Dendritic cell assay for natural immunogenicity to gene therapy agents

Abstract

Provided herein is a method for determining the natural immunogenicity of a gene therapy agent in an individual. Specifically, the method involves the use of isolated dendritic cells to detect the natural immunogenicity to a gene therapy agent. Exemplary gene therapy agents include adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, herpes simplex virus (HSV) vectors, or lipid nanoparticles.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 330,241, filed April 12, 2022, which is incorporated by reference in its entirety.

[0002] Electronic Sequence Listing Reference 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.

[0003] The present invention relates to a method for determining the natural immunogenicity to a gene therapy agent using dendritic cells. [Background technology]

[0004] The current success of gene therapy for the treatment of rare genetic diseases relies heavily on adeno-associated viral (AAV) vectors, which offer several attractive features such as tissue-specific tropism, transduction of quiescent cells, and long-term persistence of transgene expression. However, immune responses to AAV vectors pose a major challenge to successful clinical translation. All components of viral vectors (capsid, viral genome, and transgene) trigger immune responses with activation of both innate and adaptive arms of the human immune system. While adaptive immune responses to AAV triggered by B and T cells are relatively well characterized, innate immune activation by AAV is poorly understood. A major challenge in understanding the innate immune response to AAV lies in the poor reproducibility of immune signaling observed in clinical trials to ex vivo settings. What is needed are novel assays that recapitulate the innate immune signatures in several human donors in response to various AAV serotypes.

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

[0006] In some aspects, the invention provides methods of determining natural immunogenicity to a gene therapy agent in an individual, comprising: a) incubating innate immune cells from the individual with the gene therapy agent; and b) analyzing the innate immune cells for changes in expression of one or more cytokines compared to a suitable control, where the changes in expression of the one or more cytokines result in a cytokine signature, where expression of the cytokine signature after incubation with the gene therapy agent is indicative of natural immunogenicity to the gene therapy agent in the individual. 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 of the individual.

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

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

[0009] In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β. In some embodiments, the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β. In some embodiments, the expression of a cytokine in the cytokine signature is increased compared to a suitable control. In some embodiments, a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells that have not been incubated with a gene therapy agent, or a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells prior to incubation with a gene therapy agent.

[0010] In some embodiments, the invention provides a method for determining natural immunogenicity to a viral gene therapy agent in an individual, comprising: a) incubating monocytes from the individual in dendritic cell culture medium under conditions in which the monocytes differentiate into dendritic cells; b) culturing the dendritic cells in a dendritic cell culture medium for about 12 to about 36 hours at about 1×10 3 ~Approx. 1×10 5and c) analyzing the dendritic cells for changes in expression of one or more cytokines compared to a suitable control, the changes in expression of the one or more cytokines resulting in a cytokine signature, the expression of the cytokine signature after incubation with the viral gene therapy agent being indicative of natural immunogenicity to the viral gene therapy agent in the individual, the cytokine signature comprising increased expression of IL6, TNFα, and IL-1β. In some embodiments, 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 dendritic cell culture medium for about 7-8 days to differentiate the monocytes into dendritic cells. In some embodiments, the dendritic cells are obtained from 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 particles are selected from the group consisting of an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 In some embodiments, the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation, or a HBKO mutation. In some embodiments, the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR, or AAV12 ITR. In some embodiments, the one or more ITRs and capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the one or more ITRs and capsid of the AAV particle are derived from different AAV serotypes.

[0012] In some embodiments of the present invention, the viral vector is an adenovirus particle. In some embodiments, the adenovirus particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3, or a functional variant thereof.

[0013] In some embodiments of the 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, Mokara virus, rabies virus, RD114, or a functional variant thereof.

[0014] In some embodiments of the 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 invention provides a method of determining natural immunogenicity to a non-viral gene therapy agent in an individual, comprising: a) incubating monocytes from the individual in dendritic cell culture medium under conditions where 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; and c) analyzing the dendritic cells for changes in expression of one or more cytokines compared to a suitable control, where the changes in expression of the one or more cytokines result in a cytokine signature, where expression of the cytokine signature after incubation with the non-viral gene therapy agent is indicative of natural immunogenicity to the non-viral gene therapy agent in the individual, where the cytokine signature comprises 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 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 invention provides a method of determining a cytokine signature of a gene therapy agent comprising: a) incubating one or more innate immune cells from one or more individuals with the gene therapy agent; b) analyzing the one or more innate immune cells for changes in expression of one or more cytokines compared to a suitable control, wherein the changes in expression of the one or more cytokines in step b) are indicative of a cytokine signature of the gene therapy agent. In some embodiments, the innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells. In some embodiments, the one or more 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 of one or more individuals.

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

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

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

[0021] In some embodiments, the viral vector is an AAV particle. In some embodiments, the AAV particles are selected from the group consisting of an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 In some embodiments, the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation, or a HBKO mutation. In some embodiments, the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR, or AAV12 ITR. In some embodiments, the one or more ITRs and capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the one or more ITRs and capsid of the AAV particle are derived from different AAV serotypes.

[0022] In some embodiments, the viral vector is an adenovirus particle. In some embodiments, the adenovirus particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3, or a functional variant thereof.

[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, Mokara 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]FIG. 1 shows a schematic of an assay for determining the natural immunogenicity of various AAV vectors. [Diagram 2] Cytokine levels from AAV-treated cells as determined by Luminex assay are shown. AAV serotype is indicated on each plot, assayed cytokines are indicated on the left, and donor ID is indicated along the top of each plot. Colors within each plot correspond to changes in regulation compared to uninfected cells and are shown in the lower right panel. [Diagram 3] Cytokine levels from AAV-treated cells as determined by orthogonal MSD assay are shown. AAV serotype is indicated on each plot, assayed cytokines are indicated on the left, and donor ID is indicated along the top of each plot. Colors within each plot correspond to changes in regulation compared to uninfected cells and are shown in the lower right panel. [Figure 4] Experimental schematic for assaying innate immune response after LNP delivery. Peripheral blood mononuclear cells (PBMCs) were isolated from leukopak. CD14+ monocytes were purified from the PBMCs. A cocktail of differentiation factors was added to the monocytes to allow differentiation into dendritic cells, and finally maturation factors were added to obtain mature dendritic cells. The mature dendritic cells were treated with 10ug of LNPs for 24 hours. Cells were then harvested and cytotoxicity and target gene expression were measured using flow cytometry, and media was collected to assess cytokine release. [Diagram 5] We demonstrate that the dendritic cell system of the present disclosure can be used to assess the immunogenicity of LNPs without affecting cell viability. [Figure 6] We demonstrate that the dendritic cell system of the present disclosure can be used to effectively evaluate LNP transduction. Specifically, dendritic cells produced according to the present disclosure were effectively transduced with LNPs encapsulating mRNA. [Figure 7A-7D]Figure 7 shows that mRNA-encapsulated LNPs specifically secrete cytokines compared to cells that were not LNP-treated. Figure 7A shows the results of IP10 secretion. Figure 7B shows the results of MIP1b secretion. Figure 7C shows the results of CXCL9 secretion. Figure 7D shows the results of IL2 secretion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In some aspects, the invention provides a method of determining natural immunogenicity to a gene therapy agent in an individual, comprising: a) incubating innate immune cells from the individual with the gene therapy agent; and b) analyzing the innate immune cells for changes in expression of one or more cytokines compared to a suitable control, where the changes in expression of the one or more cytokines result in a cytokine signature, where expression of the cytokine signature after incubation with the gene therapy agent is indicative of natural immunogenicity to the gene therapy agent in the individual. In some embodiments, the innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells. In some embodiments, the method further comprises isolating monocytes from the individual prior to incubating the dendritic cells with the gene therapy agent, and incubating the monocytes in dendritic cell culture medium to induce dendritic cells from the monocytes. In some embodiments, the cytokine signature comprises one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the method of determining whether an individual has natural immunity to a gene therapy agent is determined prior to administration of the gene therapy agent to the individual. In some embodiments, the method of determining whether an individual has natural immunity to a gene therapy agent is determined prior to administration of the gene therapy agent to the individual, thereby identifying individuals who would benefit from administration of a modulator of a natural immune response either before, during, or after administration of the gene therapy agent.

[0029] General Technology The techniques and procedures described or referenced herein are generally well understood and may be readily understood by those of skill in the art using conventional methodology, e.g., as described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 2004). th ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,2012);Current Protocols in Molecular Biology(FMAusubel, et al.eds.,2003);the series Methods in Enzymology(Academic Press,Inc.);PCR 2:A Practical Approach(MJMacPherson,BDHames and GRTaylor eds.,1995);Antibodies,A Laboratory Manual(Harlow and Lane,eds.,1988);Culture of Animal Cells:A Manual of Basic Technique and Specialized Applications(RIFreshney,6 thed.,J.Wiley and Sons,2010);Oligonucleotide Synthesis(M.J.Gait,ed.,1984);Methods in Molecular Biology,Humana Press;Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,Academic Press,1998);Introduction to Cell and Tissue Culture(J.P.Mather and P.E.Roberts,Plenum Press,1998);Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,J.Wiley and Sons,1993-8);Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.,1996);Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987);PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(J.E.Coligan et al.,eds.,1991);Short Protocols in Molecular Biology(Ausubel et al.,eds.,J.Wiley and Sons,2002);Immunobiology(C.A.Janeway et al.,2004);Antibodies(P.Finch,1997);Antibodies:A Practical Approach(D.Catty.,ed.,IRL Press,1988-1989);Monoclonal Antibodies:A Practical Approach(P.Shepherd and C.Dean,eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., J.B. Lippincott Company, 2011), and are commonly employed using widely available methodologies.

[0030] definition As used herein, the term "IRAK degrading agent" refers to a heterobifunctional compound that binds (fully or partially) to and / or inhibits both IRAK kinase and E3 ligase with measurable affinity, resulting in ubiquitination and subsequent degradation of the IRAK kinase. In certain embodiments, the degrading agent is a DC 50 is 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. As used herein, the term "monovalent" refers to a degradable compound that does not have an E3 ligase binding moiety attached.

[0031] As used herein, the term "inhibitor" with respect to IRAK refers to a compound that binds to and / or inhibits (fully or partially) IRAK kinase with measurable affinity. In certain embodiments, an inhibitor has an IC 50 and / or the binding constant is 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.

[0032] As used herein, the term "modulator" with respect to IRAK refers to a compound that stimulates, retards, inhibits, and / or suppresses (fully or partially) the activity of IRAK kinase.

[0033] As used herein, the term "gene therapy" refers to a therapy in which expression of a nucleic acid (e.g., a gene, mRNA, etc.) in an individual's cells is altered to change the biological properties of the cells. In some examples, gene therapy involves the delivery of an exogenous nucleic acid that is expressed in the individual's cells. In some examples, gene therapy alters (e.g., decreases, inhibits, enhances) the expression of an endogenous gene in the individual's cells. In some examples, gene therapy is an in vivo therapy. In some examples, gene therapy is an ex vivo therapy (e.g., cell therapy).

[0034] As used herein, the term "gene therapy agent" refers to a nucleic acid (e.g., expression construct, miRNA, antisense, shRNA, siRNA) for modifying or manipulating the expression of one or more nucleic acids (e.g., genes, mRNA) in an individual or cell to change the biological properties of a living cell, or a nucleic acid combined with an agent used to deliver the nucleic acid to an individual or cell. Examples of gene therapy agents include, but are not limited to, viral vectors (e.g., adeno-associated virus, adenovirus, lentivirus, herpes simplex virus, baculovirus), bacterial vectors, and non-viral vectors (e.g., lipid nanoparticles (LNPs) that contain a therapeutic nucleic acid and / or encapsulate a therapeutic nucleic acid or plasmid DNA (e.g., close ended DNA) that encodes a therapeutic polypeptide).

[0035] A "vector," as used herein, refers to a recombinant plasmid or virus containing a nucleic acid that is delivered to a host cell either in vitro or in vivo.

[0036] The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically, or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a nucleic acid may contain sugar and phosphate groups (typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a nucleic acid may contain a polymer of synthetic subunits such as phosphoramidates, and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, double-stranded nucleic acid can be obtained from a single-stranded polynucleotide product of chemical synthesis by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase with an appropriate primer.

[0037] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, including, but not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host that produces the protein or by errors due to PCR amplification.

[0038] "Recombinant viral vector" refers to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one (e.g., two) inverted terminal repeats (ITRs).

[0039] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) flanked by at least one (e.g., two) AAV inverted terminal repeat (ITR). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that is infected with a suitable helper virus (or expresses suitable helper functions) and expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When the rAAV vector is integrated into a larger polynucleotide (e.g., into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the rAAV vector can be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. rAAV vectors can take any of a number of forms (such as, but not limited to, plasmids, linear artificial chromosomes), can be complexed with lipids, can be packaged within liposomes, and, in embodiments, can be encapsidated into viral particles (particularly AAV particles). rAAV vectors can be packaged into AAV viral capsids to generate "recombinant adeno-associated viral particles (rAAV particles)."

[0040] "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.

[0041] "Recombinant adenoviral vector" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of adenoviral origin) flanked by at least one adenoviral inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeats (ITR). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell expressing essential adenoviral genes (e.g., E1, E2, E4, etc.) that have been deleted from the recombinant viral genome. When a recombinant viral vector is integrated into a larger polynucleotide (e.g., into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the recombinant viral vector can be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of adenoviral packaging functions. Recombinant viral vectors can take any of a number of forms (e.g., but not limited to, plasmids, linear artificial chromosomes), can be complexed with lipids, encapsulated within liposomes, and can be encapsidated into viral particles (e.g., adenovirus particles). Recombinant viral vectors can be packaged into adenovirus viral capsids to generate "recombinant adenovirus particles."

[0042] "Recombinant lentiviral vector" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of lentiviral origin) flanked by at least one lentiviral long terminal repeat (LTR). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral long terminal repeats (LTR). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell infected with appropriate helper functions. Recombinant lentiviral vectors can be packaged into lentiviral capsids to generate "recombinant lentiviral particles".

[0043] "Recombinant herpes simplex vector (recombinant HSV vector)" refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not of HSV origin) flanked by HSV terminal repeat sequences. Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell infected with appropriate helper functions. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the recombinant viral vector can be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of HSV packaging functions. Recombinant viral vectors can take any of a number of forms (e.g., but not limited to, plasmids, linear artificial chromosomes), can be complexed with lipids, can be packaged in liposomes, and can be encapsidated into viral particles (e.g., HSV particles). Recombinant viral vectors can be packaged into HSV capsids to generate "recombinant herpes simplex viral particles."

[0044] "Solid lipid nanoparticles" (SLN, sLNP) or "lipid nanoparticles" (LNP), as used herein, refer to nanoparticles that are composed of lipids. In some instances, only one phospholipid layer is present, with the majority of the interior of the particle being composed of lipophilic materials. A payload, such as a nucleic acid, may be embedded within. In some instances, the lipid nanoparticle is a liposome that includes a lipid bilayer.

[0045] As used herein, the term "improving," when referring to gene therapy, may refer to the act of boosting, enhancing, prolonging, or otherwise increasing the expression of a therapeutic gene payload of a gene therapy agent. In some embodiments, an improved gene therapy is one in which the expression of a therapeutic gene payload of a gene therapy agent administered with an IRAK modulating agent is increased by more than about any of 10%, 25%, 50%, 75%, or 100% compared to gene therapy administered without an IRAK modulating agent. In some embodiments, an improved gene therapy is one in which the expression time of a therapeutic gene payload of a gene therapy agent administered with an IRAK modulating agent is extended by more than about any of 10%, 25%, 50%, 75%, or 100% compared to gene therapy administered without an IRAK modulating agent. In some examples, the gene therapy is improved by decreasing the immune response (e.g., innate immune response) to the gene therapy agent. In some embodiments, an improved gene therapy is one in which the immune response to the gene therapy agent administered with an IRAK modulating agent is reduced by greater than about any of 10%, 25%, 50%, 75%, or 100% as compared to gene therapy administered without the IRAK modulating agent, in some embodiments, a reduced immune response to the gene therapy agent is measured as a reduced cytokine signature following exposure of immune cells to the gene therapy agent in the presence of an IRAK modulating agent as compared to exposure of immune cells to the gene therapy agent in the absence of the IRAK modulating agent.

[0046] As used herein, the term "modulate," when referring to gene therapy, can refer to the act of changing, altering, fluctuating, improving, or otherwise modifying the presence or activity of a gene therapy agent. For example, modulating an immune response to a gene therapy agent can refer to any act that results in a change, alteration, fluctuating, improving, or otherwise modifying the immune response to a gene therapy agent (e.g., reducing, delaying, and / or eliminating an immune response (e.g., a natural immune response) to a gene therapy agent).

[0047] As used herein, the term "cytokine signature," in reference to an immune response (e.g., an innate immune response) to a gene therapy agent, refers to a change (e.g., an increase, a decrease) in expression of one or more cytokines following exposure of innate immune cells to a gene therapy agent. In some examples, the cytokines of the cytokine signature are specific to a TLR pathway (e.g., the TLR2, TLR3, TLR4, or TLR9 pathway).

[0048] Innate immune cells are white blood cells that mediate natural immunity, including basophils, dendritic cells, eosinophils, Langerhans cells, mast cells, monocytes and macrophages, neutrophils, and NK cells. Different AAV capsids can enter these innate immune cells with different efficiencies, often referred to as transduction efficiencies. Some serotypes, such as AAV1, are efficient at transducing certain immune cells, such as monocytes, while other AAVs, such as AAV6, are efficient at transducing cells, such as dendritic cells (Grimm, D et al., J. Virol., 2008, 82(12):5887-5911). However, all AAVs upon cell entry elicit an immune response. The magnitude of this immune response depends on the AAV serotype and cell type. Once AAV transduces a host immune cell, it binds to immune receptors, such as TLRs (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. 2009; 119(8): 2388-2398; Ashley SN et al., Cell. Immunol. 2019, 346: 103997). Virus activation of these TLRs results in the secretion of cytokines that establish an antiviral state in infected cells and alert neighboring cells. (Carty, M and Bowie, AG, Clin Exp Immunol, 2010, 161(3): 397-406; Lester, SN and Li, K, J Mol Biol. 2014; 426(6): 1246-1264; Fitzgerald, KA and Kagan, JC, Cell, 2020 180(6): 1044-1066).

[0049] These cytokines are also involved in the activation of the adaptive immune system, including B cells and T cells, which produce antibodies and generate cytotoxicity to kill virus-infected cells, respectively. As used herein, the upregulation or downregulation of a particular subset of cytokines is referred to as a "cytokine signature." These cytokine signatures, including one or more (e.g., three or more) cytokines, can be used as predictive markers of disease and treatment success. Examples of cytokine signatures can be found in Zuniga, J et al., Int. J. Infect. Diseases, 2020, 94:4-11, Bergamaschi, C et al., Cell Reports, 2021, 36:109504; Del Valle, DM et al., Nat. Med. 2020, 26:1636-1643.

[0050] "Heterologous" means derived from an entity that is genotypically different from another entity to which it is compared or to 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.

[0051] The term "transgene" refers to a nucleic acid that can be introduced into a cell and transcribed into RNA and, optionally, translated and / or expressed under appropriate conditions. In some aspects, a transgene confers a desired property to the cell into which it is introduced, or otherwise results in a desired therapeutic or diagnostic outcome. In another aspect, a transgene can be transcribed into a molecule that mediates RNA interference, such as a siRNA.

[0052] The terms "genome particles (gp)," "genome equivalents," or "genome copies," when used in reference to viral titer, refer to the number of virions that contain a recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation may be measured by procedures such as those described in the Examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0053] The terms "infectious unit (iu)," "infectious particle," or "replication unit," when used in reference to viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured, for example, by the infectious center assay (also known as the replication center assay) described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0054] The term "transducing unit (tu)" when used with respect to viral titer refers to the number of infectious recombinant AAV vector particles that produce a functional transgene product as measured in the Examples herein or in a functional assay such as those described in, for example, Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0055] "Inverted terminal repeat" or "ITR" sequences are a term well understood in the art and refer to relatively short sequences in inverted orientation found at the ends of viral genomes.

[0056] "AAV inverted terminal repeat (ITR)" sequences, a term well understood in the art, are sequences of approximately 145 nucleotides present at both ends of a naturally occurring single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be in either of two 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 (represented by the A, A', B, B', C, C' and D regions), allowing intrastrand base pairing to occur within this portion of the ITR.

[0057] "Terminal resolution sequence" or "trs" refers to a sequence in the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. The mutated terminal resolution sequence is refractory to cleavage by the AAV rep protein. "AAV helper functions" refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper viruses or helper virus genes that aid in AAV replication and packaging. Other AAV helper functions, such as genotoxic agents, are known in the art.

[0058] "AAV helper functions" refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper viruses or helper virus genes that aid in AAV replication and packaging. Other AAV helper functions, such as genotoxic agents, are known in the art.

[0059] AAV "helper virus" refers to a virus that allows AAV (which is a defective parvovirus) to replicate and be packaged by a host cell. Many such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses, such as vaccinia, and baculoviruses. Adenoviruses encompass many different subgroups, but adenovirus type 5 (Ad5) of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family that are also available from depositories such as the ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.

[0060] "Percent sequence identity" with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid or nucleic acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software programs, including those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, section 7.7.18, Table 7.7.1, and BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A potential alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. For the purposes of this specification, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y (where X is the number of amino acid residues scored as perfect matches by the sequence alignment program in the program alignment of A and B, and Y is the total number of amino acid residues in B). It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A.For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C having or containing a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 x fraction W / Z, where W is the number of nucleotides scored as perfect matches by a sequence alignment program in the program's alignment of C and D, and Z is the total number of nucleotides in D. It will be understood that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, then the % nucleic acid sequence identity of C to D is not equal to the % nucleic acid sequence identity of D to C.

[0061] An "effective amount" of an agent refers to an amount effective at the dosage and for the period of time necessary to achieve a desired prophylactic result. For example, an effective amount of a gene therapy agent refers to an amount effective at the dosage and for the period of time necessary to achieve a desired gene therapy result. In another example, an effective amount of an IRAK modulator can refer to an amount effective at the dosage and for the period of time necessary to achieve a desired improved gene therapy result.

[0062] A "therapeutically effective amount" of a substance / molecule (e.g., a gene therapy agent and / or an IRAK modulating agent) of the invention can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects.

[0063] The term "suitable control," when referring to a cytokine signature, refers to the expression of a cytokine in a cytokine signature from innate immune cells that have not been incubated with a gene therapy agent, or the expression of a cytokine in a cytokine signature from innate immune cells prior to incubation with a gene therapy agent.

[0064] Administration "in combination with," when it relates to a gene therapy agent and a modulator of an innate immune response (e.g., an IRAK modulating agent), includes simultaneous (concurrent), consecutive, or sequential administration in any order of the gene therapy agent and the modulator of an innate immune response (e.g., an IRAK modulating agent).

[0065] The term "concurrently" is used herein to refer to administration of a gene therapy agent and a modulator of an innate immune response (e.g., an IRAK modulator) where at least a portion of the administration overlaps in time. Thus, concurrent administration includes dosing regimens where administration of a gene therapy agent or a modulator of an innate immune response (e.g., an IRAK modulator) continues after administration of the other agent / modulator is discontinued.

[0066] As used herein, "in combination with" refers to the administration of one treatment modality in addition to another. Thus, "in combination with" refers to the administration of one treatment modality (such as a gene therapy agent, or a modulator of the innate immune response (e.g., an IRAK modulator)) to an individual before, during, or after the administration of the other treatment modality.

[0067] An "isolated" molecule (e.g., a nucleic acid or protein) or cell means that the molecule has been identified and separated and / or recovered from a component of the molecule's natural environment.

[0068] Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter per se. For example, a reference to "about X" includes a description of "X."

[0069] As used herein, the singular articles "a," "an," and "the" include plural references unless otherwise indicated.

[0070] It will be understood that aspects and embodiments of the invention described herein include aspects and embodiments that "comprise", "consist" and / or "consist essentially of".

[0071] Cellular assays to determine natural immunogenicity to gene therapy agents In some aspects, the invention provides methods of determining the natural immunogenicity to a gene therapy agent in an individual, which may be directed to any aspect of the gene therapy agent, such as the delivery vehicle (e.g., a viral capsid or lipid nanoparticle), the nucleic acid payload of the gene therapy agent, and / or any other components of the gene therapy agent.

[0072] In some aspects, the invention provides a method of determining natural immunogenicity to a gene therapy agent in an individual, comprising: a) incubating innate immune cells from the individual with the gene therapy agent; and b) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent indicates natural immunogenicity to the 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 blood (e.g., 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 the individual. As used herein, the term "inducing" dendritic cells includes differentiation of cells (e.g., monocytes) to produce dendritic cells. In some embodiments, the method further comprises isolating monocytes from the individual prior to incubating the dendritic cells with the gene therapy agent, and incubating the monocytes in dendritic cell culture medium under conditions that induce (differentiate) dendritic cells from the monocytes.

[0073] In some aspects, the invention provides a method of determining natural immunogenicity to a gene therapy agent in an individual, comprising: a) incubating dendritic cells from the individual with the gene therapy agent; and b) analyzing the dendritic cells for expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the gene therapy agent indicates natural immunogenicity to the 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 prior to incubating the dendritic cells with the gene therapy agent, and incubating the monocytes in dendritic cell culture medium to derive dendritic cells from the monocytes. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0074] In some aspects, the invention provides a method of determining natural immunogenicity to a gene therapy agent in an individual, comprising: a) isolating innate immune cells from the individual; b) incubating the innate immune cells with the gene therapy agent; and c) analyzing the innate immune cells for expression of one or more cytokines, where expression of a cytokine signature after incubation with the gene therapy agent indicates natural immunogenicity to the 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 the individual.

[0075] In some aspects, the invention provides methods of determining natural immunogenicity to a gene therapy agent in an individual, comprising: a) isolating monocytes from the individual, b) incubating the monocytes in dendritic cell culture medium to induce dendritic cells from the monocytes, c) incubating the dendritic cells with the gene therapy agent, and d) analyzing the dendritic cells for expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the gene therapy agent is indicative of natural immunogenicity to the gene therapy agent. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0076] In some aspects, the invention provides a method for determining natural immunogenicity to a viral gene therapy agent in an individual, comprising: a) incubating about 1×10 innate immune cells from the individual for about 24 hours in a culture medium comprising: 4 and b) analyzing the innate immune cells for expression of one or more cytokines, wherein 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 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 the individual.

[0077] In some aspects, the invention provides a method for determining natural immunogenicity to a viral gene therapy agent in an individual, comprising: a) incubating about 1×10 dendritic cells from the individual for about 24 hours in a culture medium comprising: 4and b) analyzing the dendritic cells for expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the viral gene therapy agent indicates natural immunogenicity to 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 prior to incubating the dendritic cells with the gene therapy agent, and incubating the monocytes in dendritic cell culture medium to derive dendritic cells from the monocytes.

[0078] In some aspects, the invention provides a method for determining the natural immunogenicity of a viral gene therapy agent in an individual, comprising: a) obtaining peripheral blood mononuclear cells (PBMCs) from the individual; b) isolating innate immune cells from the PBMCs; c) isolating the innate immune cells from about 1×10 4 and c) analyzing the innate immune cells for expression of one or more cytokines, wherein 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 innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells. In some embodiments, the innate immune cells are dendritic cells, and 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 prior to incubating the dendritic cells with the gene therapy agent, and incubating the monocytes in dendritic cell culture medium to derive dendritic cells from the monocytes.

[0079] In some embodiments, the invention provides a method for determining natural immunogenicity to a viral gene therapy agent in an individual, comprising: a) incubating monocytes from the individual in dendritic cell culture medium under conditions in which the monocytes differentiate into dendritic cells; b) culturing the dendritic cells in a dendritic cell culture medium for about 12 to about 36 hours at about 1×10 3 ~Approx. 1×10 5 and c) analyzing the dendritic cells for an altered expression (e.g., increased, decreased) of one or more cytokines compared to a suitable control, wherein the altered expression of one or more cytokines results in a cytokine signature, wherein expression of the cytokine signature after incubation with the viral gene therapy agent is indicative of natural immunogenicity to the viral gene therapy agent in the individual, the cytokine signature comprising increased expression of IL6, TNFα, and IL-1β. In some embodiments, 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 dendritic cell culture medium for about 5 to about 10 days or about 7 to 8 days to induce dendritic cells from the monocytes. In some embodiments, monocytes are incubated with dendritic cell culture medium for about any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 days to induce dendritic cells from the monocytes. In some embodiments, the dendritic cells are replated prior to incubation with the gene therapy agent in step c). In some embodiments, the dendritic cells are replated in the microwell dish prior to incubation with the gene therapy agent. In some embodiments, the dendritic cells are replated at 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 ~Approx. 1×10 5 , about 5×10 3 ~Approx. 1×10 5 , about 1×104 ~Approx. 1×10 5 , about 5×10 4 ~Approx. 1×10 5 , about 1×10 3 ~Approx. 5×10 4 , about 5×10 3 ~Approx. 5×10 4 , about 1×10 4 ~Approx. 5×10 4 , about 1×10 3 ~Approx. 1×10 4 , about 5×10 3 ~Approx. 1×10 4 , or about 1 × 10 3 ~Approx. 5×10 3 In some embodiments, the dendritic cells are incubated with the viral gene therapy agent at an MOI of any of the following: 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 more than about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 30 hours, about 36 hours, about 12 hours, about 30 hours, about 18 hours, about 30 hours, about 24 hours, about 30 hours, about 12 hours, about 24 hours, about 18 hours, about 24 hours, or about 12 hours, about 18 hours.

[0080] In some embodiments, the invention provides a method of determining natural immunogenicity to a viral gene therapy agent in an individual, comprising: a) obtaining peripheral blood mononuclear cells (PBMCs) from the individual; b) isolating CD14+ monocytes from the PBMCs; c) incubating the monocytes in dendritic cell culture medium for about 7-8 days to induce dendritic cells from the monocytes; d) re-plating the dendritic cells; and e) incubating the dendritic cells in a dendritic cell culture medium for about 24 hours at about 1×10 4and f) analyzing the dendritic cells for changes in expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the viral gene therapy agent is indicative of natural immunogenicity to the viral gene therapy agent. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0081] In some aspects, the invention provides methods of determining natural immunogenicity to a non-viral gene therapy agent in an individual comprising: a) incubating monocytes from the individual in dendritic cell culture medium under conditions where the monocytes differentiate into dendritic cells; b) incubating the dendritic cells with a non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL; and c) analyzing the dendritic cells for an altered expression (e.g., increased, decreased) of one or more cytokines compared to a suitable control, where the altered expression of the one or more cytokines results in a cytokine signature, where expression of the cytokine signature after incubation with the non-viral gene therapy agent is indicative of natural immunogenicity to the non-viral gene therapy agent in the individual, where the cytokine signature comprises 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, monocytes are incubated with dendritic cell culture medium for about 5 to about 10 days or about 7 to 8 days to induce dendritic cells from the monocytes. In some embodiments, monocytes are incubated with dendritic cell culture medium for about any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 days to induce dendritic cells from the monocytes. In some embodiments, dendritic cells are replated prior to incubation with the non-viral gene therapy agent in step c). In some embodiments, dendritic cells are replated in microwell dishes prior to incubation with the gene therapy agent. In some embodiments, the innate immune cells are incubated with the non-viral gene therapy agent at a concentration of about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 100 ng / mL, about 100 ng / mL to about 1 μg / mL, about 1 μg / mL to about 10 μg / mL, about 10 μg / mL to about 100 μg / mL, or about 100 μg / mL to about 1 mg / mL. 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 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.

[0082] In some aspects, the invention provides a method of determining natural immunogenicity to a non-viral gene therapy agent in an individual, 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; and b) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the non-viral gene therapy agent indicates natural immunogenicity to the non-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 the individual.

[0083] In some aspects, the invention provides a method of determining natural immunogenicity to a non-viral gene therapy agent in an individual, 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; and b) analyzing the dendritic cells for expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the non-viral gene therapy agent indicates natural immunogenicity to the non-viral gene therapy agent. In some embodiments, dendritic cells are isolated from peripheral blood mononuclear cells from the individual. In some embodiments, 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 prior to incubating the dendritic cells with the gene therapy agent, and incubating the monocytes in dendritic cell culture medium to derive dendritic cells from the monocytes.

[0084] In some aspects, the invention provides a method of determining natural immunogenicity to a non-viral gene therapy agent in an individual, 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 a non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL for about 24 hours; and c) analyzing the innate immune cells for expression of one or more cytokines, wherein expression of a cytokine signature after incubation with the non-viral gene therapy agent is indicative of natural immunogenicity to the non-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 dendritic cells. In some embodiments, the dendritic cells are derived from monocytes (e.g., CD14+ monocytes) of the individual.

[0085] In some aspects, the invention provides methods of determining natural immunogenicity to a non-viral gene therapy agent (e.g., LNP) in an individual, comprising: a) obtaining peripheral blood mononuclear cells (PBMCs) from the individual; b) isolating CD14+ monocytes from the PBMCs; c) incubating the monocytes in dendritic cell culture medium for about 7-8 days to induce dendritic cells from the monocytes; d) reseeding the dendritic cells; e) incubating the dendritic cells with a non-viral gene therapy agent at a concentration of about 1 ng / mL to about 1 mg / mL for about 24 hours; and f) analyzing the dendritic cells for expression of one or more cytokines, wherein expression of a cytokine signature following incubation with the gene therapy agent is indicative of natural immunogenicity to the gene therapy agent. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0086] In some embodiments, monocytes are isolated from PBMCs from an individual. In some embodiments, the monocytes are CD14+ monocytes. In some embodiments, the monocytes are incubated with dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to induce dendritic cells from the monocytes. In some embodiments, the monocytes are incubated with dendritic cell culture medium for any of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 days to induce dendritic cells from the monocytes. In some embodiments, the dendritic cells are replated prior to incubation with the gene therapy agent in step c). In some embodiments, the dendritic cells are replated in a microwell dish prior to incubation with the gene therapy agent.

[0087] In some embodiments, cells (e.g., PBMCs, monocytes, and / or dendritic cells) are isolated from a sample obtained from an individual or multiple individuals. As used herein, a "sample" obtained from an individual includes any suitable sample (e.g., blood, urine, and / or tissue from one or more individuals) that can be used to obtain PBMCs, monocytes, and / or dendritic cells. In some embodiments, cells are obtained from the blood of an individual (e.g., an individual in need of treatment (or being treated) with a gene therapy agent). In some embodiments, PBMCs are isolated from a leukopak. In some embodiments, PBMCs are isolated from blood using a Ficoll gradient. In some embodiments, the buffy coat, which contains white blood cells and platelets, is collected from the Ficoll gradient. In some embodiments, PBMCs are washed before culture. In some embodiments, PBMCs are washed 1, 2, 3, 4, 5, or more than 5 times before culture. In some embodiments, cells are washed with phosphate buffered saline before culture. In some embodiments, the PBS further comprises Fetal Bovine Serum (FBS) or Fetal Calf Serum (FCS) prior to culture. In some embodiments, the FBC and / or FCS are added to the PBS at a final concentration of about 0.1% to about 10% (v / v). In some embodiments, the FBC and / or FCS are added to the PBS at a final concentration of 1% (v / v).

[0088] In some embodiments of the invention, monocytes are isolated from PBMCs. In some embodiments, CD14+ monocytes are isolated from PBMCs. In some embodiments, monocytes (e.g., CD14+ monocytes) are isolated from PBMCs by affinity purification. In some embodiments, CD14+ monocytes are isolated from PBMCs using an antibody that specifically binds to CD14. In some embodiments, the anti-CD14 antibody is immobilized on a solid support, such as a bead or a resin. In some embodiments, the CD14+ antibody is purified from PBMCs using an anti-CD14 antibody immobilized on magnetic beads. In some embodiments, the magnetic beads are CD14 MicroBeads (Milteny Biotech).

[0089] In some embodiments of the invention, monocytes purified from PBMCs (e.g., CD14+ monocytes) 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, dendritic cells are derived from monocytes by incubating the monocytes in ImmunoCult™ DC Differentiation Medium for about 5 days. In some embodiments, ImmunoCult™ DC Differentiation Medium further comprises ImmunoCult™ DC Differentiation Nutritional Supplement. In some embodiments, ImmunoCult™ DC Differentiation Nutritional Supplement is added to the culture after about 5 days. In some embodiments, differentiated dendritic cells are harvested on about day 7 and used in the assays of the invention.

[0090] In some embodiments, innate immune cells (e.g., dendritic cells, monocytes, macrophages, or NK cells) are administered at a concentration of about 1×10 3 ~Approx. 1×10 5 Or about 1 x 10 4 In some embodiments, the innate immune cells are incubated with the viral gene therapy agent at an MOI of about 1 x 10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×105 , or 5 × 10 5 The cells are incubated with the gene therapy agent at an MOI of less than one of

[0091] 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.

[0092] In some embodiments, natural immune cells (e.g., dendritic cells, monocytes, macrophages, or NK cells) are incubated with the gene therapy agent for about 12 to about 36 hours or about 24 hours. In some embodiments, dendritic cells are incubated with the gene therapy agent for about 6 to about 48 hours, about 6 to about 36 hours, about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, about 12 to about 48 hours, about 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, about 18 to about 48 hours, about 18 to about 36 hours, about 18 to about 24 hours, about 24 to about 48 hours, about 24 to about 36 hours, or about 36 to about 48 hours.

[0093] In some embodiments, a cytokine signature is determined for a particular immune cell (e.g., dendritic cell, monocyte, macrophage, NK cell, etc.) from an individual or multiple individuals by contacting the gene therapy agent with the particular immune cell and determining a change (e.g., increase / decrease) in expression of one or more cytokines associated with an innate immune response, where a commonality in the change in expression (e.g., increase or decrease in expression) of the one or more cytokines indicates the presence of a cytokine signature. In some embodiments, the cytokine associated with the innate immune response is associated with the toll-like receptor (TLR) pathway (e.g., the TLR2 pathway, the TLR3 pathway, the TLR4 pathway, or the TLR9 pathway). In some embodiments, the cytokine signature includes a change in expression of more than any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cytokines. In some embodiments, the multiple individuals include more than any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 individuals. In some embodiments, the commonality of altered expression includes similar alterations in expression levels of cytokines in innate immune cells in greater than about 25%, 50%, 75%, or 90% of individuals in a plurality of individuals.

[0094] In some embodiments, the invention provides a method of determining a cytokine signature of a gene therapy agent comprising: a) incubating one or more innate immune cells (e.g., dendritic cells, monocytes, macrophages, NK cells, etc.) from one or more individuals with the gene therapy agent; b) analyzing the one or more innate immune cells for a change (e.g., increase / decrease) in expression of one or more cytokines compared to a suitable control, where the change in expression of the one or more cytokines in step b) is indicative of a cytokine signature of the gene therapy agent. In some embodiments, the innate immune cells are obtained from a blood sample from one or more individuals. In some embodiments, the innate immune cells are obtained from PBMCs from one or more individuals. In some embodiments, the gene therapy agent is a viral particle or lipid nanoparticle. In some embodiments, the gene therapy agent is an adeno-associated virus (AAV) particle, an adenovirus particle, a lentivirus particle, or a herpes simplex virus (HAV) particle. In some embodiments, the gene therapy agent is a lipid nanoparticle or a liposome.

[0095] In some embodiments, the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0096] In some embodiments, the cytokine signature comprises increased expression of one or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of two or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of three or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, and IL6. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, and TNFα. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, IL6, and TNFα.

[0097] In some embodiments, the innate immune cells are dendritic cells and the cytokine signature comprises increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0098] In some embodiments, the innate immune cells are dendritic cells and the cytokine signature comprises increased expression of one or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of two or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of three or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, and IL6. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, and TNFα. In some embodiments, this cytokine signature includes increased expression of IP10, MIP1b, CXCL9, IL2, IL6, and TNFα.

[0099] In some embodiments, the expression of cytokines in the cytokine signature is increased compared to the expression of cytokines in a cytokine signature from innate immune cells incubated in the absence of the gene therapy agent or is increased compared to the expression of cytokines in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent, the cytokine signature comprising increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β. In some embodiments, an increase in expression of any one of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or greater than 100% identifies an individual for treatment with a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator).

[0100] In some embodiments, the expression of cytokines in the cytokine signature is increased compared to the expression of cytokines in a cytokine signature from innate immune cells incubated in the absence of the gene therapy agent or is increased compared to the expression of cytokines in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent, the cytokine signature comprising increased expression of one or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of two or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of three or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, and IL6. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, and TNFα. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, IL6, and TNFα. In some embodiments, an increase in expression of any one of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or greater than 100% identifies an individual eligible for treatment with a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator).

[0101] In some embodiments, the expression of cytokines in the cytokine signature is increased compared to the expression of cytokines in a cytokine signature from dendritic cells incubated in the absence of the gene therapy agent or is increased compared to the expression of cytokines in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent, the cytokine signature comprising increased expression of one or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of two or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of three or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of four or more of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, IL-1β, MCP1, and MIP-1α. In some embodiments, the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β. In some embodiments, an increase in expression of any one of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or greater than 100% identifies an individual for treatment with a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator).

[0102] In some embodiments, the expression of cytokines in the cytokine signature is increased compared to the expression of cytokines in a cytokine signature from dendritic cells incubated in the absence of the gene therapy agent or is increased compared to the expression of cytokines in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent, the cytokine signature comprising increased expression of one or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of two or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of three or more of IP10, MIP1b, CXCL9, and IL2. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, and IL6. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, and TNFα. In some embodiments, the cytokine signature comprises increased expression of IP10, MIP1b, CXCL9, IL2, IL6, and TNFα. In some embodiments, an increase in expression of any one of about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, or greater than 100% identifies an individual eligible for treatment with a gene therapy agent and an innate immune response modulator (e.g., an IRAK modulator).

[0103] Gene Therapy In some aspects, the invention provides methods for determining the natural immunogenicity to a gene therapy agent in an individual improved gene therapy, for example, by identifying individuals to whom 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), simultaneously). As used herein, "modulating" the innate immune response refers to, for example, stimulating, delaying, suppressing, and / or inhibiting the innate immune response, partially or completely. In some embodiments, an IRAK modulating agent is used to inhibit the innate immune response in an individual. In some embodiments, the gene therapy agent is a viral particle or lipid nanoparticle. In some embodiments, the gene therapy agent is an adeno-associated virus (AAV) particle, an adenovirus particle, a lentivirus particle, or a herpes simplex virus (HAV) particle. In some embodiments, the gene therapy agent is a lipid nanoparticle or liposome.

[0104] AAV In some embodiments, the present invention provides a method for determining the natural immunogenicity of an individual to AAV particles. In AAV particles for gene therapy, a recombinant AAV (rAAV) genome encoding a heterologous nucleic acid (e.g., a therapeutic transgene) is encapsidated in an AAV capsid. In some embodiments, the viral genome comprises the heterologous nucleic acid and / or one or more of the following components operably linked in the direction of transcription: a control sequence including a transcription start sequence and a stop sequence, thereby forming an expression cassette.

[0105] In some embodiments, the rAAV genome comprises one or more AAV inverted terminal repeat (ITR) sequences (typically two AAV ITR sequences). For example, the expression cassette can be flanked at the 5' and 3' ends by at least one functional AAV ITR sequence. By "functional AAV ITR sequence" is meant that the ITR sequence functions as intended for AAV virion rescue, replication, and packaging. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein by reference in their entirety. To implement some aspects of the present invention, the recombinant viral genome comprises at least all of the AAV sequences essential for encapsidation into AAV capsids and the physical structure for infection by AAV particles. The AAV ITRs for use in the vectors of the present invention need not have wild-type nucleotide sequences (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801) and may be modified by nucleotide insertion, deletion, or substitution, or may be derived from any of several AAV serotypes. More than 40 serotypes of AAV are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is contemplated within the scope of the present invention.In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, caprine AAV, bovine AAV, or murine AAV ITR, or the like. In some embodiments, the nucleic acid in the AAV (e.g., rAAV vector) comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, caprine AAV, bovine AAV, or murine AAV ITR, or the like. In some embodiments, the AAV particle comprises an AAV vector encoding a heterologous transgene flanked by one or more AAV ITRs.

[0106] In some embodiments, the AAV particles are selected from the group consisting of an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 The rAAV particles comprise capsid proteins selected from E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid rAAV2 / HBoV1 (chimeric AAV / human bocavirus virus 1), AAV2HBKO capsid, AAVPHP.B capsid, or AAVPHP.eB capsid, or functional variants. A "functional variant" of an AAV capsid means that the variant capsid is capable of packaging an AAV genome to generate infectious AAV virions. In further embodiments, the rAAV particles comprise capsid proteins of an AAV serotype from clades A-F.

[0107] In some aspects, the present invention provides AAV particles comprising a recombinant self-complementary genome (e.g., a self-complementary AAV vector or a self-complementary AAV vector). AAV viral particles having self-complementary vector genomes and methods of using self-complementary rAAV genomes are described in U.S. Patent Nos. 6,596,535; 7,125,717; 7,465,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111, each of which is incorporated herein by reference in its entirety. AAV particles containing self-complementary genomes rapidly form double-stranded DNA molecules with their partially complementary sequences (e.g., complementary to the coding and non-coding strands of a heterologous nucleic acid). In some embodiments, the vector comprises a first nucleic acid sequence that encodes a heterologous nucleic acid and a second nucleic acid sequence that encodes the complement of this nucleic acid, where the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.

[0108] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are linked by a mutated ITR (e.g., a right ITR). In some embodiments, this ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO:). The mutated ITR comprises a deletion of the D region, which contains the terminal resolution sequence. As a result, upon replication of the rAAV genome, the rep protein does not cleave the viral genome at the mutated ITR, and thus a recombinant viral genome is packaged into the viral capsid, which comprises, in 5' to 3' order: the AAV ITR, the first heterologous polynucleotide sequence including the regulatory sequence, the mutated AAV ITR, the second heterologous polynucleotide in the opposite orientation to the first heterologous polynucleotide, and the third AAV ITR.

[0109] Various AAV serotypes are used to optimize the transduction of specific target cells or target specific cell types within a specific target tissue (e.g., diseased tissue). AAV particles can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes. For example, AAV particles can contain one or more ITRs and capsid from the same AAV serotype, or AAV particles can contain one or more ITRs from a different AAV serotype from the capsid of this AAV particle.

[0110] In some embodiments, the AAV capsid comprises a mutation, e.g., the capsid comprises a mutant capsid protein. In some embodiments, the mutation is a tyrosine mutation or a heparin-binding mutation. In some embodiments, the mutant capsid protein maintains the ability to form an AAV capsid. In some embodiments, the AAV particle comprises an AAV2 or AAV5 tyrosine mutant capsid (see, e.g., Zhong L. et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832), e.g., a mutation at Y444 or Y730 (numbering according to AAV2). In further embodiments, the AAV particle comprises a capsid protein of an AAV serotype from clades A-F (Gao, et al., J. Virol. 2004, 78(12):6381).

[0111] Numerous methods for the production of AAV particles for gene therapy are known in the art, including: transfection, stable cell line production, and infectious hybrid virus production systems, such as adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J. E. et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids (Urabe, M. et al., (2002) Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet. 20(R1):R2-R6). All AAV production cultures for the production of AAV particles require: 1) a suitable host cell, 2) suitable helper virus functions, 3) AAV rep and cap genes and gene products, 4) nucleic acid flanking at least one AAV ITR sequence (e.g., therapeutic nucleic acid), and 5) suitable media and media components to support AAV production. In some embodiments, the suitable host cell is a primate host cell. In some embodiments, the suitable host cell is a human-derived cell line, such as HeLa, A549, 293, or Perc.6 cells. In some embodiments, the suitable helper virus functions are provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpes virus (HSV), baculovirus, or a plasmid construct that provides helper functions. In some embodiments, the AAV rep and cap gene products can be derived from any AAV serotype. Generally, but not necessarily, the AAV rep gene product is serotype identical to the ITRs of the rAAV genome, so long as the rep gene product can function to replicate and package the rAAV genome. AAV particles can be produced using suitable media known in the art. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, AAV helper functions are provided by baculovirus and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cell).

[0112] One method for producing AAV particles is the triple transfection method. Briefly, a plasmid containing rep and capsid genes can be transfected (e.g., using calcium phosphate method) into a cell line (e.g., HEK-293 cells) together with a helper adenovirus plasmid, and the virus can be harvested and optionally purified. Thus, in some embodiments, AAV particles are produced by triple transfection of nucleic acid encoding an AAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus function into a host cell, and transfection of these nucleic acids into the host cell generates a host cell capable of producing AAV particles.

[0113] In some embodiments, AAV particles can be manufactured by a producer cell line method (see Martin et al., (2013) Human Gene Therapy Methods 24:253-269; US PG Publication No. US2004 / 0224411; and Liu, XL et al. (1999) Gene Ther. 6:293-299). Briefly, a cell line (e.g., HeLa, 293, A549, or Perc.6 cell line) can be stably transfected with a plasmid containing a vector genome including a rep gene, a capsid gene, and a promoter heterologous nucleic acid sequence. The cell line can be screened to select a lead clone for AAV production, which can then be expanded into a production bioreactor and infected with a helper virus (e.g., adenovirus or HSV) to initiate AAV production. The virus can then be harvested, the adenovirus can be inactivated (e.g., by heat) and / or removed, and the AAV particles can be purified. Thus, in some embodiments, the AAV particles were produced by a producer cell line that contains one or more of nucleic acid encoding the rAAV genome, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus functions.

[0114] In some embodiments, the nucleic acid encoding the AAV rep and cap genes and / or the AAV viral genome is stably maintained in the producer cell line. In some embodiments, the nucleic acid encoding the AAV rep and cap genes and / or the rAAV genome is introduced into the cell line on one or more plasmids to generate the producer cell line. In some embodiments, the AAV rep, AAV cap, and AAV genome are introduced into the cell on the same plasmid. In other embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell on different plasmids. In some embodiments, a cell line stably transfected with a plasmid maintains the plasmid over multiple passages of the cell line (e.g., 5, 10, 20, 30, 40, 50, or more than 50 cell passages). For example, the plasmid may replicate when the cell replicates, or the plasmid may integrate into the cell genome. A variety of sequences have been identified that allow plasmids to replicate autonomously in cells (e.g., human cells) (see, e.g., Krysan, PJ et al. (1989) Mol. Cell Biol. 9:1026-1033). In some embodiments, a plasmid may contain a selectable marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Selectable markers commonly used in mammalian cells include, but are not limited to, blastitidine, G418, hygromycin B, zeocin, puromycin, and derivatives thereof. Methods for introducing nucleic acids into cells are known in the art and include, but are not limited to, viral transduction, cationic transfection (e.g., cationic transfection using cationic polymers such as DEAE-dextran, or cationic lipids such as lipofectamine), calcium phosphate transfection, microinjection, particle bombardment, electroporation, and nanoparticle transfection (for details, see, e.g., Kim, T K and Eberwine, J H (2010) Anal. Bioanal. Chem. 397:3173-3178).

[0115] In some embodiments, the producer cell line is derived from a primate cell line (e.g., a non-human primate cell line, such as a Vero cell line or a FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the producer cell line is derived from a HeLa cell, a 293 cell, an A549 cell, or a PERC.6® (Crucell) cell. For example, the cell line is a HeLa cell line, a 293 cell line, an A549 cell line, or a PERC.6® (Crucell) cell line, or a derivative thereof, prior to introducing and / or stably maintaining / integrating nucleic acids encoding the AAV rep and cap genes and / or the rAAV genome into the cell line to generate the producer cell line.

[0116] In some embodiments, the production cell line is adapted to grow in suspension. As is known in the art, anchorage-dependent cells typically cannot grow in suspension without a substrate such as microcarrier beads. Adapting a cell line to grow in suspension can include, for example, growing the cell line in a spinner culture using agitating paddles, using a culture medium lacking calcium and magnesium ions (and optionally an antifoam agent) to prevent clumping, using a culture vessel coated with a siliconizing compound, and selecting cells in the culture (but not in large clumps or on the sides of the vessel) at each passage.

[0117] The AAV particles of the invention can be recovered from an AAV production culture by lysis of the host cells of the production culture, or by recovery of spent medium from the production culture, as described in more detail in U.S. Patent No. 6,566,118, where the cells are cultured under conditions known in the art to cause release of AAV particles from intact cells into the medium. Methods suitable 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.

[0118] In a further embodiment, the AAV particles are purified. The term "purified" as used herein includes preparations of AAV particles that are devoid of at least some of the other components that may also be present in the location where the AAV particles naturally occur or are initially prepared. Thus, for example, isolated AAV particles can be prepared using purification techniques to enrich the AAV particles from a source mixture, such as a culture lysate or a production culture supernatant. Enrichment can be measured in a variety of ways, for example, by the proportion or infectivity of DNase-resistant particles (DRP) or genome copies (gc) present in the solution, or in relation to other potential interfering substances present in the source mixture (e.g., contaminants such as production culture contaminants, or in-process contaminants such as helper viruses, medium components, and the like).

[0119] In some embodiments, the AAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters, such as a grade DOHC Millipore Millistak+HC Pod Filter, a grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 μm Filter Opticap XL1O Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or larger known in the art.

[0120] In some embodiments, harvests of AAV production cultures are further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase® digestion is performed under standard conditions known in the art (e.g., final concentrations of 1-2.5 units / ml of Benzonase® at temperatures ranging from ambient to 37° C. for periods ranging from 30 minutes to several hours).

[0121] AAV particles may be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate AAV particles; AAV capture by apatite chromatography; heat inactivation of helper virus; AAV capture by hydrophobic interaction chromatography; buffer exchange by size-exclusion chromatography (SEC); nanofiltration; and AAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps may be used alone or in various combinations or in various orders. In some embodiments, the method includes all steps in the order described below. Methods for purifying AAV particles are found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948; and WO 2010 / 148143.

[0122] Adenovirus In some embodiments, the present invention provides a method for determining natural immunity to adenoviral particles in an individual. Adenoviral vectors for gene therapy are typically adenoviral particles with a recombinant adenovirus (rAd) genome that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of adenoviral origin) between two adenoviral ITRs that are encapsidated into the adenoviral capsid. In some embodiments, the heterologous sequences encode a therapeutic transgene. In some embodiments, the rAd genome lacks or contains a defective copy of one or more E1 genes that renders adenoviral replication dysfunctional. Adenoviruses contain a linear, double-stranded DNA genome within a large (approximately 950 Å), non-enveloped, icosahedral capsid. Adenoviruses have large genomes that can incorporate more than 30 kb of heterologous sequences (e.g., in place of the E1 and / or E3 regions), making them uniquely suitable for use with larger heterologous genes. Adenoviruses are also known to infect dividing and non-dividing cells, but do not naturally integrate into the host genome (although hybrid variants may have this ability). In some embodiments, the adenovirus vector may be a first generation adenovirus vector with a heterologous sequence in place of E1. In some embodiments, the adenovirus vector may be a second generation adenovirus vector with additional mutations or deletions in E2A, E2B, and / or E4. In some embodiments, the adenovirus vector may be a third generation or disrupted adenovirus vector that lacks all viral coding genes, retains only the ITRs and packaging signal, and requires a helper adenovirus in trans for replication and packaging. Adenovirus particles have been studied for use as vectors for transient transfection of mammalian cells and as vectors for gene therapy.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.

[0123] In some embodiments, the adenoviral particle comprises a rAd genome comprising a therapeutic transgene. The use of any adenoviral serotype is considered within the scope of the present invention. In some embodiments, the adenoviral particle is derived from an adenovirus serotype, including but not limited to AdHu2, AdHu3, AdHu4, AdHu5, AdHu7, AdHu11, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, and porcine Ad3. The adenoviral particle also comprises capsid proteins. In some embodiments, the adenoviral particle comprises one or more foreign viral capsid proteins. Such combinations may be referred to as pseudotyped adenoviral particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped adenoviral particles is derived from a foreign virus or another adenovirus serotype. In some embodiments, the foreign viral capsid protein is derived from, but not limited to, reovirus type 3. Examples of vector and capsid protein combinations used in pseudotyped adenoviral particles can be found in the following references (Tatsis, N. et al. (2004) Mol. Ther. 10(4):616-629, and Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4):307-320). Different adenoviral serotypes can be used to optimize transduction of specific target cells or target specific cell types within a particular target tissue (e.g., diseased tissue). Tissues or cells targeted by specific adenovirus serotypes include, but are not limited to, lung (e.g., HuAd3), spleen and liver (e.g., HuAd37), smooth muscle, synovial cells, dendritic cells, cardiovascular cells, tumor cell lines (e.g., HuAd11), and dendritic cells (e.g., HuAd5 pseudotyped with reovirus type 3, HuAd30, or HuAd35).For further description, see Ahi, Y. et al. (2011) Curr. Gene Ther. 11(4):307-320, Kay, M. et al. (2001) Nat. Med. 7(1):33-40, and Tatsis, N. et al. (2004) Mol. Ther. 10(4):616-629.

[0124] Numerous methods for the production of adenoviral particles are known in the art. For example, in the case of a disrupted 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 the packaging signal, and both genomes can be transfected into a packaging cell line expressing a recombinase (e.g., using the Cre / loxP system), so that the adenoviral vector of interest is packaged more efficiently than the helper adenovirus (see, e.g., Alba, R. et al. (2005) Gene Ther. 12 Suppl 1:S18-27). The adenoviral vector can be harvested and purified using standard methods, such as those described herein.

[0125] Lentivirus In some embodiments, the present invention provides a method for determining the natural immunogenicity to lentiviral particles in an individual. A lentiviral vector for gene therapy is typically a lentiviral particle having a recombinant lentiviral genome that includes one or more heterologous sequences (i.e., nucleic acid sequences that are not of lentiviral origin) between two long terminal repeats (LTRs). In some embodiments, the heterologous sequence encodes a therapeutic transgene. Lentiviruses are positive-sense ssRNA retroviruses with a genome of about 10 kb. Lentiviruses integrate into the genome of dividing and non-dividing cells. Lentiviral particles can be produced, for example, by transfecting multiple plasmids (typically the lentiviral genome and genes required for replication and / or packaging are separated to prevent viral replication) into a packaging cell line, which packages the modified lentiviral genome into lentiviral particles. In some embodiments, lentiviral particles can refer to a first generation vector that lacks envelope proteins. In some embodiments, lentiviral particles can refer to a second generation vector that lacks all genes except the gag / pol and tat / rev regions. In some embodiments, lentiviral particles may refer to third generation vectors that contain only the endogenous rev, gag, and pol genes and have chimeric LTRs for transduction without the tat gene (see Dull, T. et al. (1998) J. Virol. 72:8463-71). For further explanation, see Durand, S. and Cimarelli, A. (2011) Viruses 3:132-59.

[0126] The use of any lentiviral vector is considered within the scope of the present invention. In some embodiments, the lentiviral vector is derived from a lentivirus, including but not limited to human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), visna virus (VV), and caprine arthritis encephalitis virus (CAEV). The lentiviral particle also comprises a capsid protein. In some embodiments, the lentiviral particle comprises one or more foreign viral capsid proteins. Such combinations may be referred to as pseudotyped lentiviral particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped lentiviral particle is derived from a foreign virus. In some embodiments, the foreign viral capsid protein used in the pseudotyped lentiviral particle is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with a broad range of cellular receptors, providing broad tissue tropism to pseudotyped lentiviral particles, and is thought to provide greater stability to pseudotyped lentiviral particles.In other embodiments, the exogenous viral capsid protein is derived from, but is 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), Jaagsiekte sheep retrovirus (JSRV), Moloney virus, or the like. Murine leukemia virus (MLV), gibbon ape leukemia virus (GALV), feline endogenous retrovirus (RD114), human T-lymphotropic virus 1 (HTLV-1), human foamy virus, Maedi-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 multiple nucleopolyhedro virus (AcMNPV). Examples of vector and capsid protein combinations used in pseudotyped lentiviral particles can be found, for example, in Cronin, J. et al. (2005). Curr. Gene Ther. 5(4):387-398. Different pseudotyped lentiviral particles can be used to optimize transduction of specific target cells or to target specific cell types within a particular target tissue (eg, diseased tissue).For example, tissues targeted with specific pseudotyped lentiviral particles include, but are not limited to, the 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 Mokola proteins), retina (e.g., pseudotyped with VSV-G or Mokola proteins), monocytes or muscle (e.g., pseudotyped with Mokola or Ebola proteins), hematopoietic system (e.g., pseudotyped with RD114 or GALV proteins), or cancer cells (e.g., pseudotyped with GALV or LCMV proteins). For further description, see Cronin, J. et al. (2005). Curr. Gene Ther. 5(4):387-398, and Kay, M. et al. (2001) Nat. Med. 7(1):33-40.

[0127] Numerous methods for the production of lentiviral particles are known in the art. For example, in the case of third generation lentiviral vectors, a vector containing the recombinant lentiviral genome of interest and gag and pol genes can be co-transfected with a vector containing the rev gene into a packaging cell line (e.g., 293 cell line). 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. 72: 8463-71). The lentiviral vector can be collected and purified using the methods described herein (e.g., Segura MM, et al., (2013) Expert Opin Biol Ther. 13 (7): 987-1011).

[0128] HSV In some embodiments, the present invention provides a method for determining the natural immunogenicity of an individual to HSV particles. HSV vectors for gene therapy are typically HSV particles with a recombinant HSV genome that includes one or more heterologous sequences (i.e., nucleic acid sequences that are not of HSV origin) between two terminal repeats (TRs). In some embodiments, the heterologous sequence encodes a therapeutic transgene. HSV is an enveloped, double-stranded DNA virus with a genome of about 152 kb. Advantageously, about half of the genes are non-essential and can be deleted to accommodate the heterologous sequence. HSV particles infect non-dividing cells. In addition, HSV particles naturally establish latency in neurons, travel by retrograde transport, and can be transferred across synapses, making them advantageous for transfection of neurons and / or gene therapy approaches involving the nervous system. In some embodiments, HSV particles can be replication-defective or replication-competent (e.g., capable of a single replication cycle by inactivation of one or more late genes). For further explanation, see Manservigi, R. et al. (2010) Open Virol. J. 4:123-56.

[0129] In some embodiments, the HSV particle comprises a recombinant HSV genome that includes the transgene. The use of any HSV vector is considered within the scope of the present invention. In some embodiments, the HSV vector is derived from an HSV serotype, including but not limited to HSV-1 and HSV-2. The HSV particle also comprises a capsid protein. In some embodiments, the HSV particle comprises one or more foreign viral capsid proteins. Such combinations may be referred to as pseudotyped HSV particles. In some embodiments, the foreign viral capsid protein used in the pseudotyped HSV particle is derived from a foreign virus or another HSV serotype. In some embodiments, the foreign viral capsid protein used in the pseudotyped HSV particle is vesicular stomatitis virus glycoprotein (VSV-GP). VSV-GP interacts with a wide range of cellular receptors, providing a wide range of tissue tropism for the pseudotyped HSV particle. In addition, VSV-GP is believed to provide a higher stability for the pseudotyped HSV particle. In other embodiments, the foreign viral capsid protein may be derived from various HSV serotypes. For example, HSV-1 vectors may contain one or more HSV-2 capsid proteins. Different HSV serotypes may be used to optimize transduction of specific target cells or target specific cell types within a particular target tissue (e.g., diseased tissue). Tissues or cells targeted by specific adenovirus serotypes include, but are not limited to, the central nervous system and neurons (e.g., HSV-1). For further description, see Manservigi, R. et al. (2010) Open Virol J 4:123-156, Kay, M. et al. (2001) Nat. Med. 7(1):33-40, and Meignier, B. et al. (1987) J. Infect. Dis. 155(5):921-930.

[0130] Numerous methods for the production of HSV particles are known in the art. HSV vectors can be harvested and purified using standard methods, such as those described herein. For example, in the case of replication-defective HSV vectors, the HSV genome of interest, which lacks all of the immediate early (IE) genes, can be transfected into a complementing cell line that provides genes necessary for virus production, such as ICP4, ICP27, and ICP0 (see, for example, Samaniego, LA et al. (1998) J. Virol. 72: 3307-20). HSV vectors can be harvested and purified using methods described (e.g., Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144: 63-79).

[0131] Non-viral gene therapy In some embodiments, the present invention provides a method for determining the innate immune response to a non-viral gene therapy agent. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed to a delivery system. For example, the vector can be complexed to a lipid (e.g., a cationic lipid or a neutral lipid), a liposome, a polycation, a lipid nanoparticle, or an agent that enhances cellular uptake of the nucleic acid. The nucleic acid can be complexed to an agent suitable for any of the delivery methods described herein. In some embodiments, the nucleic acid encodes a therapeutic transgene.

[0132] Lipid nanoparticles for gene therapy typically contain a vector genome encapsulated in a lipid particle or complexed with a lipid. In some embodiments, the heterologous sequence encodes a therapeutic transgene. In some embodiments, the vector genome is formulated in a lipoplex nanoparticle or liposome. In some embodiments, the lipoplex nanoparticle formulation of the gene therapy agent contains the synthetic cationic lipid (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA) and the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the DOTMA / DOPE liposome components are optimized for cellular delivery and targeting in an individual.

[0133] In some embodiments, the nucleic acid comprising the vector genome is mixed with a pharmaceutical composition comprising one or more cationic lipids (e.g., (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA)) and the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the pharmaceutical composition comprises at least one lipid. In some embodiments, the pharmaceutical composition comprises at least one cationic lipid. The cationic lipid may be monocationic or polycationic. Any cationic amphiphilic molecule (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 provided by at least one cationic lipid and the negative charge is provided by the nucleic acid. In some embodiments, the pharmaceutical composition comprises at least one helper lipid. The helper lipid may be a neutral lipid or an anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a completely synthetic lipid or lipid-like molecule that has no similarity to a natural lipid. In one embodiment, the cationic lipid and / or the helper lipid is a bilayer-forming lipid. Examples of helper lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analogue or derivative thereof, cholesterol (Chol) or an analogue or derivative thereof, and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analogue or derivative thereof.

[0134] In some embodiments, the molar ratio of at least one cationic lipid to at least one helper lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1: 1. In some embodiments, in this ratio, the molar amount of cationic lipid results from the molar amount of cationic lipid multiplied by the number of positive charges in the cationic lipid.

[0135] In some embodiments, the lipid is included 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.

[0136] Vector genome In some embodiments, the present invention provides methods for determining the innate immune response to a gene therapy agent used to deliver a therapeutic transgene to a desired target in an individual, in some embodiments, the gene therapy agent comprises a vector genome for delivery and expression of the therapeutic transgene at the desired target in the individual.

[0137] The present invention contemplates the use of gene therapy agents for the introduction of one or more nucleic acid sequences encoding therapeutic polypeptides and / or nucleic acids for packaging into viral particles (for viral gene therapy agents). The vector genome may include any element for establishing expression of a therapeutic polypeptide and / or nucleic acid, including, for example, a promoter, the ITRs of the present disclosure, a ribosome binding element, a terminator, an enhancer, a selection marker, an intron, a polyA signal, and / or an origin of replication.

[0138] In some embodiments, the therapeutic transgene encodes a therapeutic polypeptide. The therapeutic polypeptide may, for example, provide a polypeptide and / or enzyme activity that is absent or present at reduced levels in a cell or organism. Alternatively, the therapeutic polypeptide may provide a polypeptide and / or enzyme activity that indirectly counteracts an imbalance in a cell or organism. For example, a therapeutic polypeptide for a disorder associated with accumulation of a metabolite caused by a deficiency in a metabolic enzyme or activity may provide a missing metabolic enzyme or activity, or provide a replacement metabolic enzyme or activity that results in a reduction in the metabolite. Therapeutic polypeptides may also be used to reduce the activity of a polypeptide (e.g., one that is overexpressed, activated by a gain-of-function mutation, or whose activity is otherwise misregulated), for example, by acting as a dominant negative polypeptide.

[0139] The vector genome of the present invention may encode a polypeptide that is an intracellular protein, anchored in the cell membrane, retained intracellularly, or secreted by a cell transduced with a vector of the present invention. In the case of a polypeptide that is secreted by a cell receiving the vector, the polypeptide may be soluble (i.e., not attached to the cell). For example, a soluble polypeptide lacks a transmembrane region and is secreted from the cell. Techniques for identifying and removing nucleic acid sequences that encode transmembrane domains are known in the art.

[0140] In some embodiments, the vector genome of the present invention encodes a polypeptide used to treat a disease or disorder in an individual. Diseases and disorders that may be treated by the gene therapy 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.

[0141] In some embodiments, the therapeutic polypeptide is huntingtin (HTT), tau, 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 beta-glucosidase, glial derived growth factor (GDNF), brain derived growth factor (BDNF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), and / or amino acid decarboxylase (AADC), or alpha glucosidase.

[0142] 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, ribozyme, or DNAzyme. Thus, the therapeutic nucleic acid may encode an RNA that, when transcribed from the nucleic acid of the vector, can treat a disorder by interfering with the translation or transcription of abnormal or excess proteins associated with the disorder of the invention. For example, the nucleic acid of the invention may encode an RNA that treats a disorder by highly specific elimination or reduction of mRNAs that code for abnormal and / or excess proteins. Therapeutic RNA sequences include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA), and / or ribozymes (e.g., hammerhead ribozymes and hairpin ribozymes) that can treat a disorder by highly specific removal or reduction of mRNAs that code for abnormal and / or excess proteins.

[0143] In some embodiments, a therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS. Without wishing to be bound by theory, it is believed that a therapeutic polypeptide or therapeutic nucleic acid may be used to reduce or eliminate expression and / or activity of a polypeptide whose gain of function is associated with the disorder, or to enhance expression and / or activity of a polypeptide to complement a deficiency (e.g., a mutation in a gene whose expression exhibits a similar or related activity) associated with the disorder. Non-limiting examples of disorders of the invention that may be treated by a therapeutic polypeptide or therapeutic nucleic acid of the invention (exemplary genes that may be targeted or delivered are listed in parentheses for each disorder) include: stroke (e.g., caspase-3, Beclin1, Ask1, PAR1, HIF1α, PUMA, and / or any of the genes described in Fukuda, A and Badaut, J. (2013) Genes (Basel) 4:435-456), Huntington's disease (mutant HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and / or any of the genes described in Boison, D. (2010) Epilepsy), stroke (e.g., cerebrovascular accident, stroke, stroke, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, stroke syndrome, 51:1659-1668), Parkinson's disease (alpha-synuclein), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis; SOD1), Alzheimer's disease (tau, amyloid precursor protein), corticobasal degeneration or CBD(tau), corticobasal ganglionic degeneration or CBGD(tau), frontotemporal dementia or FTD(tau), progressive supranuclear palsy or PSP(tau), multiple system atrophy or MSA(alpha-synuclein), brain cancer (e.g., mutant or overexpressed cancer genes involved in brain cancer, and lysosomal storage diseases (LSDs). Disorders of the present invention can include those involving a large portion of the cerebral cortex (e.g., multiple functional areas of the cerebral cortex, multiple lobes of the cerebral cortex, and / or the entire cerebral cortex).Other non-limiting examples of disorders of the invention that may be treated with a therapeutic polypeptide or nucleic acid of the invention include traumatic brain injury, enzyme dysfunction disorders, psychiatric disorders (including post-traumatic stress syndrome), neurodegenerative diseases, and cognitive disorders (including dementia, autism, and depression). Enzyme dysfunction disorders include, but are not limited to, leukodystrophies (including Canavan disease), and any of the lysosomal storage disorders described below.

[0144] In some embodiments, the therapeutic polypeptide or therapeutic nucleic acid is used to treat lysosomal storage diseases. As is generally known in the art, lysosomal storage diseases are rare inherited metabolic disorders characterized by defective lysosomal function. Such disorders are often caused by a deficiency of enzymes required for proper mucopolysaccharide, glycoprotein, and / or lipid metabolism, resulting in pathological accumulation of cellular material stored in lysosomes. Non-limiting examples of lysosomal storage diseases of the invention that can be treated by a therapeutic polypeptide or nucleic acid of the invention (exemplary genes that can be targeted or delivered are listed in parentheses for each disorder) include: Gaucher disease type 2 or 3 (acid beta-glucosidase, GBA), GM1 gangliosidosis (beta-galactosidase-1, GLB1), Hunter disease (iduronate 2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), mannosidosis (mannosidases, e.g., alpha-D-mannosidase, MAN2B1), beta-mannosidosis (beta-mannosidase, MANBA), metachromatic leukodystrophy disease (pseudoarylsulfatase A, ARSA), mucolipidosis type II / III (N-acetylglucosamine-1-phosphotransferase, GNPTAB), Niemann-Pick disease type A (acid sphingosidase, SNPTAB), angiomyelinase, ASM), Niemann-Pick disease type C (Niemann-Pick C protein, 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 disease type D (N-acetylglucosamine-6-sulfatase, GNS), Schindler disease (alpha-N-acetylgalactosaminidase, NAGA), Sly disease (beta-glucuronidase, GUSB), Tay-Sachs disease (hexosaminidase alpha subunit, HEXA), and Wolman disease (lysosomal acid lipase, LIPA).

[0145] In some embodiments, the therapeutic polypeptide encodes: factor VIII, factor IX, myotubularin, motor neuron survival protein (SMN), retinoid isomerohydrolase (RPE65), NADH-ubiquinone oxidoreductase chain 4, chloroideremia protein (CHM), ornithine transcarbomylase, argininosuccinate synthase, β-globin, γ-globin, phenylalanine hydroxylase, adrenoleukodystrophy protein (ALD), dystrophin, truncated dystrophin, an anti-VEGF agent, or a functional variant thereof.

[0146] In some embodiments, the heterologous nucleic acid is operably linked to a promoter. Exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, the simian virus 40 (SV40) promoter, and the CK6 promoter, the transthyretin promoter (TTR), the TK promoter, the tetracycline-responsive promoter (TRE), the HBV promoter, the hAAT promoter, the LSP promoter, the chimeric liver-specific promoter (LSP), the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirus enhancer / chicken beta-actin / rabbit beta-globin promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9), the elongation factor 1-alpha promoter (EF1-alpha promoter) (Kim et al., Gene, 1990, 91(2):217-23 and Guo ...217-23). Ther., 1996, 3(9):802-10). In some embodiments, the promoter comprises a cytomegalovirus enhancer linked to a human β-glucuronidase promoter or a chicken β-actin (CBA) promoter. The promoter can be a constitutive 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. Patent Publication No. 20140335054.

[0147] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 13-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter [Invitrogen].

[0148] Inducible promoters allow for the regulation of gene expression and may be regulated by exogenously supplied compounds, by environmental factors such as temperature, or by the presence of a particular physiological state (e.g., acute phase, a particular differentiation state of cells), or only in replicating cells. Inducible promoters and induction systems are available from a variety of commercial sources. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995); Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1996)). (1998)), the RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997), and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are regulated by specific physiological conditions (e.g., temperature, acute phase, specific differentiation states of cells) or are regulated only in replicating cells.

[0149] In another embodiment, the native promoter for the transgene or a fragment thereof will be used. This native promoter may be used when it is desired that the expression of the transgene should mimic native expression. This native promoter may be used when the 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 further embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression.

[0150] In some embodiments, the regulatory sequence confers tissue-specific gene expression capability. In some cases, the tissue-specific regulatory sequence binds tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are known in the art.

[0151] In some embodiments, the vector comprises an intron. For example, in some embodiments, the intron is a chimeric intron derived from chicken beta-actin and rabbit beta-globin. In some embodiments, the intron is a minute virus of mice (MVM) intron.

[0152] In some embodiments, the vector comprises a polyadenylation (polyA) sequence. Numerous examples of polyadenylation sequences are known in the art, for example, 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.

[0153] Kits and Products The gene therapy agents described herein (e.g., AAV particles, adenoviral particles, lentiviral particles, HSV particles, lipid nanoparticles) and / or materials for isolating dendritic cells may be included in a kit or article of manufacture (e.g., designed for use in one of the methods of the invention described herein).

[0154] In some embodiments, the kit or article of manufacture further comprises instructions for using the isolated dendritic cells to assay for natural immunogenicity to a gene therapy agent. The kits or articles of manufacture described herein may further comprise other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for carrying out any of the methods described herein. Suitable packaging materials may also be included, such as any packaging material known in the art, such as vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles may further be sterilized and / or sealed.

[0155] In some embodiments, the kit or article of manufacture further comprises one or more of the buffers and / or pharma- ceutically acceptable excipients described herein (e.g., as described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991)). In some embodiments, the kit or article of manufacture comprises one or more pharma- ceutically acceptable excipients, carriers, solutions, and / or additional components described herein. The kit or article of manufacture described herein may be packaged in single unit dose or multi-dose form. The contents of the kit or article of manufacture are generally formulated as sterile and may be lyophilized or provided as a substantially isotonic solution.

[0156] Exemplary embodiments The present invention includes the embodiments enumerated below.

[0157] 1. A method for determining natural immunogenicity to a gene therapy agent in an individual, comprising: a) incubating innate immune cells from an individual with a gene therapy agent; b) analyzing the innate immune cells for altered expression of one or more cytokines relative to a suitable control, where the altered expression of the one or more cytokines results in a cytokine signature. Including, Expression of a cytokine signature following incubation with a gene therapy agent indicates natural immunogenicity to the gene therapy agent in an individual. method.

[0158] 2. The method of embodiment 1, wherein the innate immune cells are dendritic cells, monocytes, macrophages, or natural killer (NK) cells.

[0159] 3. The method of embodiment 1 or 2, wherein the innate immune cells are isolated from peripheral blood mononuclear cells from the individual.

[0160] 4. The method according to any one of embodiments 1 to 3, wherein the innate immune cells are dendritic cells.

[0161] 5. The method of embodiment 4, wherein the dendritic cells are derived from the individual's monocytes.

[0162] 6. The method of embodiment 4 or 5, further comprising isolating monocytes from the individual prior to incubating the dendritic cells with the gene therapy agent, and incubating the monocytes in dendritic cell culture medium to induce dendritic cells from the monocytes.

[0163] 7. The method of embodiment 6, wherein the monocytes are CD14+ monocytes.

[0164] 8. The method according to embodiment 6 or 7, wherein monocytes are incubated with a dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to induce dendritic cells from the monocytes.

[0165] 9. The method according to any one of the preceding embodiments, wherein the innate immune cells are re-seeded prior to incubation with the gene therapy agent in step b).

[0166] 10. The method of embodiment 9, wherein the innate immune cells are replated in the microwell dish.

[0167] 11. The gene therapy agent is a viral vector, and innate immune cells are transduced at approximately 1 x 10 3 ~Approx. 1×10 5 Or about 1 x 10 4 11. The method according to any one of embodiments 1 to 10, wherein the cells are incubated with the gene therapy agent at an MOI of 0.1 to 0.25.

[0168] 12. The method according to any one of embodiments 1 to 10, wherein the gene therapy agent is a non-viral vector and the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

[0169] 13. The method according to any one of embodiments 1 to 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.

[0170] 14. The method of any one of embodiments 1-13, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0171] 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.

[0172] 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.

[0173] 17. The method of any one of embodiments 1-15, wherein the cytokine signature comprises increased expression of IL6, TNFα, and IL-1β.

[0174] 18. The method of any one of embodiments 1 to 17, wherein expression of a cytokine in the cytokine signature is increased compared to a suitable control.

[0175] 19. The method of embodiment 18, wherein a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells that have not been incubated with the gene therapy agent, or a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

[0176] 20. The method of any one of embodiments 1-10 and 13-19, wherein the gene therapy agent is a viral vector.

[0177] 21. A method for determining natural immunogenicity to a viral gene therapy agent in an individual, comprising: a) incubating monocytes from an individual in dendritic cell culture medium under conditions such that the monocytes differentiate into dendritic cells; b) Dendritic cells are cultured at about 1 × 10 3 ~Approx. 1×10 5 incubating with the viral gene therapy agent at an MOI of c) analyzing the dendritic cells for altered expression of one or more cytokines relative to a suitable control, where the altered expression of the one or more cytokines results in a cytokine signature. Including, Expression of a cytokine signature following incubation with the viral gene therapy agent indicates natural immunogenicity to the viral gene therapy agent in the individual, the cytokine signature including increased expression of IL6, TNFα, and IL-1β. method.

[0178] 22. The method of embodiment 21, wherein the monocytes are obtained from peripheral mononuclear cells from an individual.

[0179] 23. The method of embodiment 21 or 22, wherein the monocytes are CD14+ monocytes.

[0180] 24. The method according to any one of embodiments 21 to 23, wherein the monocytes are incubated in a dendritic cell culture medium for about 7 to 8 days to differentiate the monocytes into dendritic cells.

[0181] 25. Dendritic cells were added to approximately 1 x 10 4 25. The method according to any one of embodiments 21 to 24, wherein the cells are incubated with the viral gene therapy agent at an MOI of 0.1 mg / mL or more.

[0182] 26. The method according to any one of embodiments 21 to 25, wherein the dendritic cells are incubated with the viral gene therapy agent for about 24 hours.

[0183] 27. The method of any one of embodiments 20 to 26, wherein the viral vector is an AAV particle.

[0184] 28. The AAV particle is an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 28. The method of embodiment 27, comprising an E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a murine AAV capsid rAAV2 / HBoV1 (chimeric AAV / human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid, or an AAVPHP.eB capsid, or a functional variant thereof.

[0185] 29. The method of embodiment 28, wherein the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation, or an HBKO mutation.

[0186] 30. The method of any one of embodiments 27 to 29, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), and one or more of the 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.

[0187] 31. The method of embodiment 30, wherein one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0188] 32. The method of embodiment 30, wherein one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0189] 33. The method of any one of embodiments 20 to 26, wherein the viral vector is an adenovirus particle.

[0190] 34. The method of embodiment 33, wherein the adenovirus particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, or a functional variant thereof.

[0191] 35. The method of any one of embodiments 20 to 26, wherein the viral vector is a lentiviral particle.

[0192] 36. The method of embodiment 35, wherein the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokara virus, rabies virus, RD114, or a functional variant thereof.

[0193] 37. The method of any one of embodiments 20 to 26, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0194] 38. The method of embodiment 37, wherein the HSV particles are HSV-1 particles, or HSV-2 particles, or functional variants thereof.

[0195] 39. The method of any one of embodiments 1-10 and 12-19, wherein the gene therapy agent is a lipid nanoparticle.

[0196] 40. A method for determining natural immunogenicity to a non-viral gene therapy agent in an individual, comprising: a) incubating monocytes from an individual in dendritic cell culture medium under conditions such that 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 dendritic cells for altered expression of one or more cytokines relative to a suitable control, where the altered expression of the one or more cytokines results in a cytokine signature. Including, Expression of a cytokine signature following incubation with the non-viral gene therapy agent indicates natural immunogenicity to the non-viral gene therapy agent in the individual, the cytokine signature including increased expression of IL6, TNFα, and IL-1β. method.

[0197] 41. The method of embodiment 40, wherein the monocytes are obtained from peripheral mononuclear cells from an individual.

[0198] 42. The method of embodiment 40 or 41, wherein the monocytes are CD14+ monocytes.

[0199] 43. The method according to any one of embodiments 40 to 42, wherein the monocytes are incubated in a dendritic cell culture medium for about 7 to 8 days to differentiate the monocytes into dendritic cells.

[0200] 44. The method according to any one of embodiments 40 to 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.

[0201] 45. The method of any one of the preceding embodiments, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0202] 46. ​​The method of embodiment 45, wherein the heterologous transgene is operably linked to a promoter.

[0203] 47. The method of embodiment 46, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0204] 48. A method for determining a cytokine signature of a gene therapy agent, comprising: a) incubating one or more types of innate immune cells from one or more individuals with a gene therapy agent; b) analyzing one or more innate immune cells for changes in expression of one or more cytokines compared to a suitable control. Including, The change in expression of one or more cytokines in step b) is indicative of a cytokine signature of the gene therapy agent. method.

[0205] 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.

[0206] 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.

[0207] 51. The method of any one of embodiments 48 to 50, wherein the innate immune cells are dendritic cells.

[0208] 52. The method of embodiment 51, wherein the dendritic cells are derived from monocytes of one or more individuals.

[0209] 53. The method of embodiment 51 or 52, further comprising isolating monocytes from one or more individuals prior to incubating the dendritic cells with the gene therapy agent, and incubating the monocytes in dendritic cell culture medium to induce dendritic cells from the monocytes.

[0210] 54. The method of embodiment 53, wherein the monocytes are CD14+ monocytes.

[0211] 55. The method according to embodiment 53 or 54, wherein monocytes are incubated with a dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to induce dendritic cells from the monocytes.

[0212] 56. The method according to any one of embodiments 48 to 55, wherein the innate immune cells are re-seeded prior to incubation with the gene therapy agent in step b).

[0213] 57. The gene therapy agent is a viral vector and innate immune cells are transduced at approximately 1 x 10 3 ~Approx. 1×10 5 Or about 1 x 10 4 57. The method of any one of embodiments 48 to 56, wherein the cells are incubated with the gene therapy agent at an MOI of 0.1 to 0.5 mg / mL.

[0214] 58. The method of any one of embodiments 48 to 56, wherein the gene therapy agent is a non-viral vector and the innate immune cells are incubated with the non-viral vector at a concentration of about 1 ng / mL to about 1 mg / mL.

[0215] 59. The method according to any one of embodiments 48 to 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.

[0216] 60. The method of any one of embodiments 48 to 59, wherein expression of a cytokine in the cytokine signature is increased compared to a suitable control.

[0217] 61. The method of embodiment 60, wherein a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells that have not been incubated with the gene therapy agent, or a suitable control is the expression of a cytokine in a cytokine signature from innate immune cells prior to incubation with the gene therapy agent.

[0218] 62. The method of any one of embodiments 48-57 and 59-61, wherein the gene therapy agent is a viral vector.

[0219] 63. The method of embodiment 57 or 62, wherein the viral vector is an AAV particle.

[0220] 64. The AAV particle is an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 64. The method of embodiment 63, comprising an E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a murine AAV capsid rAAV2 / HBoV1 (chimeric AAV / human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid, or an AAVPHP.eB capsid, or a functional variant thereof.

[0221] 65. The method of embodiment 64, wherein the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation, or an HBKO mutation.

[0222] 66. The method of any one of embodiments 63 to 65, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), and one or more of the 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.

[0223] 67. The method of embodiment 66, wherein one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0224] 68. The method of embodiment 66, wherein one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0225] 69. The method of embodiment 57 or 62, wherein the viral vector is an adenovirus particle.

[0226] 70. The method of embodiment 69, wherein the adenovirus particle comprises a capsid from adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, or a functional variant thereof.

[0227] 71. The method of embodiment 57 or 62, wherein the viral vector is a lentiviral particle.

[0228] 72. The method of embodiment 71, wherein the recombinant lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokara virus, rabies virus, RD114, or a functional variant thereof.

[0229] 73. The method of embodiment 57 or 62, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0230] 74. The method of embodiment 73, wherein the HSV particles are HSV-1 particles, or HSV-2 particles, or functional variants thereof.

[0231] 75. The method of any one of embodiments 48-56 and 58-61, wherein the gene therapy agent is a lipid nanoparticle.

[0232] 76. The method of any one of embodiments 48-75, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0233] 77. The method of embodiment 76, wherein the heterologous transgene is operably linked to a promoter.

[0234] 78. The method of embodiment 77, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0235] 79. A kit for use in the method according to any one of embodiments 1 to 78. EXAMPLES

[0236] The present invention will be more fully understood by referring to the following examples. However, these should not be interpreted as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in consideration thereof will be suggested to those skilled in the art, and should be included within the spirit and scope of this application and the scope of the appended claims.

[0237] Example 1: Screening of natural immunogenicity of various AAV vectors This example provides a strategy for assaying innate immune responses following AAV treatment.

[0238] Immune responses to AAV vectors pose a challenge to successful clinical application. AAV induces an immune response that involves activation of both the innate and adaptive immune systems. While the adaptive immune response to AAV is relatively well characterized, innate immune activation by AAV is less understood.

[0239] Materials and Methods Preparation of peripheral blood mononuclear cells. Blood from leukopak (AllCells) from four donors was decanted into 50 mL tubes and Dulbecco's phosphate buffered saline (DPBS) was added in a 1:1 ratio. The blood + DBPS mixture was gently pipetted into a separate 50 mL tube containing 15 mL of Ficoll (GE17-5442-02) to ensure mixing of the blood and Ficoll phases. The mixture was centrifuged at 2000 RPM (9 acceleration and no brake) for 25 minutes at room temperature. The buffy coat containing peripheral blood mononuclear cells (PBMCs) was collected, transferred to a new tube and centrifuged at 400 RCF for 5 minutes. PBMCs were washed three times with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FBS) or fetal calf serum (FCS) and counted.

[0240] Isolation of monocytes. CD14+ monocytes were isolated from PBMCs using CD14 MicroBeads (Milteny Biotech, Germany, order number 130-050-201, protocol available online on the World Wide Web at miltenyibiotec.com / upload / assets / IM0001260.PDF) according to the manufacturer's protocol. Briefly, PBMCs were incubated for 15 min at 2-8 °C for 10 7 The PBMCs were incubated with 20 μL of CD14 MicroBeads per total cells. The PBMCs were applied onto a magnetic column (Miltenyi; World Wide Web miltenyibiotec.com / US-en / products / ls-columns.html#130-042-401) and the unlabeled cells were allowed to pass through. After washing the column three times, the column was removed from the magnetic separator (Miltneyi; World Wide Web miltenyibiotec.com / US-en / products / quadromacs-separator-and-starting-kits.html#130-091-051) and placed onto a collection tube, and the magnetically labeled CD14+ monocytes were flushed out by pushing the plunger into the column.

[0241] Monocyte Differentiation. CD14+ monocytes were differentiated into dendritic cells using ImmunoCult-ACF Dendritic Cell Media, Differentiation Supplement, and Maturation Supplement (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) according to the manufacturer's protocol available online. Briefly, purified CD14+ monocytes were added to Dendritic Cell Media containing Differentiation Supplement and incubated at 37°C for 3 days. On day 3, the medium was replaced with fresh Dendritic Cell Media containing Differentiation Supplement and the cells were incubated for an additional 2 days. On day 5, Maturation Supplement was added to the cells at a 1-100 dilution (e.g., 50 μL of Supplement per 5 mL of culture). On day 7, differentiated dendritic cells were harvested.

[0242] 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; doi:10.1101 / pdb.top095513, 2020). All serotypes tested encode the same GFP transgene. Virus was purified by cesium chloride ultracentrifugation and titrated using both silver staining and quantitative polymerase chain reaction (qPCR).

[0243] process Dendritic cells were seeded in 96-well plates at 200,000 cells per well. Each treatment was performed in triplicate. Four different treatments with four different AAV vectors at 1e4 MOI were used. The cells were incubated for 24 hours in an incubator at 37°C and 5% CO2. After 24 hours, the plates were centrifuged and the medium supernatants were collected. Lipopolysaccharide LPS (300ng / mL) (Sigma Aldrich Fine Chemicals Biosciences L2630100MG) was used as a positive control, which is known to activate the receptor 4 for toll and induce cytokine production I in human monocyte-derived dendritic cells. The cell medium was then collected and centrifuged. The clarified medium was analyzed by two different methods to detect the different cytokines released from dendritic cells upon challenge with the different AAV serotypes. These two methodologies used: Multiplex Immunoassay (Luminex Bead-based assay) performed by Nanobiotec (World Wide Web nanobiotecusa.com / immunoassay) and MSD (World Wide Web mesoscale.com / products / v-plex-proinflammatory-panel-1-human-kit-k15049d / ) performed by DC3 Therapeutics https: / / www.dc3therapeutics.com / services. The experimental outline of PBMC isolation, monocyte purification, and dendritic cell differentiation is shown in Figure 1. The figure also shows how dendritic cells were treated with the various AAVs.

[0244] result All four serotypes tested produced weaker immune responses compared to LPS based on cytokines released by treated dendritic cells (Figure 2). While LPS induces significant upregulation in the majority of cytokines assayed from each donor, the various AAV serotypes tested induce less pronounced upregulation restricted to only a subset of cytokines, IL6, TNFα, IL-1β, MCP1, and MIP-1α (Figure 2). Notably, this subset of cytokines (i.e., cytokine signature) appears to be conserved across the various AAV serotypes. The data shown in Figure 2 is based on Luminex Technology, where some cytokines are above or below the limit of detection (aLOD / bLOD), therefore, we utilized MSD technology and re-examined cytokine levels from the same donor cells. As shown in Figure 2, the same results were verified using an orthogonal MSD assay (Figure 3).

[0245] Of the AAV serotypes tested, AAVrh32.33 was found to generate the most robust and consistent innate immune signature (Figures 2 and 3), as evidenced by the greater upregulation observed for IL-6, TNF-α, and IL-1β.

[0246] Consideration Successful gene therapy for the treatment of rare genetic diseases relies heavily on adeno-associated virus (AAV) viral vectors, which offer many attractive features such as tissue-specific tropism, transduction of quiescent cells, and sustained modified gene expression. However, immune responses to AAV vectors pose a major challenge to successful clinical translation. The capsid, viral genome, and transgene trigger an immune response that involves activation of both the innate and adaptive arms of the immune system. The adaptive immune response triggered by B cells and T cells is to some extent understood in the art, while innate immune activation is poorly understood. Studies based on mouse models have shown that the TLR9a DNA sensor present in the endosomes of cells detects the AAV genome and activates a signaling cascade that ultimately leads to cytokine release (Ashley, SN et al., Cell Immunol. 2019, 346:103997; (Zhu, J et al., J Clin Invest.2009;119(8):2388-2398). (Figure 1, bottom right panel). These cytokines initiate antiviral responses and trigger the activation of the adaptive immune system. A major challenge in understanding these innate immune responses is the poor reproducibility of immune responses observed in clinical trials to ex vivo settings. To circumvent this challenge and understand the innate immune response, we are currently developing a novel assay that recapitulates the innate immune signatures in different human donors in response to different AAV serotypes. We use human monocytic dendritic cells (moDCs) from healthy donors, and we sensitively detect cytokines released from these moDCs upon loading with AAV vectors.

[0247] Example 2: Screening for natural immunogenicity of LNPs This example provides a strategy for assaying innate immune responses following LNP delivery. A general overview of the process described in this example is shown in FIG.

[0248] Materials and Methods Production of differentiated dendritic cells Using the procedures described in Example 1, blood mononuclear cells were prepared, monocytes were isolated and differentiated into dendritic cells.

[0249] LNP treatment and cytokine measurements Human monocytic dendritic cells were seeded in 96-well format and treated with 10ug LNP 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 after 24 hours for downstream analysis such as cytotoxicity and target gene expression by flow cytometry. Media was collected for cytokine analysis. Cells were centrifuged at 2,000 rpm for 5 minutes and supernatants were collected for cytokine analysis. Cytokines were measured using Luminex using MILLIPLEX® Human Cytokine / Chemokine / Growth Factor Panel A 38 Plex Premixed Magnetic Bead Panel-Immunology Multiplex Assay Catalog No. HCYTA-60K-PX38 according to the manufacturer's protocol.

[0250] The reagents used in this assay are shown in Table 1.

[0251] [Table 1]

[0252] Cell viability assessment Dendritic cells (DCs) were resuspended in FACS buffer and transferred to U-bottom 96-well plates. 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 to be cell viability. Invitrogen's LIVE / DEAD™ Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation, catalog number L34975, was used for cell staining.

[0253] Target gene expression by flow cytometry 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 min and the supernatant was discarded. To analyze target gene expression, CD22 protein was diluted in FACS buffer at a final concentration of 5 μg / ml. DCs in each well were treated with 100 μl of diluted CD22 for 30 min at 4° C. Cells were washed with FACS buffer and then stained with 50 μl of flow antibody master mix (1:100 anti-hCD22 APC, 1:100 anti-hCD11c Pacific Blue, 1:100 anti-hCD11b FITC, 1:100 anti-hCD83 PE, and 1:100 Live / Dead stain APC-Cy7) for 30 min at 4° C. The cells were washed twice with FACS buffer and run on a flow cytometer (Novocyte Penteon Flow Cytometer Systems 5 Lasers, Agilent Technology).

[0254] result Cell viability Dendritic cells were harvested 24 hours after LNP treatment and flow cytometry was performed to evaluate cell viability (Figure 5). Each point in Figure 5 represents a cell derived from a human donor. The cell-only condition was without LNP treatment, and mRNA-LNP#1 and mRNA-LNP#2 were cells treated with two different LNPs, and for all donors, viability was the same as the cell-only control, indicating no cytotoxicity upon LNP treatment. The dendritic cell line can be used to evaluate LNP without affecting cell viability. One-way ANOVA was performed to measure statistical significance.

[0255] Transduction Level Dendritic cells were harvested 24 hours after LNP treatment and flow cytometry was performed to evaluate target gene expression (Figure 6). Each dot in Figure 6 represents a cell derived from a human donor. The cell-only condition was without LNP treatment, 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 with LNPs, as measured by the percentage of cells expressing mRNA. One-way ANOVA was performed to determine statistical significance. This indicates that the dendritic cell assay system of the present disclosure can be used to effectively evaluate LNP transduction.

[0256] LNP immunogenicity Media from dendritic cells was collected 24 hours after LNP treatment and Luminex analysis was performed to identify cytokine signatures. mRNA-encapsulated LNPs specifically secrete cytokines compared to non-LNP treated cells. This analysis shows that cytokines such as IP10 (Figure 7A), MIP1b (Figure 7B), CXCL9 (Figure 7C), and IL2 (Figure 7D) were upregulated across all donors compared to the respective media only controls.

Claims

1. A method for determining the innate immunogenicity of gene therapy agents in an individual, a) Isolating monocytes from the organism, and incubating the monocytes in dendritic cell culture medium to induce dendritic cells from the monocytes. b) Incubating the dendritic cells together with the gene therapy agent, c) Analyzing the innate immune cells in relation to changes in the expression of one or more cytokines compared to a suitable control, and analyzing whether a cytokine signature arises from the changes in the expression of one or more cytokines. Includes, The expression of the cytokine signature after incubation with the gene therapy agent indicates innate immunogenicity to the gene therapy agent in the individual. method.

2. The method according to claim 1, wherein the monocyte is a CD14+ monocyte.

3. The method according to claim 1, wherein the monocytes are incubated with the dendritic cell culture medium for about 5 to about 10 days or about 7 to about 8 days to induce dendritic cells from the monocytes.

4. The method according to any one of claims 1 to 3, wherein the gene therapy agent is a viral vector or a non-viral vector.

5. The method according to claim 4, wherein the viral vector is AAV.

6. The method according to claim 4, wherein the non-viral vector is lipid nanoparticles (LNPs).

7. The gene therapy agent is a viral vector, and the innate immune cells are approximately 1 × 10 3 ~Approx. 1×10 5 Or approximately 1 x 10 4 The method according to any one of claims 1 to 3, wherein the gene therapy agent is incubated with the MOI.

8. The gene therapy agent is a non-viral vector, and the innate immune cells are stimulated to approximately 1 ng / m³ The method according to any one of claims 1 to 3, wherein the non-viral vector is incubated with the non-viral vector at a concentration of L to approximately 1 mg / mL.

9. The method according to any one of claims 1 to 3, wherein the innate immune cells are incubated with the gene therapy agent for about 12 to about 36 hours or about 24 hours.

10. A method for determining the innate immunogenicity of an individual to a viral gene therapy agent, a) Incubating monocytes derived from the individual in dendritic cell culture medium under conditions that allow the monocytes to differentiate into dendritic cells. b) The dendritic cells were subjected to approximately 1 × 10⁶ times for approximately 12 to 36 hours. 3 ~Approx. 1×10 5 Incubating with the aforementioned viral gene therapy agent in the MOI, c) Analyzing the dendritic cells in comparison to a suitable control for changes in the expression of one or more cytokines, and analyzing whether a cytokine signature arises from the changes in the expression of one or more cytokines. Includes, 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, and the cytokine signature includes increased expression of IL-6, TNFα, and IL-1β. method.

11. The method according to claim 10, wherein the monocytes are obtained from peripheral mononuclear cells derived from the individual.

12. The method according to claim 11, wherein the monocyte is a CD14+ monocyte.

13. The method according to claim 11 or 12, wherein the monocytes are incubated in dendritic cell culture medium for about 7 to 8 days to differentiate the monocytes into dendritic cells.

14. The method according to claim 11 or 12, wherein the viral gene therapy agent is AAV particles.

15. A method for determining the innate immunogenicity of an individual to a nonviral gene therapy agent, a) Incubating monocytes derived from the individual in dendritic cell culture medium under conditions that allow the monocytes to differentiate into dendritic cells. b) Incubate the dendritic cells with a non-viral vector at a concentration of approximately 1 ng / mL to approximately 1 mg / mL. c) Analyzing the dendritic cells in comparison to a suitable control for changes in the expression of one or more cytokines, and analyzing whether a cytokine signature arises from the changes in the expression of one or more cytokines. Includes, The expression of cytokine signatures after incubation with the nonviral gene therapy agent indicates innate immunogenicity to the nonviral gene therapy agent in the individual, and the cytokine signatures include increased expression of IL-6, TNFα, and IL-1β. method.

16. The method according to claim 15, wherein the monocytes are obtained from peripheral mononuclear cells derived from the individual, and the monocytes are CD14+ monocytes.

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