Prediction of the immunostimulatory capacity of AAV in human preclinical models

Abstract

A composition and method are provided for predicting the intensity of the immune response induced in humans after administration of a specific rAAV vector. This method utilizes antibody opsonization of rAAV to enhance assays and elucidate and quantify the immune response in human PBMC-based assays.

Description

[Background technology]

[0001] Recombinant adeno-associated virus (rAAV) gene therapy (GT) vectors are transformative FDA-approved treatments for Leber congenital amaurosis, spinal muscular atrophy, and hemophilia B. These are early examples of what could become a new treatment paradigm for a wide range of genetic indications. In addition to these clear successes, several investigational rAAVs have encountered significant barriers to product development due to immunotoxicity, particularly when high doses are required, such as in Duchenne muscular dystrophy. The inability to adequately model adverse immune responses to rAAV has emerged as a major drawback. Among the most concerning are innate immune responses, such as those initiated via Toll-like receptor (TLR) activation and complement activation.

[0002] In four independent trials for different indications, the majority of serious clinical adverse events (SAEs), including eight Level 5 SAEs (deaths) considered investigational drug-related reported over the past two years, were unpredictable due to their inadequate modeling. The current method utilizes a mismatched rodent model requiring extensive modification, featuring 12 TLRs and 14 interferon-alpha (IFNα) subtypes, compared to 10 and 13 in humans, respectively. Recent reports suggest that susceptibility to TLR9, a key player in the host response to rAAV, is dramatically enhanced in great apes (including humans) than in lower mammals.

[0003] Cell line-based models are also used, which can adequately measure TLR9 activation by oligodeoxyribonucleotides (ODNs), but are non-physiological and not adequately stimulated by rAAVs. Ideally, a system using primary human cells, including peripheral blood mononuclear cells (PBMCs), is used to predict the immune response to rAAVs. However, PBMCs are highly variable because they are obtained from human donors and, to date, are insensitive to potentially significant differences between relevant rAAVs. Therefore, there is an acute, recognized, and unmet need for a physiologically and sensitively sensitive human model to accurately predict adverse immune responses preclinically.

[0004] The ability to accurately predict adverse immune responses in preclinical models enables the evaluation and selection of optimal lead rAAV candidates to support the application of rAAV-based gene therapy (GT) where minimizing the immune response to the product is desirable; enables the evaluation and selection of lead rAAV candidates to support the development of rAAV-based vaccine products where maximizing the immune response to the product is desirable; provides lot-to-lot consistency, safety, and efficacy verification during the production of GMP rAAV vectors; or provides a basis for companion diagnostic tools, as well as for the inclusion of human subjects in clinical trials using investigational rAAV products and licensed rAAV products.

[0005] Recombinant AAV vectors are discussed in Wright (2020) Molecular Therapy 28(3):701-703 and Hamilton and Wright (2021) Front.Immunol.12:675897.

[0006] This disclosure provides tools and methods for this analysis. [Overview of the Initiative]

[0007] Compositions and methods are provided for predicting the intensity of the immune response induced in humans after administration of a target rAAV vector. In some embodiments, predictive distinctions are made between relevant rAAV vectors, e.g., vectors having a high degree of sequence similarity. The methods of the present invention utilize antibody opsonization of rAAV to enhance assays that utilize basic physiological processes, elucidating and quantifying the immune response in human PBMC-based assays.

[0008] In some embodiments, improved assays are provided for detecting the presence and intensity of an AAV-specific cell-mediated immune response in an individual. In the method of the present invention, a sample comprising immune cells, such as dendritic cells, monocytes, T cells, Bc cells, NK cells, etc., typically but not limited to a blood sample comprising a peripheral blood mononuclear cell sample or a derivative thereof, is contacted with an effective dose of a test AAV composition in the presence of an effective amount of an AAV-specific opsonized antibody. The opsonized antibody of interest comprises an Fc region sequence that specifically binds to AAV and binds to an Fc receptor present on the immune cell. The sample is incubated for a period sufficient to allow uptake of opsonized AAV, activation of responsive immune cells, and release of IFN-α or other cytokines or immune effector molecules by the responsive cells. The level of IFN-α or other cytokines or immune effector molecules produced by the responsive immune cells indicates the level of cell-mediated responsiveness to the AAV composition. The use of opsonized antibodies in the assay to increase the sensitivity of the assay is shown herein.

[0009] In some embodiments, the immune cells are PBMCs. The immune cells may be isolated from an organism, for example, for use in determining the suitability of an AAV composition for administration to the organism in gene therapy applications. In other embodiments, the immune cells may be isolated from an organism for AAV evaluation purposes and combined in a panel of cells from various organisms. In other embodiments, the immune cells may be isolated from a non-human source, for example, for preclinical trials of an AAV composition.

[0010] In some embodiments, the immunoeffector protein is a cytokine or non-cytokine effector protein, including, but not limited to, IFN-α and pro-inflammatory cytokines such as IL-1, IL-2, IL-6, IL-12, IL-17, IL-18, IFN-γ, and TNF-α. The presence or level of the immunoeffector protein of interest can be determined at the level of the protein itself or to the extent to which the gene encoding the protein is expressed. Conveniently, assays known in the art for measuring cytokine release, including but not limited to Western blotting, flow cytometry, PCR, ELISA, Luminex, ELISpot, cyTOF, and tetramer assays, can be used in the methods of the present invention using antibody, nucleic acid, or protein-based readouts.

[0011] In some embodiments, the AAV-specific opsonized antibody is a polyclonal antibody, e.g., an antibody obtained from AAV capsid-serum positive blood, plasma, or serum from a human subject after rAAV administration. In some embodiments, the AAV-specific opsonized antibody includes AAV capsid-serum positive blood, plasma, or serum collected from a study animal after rAAV administration. In some embodiments, the AAV-specific opsonized antibody includes an AAV capsid-specific monoclonal antibody, which may be engineered or selected to have an Fc region capable of binding to a human Fc receptor in order to produce human Fc effector function. Alternatively, the monoclonal antibody may include an Fc region capable of binding to a non-human Fc receptor in order to produce Fc effector function in a non-human animal model. The monoclonal antibody may be a chimeric, e.g., a human Fc domain combined with an AAV capsid-specific mouse monoclonal (Fab)2 domain; e.g., humanized from a mouse antibody; or fully human.

[0012] The test antigen of interest for the assay comprises any AAV composition. The aim is recombinant AAV that is used or being evaluated for use in gene therapy treatment. Preferred compositions comprise an AAV genome associated with a capsid protein. One, or a plurality of test AAV compositions, e.g., a panel of test AAV compositions, can be assayed. A feature of the method is the ability to distinguish rAAV that differ only in genomic sequence.

[0013] In some embodiments, an individual is tested for responsiveness to a candidate rAAV composition by the methods disclosed herein prior to treating the individual with rAAV. An individual determined to have a low level of responsiveness can be treated with the candidate rAAV. An individual with a high responsiveness to rAAV can be treated with an alternative rAAV, or a non-AAV therapy.

[0014] The invention further provides a kit comprising the reagents and compartments necessary to perform the assay. Generally, the kit further comprises a set of instructions. The assay can also be automated or semi-automated, and the automated embodiments can be controlled by computer software.

[0015] The invention is best understood when read in conjunction with the following detailed description with the accompanying drawings. In accordance with common practice, it is emphasized that the various features of the drawings are not to scale. Conversely, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. The drawings include the following figures.

Brief Description of the Drawings

[0016] [Figure 1]AAV GT increases the serum neutralization of AAV2-CAG>GFP on Hela cells. AAV2 carrying the GFP transgene driven by the CAG promoter was incubated for 1 hour with serial dilutions of either serum from human patients who had not received AAV treatment, serum from the same patients after AAV treatment, or a mouse monoclonal antibody (A20). Then, the AAV was overlaid three times on Hela cells cultured to a 90% confluent monolayer in each well of a black opaque 96-well tissue culture plate. Four days later, the fluorescence intensity of each well was measured with a plate reader. [Figure 2] rAAV2-GFP induces 19.76 pg / mL more INFα1 than rAAV2 empty capsids from hPBMC. Human PBMC were isolated from three random healthy donors by Ficoll gradient centrifugation and seeded into 96-well U-bottom tissue culture plates. Immediately after seeding, either AAV2 empty capsids, AAV2-GFP, ODN2216, or ODN2216 with methylated CpG was overlaid three times on the PBMC. After 24 hours, the supernatant was removed, technical replicates were pooled, and ELISA was performed to measure the INFα1 concentration. [Figure 3] rAAV2-GFP induces 727.27 pg / mL more INFα1 than rAAV2 empty capsids after serum incubation following GT. Human PBMC were isolated from three random healthy donors by Ficoll gradient centrifugation and seeded into 96-well U-bottom tissue culture plates. Immediately after seeding, either AAV2 empty capsids pretreated for 1 hour with serum collected from AAV-treated recipients, AAV2-GFP pretreated for 1 hour with serum collected from AAV-treated recipients, ODN2216, or ODN2216 with methylated CpG was overlaid three times on the PBMC. After 24 hours, the supernatant was removed, technical replicates were pooled, and ELISA was performed to measure the INFα1 concentration. [Figure 4]FcR antibodies blunt serum-mediated signal amplification. Human PBMCs were isolated from six random healthy donors by Ficoll gradient centrifugation and seeded in 96-well U-bottom tissue culture plates. Anti-human Fc blocking antibody was added to one set of wells. All cells were then superimposed three times with either an empty AAV2 capsid, AAV-GFP, or AAV2-GFP pre-treated for 1 hour with serum collected from AAV-treated recipients. After 24 hours, the supernatant was removed and IL-6 concentrations were measured by ELISA. [Figure 5] Incubation with AAV-converted serum increases CpG-dependent CTL activation. Human PBMCs were isolated from three random healthy donors by Ficoll gradient centrifugation and seeded in 96-well U-bottom tissue culture plates. Immediately after seeding, the PBMCs were overlaid three times with either an empty AAV2 capsid, an empty AAV2 capsid pre-treated with serum from an AAV-treated recipient for 1 hour, AAV2-GFP, or AAV2-GFP pre-treated with serum from an AAV-treated recipient for 1 hour. After 72 hours, the cells were stained and analyzed by fluorescence-activated cell sorting (FACS) using a flow cytometer. CD3+ / CD8+ cells were investigated for the presence of CD25 and CD65 activation markers. [Figure 6] Incubation with anti-AAV mAb (A20) did not alter hPBMC-induced IFNα1. Human PBMCs were isolated from three random healthy donors by Ficoll gradient centrifugation and seeded in 96-well U-bottom tissue culture plates. Immediately after seeding, AAV2-GFP, AAV2-GFP pre-treated for 1 hour with serum from AAV-treated recipients, ODN2216, or ODN2216 with methylated CpG were layered onto the PBMCs three times. After 24 hours, the supernatant was removed, technical replicas were pooled, and INFa1 concentrations were measured by ELISA. [Figure 7]Incubation with anti-AAV mAb (A20) does not alter mPBMC-induced IL2Rα. Mouse PBMCs were extracted from 6 female and 6 male mice, isolated by Ficoll gradient centrifugation, pooled by sex, and seeded in 96-well U-bottom tissue culture plates. Immediately after seeding, cells were refracted three times with one of the following recombinant AAVs pre-treated for 1 hour: AAV2 empty capsid, AAV2 CMV > null vector, AAV2 > CAG > GFP, PMA-positive control, or serial dilution of mouse anti-AAV2 (A20). After 24 hours, supernatant was removed, technical replicates were pooled, and Luminex was performed twice. mIL2Rα results are shown. [Figure 8] hPBMCs from random donors may not respond to CpG. Human PBMCs were isolated from three random healthy donors by Ficoll gradient centrifugation and seeded in 96-well U-bottom tissue culture plates. Immediately after seeding, either ODN2216 or ODN2216 with methylated CpG was layered onto the PBMCs three times. After 24 hours, the supernatant was removed, technical replicas were pooled, and INFα1 concentrations were measured by ELISA. [Figure 9] The associated AAV vectors could not differentiate due to genomic immunogenicity. After extraction from three random donors, human PBMCs were isolated by Ficoll gradient centrifugation and seeded in 96-well U-bottom tissue culture plates. Immediately after seeding, cells were layered three times with either an AAV2 empty capsid, an AAV2 CMV > null vector, or an AAV2 > CAG > GFP. After 24 hours, the supernatant was removed and INFα1 concentration was measured by ELISA. [Figure 10]hA20 opsonization distinguishes AAVs closely related by genomic immunogenicity. After extraction from three random donors, human PBMCs were isolated by Ficoll gradient centrifugation and seeded in 96-well U-bottom tissue culture plates. Immediately after seeding, cells were layered three times with either an AAV2 empty capsid, an AAV2 CMV > null vector, or AAV2 > CAG > GFP (all pre-treated for 1 hour with chimeric anti-AAV2 mAb hA20 (mouse Fab fused to human Fc)). After 24 hours, the supernatant was removed and INFa1 concentration was measured by ELISA. [Figure 11] hA20 pre-incubation is elucidating key innate immune responses to AAV. Human PBMCs were isolated from whole blood of three random donors by Ficoll gradient centrifugation and seeded in 96-well U-bottom tissue culture plates. Immediately after seeding, cells were layered three times with either an AAV2 empty capsid, an AAV2 CMV > null vector, or AAV2 > CAG > GFP (all pre-treated for 1 hour with chimeric anti-AAV2 mAb hA20 (mouse Fab fused to human Fc)). After 24 hours, the supernatant was removed and Luminex was performed twice. mIL2RA results are shown. Results for INFα2, CXCL10, MCP3, and INFg are also shown. [Figure 12] Vector opsonization with chimeric antibodies (human Fc / mouse Fab) against AAV9 and AAVrh10 capsids facilitates robust and sensitive measurement of innate immune signaling responses in newly isolated human PBMCs. [Modes for carrying out the invention]

[0017] AAV capsid-specific cytotoxic T lymphocytes (CTLs) are CD8 +These CTLs are formed when T cells are activated and are often observed in clinical studies after systemic AAV vector administration. These CTLs can cause loss of transgene expression by disrupting transdextrins. AAV capsid-derived peptide targets for these CTLs are inherent to the characteristics of the gene delivery vehicle and cannot be eliminated. However, there is evidence that the signals that trigger these CTLs originate from unmethylated CpG motifs in the AAV vector genome. For example, there is significant hypomethylation of CpG dinucleotides present in the AAV vector genome, and activation of the innate Toll-like receptor 9 (TLR9)-MyD88 pathway in preclinical studies correlates with the frequency of CpG dinucleotides in the AAV vector genome. Clinical evidence suggests that AAV vectors with low CpG density evoke a weaker CTL response and more sustained transgene expression, while AAV vectors with high CpG density evoke a stronger CTL response and loss of transgene expression. A method for determining the relevant response to a target AAV composition is disclosed herein.

[0018] Before describing the methods and compositions of the present invention, it should be understood that the present invention is not limited to the specific methods or compositions described and can, of course, be modified. It should also be understood that the scope of the present invention is limited only by the appended claims, and that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting.

[0019] Where a range of values ​​is provided, it should be understood that, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of that range is also specifically disclosed to the tenth of a unit of the lower limit. Each smaller range between any stated value or value between any stated range and any other stated value or value between any stated range is included in the present invention. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range that includes either, neither, or both of the limitations in a smaller range is also included in the present invention, provided that it conforms to any specifically excluded limitations in the stated range. Where a stated range includes one or both of the limitations, a range that excludes either or both of the limitations that they include is also included in the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but here we describe some potential and preferred methods and materials. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials in connection with the reference of those publications. In the event of any conflict, it should be understood that this disclosure takes precedence over any disclosure in the incorporated publications.

[0021] As used herein and in the appended claims, the singular forms "a," "an," and "the" should be noted to include multiple references unless the context explicitly indicates otherwise. For example, a reference to "a cell" includes multiple such cells, and a reference to "the peptide" includes one or more peptides and their equivalents known to those skilled in the art, such as polypeptides.

[0022] The publications discussed herein are provided only for disclosures prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention has no prior rights to such publications on the grounds of prior art. Furthermore, the dates of the publications provided may differ from the actual publication dates which may need to be independently verified.

[0023] As used herein, “commercially available” compounds include those from Acros Organics (Pittsburgh PA), Aldrich Chemical (including Milwaukee WI, Sigma Chemical, and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire UK), BDH Inc. (Toronto, Canada), Bionet (Cornwall, UK), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall UK), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall UK), and Parish Chemical. It can be obtained from commercially available sources including, but not limited to, Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem, and Argonaut Technology.

[0024] The compounds may also be prepared by methods known to those skilled in the art. As used herein, “methods known to those skilled in the art” can be identified through various reference books and databases. Suitable reference books and papers that detail the synthesis of reactants useful for preparing the compounds of the present invention, or provide references to papers describing the preparations, include, for example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., New York; SRSandler et al., “Organic Functional Group Preparations”, 2nd Ed., Academic Press, New York, 1983; HOHouse, “Modern Synthetic Reactions”, 2nd Ed., WABenjamin, Inc. Menlo Park, Calif. 1972; TLGilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; and J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Specific and similar reactants can also be identified through an index of known chemicals prepared by the American Chemical Society's Chemical Abstract Service, which is available in most public and university libraries, as well as through online databases (for more information, you may contact the American Chemical Society, Washington, DC). Known chemicals that are not commercially available in catalogs can be prepared by custom chemical synthesis companies, and many standard chemical suppliers (e.g., those listed above) offer custom synthesis services.

[0025] "A comparable cell" means a cell that is of the same type as another cell that is a comparison target. Examples of comparable cells can mean cells from the same cell line or similar biological samples from different patients.

[0026] "Inhibiting" a response shall mean either reducing the likelihood of the response occurring or completely preventing its occurrence. "Inhibiting" gene expression in a cell shall mean either reducing the degree of gene expression or completely preventing such expression. "Specifically inhibiting" protein expression shall mean inhibiting the expression of that protein (a) more than the expression of any other protein, or (b) more than the expression of all proteins except for 10 or fewer other proteins.

[0027] "Treating" a disorder shall mean slowing, stopping, or reversing the progression of the disorder. In a preferred embodiment, treating a disorder shall ideally mean reversing the progression of the disorder until the disorder itself is eliminated. As used herein, improving a disorder and treating a disorder are equivalent.

[0028] "Specifically hybridizing" to a nucleic acid shall mean, with respect to a first nucleic acid, that the first nucleic acid hybridizes to a second nucleic acid that has a higher affinity than any other nucleic acid.

[0029] Terms such as "specific binding", "specifically binds" refer to the preferential binding of a molecule to another molecule or moiety in solution or in a reaction, either by non-covalent or covalent bonds. In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is 10 -5 M or less (e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12It is characterized by a KD (dissociation constant) of M or less. "Affinity" refers to the strength of the bond, and an increase in binding affinity correlates with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, used by the Biacore system. The affinity of one molecule for another is determined by measuring the bonding rate of the interaction, for example, at 25°C.

[0030] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to mammals being evaluated for and / or being treated for treatment. In one embodiment, the mammal is a human, for example, a human being for whom gene therapy is desired. The subject may be a human, but may also include other mammals, particularly mammals useful as laboratory models for human diseases, such as mice and rats.

[0031] A suitable definition of a patient sample includes blood and other fluid samples of biological origin, such as biopsy specimens or tissue cultures, or solid tissue samples such as cells derived therefrom and their offspring, including PBMCs. This definition also includes samples that have been manipulated by any method after procurement, such as treatment with reagents, washing, or concentration of a particular cell population. The sample in question is a lung lavage fluid sample. This definition also includes samples concentrated with a specific type of molecule, such as nucleic acids or polypeptides. The term "biological sample" encompasses clinical samples and also includes bone marrow, blood, plasma, serum, etc.

[0032] The isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) is a common clinical practice. Cryopreservation allows for batch processing of samples, which is convenient and improves the comparability of data. Cryopreservation also allows for the preservation of cells for future purposes. PBMC isolation is a common prerequisite for cryopreservation of blood cells, and the most common method for PBMC isolation is density gradient centrifugation using Ficoll-Hypaque, a high molecular weight carbohydrate solution.

[0033] For example, using cell preparation tubes (CPTs) containing Ficoll-Hypaque, the standard procedure for density gradient centrifugation is simplified into two methods: (1) Blood is collected in the same tube and then used to separate PBMCs; (2) The tubes are pre-loaded with Ficoll-Hypaque and separated by gel plugs to prevent interference from blood entering the tubes. After blood collection, the tubes are centrifuged, and the PBMCs and plasma are separated from the red blood cells and granulocytes by the gel plugs. This allows the rotated tubes to be shipped, maintaining the PBMCs in an environment isolated from red blood cells and granulocytes, which can improve their viability and function.

[0034] Typical evaluation of PBMC isolation protocols includes monitoring yield and purity. The latter can be determined to some extent visually, as red blood cell contamination will cause the PBMC pellet obtained after centrifugation to turn red. This is sometimes observed in CPT, and is more pronounced than in the conventional Ficoll protocol. However, these contaminated red blood cells are lost after downstream procedures such as cryopreservation and have not been observed to have any impact.

[0035] Yields can be easily determined by either manual or automated cell counting, and it has also been reported that they are nearly equivalent between CPT and the manual Ficoll method.

[0036] The term "preferred state" shall have a meaning that depends on the context in which it is used. Specifically, when used in relation to antibodies, it means a state that allows an antibody to bind to its corresponding antigen. When used in relation to nucleic acid hybridization, it means a state that allows a nucleic acid of at least 15 nucleotides to hybridize to a nucleic acid having a complementary sequence. When used in relation to drug contact with cells, it means a state that allows a drug capable of doing so to enter the cell and perform its intended function. In one embodiment, as used herein, the term "preferred state" means a physiological state.

[0037] As used herein, the terms “correlated” or “correlated with” refer to a statistical relationship between two events, including numbers, datasets, etc. For example, when numbers are involved in the events, a positive correlation (also referred to herein as “direct correlation”) means that as one increases, the other also increases. A negative correlation (also referred to herein as “inverse correlation”) means that as one increases, the other decreases.

[0038] A “dosage unit” refers to a physically distinct unit suitable for a unit dose for a particular individual being treated. Each unit may contain a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the dosage unit form may be determined by (a) the specific characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art to compound such active compounds.

[0039] "Pharmacologically acceptable excipients" means excipients that are generally safe, non-toxic, and useful in preparing desirable pharmaceutical compositions, and include excipients acceptable for veterinary use and for human pharmaceutical use. Such excipients may be solids, liquids, semi-solids, or, in the case of aerosol compositions, gases.

[0040] The terms "pharmaceutically acceptable" and "physiologically tolerable," and their grammatical variations, when referring to compositions, carriers, diluents, and reagents, are interchangeable and indicate that the material can be administered to or on humans without producing undesirable physiological effects to the extent that the administration of the composition is prohibited.

[0041] "Effective amount" means the amount of rAAV antigen or antibody that, when present in the assay, is sufficient to allow opsonization and uptake of rAAV particles.

[0042] The phrase “determining the effectiveness of a treatment” and its variations may include any method for determining whether a treatment is or will be beneficial to a subject. The term “effectiveness of a treatment” and its variations are generally indicated by the alleviation of one or more signs or symptoms associated with a disease and may be readily determined by those skilled in the art. “Effectiveness of a treatment” may also typically refer to the prevention or improvement of toxic signs and symptoms associated with standard or non-standard treatment for a disease. Determining the effectiveness of a treatment is usually indication and disease-specific and may include any method known or available in the art for determining whether a treatment is providing a beneficial effect to a patient. For example, evidence of the effectiveness of a treatment may include, but is not limited to, remission of the disease or indication. Furthermore, the effectiveness of a treatment may also include, but is not limited to, an improvement in the patient’s quality of life, an increase in the expected subject survival rate, a reduction in depression, or a reduction in the relapse rate of the indication (increase in remission time). (See, for example, Physicians' Desk Reference (2010)).

[0043] Associated vectors. As used herein, associated vectors may refer to two or more rAAV vectors that share a high degree of sequence similarity in their genomes. Two associated vectors may be at least about 99% identical, at least about 98% identical, at least about 97% identical, at least about 95% identical, or at least about 90% identical. Identity may be determined in the coding sequence of interest in the rAAV genome or across the length of the vector. In some embodiments, two associated vectors deliver the same therapeutic gene of interest but differ in codon usage or in non-coding sequences.

[0044] AAV gene therapy. Utilizing a viral vehicle to deliver genetic material into cells allows for direct targeting of pathogenic molecules and restoration of their function. The success of adeno-associated virus (AAV)-mediated gene replacement makes AAV therapy a promising gene therapy strategy. Because AAV is non-pathogenic and cannot replicate itself without a helper virus, it serves as the primary vehicle for gene therapy. It is a single-stranded DNA virus that stably and efficiently infects a wide variety of cells in multiple tissues.

[0045] In some embodiments, the vector is a recombinant adeno-associated virus (AAV) vector. AAV vectors are relatively small DNA viruses that can be incorporated into the genome of cells they infect in a stable and site-specific manner. They can infect a wide range of cells without inducing any effect on cell growth, morphology, or differentiation and do not appear to be involved in human pathology. The AAV genome has been cloned, sequenced, and characterized. It comprises approximately 4700 base pairs and includes a reverse end repeat (ITR) region of approximately 145 base pairs at each end, which functions as the origin of viral replication. The remainder of the genome is divided into two essential regions with capsid formation function: the left side of the genome containing the rep gene, which is involved in viral replication and viral gene expression, and the right side of the genome containing the cap gene, which codes for the viral capsid protein.

[0046] The application of AAV as a vector for gene therapy has developed rapidly in recent years. Wild-type AAV can infect dividing or non-dividing cells or tissues of mammals, including humans, with relatively high titers, and can also be incorporated into human cells at a specific site (on the long arm of chromosome 19) (see Kotin et al, Proc. Natl. Acad. Sci. USA, 1990.87:2211-2215, Samulski et al, EMBO J., 1991.10:3941-3950, the disclosures of which are incorporated herein by reference in their entirety). AAV vectors without rep and cap genes lose site-specific integration specificity but can still mediate the long-term stable expression of exogenous genes. AAV vectors exist in cells in two forms, one as an episome outside the chromosome and the other incorporated into the chromosome; the former is the primary form. Furthermore, AAV has not been associated with any human disease to date, nor have any changes in biological characteristics resulting from its incorporation been observed. There are 16 serotypes of AAV reported in the literature, named AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, respectively. AAV5 was originally isolated from humans (Bantel-Schaal, and H. zur Hausen. Virology, 1984. 134:52-63), while AAV1-4 and AAV6 were all discovered in the study of adenoviruses (Ursula Bantel-Schaal, Hajo Delius and Harald zur Hausen. J. Viral., 1999. 73:939-947).

[0047] AAV vectors can be prepared using any simple method. Any serotype of adeno-associated virus is preferred (e.g., Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” JRPattison, ed. (1988), Rose, Comprehensive Virology 3:1, 1974, P. Tattersall “The Evolution of Parvovirus Taxonomy” In Parvoviruses (JR Kerr, SF Cotmore. ME Bloom, RM Linden, C R Parrish, Eds.) pp. 5-14, Rudder Arnold, London, UK (2006), and DE Bowles, JE Rabinowitz, RJ Samulski “The Genus Dependovirus” (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) pp. 15-23, Rudder See Arnold, London, UK (2006), the disclosures of which are incorporated herein by reference in their entirety. Methods for purifying vectors can be found, for example, in U.S. Patents 6,566,118, 6,989,264, and 6,995,006, and in WO / 1999 / 011764 entitled “Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors,” the disclosures of which are incorporated herein by reference in their entirety. Preparation of hybrid vectors is described, for example, in PCT application PCTIUS2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. The use of AAV-derived vectors for gene transfer in vitro and in vivo is described (see, for example, International Patent Application Publications 91 / 18088 and WO93 / 09239, U.S. Patents 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety).These publications describe various AAV-derived constructs in which the rep gene and / or cap gene are deleted and replaced with the gene of interest, as well as the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into organisms). The replication-deficient recombinant AAV according to the present invention can be prepared by co-transfecting a cell line infected with a human helper virus (e.g., adenovirus) with a plasmid containing the nucleic acid sequence of interest adjacent to two AAV reverse terminal repeat (ITR) regions, and a plasmid containing the AAV capsid-forming genes (rep and cap genes). The resulting recombinant AAV is then purified by standard techniques.

[0048] In some embodiments, the vector for use in the method of the present invention is capsid-formed onto viral particles (including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16 AAV virus particles). Accordingly, the present invention is described herein. The present invention includes a method for assaying recombinant viral particles (recombinant because they contain recombinant polynucleotides) containing one of the vectors described herein. Methods for producing such particles are known in the art and are described in U.S. Patent No. 6,596,535.

[0049] AAV test samples may include crude preparations, such as cell lysates, genomic samples, or purified or partially purified capsids or particles. The term substantially pure means preparations containing up to 5% by weight of other originally related materials. In some embodiments, the genomic sequence of the AAV sample differs from the viral genomic sequence and / or the polypeptide sequence in the capsid. Such sequences include analogs and variants produced by recombinant methods, and such nucleic acid and polypeptide sequences are modified by substitution, insertion, addition, and / or deletion of one or more nucleotides in the nucleic acid sequence, resulting in substitution, insertion, addition, and / or deletion of one or more amino acid residues in the recombinant polypeptide.

[0050] Pattern Recognition Receptors. Pattern recognition receptors, or PRRs, are proteins expressed by cells of the innate immune system to recognize pathogen-associated molecular patterns, or PAMPs, associated with microbial pathogens or cellular stress. They may also be called pathogen recognition receptors or primitive pattern recognition receptors. Molecules recognized by a given PRR are called PAMPs, pathogen-associated molecular patterns, and include bacterial carbohydrates (e.g., lipopolysaccharides or LPS, mannose), nucleic acids (e.g., bacterial or viral DNA or RNA), bacterial peptides (flagellin, ax21), peptidoglycans and lipotecoinic acid (derived from Gram-positive bacteria), N-formylmethionine, lipoproteins, and fungal glucans.

[0051] PRRs are classified according to their ligand specificity, function, localization, and / or evolutionary relationships. Based on function, PRRs can be divided into endocytotic PRRs or signal transduction PRRs. Signal transduction PRRs include a large family of DNA sensors such as membrane-bound Toll-like receptors and cytoplasmic NOD-like receptors, retinoid acid-inducible gene I-like receptors, and AIM2. Endogenous PRRs promote the attachment, entrainment, and destruction of microorganisms by phagocytic cells without relaying intracellular signals. These PRRs recognize carbohydrates and include macrophage mannose receptors, glucan receptors present on all phagocytic cells, and scavenger receptors that recognize charged ligands, are found on all phagocytic cells, and mediate the removal of apoptotic cells.

[0052] Recognition of extracellular or endosomal pathogen-associated molecular patterns is mediated by an array of transmembrane proteins known as Toll-like receptors (TLRs). TLRs are members of the IL-1 receptor superfamily. Toll-like receptors trigger a range of mechanisms that lead to cytokine synthesis and secretion, as well as activation of other host defense programs crucial for the development of innate or adaptive immune responses. In mammals, these receptors are assigned numbers 1-11 (TLR1-TLR11). The interaction of TLRs with their specific PAMPs induces NF-κB signaling and MAP kinase pathways, and therefore the secretion of pro-inflammatory cytokines and costimulatory molecules. The molecules released to other cells of the immune system following TLR activation signals make TLRs important components of innate and adaptive immunity.

[0053] The Toll-like receptor 9 response is of particular interest to the assay methods of this disclosure. TLR9 is expressed in immune system cells, including dendritic cells, macrophages, natural killer cells, and other antigen-presenting cells. It binds to DNA (preferably DNA containing unmethylated CpG of bacterial or viral origin) and triggers a signaling cascade that leads to a pro-inflammatory cytokine response. TLR9 is typically activated by unmethylated CpG sequences in DNA molecules. Once activated, TLR9 migrates from the endoplasmic nettle to the Golgi organ and lysosomes, where it interacts with MyD88, a primary protein in its signaling pathway. CpG sites are relatively rare in vertebrate genomes compared to bacterial or viral DNA.

[0054] TLR9 signaling leads to the activation of cells that initiate pro-inflammatory responses, such as the production of cytokines including type I interferon, IL-6, TNF, and IL-12. In humans, the type I IFN system consists of a family of IFN proteins encoded by at least 13 IFN-alpha (IFNA) subtype genes (IFN-α1, -α2, -α4, -α5, -α6, -α7, -α8, -α10, -α13, -α14, -α16, -α17, and -α21), as well as one IFN-beta gene (IFNB), one IFN-epsilon gene, one IFN-kappa gene, and one IFN-omega gene, all of which bind to type I interferon receptors consisting of IFNAR1 and IFNAR2 chains.

[0055] Antibody opsonization. In nature, phagocytic immune cells internalize pathogens and initiate signaling pathways that often construct innate and adaptive immune responses. Once an adaptive humoral response is established, antibodies can specifically bind to pathogens, thereby a) neutralizing pathogen interactions with host cells, b) increasing phagocytic uptake through aggregation and opsonization, and c) initiating complement fixation and membrane attack complex formation, leading to cell lysis and immune mobilization.

[0056] The phenomenon of antibody opsonization to increase rAAV uptake by phagocytic cells can reveal the fundamental immune response to rAAV in PBMCs. Incubating AAV test samples with opsonized antibodies, such as humanized, humanized, or chimeric monoclonal antibodies that are specific to AAV and contain an Fc region that binds to human Fc receptors, such as human FcgR, can increase AAV uptake by plasmacytoid dendritic cells (pDCs) in PBMC compositions, thereby increasing the immune response to the virus. The immune response may include, but is not limited to, type 1 interferon secretion in response to TLR9 stimulation by rAAV containing densely packed genomic CpGs, compared to a control without genomic CpGs.

[0057] Fcg receptors (FcgRs) are the most important Fc receptors for inducing phagocytosis of opsonized particles. This Fc family includes several members such as FcgRI (CD64), FcgRIIA (CD32), FcgRIIB (CD32), FcgRIIIA (CD16a), and FcgRIIIB (CD16b), which differ in their antibody affinities due to their different molecular structures.

[0058] Antibodies, also known as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, the amino-terminal domains of the heavy and light chains being sequence-variable and therefore commonly referred to as variable region domains, or variable heavy chain (VH) domains and variable light chain (VL) domains. While the two domains conventionally associate to form a specific binding region, various non-natural structural configurations of antibodies are known and used in the art, as described herein. Antibodies may contain Fc region sequences.

[0059] A “functional” or “biologically active” antibody or antigen-binding molecule is capable of exhibiting one or more of its intrinsic activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule may have the ability to specifically bind to an antigen, and this binding may then induce or modify cellular or molecular events such as signaling transduction or enzymatic activity. A functional antibody or other binding molecule may also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule to exhibit one or more of its intrinsic activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0060] In this specification, the term “antibody” is used in its broadest sense, and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibodies consisting only of heavy chains, three-chain antibodies, single-chain Fv, nanobodies, etc., as long as they exhibit the desired biological activity, and also includes antibody fragments (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be mouse-type, human-type, humanized, chimeric, or derived from other species.

[0061] The term antibody may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or any immunologically active portion of these polypeptides, i.e., a polypeptide containing an antigen-binding site that immunely binds to an antigen or part thereof of a target of interest, such targets include cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein include the Fc sequence of an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (including engineered subclasses having modified Fc portions that provide reduced or enhanced effector cell activity). The immunoglobulins may originate from any species. In one embodiment, the immunoglobulins are primarily of human origin.

[0062] The term "variable" refers to the fact that certain parts of the variable domain have significantly different sequences within an antibody, and these differences are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The native heavy and light chain variable domains each contain four FR regions that largely adopt a beta-sheet structural arrangement, connected by three hypervariable regions that connect beta-sheet structures and, in some cases, form loops that form part of the beta-sheet structure. The hypervariable regions of each chain are held together in very close proximity by the FR regions and, together with the hypervariable region from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as antibody involvement in antibody-dependent cytotoxicity (ADCC).

[0063] As used herein, the term “hypervariable region” refers to amino acid residues of an antibody involved in antigen binding. The hypervariable region may include amino acid residues from the “complementarity-determining region” or “CDR” and / or those residues from the “hypervariable loop.” “Framework region” or “FR” residues are variable domain residues other than the hypervariable region residues as defined herein.

[0064] The target variable region contains three CDR sequences, which can be obtained from an available antibody with the desired specificity, or from an antibody developed for this purpose. Those skilled in the art will understand that several definitions of CDRs are commonly used, including Kabat's definition (see "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions" Mol Immunol. 2010;47:694-700), with Kabat's definition being the most commonly used, based on sequence variability. Chothia's definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Alternative CDR definitions of interest include Honegger, “Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool.”J Mol Biol.2001;309:657-670, Ofran et al. “Automated identification of complementarity determining regions(CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.”J Immunol.2008;181:6230-6235, Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size:implications for the rational design of antibody repertoires.”J Mol Recognit.2004;17:132-143, and Padlanet al. “Identification of specificity-determining residues in antibodies.”Faseb Examples include, but are not limited to, those disclosed in J.1995;9:133-139, each of which is specifically incorporated herein by reference.

[0065] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for naturally occurring variations that may exist in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of potentially being synthesized by other antibodies and not being contaminated. The modifier “monoclonal” indicates a characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method.

[0066] The antibodies described herein include, specifically, “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody of a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody of a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567, and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). The chimeric antibodies of interest described herein include “primatized” antibodies comprising a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0067] As used herein, “intact antibody chain” includes a full-length variable region and a full-length constant region. An intact “conventional” antibody includes an intact light chain and an intact heavy chain, as well as, for secreted IgG, a light chain constant domain (CL) and a heavy chain constant domain, CH1, hinge, CH2, and CH3. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more “effector functions” that refer to their biological activity attributable to the antibody’s Fc constant region (native sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis (ADCP), and downmodulation of cell surface receptors. Examples of constant region variants include those that alter the effector profile and those that bind to Fc receptors.

[0068] Depending on the amino acid sequence of the constant domains of their heavy chains, intact antibodies can be assigned to different "classes." Intact immunoglobulin antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structural arrangements of different classes of immunoglobulins are well known. Ig forms include hinged or hingeless forms (Roux et al (1998) J.Immunol.161:4083-4090, Lund et al (2000) Eur.J.Biochem.267:7246-7256, US2005 / 0048572, US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types called κ and λ based on the amino acid sequence of their constant domains.

[0069] The opsonized antibody intended for use in the methods of this disclosure opsonizes an AAV, e.g., rAAV, and binds to an Fc receptor, e.g., FcgR. Typically, the opsonized antibody will bind to human FcR for use in human PBMC assays. However, in some situations, it may be useful to perform assays using non-human PBMCs, in which the opsonized antibody is selected to contain an Fc region that will bind to FcR present on the non-human PBMC.

[0070] In some embodiments, monoclonal and polyclonal antibodies, such as human antibodies that may include AAV capsid serum-positive blood, plasma, or serum collected from human subjects potentially exposed to rAAV, are used. Alternatively, if the assay PBMC is non-human, AAV capsid serum-positive blood, plasma, or antibodies collected from research animals after rAAV administration can be used as opsonized antibodies.

[0071] In some embodiments, an AAV capsid-specific monoclonal antibody, or a cocktail of AAV capsid-specific monoclonal antibodies, is used as the opsonized antibody. The monoclonal antibody contains an Fc region suitable for PBMCs in the assay, and for example, a humanized, humanized, or chimeric antibody may be selected or prepared to bind to the FcR present on human PBMCs.

[0072] AAV-specific antibodies are known and commercially available in the art, and include, for example, Progen's AAV capsid protein antibodies specific to VP1, VP1 / VP2, and VP1 / VP2 / VP3. AAV particle antibodies that recognize structural epitopes of viral capsid proteins present only on fully assembled capsids are also available. These epitopes consist of distant amino acids from either the same protein or different adjacent proteins, and they may differ across different serotypes. Examples of specificities include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV12, AAVrh74, and Anc80. Engineered human anti-AAV antibodies are disclosed, for example, in US2018 / 0179265A1, which is specifically incorporated herein by reference. Alternatively, mouse anti-AAV antibodies, such as A20, may be modified by chimerization or humanization for use with human PBMCs.

[0073] Assay method The present invention provides an improved assay for detecting the presence of an AAV-specific cell-mediated immune response, which predicts the immune response generated after introduction of AAV into an individual. In the present invention, a sample containing immune cells, such as dendritic cells, monocytes, T cells, B cells, NK cells, etc., typically including but not limited to peripheral blood mononuclear cell samples, or a derivative thereof, is contacted with an effective amount of a test AAV composition in the presence of an effective amount of an AAV-specific opsonized antibody. The opsonized antibody contains an Fc region sequence that specifically binds to AAV and binds to an Fc receptor present on immune system cells. The sample is incubated for a period sufficient to allow uptake of opsonized AAV, activation of responsive T cells, and release of IFN-α or other cytokines or immune effector molecules. The level of IFN-α or other cytokines or immune effector molecules produced by immune cells indicates the level of cell-mediated responsiveness to the AAV composition. Opsonized antibodies are shown herein to increase the sensitivity of the assay.

[0074] Immune cell samples can be obtained from the individual of interest, for example, a candidate for gene therapy. Alternatively, samples may be obtained from a single individual or a panel of individuals, or commercially available, for use in a general assessment of responsiveness to the AAV test. As used herein, a sample typically refers to a cell sample containing at least T cells and antigen-presenting cells, such as dendritic cells, monocytes, and B cells. Blood samples are conveniently used as a source of complex cell populations, but other cell samples, such as bone marrow, synovial fluid, cerebrospinal fluid, bronchial lavage, and tissue samples, may be used for specific purposes. The term “blood sample” may also refer to mononuclear cell fractions, such as PBMCs, that can be isolated by conventional methods. Samples may be used directly or diluted as necessary. Assays may be carried out in any suitable physiological buffer, such as PBS, saline, HBSS, or dPBS, which may further contain sugars, such as dextrose or heparin, as known in the art.

[0075] PBMC immune cells, comprising T lymphocytes, B lymphocytes, natural killer cells, monocytes, and dendritic cells, can be obtained by minimally invasive blood collection from research animals, healthy human donors, and prospective rAAV GT recipients. After isolation by gradient centrifugation, PBMCs can be frozen or kept in cell culture dishes after exposure to stimulation, allowing for the measurement of immune responses by various assays.

[0076] The sample is typically collected in a blood collection tube. The blood collection tube includes a blood collection tube or other similar container. Conveniently, if the sample is whole blood, the blood collection tube is heparinized. Alternatively, heparin is added to the tube after the blood has been collected. Although whole blood is the preferred and most convenient sample, the present invention extends to other samples containing immune cells, such as lymph, cerebral fluid, tissue fluid, and respiratory fluids including nasal and pulmonary fluid.

[0077] The cells in the cell sample are approximately 10 4 , 2.5×10 4 , 5×10 4 , 7.5×10 4 , 10 5 , 2.4×10 5 , 5×10 5 , 7.5×10 5 , 10 6 It may be present at a concentration of 1 mononuclear cell / ml. Each assay well may contain approximately 1 μl to 1 ml of cells, for example, 1 μl, 10 μl, 100 μl, 500 μl, or more than 1 ml of cells. The MOI of rAAV relative to cells is approximately 10 2 ~about 10 6 For example, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or possibly more.

[0078] Cell samples are incubated with one or more target AAV compositions. One rAAV or a panel of rAAVs can be tested. Suitable positive and negative controls are typically included, for example, empty AAV capsids for negative controls are widely used. For example, the number of AAVs in the assay well is approximately 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 It may be, or even more.

[0079] The AAV test composition may be pre-incubated with an effective amount of opsonized antibody, or the antibody may be added to the assay essentially simultaneously with the test AAV composition. The antibody concentration will vary depending on the specific antibody used, but will generally be sufficient to provide an enhancing effect. For example, suitable monoclonal or polyclonal antibodies may be available at concentrations of approximately 0.1 μg / ml, approximately 0.5 μg / ml, approximately 1 μg / ml, approximately 5 μg / ml, approximately 50 μg / ml, approximately 100 μg / ml, approximately 500 μg / ml, or higher.

[0080] The incubation step may be 5 to 50 hours, more preferably 5 to 40 hours, even more preferably 8 to 24 hours, or any period in between.

[0081] After incubating cell samples with antigens and opsonized antibodies, the release of immunoeffector molecules, such as IFN-α, can be measured by any convenient method. Methods of interest include ELISA or other assays for quantifying the concentration of released effector molecules in the sample buffer. Alternatively, the level of mRNA encoding the immunoeffector molecule can be measured.

[0082] For example, a first antibody having specificity for the target immunoeffector molecule is covalently or passively bound to a solid surface. The solid surface is typically glass or polymer, with the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support can be in the form of a tube, beads, spheres, microplate disks, or any other surface suitable for performing the immunoassay. The binding process is well known in the art and generally consists of crosslinking, covalent bonding, or physical adsorption, and the polymer-antibody complex is washed in the preparation of the test sample. Aliquots of the sample to be tested are then added to the solid-phase complex and incubated for a sufficient period (e.g., 2 to 120 minutes, or overnight if more convenient) and under suitable conditions (e.g., about 20°C to about 40°C) to allow binding of any molecules present in the antibody sample. After the incubation period, the antibody solid phase is washed, dried, and incubated with a portion of the antigen or a second antibody specific to the first antibody. The second antibody is typically linked to a reporter molecule used to indicate binding.

[0083] Both polyclonal and monoclonal antibodies are obtained by immunization with an immunoeffector or its antigenic fragment, and both types are available for use in immunoassays. Methods for obtaining both types of serum are well known in the art. Polyclonal serum is less preferred but can be prepared relatively easily by injecting an effective amount of an immunoeffector or its antigenic portion into a suitable experimental animal, collecting the serum from the animal, and isolating the specific serum by one of the known immunoadsorption techniques. The use of monoclonal antibodies in immunoassays is particularly preferred due to their ability to be produced in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production induced by fusing an immortal cell line with lymphocytes sensitized to an immunogenic preparation can be carried out by techniques well known to those skilled in the art.

[0084] In some cases, a competitive assay may be used. In addition to the patient sample, a competitor to the antibody is added to the reaction mixture. The competitor and the antibody compete for binding to the polypeptide. Typically, the competing molecule is labeled and detected as described above, and the amount of competitive binding will be proportional to the amount of immunity present. The concentration of the competing molecule will be approximately 10 times to approximately equal to the expected maximum immunity concentration to achieve the highest sensitivity and create a linear detection range.

[0085] A reporter molecule provides an analytically discernible signal that enables the detection of an antigen-binding antibody. Detection can be either qualitative or quantitative. The reporter molecules most commonly used in this type of assay are enzymes, fluorophores or radionuclide-containing molecules (i.e., radioisotopes), and chemiluminescent molecules. In enzyme immunoassays, the enzyme is generally conjugated to a second antibody with glutaraldehyde or periodate. However, as is readily apparent, there are a wide variety of different conjugation techniques readily available to those skilled in the art. Commonly used enzymes include, among others, horseradish peroxidase, glucose oxidase, beta-galactosidase, and alkaline phosphatase. The substrate used with a particular enzyme is generally selected to produce a detectable color change upon hydrolysis by the corresponding enzyme. Examples of suitable enzymes include alkaline phosphatase and peroxidase. Fluorescent substrates can also be used, which result in a fluorescent product rather than the chromogenic substrate described above. In all cases, the enzyme-labeled antibody is added to the first antibody-antigen complex, bound, and then any excess reagent is washed away. Next, a solution containing a suitable substrate is added to the antibody-antigen-antibody complex. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which can usually be further quantified by spectrophotometric analysis to indicate the amount of antigen present in the sample.

[0086] There are many variations of these assays. One particularly useful variation is the simultaneous assay, in which all or many of the components are mixed substantially at the same time.

[0087] An alternative method utilizes a filtered immunoplaque assay, also known as the enzyme-linked immunospot assay (ELISPOT). These assays utilize relatively high concentrations of a given cellular product (such as cytokines) in the environment immediately surrounding protein-secreting cells. These cellular products are captured and detected using high-affinity antibodies. The ELISPOT assay is evaluated in "Current Protocols in Immunology, Unit 6.19, pages 6.19.1-8".

[0088] The ELISPOT assay comprises coating a membrane-backed microtiter plate with purified cytokine-specific antibodies, blocking the plate to prevent nonspecific absorption of any other proteins, incubating cytokine-secreting cells with appropriate reagents, removing the cells and reagents, adding a labeled second anti-cytokine antibody, and detecting the antibody-cytokine complex on the membrane. Therefore, the ELISPOT assay utilizes either two high-affinity cytokine-specific antibodies directed against different epitopes on the same cytokine molecule, two monoclonal antibodies, or a combination of one monoclonal antibody and one polyvalent antiserum. ELISPOT generates a spot based on a colorimetric reaction that detects cytokines secreted by a single cell. The spot represents the "footprint" of the original cytokine-producing cell. The spot is persistent and can be quantified visually, microscopically, or electronically. Detection methods using fluorescent labeling are also practiced in the art. The ELISPOT assay can be used in a clinical setting; for example, each kit can assay 24 patient samples at once (using 4 wells per sample in a 96-well plate).

[0089] An alternative method involves using polynucleotide-based assays, such as PCR, to determine the level of effector polypeptides, such as mRNA encoding IFNα, produced by immune cells in the assay.

[0090] The present invention further envisions a kit for evaluating immune responses to AAV compositions. This kit conveniently has a compartmentalized configuration with one or more compartments adapted to receive samples from subjects such as whole blood. One or another compartment may also be adapted to contain heparin if the sample is whole blood, with or without simple sugars such as dextrose. Simple sugars may also be maintained in a separate container. The kit generally contains an effective dose of opsonized antibody and may often include positive and negative control samples.

[0091] Generally, the kit is packaged for sale along with a set of instructions. The instructions generally take the form of a method for measuring a response in a subject, the method comprising: collecting a sample from the subject, wherein the sample contains cells of the immune system capable of producing immune effector molecules after stimulation with AAV; incubating the sample with an antigen; and then measuring the presence or elevation of the immune effector molecules, wherein the presence or level of the immune effector molecules indicates the subject's ability to trigger a cell-mediated immune response.

[0092] As will be apparent to those skilled in the art by reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of the invention. Any enumerated method may be performed in the order of the enumerated events, or in any other logically possible order. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments.

[0093] Although the aforementioned invention has been described in some detail by examples and illustrations for the sake of clear understanding, it will be readily apparent to those skilled in the art that certain changes and modifications may be made thereto without departing from the spirit and are not intended to limit the scope of the invention, which will be limited only by the appended claims.

[0094] Those skilled in the art will recognize, or can verify by means other than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the appended claims.

[0095] experiment The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of the methods of preparation and use of the present invention and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent all or only experiments that can be performed. While efforts have been made to ensure accuracy of the figures used (e.g., quantities, temperatures, etc.), some degree of experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or approximately atmospheric pressure.

[0096] Example 1 Toll-like receptor 9 (TLR9) innate immune receptor-mediated responses to AAV gene therapy have emerged as a significant concern. Unmethylated CpG dinucleotides, abundant in the unmodified AAV vector genome, are TLR9 ligands and are inversely correlated with clinical success. TLR9 signaling leads to type I interferon secretion and subsequent cytotoxic T lymphocyte (CTL) activation, which can contribute to serious adverse events, including thrombotic microangiopathy associated with complement activation. Unfortunately, many current methods for measuring TLR9 responses rely on limited rodent or cell line models, fail to distinguish between relevant vectors, and lack effector cell readout. Here, we present a human PBMC-based assay capable of distinguishing between closely related AAV vectors based on the magnitude of the type I interferon response induced by them and subsequent T cell activation.

[0097] HEK reporter cells are widely used to predict the TLR9 response to candidate AAV gene therapy vectors. This assay can adequately measure TLR9 activation by oligonucleotides, but up to 10 4 Stimulation by the tested MOI of AAV vector doses is insufficient. Type I interferon secretion by mouse PBMCs is minimal, and the vectors are indistinguishable from each other in preliminary experiments. Furthermore, while AAV vectors can induce statistically significant immune signals in human PBMCs, this is highly donor-dependent, making it difficult to measure differences between related vectors.

[0098] The inventors hypothesized that serum after AAV gene therapy opsonizes candidate vectors, increasing Fc receptor (FcR)-mediated uptake and type 1 IFN signaling. The inventors obtained serum from patients before and after AAV9 gene therapy and observed a dose-dependent increase in post-therapy serum-mediated neutralization of AAV2 transduction in cell cultures compared to pre-therapy serum. The inventors then exposed PBMCs from three healthy donors to either empty AAV capsids or AAV2-CAG>GFP. The inventors observed a 19.1 pg increase in INF1α secretion after 24 hours in PBMCs exposed to the GFP vector compared to empty capsids. However, when the vectors were first incubated with post-therapy serum, the inventors observed a more than 3000% increase in signal, with 727.27 pg secreted. Opsonization did not significantly affect the empty capsid signal.

[0099] The inventors then investigated the serum-mediated effect on FcR dependence and found that pretreatment of PBMCs with an FcR-blocking antibody stunted the increase in serum-mediated signaling after gene therapy. Interestingly, flow cytometry analysis of human PBMCs exposed to serum incubation vectors after gene therapy, 72 hours later, showed an increase in the expression of T cell activation markers consistent with CTL activation.

[0100] Since serologically positive human serum after AAV gene therapy is expected to be variable, we subsequently attempted to opsonize AAV2 empty capsids, AAV2-CMV>null, and AAV2-CAG>GFP using AAV monoclonal antibodies. It was a pleasant surprise to observe an opsonized vector-induced INF1α response from PBMC donors that did not respond to stimulation even by oligonucleotide-positive controls. Furthermore, the INF1α response, which would otherwise be undistinguishable by ELISA, increased in response to exposure to unmethylated genomic CpG (p<0.0001) when the vector was first opsonized, and this effect was reproduced by Luminex readout for INFγ, IL1Rα, IL6, IL8, CXCL10, MCP2, and MCP3. Opsonization of AAV vectors prior to exposure to human PBMCs enables the first human-based assay of TLR9 downstream cytokines that can distinguish different vectors based on genomic CpG content, providing a crucial tool for developing vectors that evade TLR9 recognition.

[0101] In nature, phagocytic immune cells internalize pathogens and often initiate signaling pathways that construct innate and adaptive immune responses. Once an adaptive humoral response is established, antibodies can specifically bind to pathogens, a) neutralizing pathogen interactions with host cells, b) increasing phagocytic uptake through aggregation and opsonization, and c) initiating complement fixation and membrane attack complex formation, leading to cell lysis and immune mobilization. The phenomenon of antibody opsonization can be used to increase rAAV uptake by phagocytic cells, which can reveal the fundamental immune response to rAAV in PBMCs.

[0102] Once isolated by gradient centrifugation, PBMCs can be kept in cell culture dishes after exposure to stimulation, allowing for the measurement of immune responses by various assays including Western blotting, flow cytometry, qPCR, ELISA, Luminex, and others. However, signal intensity is generally low and artifact-high for rAAV responses, and current methods are not capable of reliably distinguishing immune responses induced by subtly different vectors, such as those differing only in genomic sequence.

[0103] The inventors hypothesized that rAAV incubation using post-gene therapy (GT) serum increases uptake by plasmacytoid dendritic cells (pDCs) in PBMCs, thereby increasing type 1 interferon secretion in a state of rAAV genomic CpG density compared to controls without genomic CpG. Unmethylated CpG dinucleotides are TLR9 ligands rich in unmodified rAAV vector genomes and are inversely correlated with clinical success. TLR9 signaling leads to type 1 interferon secretion and subsequent CTL activation, which may contribute to serious adverse events, including thrombotic microangiopathy associated with complement activation.

[0104] The inventors obtained serum from patients before and after rAAV9 GT and compared it with pre-GT serum to confirm serological conversion by observing a dose-dependent increase in post-GT serum-mediated neutralization of rAAV2 transduction in cell culture (Figure 1). These data further suggest antibody cross-reactivity between rAAV2 serotypes and rAAV9 serotypes.

[0105] The inventors then exposed PBMCs from three healthy donors to either rAAV2 carrying a GFP-driven CAG promoter, or an rAAV2 empty capsid counterpart lacking a significant genome, both of which are genomes rich in unmethylated CpG pathogen-associated molecular pattern (PAMP) ligands predicted to stimulate a TLR9-mediated innate immune response. In the control group, the inventors observed a 19.1 pg increase in INFα1 secretion after 24 hours in PBMCs exposed to the GFP vector compared to the empty capsid (Figure 2). However, when the inventors first incubated rAAV with post-GT serum, they observed an approximately 3000% increase in signaling and 727.27 pg of INFα1 secretion (Figure 3). Consistent with our predictions, INFα1 secretion over 24 hours appears to depend on genomic PAMP rather than protein PAMP; therefore, opsonization did not significantly affect the signal under empty capsid conditions.

[0106] The inventors then investigated the serum-mediated effect on the dependence of the interaction between serum antibodies and cellular Fc receptors (FcR), which was confirmed when PBMC pretreatment with FcR-blocking antibodies blunted the increase in post-GT serum-mediated signaling (Figure 4). Furthermore, flow cytometry analysis revealed that human PBMCs exposed to rAAV incubated with post-GT serum showed increased expression of T cell activation markers after 72 hours, consistent with CTL effector cell activation downstream of INFα1 secretion by pDCs, compared to exposure to rAAV without serum treatment (Figure 5).

[0107] Since post-GT human serum is expected to be variable, we then investigated opsonization of the rAAV2 empty capsid and rAAV2-CAG (GFP with a commercially available anti-rAAV monoclonal antibody). Mouse monoclonal A20 antibody is well-established in rAAV studies and is known to bind to and neutralize rAAV transduction. However, the A20 pre-incubation-mediated increase in INFα1 secretion was not statistically significant compared to an unincubated rAAV control (Figure 6). Furthermore, preliminary experiments exposing mouse PBMCs to rAAV incubated with increased A20 concentrations suggest that, in this context, opsonization does not improve the signal-to-noise ratio (Figure 7). We hypothesize that, because the relevant mouse and human proteins share only 63% identity as determined by NCBI BLAST, rAAV uptake by human pDCs could not be facilitated by A20 due to incompatibility of the mouse IgG3 Fc region with human FcR. The inventors further speculate that the inability of A20 to significantly improve the signal-to-noise ratio of mouse PBMCs is due to inherent limitations of the mouse model.

[0108] Therefore, we hypothesized that by manipulating an IgG antibody combining a mouse A20 IgG3 Fab subunit and a human IgG1 Fc subunit containing a complementarity-determining region, we could achieve a chimeric monoclonal antibody (hA20) capable of consistently and reliably improving the signal-to-noise ratio after stimulation of human PBMCs with hA20-incubated rAAV.

[0109] The inventors investigated human PBMCs from three healthy donors by stimulating them with or without pre-stimulation chimeric monoclonal hA2O incubation using three rAAV:rAAV2 empty capsids, an rAAV2-CMV null vector carrying an intermediate concentration of genomic CpG, and rAAV2-CAG GFP. For each donor, 2.5 × 10⁶ nucleotides suspended in 100 μl of medium (RPMI + 10% FBS + 1% PennStrep) were used. 4 Individual PBMCs were seeded into a U-bottomed, pre-treated 96-well plate. Immediately, with or without a 1-hour pre-incubation with 12.75 mg of hA20, 2.5 × 10⁶ cells were incubated. 9 rAAV(MOI=10) 4 100 µl of culture medium containing ) was added to three wells in a triple-plate system. The TLR9 agonist ODN2216 and CpG-free ODN2216 were added to the control wells. After 24 hours, the plates were centrifuged at 400 rfu for 7 minutes to pellet the cells, the supernatant was collected, and the INFα1 secretion was assayed.

[0110] As expected, the immune response was highly donor-dependent, with supernatants from all three donors containing increased INFα1 in the ODN2216-positive control condition compared to ODN2216 without CpG, although statistical significance was limited to one donor (Figure 8). Furthermore, the differences in PBMC supernatant INFα1 concentrations induced by the three untreated rAAVs were not statistically significant in the 8 / 9 comparison (Figure 9). However, we observed an opsonized vector-induced INFα1 response with hA20 from PBMC donors that had not previously responded to stimulation with either untreated rAAVs or oligonucleotide-positive controls.

[0111] Furthermore, the INFα1 response increased in response to exposure to unmethylated genomic CpG (p<0.0001) when the vector was first incubated with hA20 (Figure 10), and similar effects were observed with Luminex readout of MCP3, CXCL10, and INFγ (Figure 11).

[0112] As shown in Figure 12, peripheral blood mononuclear cells (PBMCs) were isolated from random healthy donors using Ficoll density gradient centrifugation of whole blood. PBMCs suspended in complete medium (RPMI+FBS+P / S) were seeded in triple rows for each experimental condition into U-bottomed, tissue-culture-treated 96-well plates at 250,000 cells / well. In parallel, AAV vectors were pre-incubated with a capsid-specific chimeric antibody (human Fc / mouse Fab) and then added to the PBMCs. 9 Individual viral genomes (MOI10) 4 The final concentration of the antibody was 2 mg per well. The seeded plates were incubated for 24 hours under standard human cell culture conditions, then the cells were centrifuged at 600 g for 5 minutes, and the supernatant was collected for ELISA analysis of IFN secretion.

[0113] These data demonstrate that vector opsonization using chimeric antibodies (human Fc / mouse Fab) against AAV9 and AAVrh10 capsids facilitates robust and sensitive measurement of innate immune signaling responses on newly isolated human PBMCs. AAV-EC is an AAV2 capsid lacking a significant genome. Both AAV9 and AAVrh10 capsids possess a GFP genome rich in CpG dinucleotides, which is a known pathogen-associated molecular pattern predicted to stimulate Toll-like receptor 9 signaling and IFN-α secretion. Therefore, the data demonstrate genome-dependent innate immune response signaling.

[0114] Our method can be employed to thoroughly elucidate the human immune response to rAAV via TLR9 or via other receptors such as TLR2 and TLR4, which are ligands for viral proteins. Opsonization of rAAV vectors prior to exposure to human PBMCs enables, for the first time, a human-based assay that can distinguish vectors closely associated with innate immune responses, revealing a key tool for developing vectors that evade immune recognition, verifying the consistency of production lots, and modeling the immune response to rAAV using primary cells isolated from future GT recipients.

[0115] The foregoing merely illustrates the principles of the present invention. Those skilled in the art will understand that various arrangements embodying the principles of the present invention and falling within its spirit and scope can be devised, although these are not expressly described or shown herein. Furthermore, all examples and conditional statements enumerated herein are intended primarily to assist the reader in understanding the principles of the present invention and the concepts to which the inventors have contributed to the development of the art, and should be interpreted not as limitations to such specifically enumerated examples and conditions. Moreover, all descriptions herein enumerating the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and those to be developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments illustrated and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims.

[0116] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 461,533, filed on 24 April 2023, the contents of which are incorporated herein by reference in their entirety.

Claims

1. A method for determining the immunostimulatory activity of an AAV composition, wherein the method is (i) a test AAV composition, (ii) an opsonized anti-AAV antibody, and (iii) a population of immune cells are combined in an assay. Incubating immune cells for a sufficient period of time to activate them and release immune effector molecules. This includes measuring the levels of the released immune effector molecules, A method wherein the level of immune effector molecules produced by the immune cells indicates the level of immunostimulatory activity of the test AAV composition.

2. The method according to claim 1, wherein the AAV composition comprises a recombinant AAV capsid.

3. The method according to claim 1 or 2, wherein multiple assays are performed.

4. The method according to claim 3, wherein multiple test AAV compositions are assayed.

5. The method according to claim 4, wherein the plurality of test AAV compositions have different viral genome sequences.

6. The method according to any one of claims 1 to 5, wherein the population of immune cells is peripheral blood mononuclear cells (PBMCs).

7. The method according to claim 6, wherein the PBMC is a human cell.

8. The method according to claim 7, wherein the opsonized anti-AAV antibody comprises a human Fc region sequence.

9. The method according to claim 7 or 8, wherein the PBMC is obtained from an individual to determine the AAV immunostimulatory capacity before therapeutic administration of the AAV.

10. The method according to claim 7 or 8, wherein the AAV composition is a candidate for therapeutic gene therapy.

11. The method according to claim 7 or 8, wherein the AAV composition is a vaccine candidate.

12. The method according to claim 6, wherein the PBMC is a non-human mammalian cell.

13. The method according to any one of claims 1 to 12, wherein the immune effector molecule is a cytokine.

14. The method according to claim 13, wherein the cytokine is a pro-inflammatory cytokine.

15. The method according to any one of claims 1 to 14, wherein the immune effector molecule is a type I interferon.

16. The method according to any one of claims 1 to 15, wherein the immune effector molecule is interferon alpha (IFN-α).

17. The method according to any one of claims 1 to 16, wherein the opsonized antibody comprises AAV capsid serum-positive blood, plasma, or serum collected from a human subject after rAAV administration.

18. The method according to any one of claims 1 to 16, wherein the AAV-specific opsonized antibody comprises AAV capsid serum-positive blood, plasma, or the like collected from a research animal after rAAV administration.

19. The method according to any one of claims 1 to 16, wherein the AAV-specific opsonized antibody comprises an AAV capsid-specific monoclonal antibody.

20. The method according to any one of claims 1 to 16, wherein the AAV-specific opsonized antibody comprises an AAV capsid-specific monoclonal antibody that has been manipulated or selected to bind to a human Fc receptor via its Fc domain or to bind to a non-human Fc receptor.

21. The method according to claim 20, wherein the monoclonal antibody is a chimeric, humanized, or human-type antibody containing a human Fc region sequence.

22. The method according to any one of claims 1 to 21, wherein the level of an immunoeffector protein is determined by the presence of the protein or by the presence of mRNA encoding the protein.

23. The method according to claim 22, wherein the level of the released immunoeffector molecule is determined by Western blotting, flow cytometry, PCR, ELISA, Luminex, ELISApot, cyTOF, or tetramer assay.

24. The method according to any one of claims 1 to 23, further comprising administering to an individual a test AAV composition determined to have low immunostimulatory activity.

25. A kit for use in the method according to any one of claims 1 to 24.

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