Dendritic cell assays for innate immunogenicity against gene therapeutics

Abstract

Provided herein are methods for determining the innate immunogenicity of a gene therapeutic agent in an individual. In particular, these methods relate to the use of isolated dendritic cells for detecting innate immunogenicity against gene therapeutics. Exemplary gene therapeutics include adeno-associated virus (AAV) vectors, adenoviral vectors, lentiviral vectors, herpes simplex virus (HSV) vectors, or lipid nanoparticles.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 330,241, filed on April 12, 2022, which is incorporated by reference in its entirety. Reference to an electronic sequence listing

[0002] The contents of the electronic Sequence Listing (159792018140SEQLIST.xml; size: 2,016 bytes; and creation date: April 12, 2023) are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to methods of determining innate immunogenicity to gene therapy agents using dendritic cells. Background Art

[0004] The current success of gene therapy for the treatment of rare genetic diseases relies heavily on adeno-associated virus (AAV) vectors, which provide several attractive features, including tissue-specific tropism, transduction of quiescent cells, and long-term persistence of transgene expression. However, the immune response to AAV vectors poses a major challenge to successful clinical transformation. All components of viral vectors (capsid, viral genome, and transgene) trigger immune responses involving the activation of both innate and adaptive immunity of the human immune system. The adaptive immune response to AAV triggered by B and T cells has been relatively well characterized; however, little is known about the innate immune activation of AAV. The main challenge of the innate immune response to AAV that we understand is the poor reproducibility of immune signaling observed in clinical trials in an ex vivo environment. What is needed is a new assay that reproduces the innate immune characteristics of several human donors in response to various AAV serotypes.

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

[0006] In some aspects, the present invention provides a method for determining the innate immunogenicity of a gene therapy agent in an individual, the method comprising a) incubating an innate immune cell from the individual with the gene therapy agent, b) analyzing the expression of one or more cytokines in the innate immune cell compared to a suitable control, whereby the expression of the one or more cytokines produced a cytokine signature, wherein the expression of the cytokine signature after incubation with the gene therapy agent indicates the innate immunogenicity of the gene therapy agent in the individual. In some embodiments, the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell. In some embodiments, the innate immune cell is isolated from a peripheral blood mononuclear cell from the individual. In some embodiments, the innate immune cell is a dendritic cell. In some embodiments, the dendritic cell is derived from a monocyte of the individual.

[0007] In some embodiments of the present invention, the method further comprises isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent. In some embodiments, the monocytes are CD14+ monocytes. In some embodiments, the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes. In some embodiments, the innate immune cells are re-plated before incubating with the gene therapy agent of step b). In some embodiments, the innate immune cells are re-plated in a microwell dish.

[0008] In some embodiments of the invention, the gene therapy agent is a viral vector, and wherein the innate immune cells are coupled to the gene therapy agent at a rate of about 1×10 3 About 1×10 5 or about 1×10 4 In some embodiments, the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL. In some embodiments, the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

[0009] In some embodiments, the cytokine signature includes increased expression of IL6, TNFα and IL-1β. In some embodiments, the cytokine signature includes increased expression of one or more of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα and IL-1β. In some embodiments, compared with a suitable control, the expression of these cytokines in the cytokine signature increases. In some embodiments, the suitable control is the expression of these cytokines in the cytokine signature of innate immune cells not incubated with the gene therapy agent, or wherein the suitable control is the expression of these cytokines in the cytokine signature of innate immune cells before incubation with the gene therapy agent.

[0010] In some aspects, the invention provides a method of determining innate immunogenicity to a viral gene therapy agent in an individual, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions wherein the monocytes differentiate into dendritic cells, b) incubating the dendritic cells with the viral gene therapy agent at a concentration of about 1×10 3 About 1×10 5 The invention relates to a method for incubating the dendritic cells with an MOI of about 12 to about 36 hours, c) analyzing the altered expression of one or more cytokines in the dendritic cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, wherein the expression of the cytokine signature after incubation with the viral gene therapy agent indicates innate immunogenicity to the viral gene therapy agent in the individual, wherein the cytokine signature includes increased expression of IL6, TNFα, and IL-1β. In some embodiments, the monocytes are obtained from peripheral mononuclear cells from the individual. In some embodiments, the monocytes are CD14+ monocytes. In some embodiments, the monocytes are incubated in a dendritic cell culture medium for about 7-8 days to differentiate the monocytes into dendritic cells. In some embodiments, the dendritic cells are incubated with the viral gene therapy agent at a concentration of about 1×10 4 In some embodiments, the dendritic cells are incubated with the viral gene therapy agent for about 24 hours.

[0011] In some embodiments of the invention, the viral vector is an AAV particle. In some embodiments, the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human Bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof. In some embodiments, the AAV capsid comprises a tyrosine mutation, a heparin binding mutation or an HBKO mutation. In some embodiments, the AAV virus particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR or AAV12 ITR. In some embodiments, the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0012] In some embodiments of the invention, the viral vector is an adenoviral particle. In some embodiments, the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4,, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad 3, canine Ad 2, ovine Ad or porcine Ad 3, or a functional variant thereof.

[0013] In some embodiments of the present invention, the viral vector is a lentiviral particle. In some embodiments, the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

[0014] In some embodiments of the present invention, 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.

[0015] In some aspects, the present invention provides a method for determining the innate immunogenicity of a non-viral gene therapy agent in an individual, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions where the monocytes differentiate into dendritic cells, b) incubating the dendritic cells with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL, c) analyzing the altered expression of one or more cytokines in the dendritic cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, wherein the expression of the cytokine signature after incubation with the non-viral gene therapy agent indicates the innate immunogenicity of the non-viral gene therapy agent in the individual, wherein the cytokine signature includes increased expression of IL6, TNFα and IL-1β. In some embodiments, the monocytes are obtained from peripheral mononuclear cells from the individual. In some embodiments, the monocytes are CD14+ monocytes. In some embodiments, the monocytes are incubated in a dendritic cell culture medium for about 7-8 days to differentiate the monocytes into dendritic cells. In some embodiments, the dendritic cells are incubated with the non-viral gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

[0016] 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.

[0017] In some aspects, the present invention provides a method for determining the cytokine signature of a gene therapy agent, the method comprising a) incubating one or more innate immune cells from one or more individuals with the gene therapy agent, b) analyzing the expression of changes in one or more cytokines in the one or more innate immune cells compared to a suitable control, wherein the expression of the changes in one or more cytokines in step b) indicates the cytokine signature of the gene therapy agent. In some embodiments, the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell. In some embodiments, the one or more innate immune cells are separated from peripheral blood mononuclear cells from the individual. In some embodiments, the innate immune cell is a dendritic cell. In some embodiments, the dendritic cell is derived from the monocyte of the one or more individuals.

[0018] In some embodiments of the invention, the method further comprises isolating monocytes from the one or more individuals, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent. In some embodiments, the monocytes are CD14+ monocytes. In some embodiments, the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes. In some embodiments, the innate immune cells are re-plated before incubating with the gene therapy agent of step b).

[0019] In some embodiments, the gene therapy agent is a viral vector, and wherein the innate immune cells are coupled to the gene therapy agent at a concentration of about 1×10 3 to about 1×10 5 or about 1×10 4 In some embodiments, the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL. In some embodiments, the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

[0020] In some embodiments of the invention, the expression of the cytokines in the cytokine signature is increased compared to a suitable control. In some embodiments, the suitable control is the expression of the cytokines in the cytokine signature from innate immune cells that have not been incubated with the gene therapy agent, or wherein the suitable control is the expression of the cytokines in the cytokine signature from innate immune cells prior to incubation with the gene therapy agent. In some embodiments, the gene therapy agent is a viral vector or a non-viral vector.

[0021] In some embodiments, the viral vector is an AAV particle. In some embodiments, the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAVDJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human Bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof. In some embodiments, the AAV capsid comprises a tyrosine mutation, a heparin binding mutation or an HBKO mutation. In some embodiments, the AAV virus particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR or AAV12 ITR. In some embodiments, the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0022] In some embodiments, the viral vector is an adenoviral particle. In some embodiments, the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4,, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3, or a functional variant thereof.

[0023] In some embodiments, the viral vector is a lentiviral particle. In some embodiments, the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

[0024] 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.

[0025] 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.

[0026] In some embodiments, the invention provides kits for use in any of the methods of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of the assay used to determine the innate immunogenicity of different AAV vectors is shown.

[0028] Figure 2 Cytokine levels from AAV-treated cells as determined by Luminex assay are shown. AAV serotype is indicated above each graph; assayed cytokines are shown on the left; donor ID is shown along the top of each graph. The colors within each graph correspond to the change in regulation compared to uninfected cells and are indicated in the lower right graph.

[0029] Figure 3 Cytokine levels from AAV-treated cells as determined by orthogonal MSD assays are shown. AAV serotype is indicated above each graph; assayed cytokines are shown on the left; donor ID is shown along the top of each graph. The colors within each graph correspond to the change in regulation compared to uninfected cells and are indicated in the lower right graph.

[0030] Figure 4The experimental schematic diagram for measuring the innate immune response after LNP delivery is shown. From the peripheral blood mononuclear cells (PBMC) separated from the enriched leukocyte apheresis product (leukopak). From (PBMC) purifying CD14+ monocytes. Add differentiation factor mixture to these monocytes to allow differentiation into dendritic cells, and finally add maturation factors to obtain mature dendritic cells. Mature dendritic cells are processed 24h with the LNP of 10ug. Then harvest cells for using flow cytometry to measure cytotoxicity and target gene expression, and collect culture medium for assessing cytokine release.

[0031] Figure 5 It was shown that the dendritic cell system of the present disclosure can be used to assess the immunogenicity of LNPs without affecting cell viability.

[0032] Figure 6 It is shown that the dendritic cell system of the present disclosure can be used to effectively evaluate LNP transduction. In particular, dendritic cells generated according to the present disclosure are effectively transduced by LNPs encapsulating mRNA.

[0033] Figure 7A-7D Cytokine-specific expression of LNPs encapsulating mRNA compared to cells not treated with LNPs is shown. Fig. 7A Results for IP10 secretion are shown. Figure 7B Results for MIP1b secretion are shown. Figure 7C Results for CXCL9 secretion are shown. Fig.7D Results for IL2 secretion are shown. DETAILED DESCRIPTION

[0034] In some aspects, the present invention provides a method for determining the innate immunogenicity of a gene therapy agent in an individual, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the expression of one or more cytokines in the innate immune cells compared to a suitable control, whereby the expression of the one or more cytokines changes to produce a cytokine signature, wherein the expression of the cytokine signature after incubation with the gene therapy agent indicates the innate immunogenicity of the gene therapy agent in the individual. In some embodiments, the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell. In some embodiments, the method further comprises isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent. In some embodiments, the cytokine signature includes one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the method for determining whether an individual has innate immunity to a gene therapy agent is determined before administering the gene therapy agent to the individual. In some embodiments, the method of determining whether an individual has innate immunity to a gene therapy agent is determined prior to administering the gene therapy agent to the individual, thereby identifying individuals who would benefit from administration of a modulator of the innate immune response before, during, or after administration of the gene therapy agent. General Technology

[0035] The techniques and procedures described or referenced herein are generally well known to those skilled in the art and are routinely employed using conventional methods, such as the widely used methods described in the following references: Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications [Cultivation of Animal Cells: A Handbook of Basic Techniques and Specialized Applications] (RI Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis [Oligonucleotide Synthesis] (M. J. Gait, ed., 1984); Methods in Molecular Biology [Molecular Biology Methods], Humana Press [Humana Press]; Cell Biology: A Laboratory Notebook [Cell Biology: Laboratory Notebook] (J. E. Cellis, ed., Academic Press [Academic Press], 1998); Introduction to Cell and Tissue Culture [Introduction to Cell and Tissue Culture] (J. P. Heathen and P. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures [Cell and Tissue Culture: Laboratory Procedures] (A. Doyle, J. B. Griffiths and D. G. Smith, ed., Plenum Press, 1998);Newell, ed., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 1993-8); Sons [John Wiley & Sons, 2002); Immunobiology [Immunobiology] (CA Janeway et al., 2004); Antibodies [Antibodies] (P. Finch, 1997); Antibodies: A Practical Approach [Antibodies: A Practical Approach] (D. Catty. ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach [Monoclonal Antibodies: A Practical Approach] (P. Shepherd and C. Dean eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual [Using Antibodies: A Laboratory Manual] (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies [Antibodies] (M. Zanetti and JD Capra eds., Harwood Academic Press, 1996); Publishers [Harwood Academic Publishers, 1995]; and Cancer: Principles and Practice of Oncology [Cancer: Principles and Practice of Oncology] (VT DeVita et al., eds., JB Lippincott Company [JBLippincott & Co., 2011). definition

[0036] As used herein, the term "IRAK degrader" is a heterobifunctional compound that binds to and / or inhibits (completely or partially) both IRAK kinase and E3 ligase with measurable affinity, resulting in ubiquitination and subsequent degradation of IRAK kinase. In certain embodiments, the degrader has a DC of less than about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. 50 As used herein, the term "monovalent" refers to a degrader compound that does not contain an additional E3 ligase binding moiety.

[0037] As used herein, the term "inhibitor" with respect to IRAK is a compound that binds to and / or inhibits (completely or partially) the IRAK kinase with measurable affinity. In certain embodiments, the inhibitor has an IC of less than about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. 50 and / or binding constant.

[0038] As used herein, the term "modulator" with respect to IRAK is a compound that stimulates, delays, inhibits and / or suppresses (completely or partially) the activity of IRAK kinase.

[0039] As used herein, the term "gene therapy" refers to a therapy that modifies the expression of nucleic acids (e.g., genes, mRNA, etc.) in the cells of an individual to change the biological properties of the cells. In some instances, gene therapy includes delivering exogenous nucleic acids to be expressed in the cells of an individual. In some instances, gene therapy changes (e.g., degrades, inhibits, enhances) the expression of endogenous genes in the cells of an individual. In some instances, gene therapy is an in vivo therapy. In some instances, gene therapy is an ex vivo therapy (e.g., cell therapy).

[0040] As used herein, the term "gene therapy agent" refers to a nucleic acid (e.g., expression construct, miRNA, antisense, shRNA, siRNA) or a combination of a nucleic acid and an agent that is used to deliver nucleic acid to an individual or cell to modify or manipulate the expression of one or more nucleic acids (e.g., gene, mRNA) in the individual or cell to change the biological properties of living cells. 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 (LNPs) encapsulating therapeutic nucleic acids or encapsulating plasmid DNA (e.g., end-blocked DNA) containing therapeutic nucleic acids and / or encoding therapeutic polypeptides).

[0041] As used herein, "vector" refers to a recombinant plasmid or virus comprising a nucleic acid to be delivered into a host cell in vitro or in vivo.

[0042] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, the term includes but is not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of the nucleic acid can include sugar and phosphate groups (as can be found in RNA or DNA generally), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the nucleic acid can include a polymer of synthetic subunits such as aminophosphorylates, therefore oligodeoxynucleoside aminophosphorylates (P-NH2) or mixed aminophosphorylates-phosphodiester oligomers. In addition, double-stranded nucleic acids can be obtained from chemically synthesized single-stranded polynucleotide products in the following manner: by synthesizing complementary chains and annealing the chains under appropriate conditions, or by using DNA polymerase to synthesize complementary chains from scratch with appropriate primers.

[0043] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. This definition encompasses both full-length proteins and fragments thereof. The term also includes post-translational modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications (such as deletions, additions and substitutions, which are generally conservative in nature) relative to the native sequence, as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host producing the protein or due to errors in PCR amplification.

[0044] "Recombinant viral vector" refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (ie, nucleic acid sequences of non-viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one, for example two, inverted terminal repeats (ITRs).

[0045] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences from non-AAV sources), which are flanked by at least one, for example, two AAV inverted terminal repeats (ITRs). When present in a host cell that has been infected with a suitable helper virus (or is expressing a suitable helper function) and is expressing AAVrep and cap gene products (i.e., AAV Rep and Cap proteins), such rAAV vectors can be replicated and packaged into infectious viral particles. When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or another vector such as a plasmid for cloning or transfection), the rAAV vector can be referred to as a "front vector", which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. The rAAV vector can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles (particularly AAV particles) in an embodiment. rAAV vectors can be packaged into AAV viral capsids to produce "recombinant adeno-associated virus particles (rAAV particles)".

[0046] "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.

[0047] "Recombinant adenoviral vector" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-adenoviral origin), which are flanked by at least one adenoviral inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeats (ITR). When present in a host cell expressing essential adenoviral genes (e.g., E1 gene, E2 gene, E4 gene, etc.) deleted from the recombinant viral genome, such recombinant viral vectors can be replicated and packaged into infectious viral particles. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid for cloning or transfection), the recombinant viral vector can be referred to as a "pre-vector", which can be "rescued" by replication and encapsidation in the presence of an adenoviral packaging function. The recombinant viral vector can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles (e.g., adenoviral particles). The recombinant viral vector can be packaged into an adenoviral capsid to produce a "recombinant adenoviral particle".

[0048] "Recombinant lentiviral vector" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-lentiviral origin) flanked by at least one lentiviral terminal repeat (LTR). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral terminal repeats (LTR). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with appropriate helper functions. Recombinant lentiviral vectors can be packaged into lentiviral capsids to produce "recombinant lentiviral particles".

[0049] "Recombinant herpes simplex virus vector (recombinant HSV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences not derived from HSV), which are flanked by HSV terminal repeats. When present in a host cell that has been infected with a suitable helper function, such a recombinant viral vector can be replicated and packaged into infectious viral particles. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid for cloning or transfection), the recombinant viral vector can be referred to as a "provector", which can be "rescued" by replication and encapsidation in the presence of an HSV packaging function. The recombinant viral vector can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles (e.g., HSV particles). The recombinant viral vector can be packaged into an HSV capsid to produce a "recombinant herpes simplex virus particle".

[0050] As used herein, "solid lipid nanoparticles" (SLN, sLNP) or "lipid nanoparticles" (LNP) refer to nanoparticles composed of lipids. In some instances, there is only one phospholipid layer, and the majority of the interior of the particle is composed of lipophilic substances. Payloads (such as nucleic acids) can be embedded inside. In some instances, the lipid nanoparticle is a liposome comprising a lipid bilayer.

[0051] As used herein, the term "improvement" when it comes to gene therapy can refer to the act of strengthening, improving, prolonging or otherwise increasing the expression of the therapeutic gene payload of the gene therapy agent. In some embodiments, the improved gene therapy is such a gene therapy, wherein the expression of the therapeutic gene payload of the gene therapy agent administered with the IRAK modulator is increased by greater than about 10%, 25%, 50%, 75% or 100% compared to the gene therapy not administered with the IRAK modulator. In some embodiments, the improved gene therapy is such a gene therapy, wherein the expression time of the therapeutic gene payload of the gene therapy agent administered with the IRAK modulator is extended by greater than about 10%, 25%, 50%, 75% or 100% compared to the gene therapy not administered with the IRAK modulator. In some instances, the gene therapy is improved by reducing the immune response (e.g., innate immune response) to the gene therapy agent. In some embodiments, the improved gene therapy is a gene therapy wherein the immune response to the gene therapy administered with the IRAK modulator is reduced by greater than about any of 10%, 25%, 50%, 75%, or 100% compared to the gene therapy administered without the IRAK modulator. In some embodiments, the reduction in immune response to the gene therapy agent is measured as a reduction in cytokine signature after exposure of the gene therapy agent to immune cells in the presence of the IRAK modulator compared to exposure of the gene therapy agent to immune cells in the absence of the IRAK modulator.

[0052] As used herein, the term "modulate" in reference to gene therapy can refer to an action that changes, alters, modulates, improves, or otherwise modifies the presence or activity of a gene therapy agent. For example, modulating an immune response to a gene therapy agent can refer to any action that results in a change, alteration, modification, improvement, or otherwise modifies the immune response to a gene therapy agent (e.g., reducing, delaying, and / or eliminating an immune response (e.g., an innate immune response) to a gene therapy agent).

[0053] As used herein, the term "cytokine signature" when referring to an immune response (e.g., an innate immune response) to a gene therapy agent refers to the expression of an alteration (e.g., increase, decrease) of one or more cytokines after exposing innate immune cells to a gene therapy agent. In some instances, the cytokines of the cytokine signature are specific to a TLR pathway (e.g., a TLR2, TLR3, TLR4, or TLR9 pathway).

[0054] Innate immune cells are leukocytes that mediate innate immunity, and include basophils, dendritic cells, eosinophils, Langerhans cells, mast cells, monocytes and macrophages, neutrophils and NK cells. Different AAV capsids can enter these innate immune cells with different efficiencies (commonly referred to as transduction efficiencies). Some serotypes (such as AAV1) are effective in transducing certain immune cells like monocytes, while other AAVs (such as AAV6) are effective in transducing cells like dendritic cells (Grimm, D et al., J. Virol. [Journal of Virology], 2008, 82 (12): 5887-5911). However, all AAVs cause immune responses after entering cells. The extent of this immune response depends on the AAV serotype and cell type. Once AAV transduces host immune cells, they engage immune receptors, such as TLR (i.e., TLR9). Several studies using mouse models have revealed that TLR9 is a key DNA sensor that contributes to AAV immunogenicity (Zhu, J et al., J Clin Invest. [Clinical Research Journal] 2009; 119 (8): 2388-2398; Ashley SN et al., Cell. Immunol. [Cellular Immunology] 2019, 346: 103997). Once these TLRs are activated by the virus, they secrete cytokines that establish an antiviral state within the infected cell and alert neighboring cells. (Carty, M and Bowie, AG, Clin Exp Immunol, 2010, 161(3):397-406; Lester, SN and Li, K, J Mol Biol. 2014; 426(6):1246-1264; Fitzgerald, KA and Kagan, JC, Cell, 2020180(6):1044-1066).

[0055] These cytokines are also responsible for activating the adaptive immune system, which includes B cells and T cells that produce antibodies and produce cytotoxicity to kill virus-infected cells, respectively. As used herein, the upregulation or downregulation of certain subsets of cytokines is referred to as "cytokine signature". These cytokine signatures comprising one or more (e.g., three or more) cytokines can be used as predictive markers for disease and therapy success. Examples of cytokine signatures can be found in Zuniga, J et al., Int. J. Infect. Diseases [International Journal of Infectious Diseases], 2020, 94: 4-11, Bergamaschi, C et al., Cell Reports [Cell Report], 2021, 36: 109504; Del Valle, DM et al., Nat. Med. [Natural Medicine] 2020, 26: 1636-1643.

[0056] "Heterologous" means an entity that is genotypically different from the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a nucleic acid introduced into a different cell type by genetic engineering techniques is a heterologous nucleic acid (and may encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0057] The term "transgenic" refers to a nucleic acid that is introduced into a cell and can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In some aspects, it imparts desired properties to the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic outcome. On the other hand, it can be transcribed into a molecule that mediates RNA interference, such as siRNA.

[0058] The terms "genomic particles (gp)", "genomic equivalents" or "genomic copies" as used in relation to viral titer refer to the number of virions containing the recombinant AAV DNA genome, without regard to infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by procedures such as those described in the Examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther. [Human Gene Therapy], 10: 1031-1039; Veldwijk et al. (2002) Mol. Ther. [Molecular Therapy], 6: 272-278.

[0059] The terms "infectious unit (iu)", "infectious particles" or "replication units" as used in reference to viral titers refer to the number of infectious and replicative recombinant AAV vector particles measured by the infectious center assay (also called the replication center assay), as described, for example, in McLaughlin et al. (1988) J. Virol., 62: 1963-1973.

[0060] The term "transducing unit (tu)" as used in reference to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured in a functional assay such as described in the Examples herein or, for example, in Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al. (1996) J. Virol., 70: 520-532 (LFU assay).

[0061] "Inverted terminal repeat" or "ITR" sequence is a term well known in the art and refers to relatively short sequences in opposite orientation found at the ends of the viral genome.

[0062] The "AAV inverted terminal repeat (ITR)" sequence is a term well known in the art and is a sequence of approximately 145 nucleotides present at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can exist in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (designated as region A, region A', region B, region B', region C, region C', and region D) that allow intrastrand base pairing to occur within this portion of the ITR.

[0063] "Terminal dissociation sequence" or "trs" is a sequence in the D region of AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. Mutant terminal dissociation sequences are resistant to cleavage by the AAV rep protein. "AAV helper function" refers to a function that allows AAV to be replicated and packaged by a host cell. AAV helper function can be provided in any of a variety of forms, including but not limited to helper viruses or helper viral genes that assist in AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0064] "AAV helper function" refers to a function that allows AAV to be replicated and packaged by a host cell. AAV helper function can be provided in any of a variety of forms, including but not limited to a helper virus or helper virus gene that assists in AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0065] The "helper virus" of AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by host cells. Many such helper viruses have been identified, including adenovirus, herpes virus, pox virus (such as vaccinia) and baculovirus. Adenovirus covers many different subgroups, but the 5-type adenovirus (Ad5) of subgroup C is the most commonly used. Many adenoviruses in human, non-human mammal and avian sources are known and can be obtained from depository institutions such as ATCC. The viruses of the herpes family that can also be obtained from depository institutions such as ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and pseudorabies virus (PRV). The baculovirus that can be obtained from depository institutions includes Autographa californica nuclear polyhedrosis virus.

[0066] "Percentage (%) of sequence identity" with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues or nucleotides in a reference polypeptide or nucleic acid sequence after aligning the sequences and introducing spaces (if necessary) to obtain the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology [Molecular Biology Experiment Manual] (Ausubel et al., 1987), Supplement 30, Section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Potential alignment programs are ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequence. For the purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, and or to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, and or to a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as identical matches by a sequence alignment program in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. For the purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, and or for a given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C having or comprising a certain % nucleic acid sequence identity to, and or for a given nucleic acid sequence D) is calculated as follows: 100 multiplied by a fraction W / Z, where W is the number of nucleotides that are scored as identical matches by a sequence alignment program in the program's alignment of C and D, and where Z is the total number of nucleotides in D. It should be understood that when the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not be equal to the % nucleic acid sequence identity of D to C.

[0067] An "effective amount" of an agent is an amount effective to achieve the desired therapeutic outcome at the dosage and time period necessary. For example, an effective amount of a gene therapy agent is an amount effective to achieve the desired gene therapy outcome at the dosage and time period necessary. In another example, an effective amount of an IRAK modulator can be an amount effective to achieve the desired outcome of improved gene therapy at the dosage and time period necessary.

[0068] A "therapeutically effective amount" of a substance / molecule of the invention (e.g., a gene therapy agent and / or an IRAK modulator) can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects.

[0069] The term "appropriate control" when referring to a cytokine signature is the expression of cytokines in a cytokine signature from innate immune cells that have not been incubated with the gene therapy agent, or the expression of cytokines in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

[0070] Administration "in combination with" with respect to a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator) includes simultaneous (concurrent), consecutive, or sequential administration of the gene therapy agent and the innate immune response modulator (e.g., an IRAK modulator) in any order.

[0071] The term "concurrently" is used herein to refer to the administration of a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator) wherein at least a portion of the administration overlaps in time. Thus, concurrent administration includes dosing regimens when administration of a gene therapy agent or an innate immune response modulator (e.g., an IRAK modulator) continues after administration of the other agent / modulator has ceased.

[0072] As used herein, "in combination with" means administering another therapeutic modality in addition to one therapeutic modality. Thus, "in combination with" means administering another therapeutic modality (gene therapy agent or innate immune response modulator (e.g., IRAK modulator)) before, during, or after one therapeutic modality is administered to an individual.

[0073] An "isolated" molecule (eg, nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0074] References herein to "about" a value or parameter include (and describe) embodiments involving that value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X".

[0075] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0076] It should be understood that the aspects and embodiments of the present invention described herein include "comprising the aspects and embodiments," "consisting of," and / or "consisting essentially of," the aspects and embodiments. Cellular Assays for Determining Innate Immunogenicity to Gene Therapeutic Agents

[0077] In some aspects, the invention provides methods for determining innate immunogenicity to a gene therapy agent in an individual, wherein the innate immunogenicity can be to any aspect of the gene therapy agent; for example, the delivery vehicle (e.g., a viral capsid or lipid nanoparticle), the nucleic acid payload of the gene therapy agent, and / or any other component of the gene therapy agent.

[0078] In some aspects, the present invention provides a method for determining the innate immunogenicity of a gene therapy agent in an individual, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the expression of one or more cytokines in these innate immune cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent indicates the innate immunogenicity of the gene therapy agent. In some embodiments, the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell. In some embodiments, innate immune cells are isolated from blood (e.g., peripheral blood mononuclear cells) from an individual. In some embodiments, the innate immune cell is a dendritic cell. In some embodiments, the dendritic cell is derived from a monocyte (e.g., CD14+ monocyte) of an individual. As used herein, the term "derived" dendritic cell includes differentiation of cells (e.g., monocytes) to produce dendritic cells. In some embodiments, the method further comprises isolating monocytes from the individual and incubating the monocytes in a dendritic cell culture medium under conditions wherein dendritic cells are derived (differentiated) from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0079] In some aspects, the present invention provides a method for determining the innate immunogenicity of a gene therapy agent in an individual, the method comprising a) incubating dendritic cells from the individual with the gene therapy agent, b) analyzing the expression of one or more cytokines in the dendritic cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent indicates the innate immunogenicity for the gene therapy agent. In some embodiments, the dendritic cells are separated from peripheral blood mononuclear cells from the individual. In some embodiments, the dendritic cells are derived from monocytes (e.g., CD14+ monocytes) of the individual. In some embodiments, the method further comprises isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, and IL-1β.

[0080] In some aspects, the present invention provides a method for determining the innate immunogenicity for a gene therapy agent in an individual, the method comprising a) isolating innate immune cells from the individual, b) incubating the innate immune cells with the gene therapy agent, c) analyzing the expression of one or more cytokines in the innate immune cells, wherein the expression of the cytokine signature after incubation with the gene therapy agent indicates the innate immunogenicity for the gene therapy agent. In some embodiments, the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell. In some embodiments, the innate immune cell is isolated from peripheral blood mononuclear cells from the individual. In some embodiments, the innate immune cell is a dendritic cell. In some embodiments, the dendritic cell is derived from a monocyte (e.g., CD14+ monocyte) of an individual.

[0081] In some aspects, the invention provides a method for determining the innate immunogenicity of a gene therapy agent in an individual, the method comprising a) isolating monocytes from the individual, b) incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, c) incubating the dendritic cells with the gene therapy agent, d) analyzing the expression of one or more cytokines in the dendritic cells, wherein the expression of a cytokine signature after incubation with the gene therapy agent indicates innate immunogenicity to the gene therapy agent. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, and IL-1β.

[0082] In some aspects, the present invention provides a method for determining innate immunogenicity to a viral gene therapy agent in an individual, the method comprising: a) incubating innate immune cells from the individual with the viral gene therapy agent at a concentration of about 1×10 4In some embodiments, the innate immune cells are incubated with an MOI of about 24 hours, and b) analyzing the expression of one or more cytokines in these innate immune cells, wherein the expression of the cytokine signature after incubation with the viral gene therapy agent indicates the innate immunogenicity for the viral gene therapy agent. 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 monocytes (e.g., CD14+ monocytes) of an individual.

[0083] In some aspects, the present invention provides a method for determining innate immunogenicity to a viral gene therapy agent in an individual, the method comprising: a) incubating dendritic cells from the individual with the viral gene therapy agent at a concentration of about 1×10 4 The method further comprises incubating the dendritic cells with an MOI of about 24 hours, b) analyzing the expression of one or more cytokines in the dendritic cells, wherein the expression of the cytokine signature after incubation with the viral gene therapy agent indicates the innate immunogenicity of the viral gene therapy agent. In some embodiments, the dendritic cells are isolated from peripheral blood mononuclear cells from the individual. In some embodiments, the dendritic cells are derived from monocytes (e.g., CD14+ monocytes) of the individual. In some embodiments, the method further comprises isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0084] In some aspects, the present invention provides a method for determining the innate immunogenicity of a viral gene therapy agent in an individual, the method comprising: a) obtaining peripheral blood mononuclear cells (PBMCs) from the individual, b) isolating innate immune cells from the PBMCs, c) incubating the innate immune cells with the viral gene therapy agent at a concentration of about 1×10 4 The method further comprises isolating monocytes from the individual and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0085] In some aspects, the invention provides a method of determining innate immunogenicity to a viral gene therapy agent in an individual, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions wherein the monocytes differentiate into dendritic cells, b) incubating the dendritic cells with the viral gene therapy agent at a concentration of about 1×10 3 About 1×10 5 MOI of about 12 to about 36 hours, c) analyzing the changes (e.g., increased, decreased) expression of one or more cytokines in these dendritic cells compared to a suitable control, whereby the expression of the changes in the one or more cytokines produces a cytokine signature, wherein the expression of the cytokine signature after incubation with the viral gene therapy agent indicates the innate immunogenicity of the individual for the viral gene therapy agent, wherein the cytokine signature includes the increased expression of IL6, TNFα and IL-1β. In some embodiments, these monocytes are obtained from peripheral mononuclear cells from the individual. In some embodiments, these monocytes are CD14+ monocytes. In some embodiments, these monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from these monocytes. In some embodiments, monocytes are incubated with the dendritic cell culture medium for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 days to derive dendritic cells from monocytes. In some embodiments, the dendritic cells are re-plated prior to incubation with the gene therapy agent of step c). In some embodiments, the dendritic cells are re-plated into a microwell dish prior to incubation with the gene therapy agent. In some embodiments, the dendritic cells are incubated with the viral gene therapy agent at a concentration of about 1×10 3 , about 5×10 3 , about 1×10 4 , about 5×10 4 or about 1×10 5 In some embodiments, dendritic cells are incubated with the viral gene therapy agent at an MOI of about 1×10 3 About 1×10 5 , about 5×10 3 About 1×10 5 , about 1×10 4 About 1×10 5 , about 5×10 4 About 1×10 5 , about 1×10 3 About 5×10 4 , about 5×10 3 About 5×10 4 , about 1×10 4 About 5×104 , about 1×10 3 About 1×10 4 , about 5×10 3 About 1×10 4 or about 1×10 3 About 5×10 3 In some embodiments, the dendritic cells are incubated with the viral gene therapy agent for more than about 12 hours, about 18 hours, about 24 hours, about 30 hours, or about 36 hours. In some embodiments, the dendritic cells are incubated with the viral gene therapy agent for about 12 hours to about 36 hours, about 18 hours to about 36 hours, about 24 hours to about 36 hours, about 30 hours to about 36 hours, about 12 hours to about 30 hours, about 18 hours to about 30 hours, about 24 hours to about 30 hours, about 12 hours to about 24 hours, about 18 hours to about 24 hours, or about 12 hours to about 18 hours.

[0086] In some aspects, the present invention provides a method for determining innate immunogenicity against a viral gene therapy agent in an individual, the method comprising: a) obtaining peripheral blood mononuclear cells (PBMCs) from the individual, b) isolating CD14+ monocytes from the PBMCs, c) incubating the monocytes in a dendritic cell culture medium for about 7-8 days to derive dendritic cells from the monocytes, d) re-plating the dendritic cells, e) incubating the dendritic cells with the viral gene therapy agent at a concentration of about 1×10 4 f) analyzing the expression of one or more cytokines in the dendritic cells, wherein the expression of a cytokine signature after incubation with the viral gene therapy agent indicates innate immunogenicity to the viral gene therapy agent. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, and IL-1β.

[0087] In some aspects, the present invention provides a method for determining innate immunogenicity for a non-viral gene therapy agent in an individual, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions in which the monocytes differentiate into dendritic cells, b) incubating the dendritic cells with the non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL, c) analyzing the altered (e.g., increased, decreased) expression of one or more cytokines in the dendritic cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, wherein the expression of the cytokine signature after incubation with the non-viral gene therapy agent indicates innate immunogenicity for the non-viral gene therapy agent in the individual, wherein the cytokine signature includes increased expression of IL6, TNFα, and IL-1β. In some embodiments, the monocytes are obtained from peripheral mononuclear cells from the individual. In some embodiments, the monocytes are CD14+ monocytes. In some embodiments, the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes. In some embodiments, the monocytes are incubated with the dendritic cell culture medium for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days to derive dendritic cells from the monocytes. In some embodiments, the dendritic cells are re-plated prior to incubation with the non-viral gene therapy agent of step c). In some embodiments, the dendritic cells are re-plated into a microwell dish prior to incubation with the gene therapy agent. In some embodiments, the innate immune cells are incubated with a non-viral gene therapy agent at a concentration of about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 100 ng / mL, about 100 ng / mL to about 1 μg / mL, about 1 μg / mL to about 10 μg / mL, about 10 μg / mL to about 100 μg / mL, or about 100 μg / mL to about 1 mg / mL. In some embodiments, the dendritic cells are incubated with the non-viral gene therapy agent for more than about 12 hours, about 18 hours, about 24 hours, about 30 hours, or about 36 hours. In some embodiments, dendritic cells are incubated with the non-viral gene therapy agent for any of between about 12 hours and about 36 hours, between about 18 hours and about 36 hours, between about 24 hours and about 36 hours, between about 30 hours and about 36 hours, between about 12 hours and about 30 hours, between about 18 hours and about 30 hours, between about 24 hours and about 30 hours, between about 12 hours and about 24 hours, between about 18 hours and about 24 hours, or between about 12 hours and about 18 hours.

[0088] In some aspects, the present invention provides a method for determining the innate immunogenicity of a non-viral gene therapy agent in an individual, the method comprising: a) incubating innate immune cells from the individual with the non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL for about 24 hours, b) analyzing the expression of one or more cytokines in these innate immune cells, wherein the expression of the cytokine signature after incubation with the non-viral gene therapy agent indicates the innate immunogenicity of the non-viral gene therapy agent. In some embodiments, the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell. In some embodiments, the innate immune cell is isolated from a peripheral blood mononuclear cell from the individual. In some embodiments, the innate immune cell is a dendritic cell. In some embodiments, the dendritic cell is derived from a monocyte (e.g., CD14+ monocyte) of an individual.

[0089] In some aspects, the present invention provides a method for determining the innate immunogenicity of a non-viral gene therapy agent in an individual, the method comprising: a) incubating dendritic cells from the individual with the non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL for about 24 hours, b) analyzing the expression of one or more cytokines in the dendritic cells, wherein the expression of the cytokine signature after incubation with the non-viral gene therapy agent indicates the innate immunogenicity of the non-viral gene therapy agent. In some embodiments, the dendritic cells are isolated from peripheral blood mononuclear cells from the individual. In some embodiments, the dendritic cells are derived from monocytes (e.g., CD14+ monocytes) of the individual. In some embodiments, the method further comprises isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0090] In some aspects, the present invention provides a method for determining the innate immunogenicity of a non-viral gene therapy agent in an individual, the method comprising: a) obtaining peripheral blood mononuclear cells (PBMCs) from the individual, b) isolating innate immune cells from the PBMCs, c) incubating the innate immune cells with the non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL for about 24 hours, c) analyzing the expression of one or more cytokines in the innate immune cells, wherein the expression of the cytokine signature after incubation with the non-viral gene therapy agent indicates innate immunogenicity for the non-viral gene therapy agent. In some embodiments, the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell. In some embodiments, the innate immune cell is a dendritic cell. In some embodiments, the dendritic cell is derived from a monocyte (e.g., CD14+ monocyte) of an individual.

[0091] In some aspects, the present invention provides a method for determining innate immunogenicity to a non-viral gene therapy agent (e.g., LNP) in an individual, the method comprising: a) obtaining peripheral blood mononuclear cells (PBMC) from the individual, b) isolating CD14+ monocytes from the PBMCs, c) incubating the monocytes in a dendritic cell culture medium for about 7-8 days to derive dendritic cells from the monocytes, d) re-plating the dendritic cells, e) incubating the dendritic cells with the non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL for about 24 hours, f) analyzing the expression of one or more cytokines in the dendritic cells, wherein the expression of a cytokine signature after incubation with the gene therapy agent indicates innate immunogenicity to the gene therapy agent. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, and IL-1β.

[0092] In certain embodiments, monocytes are isolated from PBMCs from the individual. In certain embodiments, these monocytes are CD14+ monocytes. In certain embodiments, these monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from these monocytes. In certain embodiments, monocytes are incubated with the dendritic cell culture medium for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 days to derive dendritic cells from monocytes. In certain embodiments, before incubating with the gene therapy agent of step c), dendritic cells are re-plated. In certain embodiments, before incubating with the gene therapy agent, dendritic cells are re-plated in a microwell dish.

[0093] In some embodiments, cells (e.g., PBMC, monocytes and / or dendritic cells) are isolated from a sample obtained from one or more individuals. As used herein, "samples" obtained from individuals include any suitable samples (such as blood, urine and / or tissues) that can be used to obtain PBMC, monocytes and / or dendritic cells from one or more individuals. In some embodiments, cells are obtained from the blood of an individual (e.g., an individual who needs to be treated with a gene therapy agent (or has been treated with a gene therapy agent)). In some embodiments, PBMCs are separated from enriched leukocyte apheresis products. In some embodiments, PBMCs are separated from blood using a Ficoll gradient. In some embodiments, buffy coats containing leukocytes and platelets are collected from a Ficoll gradient. In some embodiments, PBMCs are washed before culture. In some embodiments, PBMCs are washed once, twice, three times, four times, five times or more than five times before culture. In some embodiments, cells are washed with phosphate buffered saline before culture. In some embodiments, PBS further contains fetal bovine serum (FBS) or fetal calf serum (FCS) before culture. In some embodiments, FBC and / or FCS are added to PBS at a final concentration of between about 0.1% to about 10% (v / v). In some embodiments, FBC and / or FCS are added to PBS at a final concentration of 1% (v / v).

[0094] In some embodiments of the present invention, monocytes are isolated from PBMC. In some embodiments, CD14+ monocytes are isolated from PBMC. In some embodiments, monocytes (e.g., CD14+) monocytes are isolated from PBMC by affinity purification. In some embodiments, CD14+ monocytes are isolated from PBMC using antibodies that specifically bind to CD14. In some embodiments, anti-CD14 antibodies are attached to a solid support (e.g., beads or resin). In some embodiments, CD14+ antibodies are purified from PBMC using anti-CD14 antibodies attached to magnetic beads. In some embodiments, magnetic beads are CD14 microbeads (Miltenyi Biotech).

[0095] In some embodiments of the invention, monocytes (e.g., CD14+ monocytes) purified from PBMCs are differentiated into dendritic cells. In some embodiments, monocytes from PBMCs are differentiated into dendritic cells by incubating the monocytes in the presence of cytokines that favor differentiation into dendritic cells. In some embodiments, monocytes are differentiated into dendritic cells by incubating the monocytes in the presence of ImmunoCult TM Dendritic cells are derived from monocytes by incubation in DC differentiation medium for about five days. TMThe DC differentiation medium further comprises ImmunoCult TM In some embodiments, after about five days, ImmunoCult TM DC maturation supplements are added to the culture.In some embodiments, differentiated dendritic cells are harvested at about day 7 for use in the assays of the invention.

[0096] In some embodiments, innate immune cells (e.g., dendritic cells, monocytes, macrophages, or NK cells) are co-administered with a viral gene therapy agent at a concentration of about 1×10 3 About 1×10 5 or about 1×10 4 In some embodiments, the innate immune cells are incubated with the gene therapy agent at an MOI of less than about 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 or 5×10 5 Incubate with the cells at any MOI.

[0097] In some embodiments, innate immune cells (e.g., dendritic cells, monocytes, macrophages, or NK cells) are incubated with a non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL. In some embodiments, innate immune cells are incubated with a non-viral gene therapy agent at a concentration of about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 100 ng / mL, about 100 ng / mL to about 1 μg / mL, about 1 μg / mL to about 10 μg / mL, about 10 μg / mL to about 100 μg / mL, or about 100 μg / mL to about 1 mg / mL.

[0098] In some embodiments, the innate immune cells (e.g., dendritic cells, monocytes, macrophages, or NK 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.

[0099] In some embodiments, by contacting specific immune cells from one or more individuals with a gene therapy agent and determining the altered (e.g., increased / decreased) expression of one or more cytokines associated with an innate immune response, a cytokine signature in a specific immune cell (e.g., dendritic cells, monocytes, macrophages, NK cells, etc.) is determined for a gene therapy agent, wherein the commonality of the expression change (e.g., increased or decreased expression) of the one or more cytokines indicates the presence of a cytokine signature. In some embodiments, the cytokines associated with an innate immune response are associated with a toll-like receptor (TLR) pathway (e.g., TLR2, TLR3, TLR4, or TLR9 pathway). In some embodiments, the cytokine signature includes changes in the expression of any one of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cytokines. In some embodiments, a plurality of individuals include any one of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 individuals. In some embodiments, the commonality of expression changes comprises similar changes in the expression levels of a cytokine in innate immune cells in greater than about 25%, 50%, 75%, or 90% of the individuals across a plurality of individuals.

[0100] In some embodiments, the present invention provides a method for determining the cytokine characteristics of a gene therapy agent, the method comprising a) incubating one or more innate immune cells (e.g., dendritic cells, monocytes, macrophages, NK cells, etc.) from one or more individuals with a gene therapy agent, b) analyzing the altered (e.g., increased / decreased) expression of one or more cytokines in the one or more innate immune cells compared to a suitable control, wherein the altered expression of one or more cytokines in step b) indicates the cytokine characteristics of the gene therapy agent. In some embodiments, innate immune cells are obtained from a blood sample from the one or more individuals. In some embodiments, innate immune cells are obtained from PBMCs from the one or more individuals. In some embodiments, the gene therapy agent is a viral particle or a lipid nanoparticle. In some embodiments, the gene therapy agent is an adeno-associated virus (AAV) particle, an adenovirus particle, a lentiviral particle, or a herpes simplex virus (HAV) particle. In some embodiments, the gene therapy agent is a lipid nanoparticle or a liposome.

[0101] In some embodiments, the cytokine signature includes the expression of one or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of two or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of three or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of four or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In some embodiments, the cytokine signature includes the expression of increases in IL6, TNFα and IL-1β.

[0102] In some embodiments, the cytokine signature includes the increased expression of one or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the increased expression of two or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the increased expression of three or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the increased expression of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the increased expression of IP10, MIP1b, CXCL9, IL2 and IL6. In some embodiments, the cytokine signature includes the increased expression of IP10, MIP1b, CXCL9, IL2 and TNFα. In some embodiments, the cytokine signature includes the increased expression of IP10, MIP1b, CXCL9, IL2, IL6 and TNFα.

[0103] In certain embodiments, the innate immune cell is a dendritic cell, and the cytokine signature includes the expression of one or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In certain embodiments, the cytokine signature includes the expression of two or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In certain embodiments, the cytokine signature includes the expression of three or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In certain embodiments, the cytokine signature includes the expression of four or more increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In certain embodiments, the cytokine signature includes the expression of increases in IL6, TNFα, IL-1β, MCP1 and MIP-1α. In certain embodiments, the cytokine signature includes the expression of increases in IL6, TNFα and IL-1β.

[0104] In certain embodiments, the innate immune cell is a dendritic cell, and the cytokine signature includes the expression of one or more increases in IP10, MIP1b, CXCL9 and IL2. In certain embodiments, the cytokine signature includes the expression of two or more increases in IP10, MIP1b, CXCL9 and IL2. In certain embodiments, the cytokine signature includes the expression of three or more increases in IP10, MIP1b, CXCL9 and IL2. In certain embodiments, the cytokine signature includes the expression of increases in IP10, MIP1b, CXCL9 and IL2. In certain embodiments, the cytokine signature includes the expression of increases in IP10, MIP1b, CXCL9, IL2 and IL6. In certain embodiments, the cytokine signature includes the expression of increases in IP10, MIP1b, CXCL9, IL2 and TNFα. In certain embodiments, the cytokine signature includes the expression of increases in IP10, MIP1b, CXCL9, IL2, IL6 and TNFα.

[0105] In some embodiments, compared to the expression of cytokines in the cytokine signature of innate immune cells incubated in the absence of a gene therapy agent, or compared to the expression of cytokines in the cytokine signature of innate immune cells before incubation with a gene therapy agent, the expression of cytokines in the cytokine signature increases, wherein the cytokine signature includes the expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes the expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes the expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes the expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes the expression of an increase in IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, and IL-1β. In some embodiments, any one of an increase in expression of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or more than 100% identifies an individual treated with a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator).

[0106] In some embodiments, the expression of cytokines in the cytokine signature of innate immune cells incubated in the absence of a gene therapy agent is compared to the expression of cytokines in the cytokine signature of innate immune cells before incubation with a gene therapy agent, or compared to the expression of cytokines in the cytokine signature of innate immune cells before incubation with a gene therapy agent, the expression of cytokines in the cytokine signature is increased, wherein the cytokine signature includes the expression of one or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the expression of two or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the expression of three or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the expression of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the expression of IP10, MIP1b, CXCL9, IL2 and IL6. In some embodiments, the cytokine signature includes the expression of IP10, MIP1b, CXCL9, IL2 and TNFα. In some embodiments, the cytokine signature includes increased expression of IP10, MIP1b, CXCL9, IL2, IL6, and TNFα. In some embodiments, any one of an increase in expression of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or more than 100% identifies an individual treated with a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator).

[0107] In some embodiments, compared to the expression of cytokines in the cytokine signature of dendritic cells incubated in the absence of a gene therapy agent, or compared to the expression of cytokines in the cytokine signature of innate immune cells before incubation with a gene therapy agent, the expression of cytokines in the cytokine signature increases, wherein the cytokine signature includes the expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes the expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes the expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes the expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes the expression of an increase in IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature includes increased expression of IL6, TNFα, and IL-1β. In some embodiments, any one of an increase in expression of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or more than 100% identifies an individual treated with a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator).

[0108] In some embodiments, the expression of cytokines in the cytokine signature of dendritic cells incubated in the absence of a gene therapy agent is compared to the expression of cytokines in the cytokine signature of innate immune cells before incubation with a gene therapy agent, or compared to the expression of cytokines in the cytokine signature of innate immune cells before incubation with a gene therapy agent, wherein the cytokine signature includes the increased expression of one or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the increased expression of two or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the increased expression of three or more of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the increased expression of IP10, MIP1b, CXCL9 and IL2. In some embodiments, the cytokine signature includes the increased expression of IP10, MIP1b, CXCL9, IL2 and IL6. In some embodiments, the cytokine signature includes the increased expression of IP10, MIP1b, CXCL9, IL2 and TNFα. In some embodiments, the cytokine signature includes increased expression of IP10, MIP1b, CXCL9, IL2, IL6, and TNFα. In some embodiments, any one of an increase in expression of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or more than 100% identifies an individual treated with a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator). Gene therapy agents

[0109] In some aspects, the present invention provides methods for determining the innate immunogenicity of a gene therapy agent in an improved gene therapy for an individual; for example, by identifying an individual in which an agent that modulates the innate immune response to the gene therapy agent is administered in combination with the gene therapy agent (sequentially (before or after the latter), simultaneously). As used herein, "modulating" an innate immune response refers to, for example, partial or complete stimulation, delayed suppression, and / or inhibition of an innate immune response. In some embodiments, an IRAK modulator is used to inhibit an innate immune response in an individual. In some embodiments, the gene therapy agent is a viral particle or a lipid nanoparticle. In some embodiments, the gene therapy agent is an adeno-associated virus (AAV) particle, an adenovirus particle, a lentivirus particle, or a herpes simplex virus (HAV) particle. In some embodiments, the gene therapy agent is a lipid nanoparticle or a liposome. AAV

[0110] In some embodiments, the present invention provides a method for determining the innate immunogenicity of AAV particles in an individual. In the AAV particles used for gene therapy, the recombinant AAV (rAAV) genome encoding heterologous nucleic acids (e.g., therapeutic transgenes) is encapsidated in the AAV capsid. In some embodiments, the viral genome includes one or more of the heterologous nucleic acids and / or the following components that are operably connected in the transcription direction: control sequences (including transcription start sequences and termination sequences), thereby forming an expression cassette.

[0111] In some embodiments, the rAAV genome comprises one or more AAV inverted terminal repeat (ITR) sequences (typically two AAV ITR sequences). For example, the expression cassette can be flanked at least one functional AAV ITR sequence at the 5' and 3' ends. "Functional AAV ITR sequence" means that the ITR sequence functions as intended for rescue, replication and packaging of AAV virions. See Davidson et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 2000, 97 (7) 3428-32; Passini et al., J. Virol. [Journal of Virology], 2003, 77 (12): 7034-40; and Pechan et al., Gene Ther. [Gene Therapy], 2009, 16: 10-16, all of which are incorporated herein by reference in their entirety. For the purpose of implementing some aspects of the present invention, the recombinant viral genome comprises at least all AAV sequences necessary for encapsidation into an AAV capsid and the physical structure for infection by AAV particles. The AAV ITRs for use in the vectors of the invention need not have wild-type nucleotide sequences (e.g., as described in Gene Ther., 1994, 5:793-801), and may be altered by insertion, deletion, or substitution of nucleotides, or the AAV ITRs may be derived from any of several AAV serotypes. More than 40 AAV serotypes are currently known, and new serotypes and variants of existing serotypes are continually being identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is contemplated as being within the scope of the invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype including but not limited to the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, goat AAV, bovine AAV or mouse AAV ITR, etc.In some embodiments, the nucleic acid in the AAV (e.g., rAAV vector) includes the following ITRs: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, goat AAV, bovine AAV, or mouse AAV ITRs, etc. In some embodiments, the AAV particle comprises an AAV vector encoding a heterologous transgene flanked by one or more AAV ITRs.

[0112] In some embodiments, the AAV particle comprises a capsid protein selected from the group consisting of: AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human Bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof. "Functional variant" of AAV capsid means that the variant capsid is able to package the AAV genome to produce infectious AAV virions. In another embodiment, the rAAV particle comprises a capsid protein of an AAV serotype from clade AF.

[0113] In some aspects, the present invention provides AAV particles comprising a recombinant self-complementary genome (e.g., a self-complementary or self-complementary AAV vector). rAAV virus particles with self-complementary vector genomes and methods of using self-complementary AAV genomes are described in U.S. Patent Nos. 6,596,535; 7,125,717; 7,465,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111, each of which is incorporated herein by reference in its entirety. AAV particles comprising a self-complementary genome will rapidly form double-stranded DNA molecules by virtue of their partially complementary sequences (e.g., complementary coding strands and non-coding strands of heterologous nucleic acids). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding the complement of the nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.

[0114] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are connected by a mutated ITR (e.g., right ITR). In some embodiments, the ITR comprises a polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO:). The mutated ITR comprises a deletion of the D region containing the terminal dissociation sequence. Therefore, when the rAAV genome is replicated, the rep protein will not cut the viral genome at the mutated ITR, and therefore, the recombinant viral genome comprising the following items in 5' to 3' order will be packaged in the viral capsid: AAV ITR, a first heterologous polynucleotide sequence comprising a regulatory sequence, a mutated AAV ITR, a second heterologous polynucleotide in the opposite orientation to the first heterologous polynucleotide, and a third AAV ITR.

[0115] Different AAV serotypes are used to optimize transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). AAV can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes. For example, an AAV particle can contain one or more ITRs and capsids derived from the same AAV serotype, or an AAV particle can contain one or more ITRs derived from an AAV serotype different from the capsid of the AAV particle.

[0116] In some embodiments, the AAV capsid comprises a mutation, for example, the capsid comprises a mutant capsid protein. In some embodiments, the mutation is a tyrosine mutation or a heparin binding mutation. In some embodiments, the mutant capsid protein retains the ability to form an AAV capsid. In some embodiments, the AAV particles comprise AAV2 or AAV5 tyrosine mutant capsids (see, e.g., Zhong L. et al., (2008) Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States] 105 (22): 7827-7832), such as mutations in Y444 or Y730 (numbered according to AAV2). In another embodiment, the AAV particles comprise capsid proteins of AAV serotypes from clade AF (Gao et al., J. Virol. [Journal of Virology] 2004, 78 (12): 6381).

[0117] A variety of methods are known in the art for producing AAV particles for gene therapy, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids (Urabe, M. et al., (2002) Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet. 20(R1):R2-R6). AAV production cultures for producing AAV particles all require; 1) suitable host cells, 2) suitable helper virus functions, 3) AAV rep and cap genes and gene products; 4) nucleic acids flanked by at least one AAV ITR sequence (such as therapeutic nucleic acids); and 5) suitable culture media and culture media components that support AAV production. In some embodiments, suitable host cells are primate host cells. In some embodiments, suitable host cells are cell lines of human origin, such as HeLa, A549, 293 or Perc.6 cells. In some embodiments, suitable helper virus functions are provided by wild-type or mutant adenoviruses (such as temperature-sensitive adenoviruses), herpes viruses (HSV), baculoviruses, or plasmid constructs that provide helper functions. In some embodiments, AAV rep and cap gene products can be from any AAV serotype. Generally, but not necessarily, the AAV rep gene product has the same serotype as the ITR of the rAAV genome, as long as the rep gene product can play a role in replicating and packaging the rAAV genome. Suitable culture media known in the art can be used to produce AAV particles. In some embodiments, the AAV helper function is provided by adenovirus or HSV. In some embodiments, the AAV helper functions are provided by a baculovirus and the host cell is an insect cell (eg, a Spodoptera frugiperda (Sf9) cell).

[0118] One method for producing AAV particles is a triple transfection method. In short, a plasmid containing a rep gene and a capsid gene can be transfected (e.g., using a calcium phosphate method) into a cell line (e.g., HEK-293 cells) together with a helper adenovirus plasmid, and the virus can be collected and optionally purified. Therefore, in some embodiments, AAV particles are produced by triple transfection of a nucleic acid encoding an AAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding an AAV helper virus function into a host cell, wherein the nucleic acid is transfected into the host cell to produce a host cell capable of producing AAV particles.

[0119] In some embodiments, AAV particles can be produced by a production cell line method (see Martin et al., (2013) Human Gene Therapy Methods [Human Gene Therapy Methods] 24: 253-269; U.S. Patent Pre-Grant Publication No. US2004 / 0224411; and Liu, XL et al. (1999) Gene Ther. [Gene Therapy] 6: 293-299). Briefly, a cell line (e.g., HeLa, 293, A549 or Perc.6 cell line) can be stably transfected with a plasmid containing a rep gene, a capsid gene, and a vector genome comprising a promoter-heterologous nucleic acid sequence. The cell line can be screened to select a leading clone for AAV production, which can then be amplified into a production bioreactor and infected with a helper virus (e.g., adenovirus or HSV) to initiate AAV production. The virus can then be harvested, the adenovirus can be inactivated (e.g., by heating) and / or removed, and the AAV particles can be purified. Thus, in some embodiments, AAV particles are produced by a producer cell line comprising one or more of: nucleic acid encoding the rAAV genome, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

[0120] In some embodiments, nucleic acids encoding AAV rep and cap genes and / or AAV viral genomes are stably maintained in production cell lines. In some embodiments, nucleic acids encoding AAV rep and cap genes and / or rAAV genomes are introduced into cell lines on one or more plasmids to produce production cell lines. In some embodiments, AAV rep, AAV cap and AAV genomes are introduced into cells on the same plasmid. In other embodiments, AAV rep, AAV cap and rAAV genomes are introduced into cells on different plasmids. In some embodiments, cell lines stably transfected with plasmids maintain plasmids in multiple passages (e.g., 5, 10, 20, 30, 40, 50 or more than 50 cell passages) of cell lines. For example, one or more plasmids can be replicated as cells replicate, or one or more plasmids can be integrated into the cell genome. A variety of sequences that enable plasmids to replicate autonomously in cells (e.g., human cells) have been identified (see, e.g., Krysan, PJ et al. (1989) Mol. Cell Biol. [Molecular Cell Biology] 9: 1026-1033). In certain embodiments, one or more plasmids may contain a selective marker (e.g., an antibiotic resistance marker) that allows the cells maintaining the plasmid to be selected. Selective markers commonly used in mammalian cells include, but are not limited to, blasticidin, G418, hygromycin B, bleomycin, puromycin and derivatives thereof. Methods for introducing nucleic acid into cells are known in the art, and include, but are not limited to, viral transduction, cationic transfection (e.g., using cationic polymers such as DEAE-dextran or cationic lipids such as lipofectamine), calcium phosphate transfection, microinjection, particle bombardment, electroporation and nanoparticle transfection (for more details, see, for example, Kim, TK and Eberwine, JH (2010) Anal. Bioanal. Chem. [Analysis and Bioanalytical Chemistry] 397: 3173-3178).

[0121] In some embodiments, the production cell line is derived from a primate cell line (e.g., a non-human primate cell line, such as a Vero or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the production cell line is derived from HeLa, 293, A549, or (Crucell) cells. For example, the cell line is HeLa, 293, A549 or (Crusell) cell line or its derivatives.

[0122] In some embodiments, the production cell line is suitable for growth in suspension. As known in the art, anchorage-dependent cells typically cannot grow in suspension without a matrix (such as microcarrier beads). Adapting the cell line to growth in suspension can include, for example, growing the cell line in a rotary culture with a stirring paddle, using a culture medium lacking calcium and magnesium ions to prevent agglomeration (and optionally a defoaming agent), using a culture vessel coated with a siliconized compound, and selecting cells in the culture (rather than in a large mass or on the side of the vessel) at each passage.

[0123] The AAV particles of the invention can be harvested from AAV production cultures by lysing the host cells of the production culture or by harvesting spent culture medium from the production culture, provided that the cells are cultured under conditions known in the art to cause release of AAV particles from intact cells into the culture medium, as described more fully in U.S. Pat. No. 6,566, 118. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0124] In another embodiment, the AAV particles are purified. As used herein, the term "purified" includes the following preparations of AAV particles, which lack at least some other components that may also be present in the natural presence of the AAV particles or the place where they were originally prepared. Thus, for example, the separated AAV particles can be prepared by enriching them from a source mixture (such as a culture lysate or a production culture supernatant) using purification techniques. Enrichment can be measured in a variety of ways, such as by the ratio of DNase-resistant particles (DRP) or genome copies (gc) present in the solution, or by infectivity, or enrichment can be measured relative to a second potential interfering substance present in the source mixture, the second potential interfering substance such as a contaminant, including a production culture contaminant or a process contaminant, including a helper virus, a culture medium component, etc.

[0125] In some embodiments, the AAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtering through a series of depth filters, including, for example, DOHC grade Millipore Millistak + HC Pod filter, A1HC grade Millipore Millistak + HC Pod filter, and 0.2 μm Filter Opticap XL10 Millipore Express SHC hydrophilic membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtering through any cellulose acetate filter of 0.2 μm or greater pore size known in the art.

[0126] In some embodiments, The AAV production culture harvest is further processed to digest any high molecular weight DNA present in the production culture. Digestion was performed under standard conditions known in the art, including, for example, 1-2.5 units / ml of The concentration of 50% HCl is 0.1% HCl and the ... temperature ranges from ambient temperature to 37°C for a period of time ranging from 30 minutes to several hours.

[0127] AAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating AAV particles; AAV capture by apatite chromatography; heat inactivation of helper virus; AAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and AAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. In some embodiments, the method includes all steps in the order described below. Methods for purifying AAV particles can be found in, for example, Xiao et al., (1998) Journal of Virology 72: 2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO 2010 / 148143. Adenovirus

[0128] In some embodiments, the present invention provides methods for determining innate immunity to adenoviral particles in an individual. Adenoviral vectors for gene therapy are typically adenoviral particles having a recombinant adenoviral (rAd) genome that contains one or more heterologous sequences (i.e., nucleic acid sequences of non-adenoviral origin) between two adenoviral ITRs encapsidated into an adenoviral capsid. In some embodiments, the heterologous sequence encodes a therapeutic transgene. In some embodiments, the rAd genome lacks one or more E1 genes or contains a defective copy of one or more E1 genes, which renders the adenovirus replication-defective. Adenoviruses are large (approximately ) includes a linear double-stranded DNA genome within a non-enveloped icosahedral capsid. Adenoviruses have large genomes that can incorporate heterologous sequences of more than 30 kb (e.g., replacing E1 and / or E3 regions), making them uniquely suited for use with larger heterologous genes. They are also known to infect dividing and non-dividing cells and do not naturally integrate into the host genome (although hybrid variants may have this ability). In some embodiments, the adenoviral vector may be a first-generation adenoviral vector with a heterologous sequence replacing E1. In some embodiments, the adenoviral vector may be a second-generation adenoviral vector with additional mutations or deletions in E2A, E2B, and / or E4. In some embodiments, the adenoviral vector may be a third-generation or internally destroyed (gutted) adenoviral vector that lacks all viral encoding genes (retaining only ITRs and packaging signals) and requires a trans-acting helper adenovirus for replication and packaging. Adenoviral particles have been studied for use as vectors for transient transfection of mammalian cells and as gene therapy vectors. For further description, see, e.g., Danthinne, X. and Imperiale, MJ (2000) Gene Ther. 7:1707-14; and Tatsis, N. and Ertl, HC (2004) Mol. Ther. 10:616-29.

[0129] In some embodiments, the adenoviral particles comprise a rAd genome containing a therapeutic transgene. The use of any adenoviral serotype is considered to be within the scope of the present invention. In some embodiments, the adenoviral particles are derived from adenoviral serotypes including but not limited to AdHu2, AdHu 3, AdHu4, AdHu5, AdHu7, AdHu11, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3 type, canine Ad 2 type, sheep Ad and porcine Ad 3 type. The adenoviral particles also comprise capsid proteins. In some embodiments, the adenoviral particles include one or more foreign viral capsid proteins. Such combinations can be referred to as pseudotyped adenoviral particles. In some embodiments, the foreign viral capsid proteins used in the pseudotyped adenoviral particles are derived from foreign viruses or derived from another adenoviral serotype. In some embodiments, foreign viral capsid proteins are derived from (including but not limited to) reovirus type 3. Examples of vector and capsid protein combinations used in pseudotyped adenoviral particles can be found in the following references (Tatsis, N. et al. (2004) Mol. Ther. [Molecular Therapy] 10 (4): 616-629; and Ahi, Y. et al. (2011) Curr. Gene Ther. [Current Gene Therapy] 11 (4): 307-320). Different adenoviral serotypes can be used to optimize transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). Tissues or cells targeted by specific adenoviral serotypes include, but are not limited to, lung (e.g., HuAd3), spleen and liver (e.g., HuAd37), smooth muscle, synoviocytes, dendritic cells, cardiovascular cells, tumor cell lines (e.g., HuAd11), and dendritic cells (e.g., HuAd5, HuAd30, or HuAd35 pseudotyped with reovirus type 3). For further description, see Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4):307-320; Kay, M. et al. (2001) Nat. Med. 7(1):33-40; and Tatsis, N. et al. (2004) Mol. Ther. 10(4):616-629.

[0130] Many methods for producing adenoviral particles are known in the art. For example, for an internally destroyed adenoviral vector, the adenoviral vector genome and the helper adenoviral genome can be transfected into a packaging cell line (e.g., a 293 cell line). In some embodiments, the helper adenoviral genome can contain recombination sites flanking its packaging signal, and the two genomes can be transfected into a packaging cell line expressing a recombinase (e.g., a Cre / loxP system can be used), so that the adenoviral vector of interest is more efficiently packaged than the helper adenovirus (see, e.g., Alba, R. et al. (2005) Gene Ther. [Gene Therapy] 12 Supplement 1: S18-27). Adenoviral vectors can be harvested and purified using standard methods (such as those described herein). Lentivirus

[0131] In some embodiments, the present invention provides a method for determining the innate immunogenicity of lentiviral particles in an individual. The lentiviral vector for gene therapy is typically a lentiviral particle with a recombinant lentiviral genome, which contains one or more heterologous sequences (i.e., nucleic acid sequences of non-lentiviral origin) between two long terminal repeats (LTR). In some embodiments, the heterologous sequence encodes a therapeutic transgene. Slow virus is a positive ssRNA retrovirus with a genome of about 10kb. Slow virus is integrated into the genome of dividing and non-dividing cells. Slow virus particles can be produced, for example, by transfecting multiple plasmids (typically lentiviral genomes and genes required for replication and / or packaging are separated to prevent viral replication) into a packaging cell line, which packages the modified lentiviral genome into lentiviral particles. In some embodiments, lentiviral particles can refer to first-generation vectors lacking envelope proteins. In some embodiments, lentiviral particles can refer to second-generation vectors lacking all genes except gag / pol and tat / rev regions. In some embodiments, the lentiviral particle may refer to a third generation vector that contains only endogenous rev, gag, and pol genes and has a chimeric LTR for transduction without a tat gene (see Dull, T. et al. (1998) J. Virol. [Journal of Virology] 72: 8463-71). For further description, see Durand, S. and Cimarelli, A. (2011) Viruses [Virus] 3: 132-59.

[0132] The use of any lentiviral vector is considered to be within the scope of the present invention. In some embodiments, the lentiviral vector is derived from a lentivirus including but not limited to human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), visnavirus (VV) and caprine arthritis encephalitis virus (CAEV). The lentiviral particle also comprises a capsid protein. In some embodiments, the lentiviral particle includes one or more foreign viral capsid proteins. Such combinations can be referred to as pseudotyped lentiviral particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped lentiviral particles is derived from a foreign virus. In some embodiments, the foreign viral capsid protein used in the pseudotyped lentiviral particles is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with ubiquitous cell receptors to provide a wide range of tissue tropism for pseudotyped lentiviral particles. Furthermore, VSV-GP is thought to provide increased stability to pseudotyped lentiviral particles. In other embodiments, the foreign viral capsid protein is derived from, including but not limited to, Chandipura virus, rabies virus, Mokola virus, lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Sindbis virus, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Reston ebola virus, Zaire ebola virus, Marburg virus, Lassa virus, avian leukosis virus (ALV), sheep lung adenomatous retrovirus (JSRV), Moloney murine leukemia virus (MLV), Gibbon ape leukemia virus (GALV), feline endogenous retrovirus (RD114), human T-lymphotropic virus 1 (HTLV-1), human foamy virus, Mae-Visna virus (MVV), SARS-CoV, Sendai virus, respiratory syncytial virus (RSV), human parainfluenza virus type 3, hepatitis C virus (HCV), influenza virus, fowl plague virus (FPV) or Autographa californica nuclear polyhedrosis virus (AcMNPV). Examples of vector and capsid protein combinations used in pseudotyped lentiviral particles can be found, for example, in Cronin, J. et al. (2005). Curr. Gene Ther. [Current Gene Therapy] 5(4): 387-398. Different pseudotyped lentiviral particles can be used to optimize transduction of specific target cells or to target specific cell types within a specific target tissue (e.g., diseased tissue).For example, tissues targeted by specifically pseudotyped lentiviral particles include, without limitation, liver (e.g., pseudotyped with VSV-G, LCMV, RRV or SeV F proteins), lung (e.g., pseudotyped with Ebola, Marburg, SeV F and HN or JSRV proteins), pancreatic islet cells (e.g., pseudotyped with LCMV proteins), central nervous system (e.g., pseudotyped with VSV-G, LCMV, rabies or Merckola proteins), retina (e.g., pseudotyped with VSV-G or Merckola proteins), monocytes or muscle (e.g., pseudotyped with Merckola or Ebola proteins), hematopoietic system (e.g., pseudotyped with RD114 or GALV proteins), or cancer cells (e.g., pseudotyped with GALV or LCMV proteins). For further description, see Cronin, J. et al. (2005). Curr. Gene Ther. 5(4):387-398; and Kay, M. et al. (2001) Nat. Med. 7(1):33-40.

[0133] Many methods for producing lentiviral particles are known in the art. For example, for third-generation lentiviral vectors, a vector containing a recombinant lentiviral genome of interest with gag and pol genes can be co-transfected into a packaging cell line (e.g., 293 cell line) together with a vector containing the rev gene. The recombinant lentiviral genome of interest also contains a chimeric LTR that promotes transcription in the absence of Tat (see Dull, T. et al. (1998) J. Virol. [Journal of Virology] 72: 8463-71). The lentiviral vector can be harvested and purified using the methods described herein (e.g., Segura MM et al., (2013) Expert Opin Biol Ther. [Expert Opinion on Biological Therapy] 13 (7): 987-1011). HSV

[0134] In some embodiments, the present invention provides a method for determining the innate immunogenicity of HSV particles in an individual. The HSV vector for gene therapy is typically an HSV particle with a recombinant HSV genome, which contains one or more heterologous sequences (i.e., nucleic acid sequences from non-HSV sources) between two terminal repeats (TRs). In some embodiments, the heterologous sequence encodes a therapeutic transgene. HSV is an enveloped double-stranded DNA virus with an approximately 152kb genome. Advantageously, about half of its genes are non-essential and can be deleted to accommodate heterologous sequences. HSV particles infect non-dividing cells. In addition, they naturally establish latency in neurons, travel by retrograde translocation, and can be transferred across synapses, making them conducive to transfection of neurons and / or conducive to gene therapy methods involving the nervous system. In some embodiments, HSV particles can be replication-deficient or replication-type (e.g., capable of performing a single replication cycle by inactivating one or more late genes). For further description, see Manservigi, R. et al. (2010) Open Virol. J. [Open Virology Journal] 4: 123-56.

[0135] In some embodiments, the HSV particles include a recombinant HSV genome containing a transgene. The use of any HSV vector is considered to be within the scope of the present invention. In some embodiments, the HSV vector is derived from an HSV serotype, including but not limited to HSV-1 and HSV-2. The HSV particles also include capsid proteins. In some embodiments, the HSV particles include one or more foreign virus capsid proteins. Such combinations can be referred to as pseudotyped HSV particles. In some embodiments, the foreign virus capsid proteins used in the pseudotyped HSV particles are derived from foreign viruses or derived from another HSV serotype. In some embodiments, the foreign virus capsid proteins used in the pseudotyped HSV particles are vesicular stomatitis virus glycoproteins (VSV-GP). VSV-GP interacts with ubiquitous cell receptors to provide a wide range of tissue tropism for pseudotyped HSV particles. In addition, VSV-GP is considered to provide higher stability for pseudotyped HSV particles. In other embodiments, foreign virus capsid proteins can be from different HSV serotypes. For example, an HSV-1 vector can contain one or more HSV-2 capsid proteins. Different HSV serotypes can be used to optimize transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). Tissues or cells targeted by specific adenovirus serotypes include, but are not limited to, the central nervous system and neurons (e.g., HSV-1). For further description, see Manservigi, R. et al. (2010) Open Virol J [Open Virology Journal] 4: 123-156; Kay, M. et al. (2001) Nat. Med. [Natural Medicine] 7 (1): 33-40; and Meignier, B. et al. (1987) J. Infect. Dis. [Infectious Diseases Journal] 155 (5): 921-930.

[0136] Many methods for producing HSV particles are known in the art. Standard methods (such as those described herein) can be used to harvest and purify HSV vectors. For example, for replication-defective HSV vectors, the target HSV genome lacking all immediate early (IE) genes can be transfected into a complementary cell line that provides genes (such as ICP4, ICP27, and ICP0) required for virus production (see, for example, Samaniego, LA et al. (1998) J. Virol. [Journal of Virology] 72: 3307-20). The described method (e.g., Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology [Herpes Simplex Virus Methods in Molecular Biology] 1144: 63-79) can be used to harvest and purify HSV vectors. Non-viral gene therapy agents

[0137] In certain embodiments, the invention provides methods for determining the innate immune response for non-viral gene therapy agents. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids compounded with delivery systems. For example, carriers can be compounded with lipids (e.g., cationic lipids or neutral lipids), liposomes, polycations, lipid nanoparticles, or agents that enhance cell uptake of nucleic acids. Nucleic acids can be compounded with agents suitable for any delivery method described herein. In certain embodiments, nucleic acid encoding therapeutic transgenes.

[0138] Lipid nanoparticles for gene therapy typically include a vector genome encapsulated in a lipid particle or a vector genome complexed with lipids. In some embodiments, the heterologous sequence encodes a therapeutic transgene. In some embodiments, the vector genome is formulated in a cationic liposome / nucleic acid complex (lipoplex) nanoparticle or liposome. In some embodiments, the cationic liposome / nucleic acid complex nanoparticle formulation for gene therapy agents includes a synthetic cationic lipid (R)-N,N,N-trimethyl-2,3-dioleoyloxy-1-propylammonium chloride (DOTMA) and a phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the DOTMA / DOPE liposome component is optimized for delivery and targeting cells in an individual.

[0139] In some embodiments, a nucleic acid comprising a vector genome is mixed with a pharmaceutical composition comprising one or more cationic lipids, including, for example, (R)-N,N,N-trimethyl-2,3-dioleoyloxy-1-propylammonium chloride (DOTMA) and phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the pharmaceutical composition comprises at least one lipid. In some embodiments, the pharmaceutical composition comprises at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphiphilic molecule (e.g., a molecule comprising at least one hydrophilic and lipophilic portion) is a cationic lipid within the meaning of the present invention. In some embodiments, the positive charge is contributed by at least one cationic lipid, and the negative charge is contributed by the nucleic acid. In some embodiments, the pharmaceutical composition comprises at least one auxiliary lipid. The auxiliary lipid can be a neutral lipid or an anionic lipid. The auxiliary lipid can be a natural lipid (e.g., phospholipid) or an analog of a natural lipid, or a completely synthetic lipid or lipid-like molecule that has no similarity to a natural lipid. In one embodiment, the cationic lipid and / or the auxiliary lipid are lipids that form a bilayer. Examples of helper lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analog or derivative thereof, cholesterol (Chol) or an analog or derivative thereof, and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analog or derivative thereof.

[0140] In some embodiments, the molar ratio of at least one cationic lipid to at least one helper lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1: 1. In some embodiments, at this ratio, the molar amount of the cationic lipid is generated by multiplying the molar amount of the cationic lipid by the number of positive charges in the cationic lipid.

[0141] In some embodiments, the lipid is contained in a vesicle that encapsulates the vector genome. The vesicle can be a multilamellar vesicle, a unilamellar vesicle, or a mixture thereof. The vesicle can be a liposome. Vector genome

[0142] In some embodiments, the invention provides methods for determining an innate immune response to a gene therapy agent for delivering a therapeutic transgene to a desired target in an individual. In some embodiments, the gene therapy agent comprises a vector genome for delivering and expressing a therapeutic transgene in a desired target in an individual.

[0143] The present invention contemplates the use of gene therapy agents for the introduction of one or more nucleic acid sequences encoding therapeutic polypeptides and / or nucleic acids for packaging into viral particles (for viral gene therapy agents). The vector genome may include any element that establishes expression of the therapeutic polypeptide and / or nucleic acid, such as a promoter, ITRs of the present disclosure, ribosome binding elements, terminators, enhancers, selectable markers, introns, poly A signals, and / or replication origins.

[0144] In certain embodiments, therapeutic transgenic encoding therapeutic polypeptide.Therapeutic polypeptide can for example provide polypeptide and / or enzymatic activity that does not exist in cell or organism or exists with the level of reduction.Alternately, therapeutic polypeptide can provide polypeptide and / or enzymatic activity that indirectly offsets the imbalance in cell or organism.For example, the therapeutic polypeptide that is used for the obstacle relevant to the accumulation of metabolites caused by metabolic enzyme or activity defect can provide the metabolic enzyme or activity of missing, or it can provide the alternative metabolic enzyme or activity that causes metabolite reduction.Therapeutic polypeptide can also be used for reducing the activity of polypeptide (for example, overexpressed, activated by functional acquisition mutation or its activity is otherwise misregulated) by for example acting as dominant negative polypeptide.

[0145] The vector genome of the present invention can encode polypeptides that are intracellular proteins, anchored in the cell membrane, retained in the cell, or secreted by cells transduced with the vector of the present invention. With respect to polypeptides secreted by cells receiving the vector; these polypeptides can be soluble (i.e., not attached to the cell). For example, soluble polypeptides lack a transmembrane region and are secreted from the cell. Techniques for identifying and removing nucleic acid sequences encoding transmembrane domains are known in the art.

[0146] In some embodiments, the vector genome of the present invention encodes a polypeptide for treating a disease or disorder in an individual. Diseases and disorders treated by the gene therapy agents of the present invention include, but are not limited to, Huntington's disease (HD), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), metachromatic leukodystrophy (MLD), amyotrophic lateral sclerosis (ALS), age-related macular degeneration (AMD), congenital muscular dystrophy (CMD), phenylketonuria (PKU), muscular dystrophy (MD), A1AT deficiency, focal segmental glomerulosclerosis (FSGS), cystinuria, hemophilia A, hemophilia B, Gaucher disease (GBA), Parkinson's disease (PD) and Pompe disease.

[0147] In some embodiments, the therapeutic polypeptide is huntingtin protein (HTT), tau protein, amyloid precursor protein, alpha-synuclein, pseudoarylsulfatase (ARSA), superoxide dismutase 1 (SOD1), phenylalanine hydroxylase (PAH), dystrophin, alpha-1-antitrypsin (A1AT), cysteine ​​transporter, factor VIII (FVIII), factor IX (FIX), acid β-glucosidase, glial-derived growth factor (GDNF), brain-derived growth factor (BDNF), tyrosine hydroxylase (TH), GTP cyclohydrolase (GTPCH) and / or amino acid decarboxylase (AADC) or alpha-glucosidase.

[0148] In some embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid may include but is not limited to siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozymes or deoxyribozymes. Therefore, the therapeutic nucleic acid may encode an RNA that, when transcribed from the nucleic acid of the vector, can treat the disorder by interfering with the translation or transcription of an abnormal or excessive protein associated with the disorder of the present invention. For example, the nucleic acid of the present invention may encode an RNA that treats the disorder by highly specifically eliminating or reducing the mRNA encoding abnormal and / or excessive protein. Therapeutic RNA sequences include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA) and / or ribozymes (such as hammerhead and hairpin ribozymes), which can treat the disorder by highly specifically eliminating or reducing the mRNA encoding abnormal and / or excessive protein.

[0149] In some embodiments, therapeutic polypeptides or therapeutic nucleic acids are used to treat CNS disorders. Without wishing to be bound by theory, it is believed that therapeutic polypeptides or therapeutic nucleic acids can be used to reduce or eliminate the expression and / or activity of polypeptides whose gain of function is associated with disorders, or to enhance the expression and / or activity of polypeptides to supplement defects associated with disorders (e.g., mutations in genes whose expression shows similar or related activities). Non-limiting examples of disorders of the present invention that can be treated by therapeutic polypeptides or therapeutic nucleic acids of the present invention (exemplary genes that can be targeted or provided are provided in parentheses for each disorder) include stroke (e.g., caspase-3, Beclin1, Ask1, PAR1, HIF1α, PUMA, and / or any gene described in Fukuda, AM and Badaut, J. (2013) Genes (Basel) 4: 435-456), Huntington's disease (mutant HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and / or Boison, D. (2 010) Epilepsy [Epilepsia] 51: 1659-1668), Parkinson's disease (alpha-synuclein), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis; SOD1), Alzheimer's disease (tau protein, amyloid precursor protein), corticobasal degeneration or CBD (tau protein), corticobasal ganglionic degeneration or CBGD (tau protein), frontotemporal dementia or FTD (tau protein), progressive supranuclear palsy or PSP (tau protein), multiple system atrophy or MSA (alpha-synuclein), brain cancer (e.g., mutant or overexpressed oncogenes involved in brain cancer), and lysosomal storage diseases (LSD). Disorders of the invention can include those involving large areas of the cortex (e.g., more than one functional area of ​​the cortex, more than one lobe of the cortex, and / or the entire cortex). Other non-limiting examples of disorders of the invention that can be treated by the therapeutic polypeptides or therapeutic nucleic acids of the invention include traumatic brain injury, enzyme dysfunction disorders, mental disorders (including post-traumatic stress syndrome), neurodegenerative diseases, and cognitive disorders (including dementia, autism, and depression). Enzyme dysfunction disorders include, but are not limited to, any of the leukodystrophies (including Canavan's disease) and the lysosomal storage diseases described below.

[0150] In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat lysosomal storage diseases. As is generally known in the art, lysosomal storage diseases are rare inherited metabolic disorders characterized by defective lysosomal function. Such disorders are usually caused by a deficiency of enzymes required for proper mucopolysaccharide, glycoprotein and / or lipid metabolism, resulting in pathological accumulation of lysosomal stored cellular material. Non-limiting examples of lysosomal storage diseases of the invention that can be treated by the therapeutic polypeptides or therapeutic nucleic acids of the invention (exemplary genes that can be targeted or provided are provided in parentheses for each disorder) include Gaucher disease type 2 or type 3 (acid β-glucosidase, GBA), GM1 gangliosidosis (β-galactosidase-1, GLB1), Hunter disease (iduronate-2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), alpha mannosidase (alpha mannosidase, such as α-D-mannosidase, MAN2B1), beta mannosidase (β-mannosidase, MANBA), metachromatic leukodystrophy (pseudoarylsulfatase A, ARSA), mucolipidosis type II / III (N-acetylglucosamine-1-phosphotransferase, GNPTAB), Niemann-Pick type A disease (acid sphingomyelinase, ASM), Niemann-Pick disease type C (Niemann-Pick protein C, NPC1), Pompe disease (acid alpha-1,4-glucosidase, GAA), Sandhoff disease (hexosaminidase beta subunit, HEXB), Sanfilippo disease type A (N-sulfoglucosamine sulfohydrolase, MPS3A), Sanfilippo disease type B (N-alpha-acetylglucosaminidase, NAGLU), Sanfilippo disease type C (heparin Acetyl-CoA: alpha-glucosaminidase N-acetyltransferase, MPS3C), Sanfilippo type D disease (N-acetylglucosamine-6-sulfatase, GNS), Schendler disease (alpha-N-acetylgalactosaminidase, NAGA), Slae disease (beta-glucuronidase, GUSB), Tay-Sachs disease (hexosaminidase alpha subunit, HEXA), and Wolman disease (lysosomal acid lipase, LIPA).

[0151] In some embodiments, the therapeutic polypeptide encodes factor VIII, factor IX, myotubularin, survival motor neuron protein (SMN), retinoid isomerohydrolase (RPE65), NADH-ubiquinone oxidoreductase chain 4, choroideremia protein (CHM), ornithine transcarbamylase, arginylsuccinate synthetase, β-globin, γ-globin, phenylalanine hydroxylase, adrenoleukodystrophy protein (ALD), dystrophin, truncated dystrophin, anti-VEGF agent or a functional variant thereof.

[0152] In some embodiments, the heterologous nucleic acid is operably linked to a promoter. Exemplary promoters include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter and CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG promoter; Niwa et al., Gene [Gene], 1991, 108 (2): 193-9) and elongation factor 1-alpha promoter (EFl-alpha) promoter (Kim et al., Gene [Gene], 1990, 91 (2): 217-23 and Guo ... Ther. [Gene Therapy], 1996, 3(9):802-10). In some embodiments, the promoter comprises a human β-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β-actin (CBA) promoter. The promoter may be a constitutive promoter, an inducible promoter, or a repressible promoter. In some embodiments, the present invention provides a recombinant vector comprising a nucleic acid encoding a heterologous transgene of the present disclosure operably linked to a CBA promoter. Exemplary promoters and descriptions can be found, for example, in U.S. Pre-Grant Publication 20140335054.

[0153] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 13-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter [Invitrogen].

[0154] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors (such as temperature), or the presence of a specific physiological state, such as the acute phase, a specific differentiation state of the cell, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 2004). 98 / 10088); ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), tetracycline repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetracycline inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al. =Also, Curr. Opin. Chem. Biol. [Current Opinion in Chemical Biology], 2:512-518 (1998)), the RU486 inducible system (Wang et al., Nat. Biotech. [Nature Biotechnology], 15:239-243 (1997) and Wang et al., Gene Ther. [Gene Therapy], 4:432-441 (1997)) and the rapamycin inducible system (Magari et al., J. Clin. Invest. [Journal of Clinical Research], 100:2865-2872 (1997)). Other types of inducible promoters useful in this context are promoters that are regulated by specific physiological states, such as temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells.

[0155] In another embodiment, a natural promoter or fragment thereof for the transgene will be used. A natural promoter may be used when it is desired that expression of the transgene should mimic natural expression. A natural promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In additional embodiments, other natural expression control elements (such as enhancer elements, polyadenylation sites, or Kozak consensus sequences) may also be used to mimic natural expression.

[0156] In certain embodiments, regulatory sequences confer tissue-specific gene expression capability. In some cases, tissue-specific regulatory sequences are combined with tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art.

[0157] In some embodiments, the vector comprises an intron. For example, in some embodiments, the intron is a chimeric intron derived from chicken β-actin and rabbit β-globin. In some embodiments, the intron is a minute virus of mice (MVM) intron.

[0158] In some embodiments, the vector comprises a polyadenylation (poly A) sequence. Various examples of polyadenylation sequences are known in the art, such as the bovine growth hormone (BGH) poly (A) sequence (see, e.g., Accession No. EF592533), the SV40 polyadenylation sequence, and the HSV TK pA polyadenylation sequence. Kits and Products

[0159] The gene therapy agent (e.g., AAV particle, adenoviral particle, lentiviral particle, HSV particle or lipid nanoparticle) and / or material for isolating dendritic cells as described herein can be contained in a kit or article of manufacture designed for use in one of the methods of the invention as described herein.

[0160] In some embodiments, the kit or article further includes instructions for determining the innate immunogenicity of the gene therapy agent using isolated dendritic cells. The kit or article described herein may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any method described herein. Suitable packaging materials may also be included, and may be any packaging materials known in the art, including, for example, vials (such as sealed vials), vessels, ampoules, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These products may be further sterilized and / or sealed.

[0161] In some embodiments, the kit or article of manufacture further contains one or more of the buffers and / or pharmaceutically acceptable excipients described herein (e.g., as described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991). In some embodiments, the kit or article of manufacture includes one or more pharmaceutically acceptable excipients, carriers, solutions, and / or additional ingredients described herein. The kits or articles of manufacture described herein may be packaged in single unit dose or in multiple dose form. The contents of the kit or article of manufacture are generally formulated sterile and may be lyophilized or provided as a substantially isotonic solution. Exemplary Embodiments

[0162] The present invention includes the following enumerated embodiments.

[0163] 1. A method for determining innate immunogenicity in an individual to a gene therapy agent, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the altered expression of one or more cytokines in the innate immune cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, Wherein expression of the cytokine signature following incubation with the gene therapy agent is indicative of innate immunogenicity to the gene therapy agent in the individual.

[0164] 2. The method of embodiment 1, wherein the innate immune cell is a dendritic cell, a monocyte, a macrophage or a natural killer (NK) cell.

[0165] 3. The method of embodiment 1 or 2, wherein the innate immune cells are isolated from peripheral blood mononuclear cells from the individual.

[0166] 4. The method of any one of embodiments 1-3, wherein the innate immune cell is a dendritic cell.

[0167] 5. The method of embodiment 4, wherein the dendritic cells are derived from monocytes of the individual.

[0168] 6. The method of embodiment 4 or 5, further comprising isolating monocytes from the individual, incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0169] 7. The method of embodiment 6, wherein the monocytes are CD14+ monocytes.

[0170] 8. The method of embodiment 6 or 7, wherein the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes.

[0171] 9. The method of any one of embodiments 1-8, wherein the innate immune cells are re-plated prior to incubation with the gene therapy agent of step b).

[0172] 10. The method of embodiment 9, wherein the innate immune cells are re-plated into microwell dishes.

[0173] 11. The method of any one of embodiments 1-10, wherein the gene therapy agent is a viral vector, and wherein the innate immune cells are combined with the gene therapy agent at a rate of about 1×10 3 About 1×10 5 or about 1×10 4 MOI was determined and incubated together.

[0174] 12. The method of any one of embodiments 1-10, wherein the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

[0175] 13. The method of any one of embodiments 1-12, wherein the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

[0176] 14. The method of any one of embodiments 1-13, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0177] 15. The method of any one of embodiments 1-14, wherein the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α.

[0178] 16. The method of any one of embodiments 1-15, wherein the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α.

[0179] 17. The method of any one of embodiments 1-15, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0180] 18. The method of any one of embodiments 1-17, wherein expression of the cytokines in the cytokine signature is increased compared to a suitable control.

[0181] 19. The method of embodiment 18, wherein the appropriate control is the expression of these cytokines in the cytokine signature of innate immune cells that have not been incubated with the gene therapy agent, or wherein the appropriate control is the expression of these cytokines in the cytokine signature of innate immune cells prior to incubation with the gene therapy agent.

[0182] 20. The method of any one of embodiments 1-10 and 13-19, wherein the gene therapy agent is a viral vector.

[0183] 21. A method for determining innate immunogenicity in an individual to a viral gene therapy agent, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions wherein the monocytes differentiate into dendritic cells, b) Incubate these dendritic cells with the viral gene therapy agent at a concentration of about 1×10 3 About 1×10 5 MOI for about 12 to about 36 hours. c) analyzing the altered expression of one or more cytokines in the dendritic cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, Wherein expression of the cytokine signature following incubation with the viral gene therapy agent indicates innate immunogenicity to the viral gene therapy agent in the individual, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0184] 22. The method of embodiment 21, wherein the monocytes are obtained from peripheral mononuclear cells from the individual.

[0185] 23. A method as described in Example 21 or 22, wherein the monocytes are CD14+ monocytes.

[0186] 24. The method of any one of embodiments 21-23, wherein the monocytes are incubated in dendritic cell culture medium for about 7-8 days to differentiate the monocytes into dendritic cells.

[0187] 25. The method of any one of embodiments 21-24, wherein the dendritic cells are administered with the viral gene therapy agent at a concentration of about 1×10 4 MOI was determined and incubated together.

[0188] 26. The method of any one of embodiments 21-25, wherein the dendritic cells are incubated with the viral gene therapy agent for about 24 hours.

[0189] 27. The method of any one of embodiments 20-26, wherein the viral vector is an AAV particle.

[0190] 28. The method of embodiment 27, wherein the AAV particles comprise AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

[0191] 29. The method of embodiment 28, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

[0192] 30. The method of any one of embodiments 27-29, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, AAVrh8 ITRs, AAV9 ITRs, AAV10 ITRs, AAVrh10 ITRs, AAV11 ITRs, or AAV12 ITRs.

[0193] 31. The method of embodiment 30, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0194] 32. The method of embodiment 30, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0195] 33. The method of any one of embodiments 20-26, wherein the viral vector is an adenoviral particle.

[0196] 34. The method of embodiment 33, wherein the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4,, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3 or a functional variant thereof.

[0197] 35. The method of any one of embodiments 20-26, wherein the viral vector is a lentiviral particle.

[0198] 36. The method of embodiment 35, wherein the recombinant lentiviral particles are pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

[0199] 37. The method of any one of embodiments 20-26, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0200] 38. A method as described in Example 37, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

[0201] 39. The method of any one of embodiments 1-10 and 12-19, wherein the gene therapy agent is a lipid nanoparticle.

[0202] 40. A method of determining innate immunogenicity in an individual to a non-viral gene therapy agent, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions wherein the monocytes differentiate into dendritic cells, b) incubating the dendritic cells with a non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL, c) analyzing the altered expression of one or more cytokines in the dendritic cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, Wherein expression of the cytokine signature following incubation with the non-viral gene therapy agent indicates innate immunogenicity to the non-viral gene therapy agent in the individual, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0203] 41. The method of embodiment 40, wherein the monocytes are obtained from peripheral mononuclear cells from the individual.

[0204] 42. A method as described in Example 40 or 41, wherein the monocytes are CD14+ monocytes.

[0205] 43. The method of any one of embodiments 40-42, wherein the monocytes are incubated in a dendritic cell culture medium for about 7-8 days to differentiate the monocytes into dendritic cells.

[0206] 44. The method of any one of embodiments 40-43, wherein the dendritic cells are incubated with the non-viral gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

[0207] 45. The method of any one of embodiments 1-44, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0208] 46. ​​The method of embodiment 45, wherein the heterologous transgene is operably linked to a promoter.

[0209] 47. The method of embodiment 46, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0210] 48. A method for determining the cytokine signature of a gene therapy agent, the method comprising a) incubating one or more innate immune cells from one or more individuals with the gene therapy agent, b) analyzing the altered expression of one or more cytokines in the one or more innate immune cells compared to a suitable control, Wherein the altered expression of one or more cytokines in step b) is indicative of a cytokine signature of the gene therapy agent.

[0211] 49. The method of embodiment 48, wherein the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell.

[0212] 50. The method of embodiment 48 or 49, wherein the one or more innate immune cells are isolated from peripheral blood mononuclear cells from the individual.

[0213] 51. The method of any one of embodiments 48-50, wherein the innate immune cell is a dendritic cell.

[0214] 52. The method of embodiment 51, wherein the dendritic cells are derived from monocytes of the one or more individuals.

[0215] 53. The method of embodiment 51 or 52, further comprising isolating monocytes from the one or more individuals, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

[0216] 54. A method as described in Example 53, wherein the monocytes are CD14+ monocytes.

[0217] 55. The method of embodiment 53 or 54, wherein the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes.

[0218] 56. The method of any one of embodiments 48-55, wherein the innate immune cells are re-plated prior to incubation with the gene therapy agent of step b).

[0219] 57. The method of any one of embodiments 48-56, wherein the gene therapy agent is a viral vector, and wherein the innate immune cells are coupled to the gene therapy agent at a rate of about 1×10 3 About 1×10 5 or about 1×10 4 MOI was determined and incubated together.

[0220] 58. The method of any one of embodiments 48-56, wherein the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

[0221] 59. The method of any one of embodiments 48-58, wherein the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

[0222] 60. The method of any one of embodiments 48-59, wherein expression of the cytokines in the cytokine signature is increased compared to a suitable control.

[0223] 61. The method of embodiment 60, wherein the appropriate control is the expression of these cytokines in the cytokine signature of innate immune cells that have not been incubated with the gene therapy agent, or wherein the appropriate control is the expression of these cytokines in the cytokine signature of innate immune cells prior to incubation with the gene therapy agent.

[0224] 62. The method of any one of embodiments 48-57 and 59-61, wherein the gene therapy agent is a viral vector.

[0225] 63. A method as described in Example 57 or 62, wherein the viral vector is an AAV particle.

[0226] 64. A method as described in Example 63, wherein the AAV particles comprise AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

[0227] 65. The method of embodiment 64, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

[0228] 66. The method of any one of embodiments 63-65, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR, or AAV12 ITR.

[0229] 67. The method of embodiment 66, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0230] 68. The method of embodiment 66, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0231] 69. A method as described in Example 57 or 62, wherein the viral vector is an adenoviral particle.

[0232] 70. The method of embodiment 69, wherein the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4,, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3, or a functional variant thereof.

[0233] 71. The method of embodiment 57 or 62, wherein the viral vector is a lentiviral particle.

[0234] 72. The method of embodiment 71, wherein the recombinant lentiviral particles are pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

[0235] 73. The method of embodiment 57 or 62, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0236] 74. A method as described in Example 73, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

[0237] 75. The method of any one of embodiments 48-56 and 58-61, wherein the gene therapy agent is a lipid nanoparticle.

[0238] 76. The method of any one of embodiments 48-75, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0239] 77. The method of embodiment 76, wherein the heterologous transgene is operably linked to a promoter.

[0240] 78. The method of embodiment 77, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0241] 79. A kit for use in the method of any one of embodiments 1-78. Examples

[0242] The present invention will be more fully understood by reference to the following examples. However, these examples should not be construed as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and the various modifications or changes made according to the examples and embodiments will be conceived by those skilled in the art and should be included in the spirit and scope of the present application and within the scope of the appended claims. Example 1: Screening of different AAV vectors for innate immunogenicity

[0243] This example provides a strategy for measuring the innate immune response following AAV treatment.

[0244] Immune responses to AAV vectors pose a challenge to successful clinical applications. AAV triggers an immune response involving activation of both the innate and adaptive immune systems. Although the adaptive immune response to AAV is relatively well characterized, little is known about innate immune activation caused by AAV. Materials and methods

[0245] Preparation of peripheral blood mononuclear cells.The blood from four donors from enriched leukocyte apheresis product (Ausai Sisi company (AllCells)) is decanted in 50mL tubes, and Dulbecco's phosphate buffered saline (DPBS) is added with a ratio of 1:1.Blood is added with DBPS and the mixture is pipetted gently in a separate 50mL tube containing 15mL Ficoll (GE17-5442-02), to ensure that blood and Ficoll do not mix.The mixture is centrifuged at room temperature with 2000RPM for 25 minutes (9 accelerations and no braking).Collect the buffy coat containing peripheral blood mononuclear cells (PBMC), transfer it to a new tube, and centrifuge for five minutes with 400RCF.PBMC is washed three times and counted with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FBS) or fetal calf serum (FCS).

[0246] Isolation of monocytes. CD14+ monocytes were isolated from PBMCs using CD14 microbeads according to the manufacturer's protocol (Miltenyi Biotec, Germany, order number 130-050-201, protocol available online at www.miltenyibiotec.com / upload / assets / IM0001260.PDF). Briefly, PBMCs were incubated with 20 μL CD14 microbeads / 10 μL at 2°C-8°C. 7 Total cells were incubated together for 15 minutes. PBMCs were applied to a magnetic column (Miltenyi Biotec; World Wide Web miltenyibiotec.com / US-en / products / ls-columns.html#130-042-401), and unlabeled cells were allowed to pass through. After the column was washed three times, the column was taken out from a magnetic separator (Miltenyi Biotec; World Wide Web miltenyibiotec.com / US-en / products / quadromacs-separator-and-starting-kits.html#130-091-051), placed on a collection tube, and magnetically labeled CD14+ monocytes were flushed out by pushing the plunger into the column.

[0247] Differentiation of monocytes. Using ImmunoCult-ACF dendritic cell culture medium, differentiation supplements and maturation supplements (Stem Cell Technologies, Catalog Nos. 10986, 10988 and 10989; World Wide Web cdn.stemcell.com / media / Files / pis / DX20521-PIS_1_2_0.pdf?_ga=2.81451927.1035383195.1642105700-1174975582.1603298321), CD14+ monocytes were differentiated into dendritic cells according to the manufacturer's protocol available online. In brief, purified CD14+ monocytes were added to dendritic cell culture medium containing differentiation supplements and incubated at 37°C for three days. On the 3rd day, the culture medium was replaced with fresh dendritic cell culture medium containing differentiation supplements, and the cells were incubated for another two days. On day 5, maturation supplement was added to the cells at a 1 in 100 dilution (eg, 50 μL supplement / 5 mL culture). Differentiated dendritic cells were harvested on day 7.

[0248] rAAV production and titration. rAAV vectors (AAV1, AAV2, AAVDJ and AAVrh32.33) were produced using a standard triple transfection method (Sena-Esteves and Gao, Cold Spring Harb Protoc [Cold Spring Harb Protocol]; doi: 10.1101 / pdb.top095513, 2020). All serotypes tested encode the same GFP transgene. The virus was purified by cesium chloride ultracentrifugation and titrated using both silver staining and quantitative polymerase chain reaction (qPCR). deal with

[0249] Dendritic cells were plated in 96-well plates at 200,000 cells / well. Each treatment was performed in triplicate. Four different treatments were performed using four different AAV vectors at 1e4 MOI. The cells were incubated in an incubator at 37°C, 5% CO2 for 24 hours. After 24h, the plate was centrifuged and the culture supernatant was collected. Lipopolysaccharide LPS (300ng / mL) (Sigma Aldrich Fine Chemicals Biosciences L2630100MG) was used as a positive control, which is known to activate toll-like receptor 4 and induce cytokine production in human monocyte-derived dendritic cells. Cell culture medium was then collected and centrifuged. The clarified culture medium was analyzed by two different methods to detect different cytokines released from dendritic cells after being stimulated with different AAV serotypes. Both methods used a multiplex immunoassay (Luminex bead-based assay) performed by Nanobiotec (www. nanobiotecusa.com / immunoassay) and MSD (www. mesoscale.com / products / v-plex-proinflammatory-panel-1-human-kit-k15049d / ) performed by DC3 therapeutics https: / / www.dc3therapeutics.com / services. An experimental outline of PBMC isolation, monocyte purification, and dendritic cell differentiation is presented in Figure 1 This figure also shows how dendritic cells are treated with different AAVs. result

[0250] Based on cytokine release from treated dendritic cells, all four serotypes tested produced weaker immune responses than LPS ( Figure 2While LPS induced a significant upregulation of most cytokines assayed from each donor, the different AAV serotypes tested induced a less significant upregulation that was limited to a subset of cytokines: IL6, TNFα, IL-1β, MCP1, and MIP-1α ( Figure 2 Notably, this cytokine subset (ie, cytokine signature) appears to be conserved across different AAV serotypes. Figure 2 The data presented in the article are based on Luminex technology, where some cytokines were above or below the limit of detection (aLOD / bLOD), therefore, we utilized MSD technology and re-examined the cytokine levels from the same donor cells. Figure 2 As shown, the same results were confirmed using orthogonal MSD measurement ( Figure 3 ).

[0251] Among the AAV serotypes tested, AAVrh32.33 was found to produce the most robust and consistent innate immune signature ( Figure 2 and Figure 3 ). This was evidenced by the greater upregulation observed for IL-6, TNF-α, and IL-1β. discuss

[0252] The success of gene therapy for the treatment of rare genetic diseases relies heavily on adeno-associated virus (AAV) viral vectors, which provide many attractive features, including tissue-specific tropism, transduction of quiescent cells, and maintenance of modified gene expression. However, the immune response to AAV vectors poses a major challenge to successful clinical transformation. Capsids, viral genomes, and transgenes trigger immune responses involving the activation of both innate and adaptive immunity of the immune system. The adaptive immune response triggered by B cells and T cells has been understood to some extent in the art, but innate immune activation is poorly understood. Studies based on mouse models indicate that TLR9a DNA sensors present in endosomes detect AAV genomes and activate signal transduction cascades that ultimately lead to cytokine release (Ashley, SN et al., Cell Immunol. [Cellular Immunology] 2019, 346: 103997; Zhu, J et al., J Clin Invest. [Clinical Research Journal] 2009; 119 (8): 2388-2398). ( Figure 1, lower right). These cytokines trigger an antiviral response and trigger the activation of the adaptive immune system. The main challenge in understanding these innate immune responses is the poor reproducibility of the immune responses observed in clinical trials in an ex vivo setting. In order to circumvent this challenge and understand the innate immune response, we have now developed a novel assay that reproduces the innate immune signatures in response to various AAV serotypes in various human donors. We have used human monocytic dendritic cells (moDC) from healthy donors, and we sensitively detect cytokines released from these moDCs after stimulation with AAV vectors. Example 2: Screening of LNPs for innate immunogenicity

[0253] This example provides a strategy for determining the innate immune response after LNP delivery. An overall schematic diagram of the method described in the example is depicted in Figure 4 middle. Materials and methods Generation of differentiated dendritic cells

[0254] Blood monocytes were prepared using the method described in Example 1, and the monocytes were isolated and differentiated into dendritic cells. LNP treatment and cytokine measurements

[0255] Human monocytic dendritic cells were plated in a 96-well format and treated with 10 ug LNPs at 37°C using standard protocols (see https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5577173 / and https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5646367 / ) and harvested 24 hours later for downstream analysis via flow cytometry, such as cytotoxicity and target gene expression. The culture medium was collected for cytokine analysis. The cells were centrifuged at 2,000 rpm for 5 minutes and the supernatant was collected for cytokine analysis. The luminex was used Human Cytokine / Chemokine / Growth Factor Panel A 38Plex Premixed Magnetic Bead Panel - Immunology Multiplex Assays Catalog No. HCYTA-60K-PX38 Cytokines were measured according to the manufacturer's protocol. The reagents used in the assay are shown in Table 1. Table 1: List of reagents and suppliers Cell viability assessment

[0256] Dendritic cells (DCs) were resuspended in FACS buffer and transferred to a U-bottom 96-well plate. The cells were centrifuged at 2,000 rpm for 5 minutes and the supernatant was discarded. To analyze cell viability, live / dead negative stained cells were gated and the percentage was determined as cell viability. LIVE / DEAD from Invitrogen was used. TM Cells can be stained using the Fixable Near Infrared Dead Cell Stain Kit (for 633 or 635 nm excitation, catalog number L34975). Target gene expression by flow cytometry

[0257] Dendritic cells (DC) are resuspended in FACS buffer and transferred to a U-shaped bottom 96-well plate. The cells are centrifuged at 2,000 rpm for 5 minutes, and the supernatant is discarded. In order to analyze the expression of the target gene, the CD22 protein is diluted to a final concentration of 5 μg / ml with FACS buffer. The DC in each well is treated with 100 μl of diluted CD22 for 30 minutes at 4°C. The cells are washed with FACS buffer and then stained with 50 μl of flow antibody premix (1:100 anti-hCD22APC, 1:100 anti-hCD11c Pacific Blue, 1:100 anti-hCD11b FITC, 1:100 anti-hCD83PE, and 1:100 live / dead staining APC-Cy7) at 4°C for 30 minutes. Cells were washed twice with FACS buffer and run on a flow cytometer (Novocyte Penteon Flow Cytometer System 5 Laser, Agilent Technology). result Cell viability

[0258] Dendritic cells were harvested 24 h after LNP treatment and flow cytometry was performed to assess cell viability ( Figure 5 ). Figure 5 Each point in represents the cell derived from human donor.Only cell condition is not treated with LNP, and mRNA-LNP#1 and mRNA-LNP#2 are cells treated with two different LNPs, and for all donors, viability is the same as cell control only, which shows that LNP has no cytotoxicity after treatment. Dendritic cell system can be used to evaluate LNP without affecting cell viability. One-way ANOVA is carried out to measure statistical significance. Transduction level

[0259] Dendritic cells were harvested 24 h after LNP treatment and flow cytometry was performed to assess target gene expression ( Figure 6 ). Figure 6Each point in represents a cell derived from a human donor. The cell-only condition was not treated with LNPs, and mRNA-LNP#1 and mRNA-LNP#2 were cells treated with two different LNPs encapsulating the same mRNA. Dendritic cells from all different donors were transduced by LNPs, as measured by the percentage of mRNA expressing cells. A one-way ANOVA was performed to measure statistical significance. This suggests that the disclosed dendritic cell assay system can be used to effectively evaluate LNP transduction. LNP immunogenicity

[0260] Culture medium from dendritic cells was collected 24 hours after LNP treatment and Luminex analysis was performed to identify cytokine signatures. Compared to cells not treated with LNP, LNPs encapsulating mRNA specifically secreted cytokines. Analysis showed that cytokines such as IP10 ( Fig. 7A )、MIP1b( Figure 7B )、CXCL9( Figure 7C ) and IL2( Fig.7D ) were all raised.

Claims

1. A method for determining innate immunogenicity in an individual to a gene therapy agent, the method comprising a) incubating innate immune cells from the individual with the gene therapy agent, b) analyzing the altered expression of one or more cytokines in the innate immune cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, Wherein expression of the cytokine signature following incubation with the gene therapy agent is indicative of innate immunogenicity to the gene therapy agent in the individual.

2. The method of claim 1, wherein the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell.

3. The method of claim 1 or 2, wherein the innate immune cells are isolated from peripheral blood mononuclear cells from the individual.

4. The method of any one of claims 1-3, wherein the innate immune cell is a dendritic cell. The method of claim 4 , wherein the dendritic cells are derived from monocytes of the individual.

6. The method of claim 4 or 5, further comprising isolating monocytes from the individual, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

7. The method of claim 6, wherein the monocytes are CD14+ monocytes.

8. The method of claim 6 or 7, wherein the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes.

9. The method of any one of claims 1-8, wherein the innate immune cells are replated prior to incubation with the gene therapy agent of step b).

10. The method of claim 9, wherein the innate immune cells are re-plated into microwell dishes.

11. The method of any one of claims 1-10, wherein the gene therapy agent is a viral vector or a non-viral vector.

12. The method of claim 11, wherein the viral vector is AAV.

13. The method of claim 11, wherein the non-viral vector is a lipid nanoparticle (LNP).

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

15. The method of any one of claims 1-10, wherein the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

16. The method of any one of claims 1-15, wherein the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

17. The method of any one of claims 1-16, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

18. The method of any one of claims 1-17, wherein the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α.

19. The method of any one of claims 1-18, wherein the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α.

20. The method of any one of claims 1-18, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

21. The method of any one of claims 1-16, wherein the cytokine signature comprises increased expression of one or more of IP10, MIP1b, CXCL9, and IL2.

22. The method of any one of claims 1-16, wherein the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, and IL2.

23. The method of any one of claims 1-22, wherein expression of the cytokines in the cytokine signature is increased compared to a suitable control.

24. The method of claim 23, wherein the appropriate control is the expression of the cytokines in a cytokine signature from innate immune cells that have not been incubated with the gene therapy agent, or wherein the appropriate control is the expression of the cytokines in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

25. The method of any one of claims 1-13 and 16-24, wherein the gene therapy agent is a viral vector.

26. A method of determining innate immunogenicity in an individual to a viral gene therapy agent, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions wherein the monocytes differentiate into dendritic cells, b) Incubate these dendritic cells with the viral gene therapy agent at a concentration of about 1×10 3 About 1×10 5 MOI for about 12 to about 36 hours. c) analyzing the altered expression of one or more cytokines in the dendritic cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, Wherein expression of the cytokine signature following incubation with the viral gene therapy agent indicates innate immunogenicity to the viral gene therapy agent in the individual, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

27. The method of claim 26, wherein the monocytes are obtained from peripheral mononuclear cells from the individual.

28. The method of claim 26 or 27, wherein the monocytes are CD14+ monocytes.

29. The method of any one of claims 26-28, wherein the monocytes are incubated in dendritic cell culture medium for about 7-8 days to differentiate the monocytes into dendritic cells.

30. The method of any one of claims 26-29, wherein the dendritic cells are administered with the viral gene therapy agent at a concentration of about 1×10 4 MOI was determined and incubated together.

31. The method of any one of claims 26-30, wherein the dendritic cells are incubated with the viral gene therapy agent for about 24 hours.

32. The method of any one of claims 25-31, wherein the viral vector is an AAV particle.

33. The method of claim 32, wherein the AAV particles comprise AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

34. The method of claim 33, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

35. The method of any one of claims 32-34, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, AAVrh8 ITRs, AAV9 ITRs, AAV10 ITRs, AAVrh10 ITRs, AAV11 ITRs, or AAV12 ITRs.

36. The method of claim 35, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

37. The method of claim 35, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

38. The method of any one of claims 25-31, wherein the viral vector is an adenoviral particle.

39. The method of claim 38, wherein the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3, or a functional variant thereof.

40. The method of any one of claims 25-31, wherein the viral vector is a lentiviral particle.

41. The method of claim 40, wherein the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

42. The method of any one of claims 25-31, wherein the viral vector is a herpes simplex virus (HSV) particle.

43. The method of claim 42, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

44. The method of any one of claims 1-10 and 15-20, wherein the gene therapy agent is a lipid nanoparticle.

45. The method of claim 21 or claim 22, wherein the gene therapy agent is a lipid nanoparticle.

46. ​​A method of determining innate immunogenicity in an individual to a non-viral gene therapy agent, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions wherein the monocytes differentiate into dendritic cells, b) incubating the dendritic cells with a non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL, c) analyzing the altered expression of one or more cytokines in the dendritic cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, Wherein expression of the cytokine signature following incubation with the non-viral gene therapy agent indicates innate immunogenicity to the non-viral gene therapy agent in the individual, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

47. The method of claim 46, wherein the monocytes are obtained from peripheral mononuclear cells from the individual.

48. The method of claim 46 or 47, wherein the monocytes are CD14+ monocytes.

49. The method of any one of claims 46-48, wherein the monocytes are incubated in dendritic cell culture medium for about 7-8 days to differentiate the monocytes into dendritic cells.

50. The method of any one of claims 46-49, wherein the dendritic cells are incubated with the non-viral gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

51. The method of any one of claims 1-50, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

52. The method of claim 51, wherein the heterologous transgene is operably linked to a promoter.

53. The method of claim 52, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

54. A method for determining innate immunogenicity against lipid nanoparticles (LNPs) in an individual, the method comprising a) incubating monocytes from the individual in a dendritic cell culture medium under conditions wherein the monocytes differentiate into dendritic cells, b) incubating the dendritic cells with the LNP, c) analyzing the altered expression of one or more cytokines in the dendritic cells compared to a suitable control, whereby the altered expression of the one or more cytokines produces a cytokine signature, Wherein expression of the cytokine signature following incubation with the LNP is indicative of innate immunogenicity to the non-viral gene therapeutic agent in the individual.

55. The method of claim 54, wherein the cytokine signature comprises increased expression of one or more of IP10, MIP1b, CXCL9, and IL2.

56. The method of claim 54, wherein the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, and IL2.

57. A method for determining the cytokine signature of a gene therapy agent, the method comprising a) incubating one or more innate immune cells from one or more individuals with the gene therapy agent, b) analyzing the altered expression of one or more cytokines in the one or more innate immune cells compared to a suitable control, Wherein the altered expression of one or more cytokines in step b) is indicative of a cytokine signature of the gene therapy agent.

58. The method of claim 57, wherein the innate immune cell is a dendritic cell, a monocyte, a macrophage, or a natural killer (NK) cell.

59. The method of claim 57 or 58, wherein the one or more innate immune cells are isolated from peripheral blood mononuclear cells from the individual.

60. The method of any one of claims 57-59, wherein the innate immune cell is a dendritic cell.

61. The method of claim 60, wherein the dendritic cells are derived from monocytes of the one or more individuals.

62. The method of claim 60 or 61, further comprising isolating monocytes from the one or more individuals, and incubating the monocytes in a dendritic cell culture medium to derive dendritic cells from the monocytes, and then incubating the dendritic cells with the gene therapy agent.

63. The method of claim 62, wherein the monocytes are CD14+ monocytes.

64. The method of claim 63 or 63, wherein the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to derive dendritic cells from the monocytes.

65. The method of any one of claims 57-64, wherein the innate immune cells are replated prior to incubation with the gene therapy agent of step b).

66. The method of any one of claims 57-65, wherein the gene therapy agent is a viral vector, and wherein the innate immune cells are coupled to the gene therapy agent at a rate of about 1×10 3 About 1×10 5 or about 1×10 4 MOI was determined and incubated together.

67. The method of any one of claims 57-65, wherein the gene therapy agent is a non-viral vector, and wherein the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

68. The method of any one of claims 57-67, wherein the innate immune cells are incubated with the gene therapy agent for about 12 hours to about 36 hours or about 24 hours.

69. The method of any one of claims 57-68, wherein expression of the cytokines in the cytokine signature is increased compared to a suitable control.

70. The method of claim 69, wherein the appropriate control is the expression of the cytokines in a cytokine signature from innate immune cells that have not been incubated with the gene therapy agent, or wherein the appropriate control is the expression of the cytokines in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

71. The method of any one of claims 57-65 and 68-70, wherein the gene therapy agent is a viral vector.

72. The method of claim 66 or 71, wherein the viral vector is an AAV particle.

73. The method of claim 72, wherein the AAV particles comprise AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, AAVrh32.33 capsid, AAV-XL32 capsid, AAV-XL32.1 capsid, AAV LK03 capsid, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV DJ8 capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or a functional variant thereof.

74. The method of claim 73, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

75. The method of any one of claims 72-74, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, AAVrh8 ITRs, AAV9 ITRs, AAV10 ITRs, AAVrh10 ITRs, AAV11 ITRs, or AAV12 ITRs.

76. The method of claim 75, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

77. The method of claim 75, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

78. The method of claim 66 or 71, wherein the viral vector is an adenoviral particle.

79. The method of claim 78, wherein the adenoviral particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad or porcine Ad type 3, or a functional variant thereof.

80. The method of claim 66 or 71, wherein the viral vector is a lentiviral particle.

81. The method of claim 80, wherein the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or a functional variant thereof.

82. The method of claim 66 or 71, wherein the viral vector is a herpes simplex virus (HSV) particle.

83. The method of claim 82, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

84. The method of any one of claims 57-65 and 67-70, wherein the gene therapy agent is a lipid nanoparticle.

85. The method of any one of claims 67-84, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

86. The method of claim 85, wherein the heterologous transgene is operably linked to a promoter.

87. The method of claim 86, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

88. A kit for use in the method of any one of claims 1-87.

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