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. A pharmaceutical composition according to any one of claims 1 to 4, comprising the compound or a pharmaceutically acceptable salt thereof.

6. The IRAK-4 degrading agent or IRAK-4 inhibitor is a compound of formula (III) or a compound of formula (IV): 【Transformation 7】 A pharmaceutical composition according to any one of claims 1 to 4, comprising a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to any one of claims 1 to 4, wherein the IRAK-4 degrading agent or IRAK-4 inhibitor is PROTAC IRAK-4 degrading agent 1 or PF06650833.

8. The pharmaceutical composition according to any one of claims 1 to 4, wherein the viral vector is an AAV particle.

9. The AAV particles are 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 The pharmaceutical composition according to claim 8, comprising 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 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

10. The pharmaceutical composition according to claim 9, wherein the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation, or an HBKO mutation.

11. The pharmaceutical composition according to claim 8, wherein the AAV virus particle comprises an AAV genome containing one or more inverted terminal repeats (ITRs), and 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.

12. The pharmaceutical composition according to any one of claims 1 to 4, wherein the viral vector is an adenovirus particle.

13. The pharmaceutical composition according to any one of claims 1 to 4, wherein the viral vector is a lentiviral particle.

14. The pharmaceutical composition according to any one of claims 1 to 4, wherein the viral vector is a herpes simplex virus (HSV) particle.

15. The pharmaceutical composition according to any one of claims 1 to 4, wherein the IRAK-4 degrading agent or IRAK-4 inhibitor is administered orally.

16. A pharmaceutical composition for use in delivering a viral vector to the cells of an individual that requires it, wherein the use is a) Incubating innate immune cells from the individual with the viral vector; and b) Analyzing the innate immune cells for the expression of one or more cytokines, wherein the expression of cytokine signatures after incubation with the viral vector is used to identify and analyze individuals with innate immunity to the gene therapy agent. c) Administering an IRAK-4 inhibitor or an IRAK-4 degrading agent to the individual identified in step b), d) Administering the viral vector to the individual identified in step b) A pharmaceutical composition containing the following:

17. The pharmaceutical composition for use according to claim 16, wherein the innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells.

18. The method according to claim 16 or 17, wherein the viral vector is an AAV particle.

19. Use of a composition in the manufacture of a pharmaceutical product for modulating the immune response to a viral vector in an individual requiring gene therapy, wherein the composition comprises a viral vector, and the composition is formulated for use in combination with an IRAK-4 degrader or an IRAK-4 inhibitor.

20. The use according to claim 19, wherein the gene therapy agent is AAV particles.

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