Compositions and methods for delivery of AAV vectors

Abstract

The present disclosure relates to compositions and methods for the dosing of subjects previously administered an AAV gene therapy dose with a subsequent AAV gene therapy dose. Several embodiments provided for herein relate to determining the subject's antibody titers to the previously administered AAV serotype and depending on the results providing immunomodulators to permit tissue transduction by the subsequent AAV gene therapy dose.

Description

AAVAN.110WO2 PATENTCOMPOSITIONS AND METHODS FOR DELIVERY OF AAV VECTORSRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Nos.63 / 745810, filed on January 16, 2025, 63 / 788620, filed on April 14, 2025, and 63 / 806570, filed on May 15, 2025, which are hereby incorporated by reference in their entirety.

[0002] This application incorporates the material provided in the accompanying XML file entitled SequenceListing_AAVAN110WO2.xml, created January 16, 2026, which is 10,984 bytes in size.BACKGROUND

[0003] The present disclosure relates to compositions and methods for delivery of AAV gene therapy vectors to target tissues in subjects previously administered AAV-gene therapy.

[0004] After administration of an AAV gene therapy, patients may develop a humoral immune response resulting in high-titer anti-AAV antibodies, which cross-react with AAV serotypes not used to administer the prior administration thus precluding the cross-reactive serotypes from delivering subsequent gene therapy doses. Thus, treatment options are limited for previously dosed patients in need of a subsequent AAV gene therapy dose.SUMMARY

[0005] AAV vectors used for gene therapy may elicit a host humoral immune response, which produces antibodies to the capsid proteins of the AAV vector. AAV gene therapy may be excluded as a treatment option due to the presence of anti-AAV antibodies in subjects due to safety concerns (e.g., an immune response to the therapy) and efficacy concerns due to the inhibition of transduction. Additionally, AAV gene therapy-dosed subjects often have high titers of antibodies that cross react with AAV serotypes not used to deliver theirinitial dose, which limits treatment options for patients in need of a subsequent AAV gene therapy dose.

[0006] Disclosed herein are approaches for delivery of AAV gene therapy vectors to target tissues in patients previously dosed with an AAV gene therapy vector.

[0007] Some embodiments provided herein relate to methods of treating a subject in need of a subsequent AAV gene therapy dose by administering a subsequent dose of an AAV gene therapy vector, and uses thereof. In some embodiments, the subject was previously administered AAV gene therapy. In some embodiments, the method includes identifying a subject previously administered AAV gene therapy that is in need of a subsequent AAV gene therapy dose. In some embodiments, the method includes determining the subject’s antibody titer against the subsequent AAV gene therapy dose. In some embodiments, the methods include characterizing the subject as seronegative for the subsequent AAV gene therapy dose. In some embodiments, if the subject is seronegative for a subsequent AAV gene therapy dose, a subsequent AAV gene therapy dose is administered to the subject. In some embodiments, the subject was previously administered an AAV gene therapy vector having a serotype such as AAVrh74, AAVrhlO, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-SLB101, or AAV10. In some embodiments, the previously administered AAV gene therapy vector is an AAVrh74 serotype, an AAV-SLB101 serotype, or an AAV8 serotype. In some embodiments, the subsequent AAV gene therapy dose is an AAV-SLB101 serotype or an AAVrh74 serotype.

[0008] In some embodiments, the subject is not seropositive for or has a titer below a quantifiable limit of neutralizing and / or non-neutralizing antibodies against the subsequent AAV gene therapy dose. In some embodiments, the subject is not seropositive for or has a titer below the quantifiable limit of neutralizing antibodies against the subsequent AAV gene therapy. In some embodiments, the subject is not seropositive for or has below the quantifiable limit of non-neutralizing antibodies against the subsequent AAV gene therapy. In some embodiments, the subject was administered a previous AAV gene therapy between about 1 month and 15 years previously. In some embodiments, the subject was administered a previous AAV gene therapy dose by systemic IV infusion. In some embodiments, the subject was administered the previous dose intrathecally or directly into the brain. In some embodiments, the subject was administered the previous dose interpleurally.

[0009] In some embodiments, the subject has a neuromuscular disorder, such as a muscular dystrophy, e.g., Duchenne muscular dystrophy or Friedrich’s ataxia (FA). In some embodiments, the subject has a cardiac disorder, such as dilated cardiac myopathy (DCM), hypertrophic cardiac myopathy (HCM), or catecholaminergic polymorphic ventricular tachycardia (CPVT).

[0010] In some embodiments, the subject in need of a subsequent AAV gene therapy is seropositive for or has a detected titer of the subsequent AAV gene therapy. In some embodiments, the subject that is seropositive for or has a detected titer of the subsequent AAV gene therapy is administered an immunomodulator before being administered the subsequent AAV gene therapy. In some embodiments, the immunomodulator is an IdeS or an FcRn blocker or FcRn inhibitor (FcRni). In some embodiments, the immunomodulator is an IdeS. In some embodiments, the immunomodulator is an FcRni. In some embodiments, the immunomodulators are administered to the subject from between 1 day to 1 year before the subsequent AAV gene therapy is administered. In some embodiments, the subsequent AAV gene therapy is administered after digested IgG products are cleared from the subject’s circulation.

[0011] Some embodiments provided herein relate to methods of treating a subject in need of a gene therapy, and uses thereof. In some embodiments, the methods include determining an anti- A AV- S LB 101 antibody titer from the subject. In some embodiments, the methods include characterizing the subject as seronegative for the anti-AAV-SLBlOl antibodies. In some embodiments, the methods include administering a gene therapy comprising an AAV-SLB101 capsid to the subject. In some embodiments, the subject was previously administered a gene therapy comprising an AAVrh74. In some embodiments, the anti-AAV-SLBlOl antibodies are total antibodies (TAbs). In some embodiments, the anti-AAV-SLB101 antibodies are neutralizing antibodies (NAbs). In some embodiments, the subject was previously administered the gene therapy comprising an AAVrh74 capsid between about 1 month and about 15 years before being administered the gene therapy comprising an AAV-SLB101 capsid. In some embodiments, the subject was administered the gene therapy comprising an AAVrh74 capsid by intravenous administration. In some embodiments, the subject was administered the AAV gene therapy comprising an AAVrh74 capsid intrathecally. In some embodiments, the subject was administered the AAV gene therapy comprising anAAVrh74 capsid interpleurally. In some embodiments, the subject has a neuromuscular or cardiac disease or disorder. In some embodiments, the neuromuscular disease or disorder is Duchenne muscular dystrophy or Friedrich’s ataxia.

[0012] Some embodiments provided herein relate to methods of treating a subject in need of a gene therapy, and uses thereof. In some embodiments, the methods include determining an anti-AAV-rh74 antibody titer from the subject. In some embodiments, the methods include characterizing the subject as seronegative for the anti-AAV-rh74 antibodies. In some embodiments, the methods include administering a gene therapy comprising an AAV-rh74 capsid to the subject. In some embodiments, the subject was previously administered a gene therapy comprising an SLB101 capsid. In some embodiments, the anti-AAV-rh74 antibodies are total antibodies (TAbs). In some embodiments, the anti-AAV-rh74 antibodies comprise neutralizing antibodies (NAbs). In some embodiments, the subject was previously administered the gene therapy comprising an AAV-SLB101 capsid between about 1 month and about 15 years before being administered the gene therapy comprising an AAV-rh74 capsid. In some embodiments, the subject was administered the gene therapy comprising an AAV-SLB101 capsid by intravenous administration. In some embodiments, the subject was administered the AAV gene therapy comprising an AAV-SLB101 capsid intrathecally. In some embodiments, the subject was administered the AAV gene therapy comprising an AAV-SLB101 capsid interpleurally. In some embodiments, the subject has a neuromuscular or cardiac disease or disorder. In some embodiments, the neuromuscular disease or disorder is Duchenne muscular dystrophy or Friedrich’s ataxia.

[0013] Some embodiments provided herein relate to methods of treating a subject in need of a subsequent gene therapy comprising an rAAV vector and having antibodies that specifically bind the capsid of the rAAV viral particle comprising the rAAV vector, and uses thereof. In some embodiments, the methods include administering to the subject a therapeutically effective amount of an immunomodulator. In some embodiments, the methods include administering to the subject a therapeutically effective amount of the subsequent gene therapy. In some embodiments, the subject was previously treated with a first gene therapy. In some embodiments, the first and the subsequent gene therapies are of different serotypes. In some embodiments, the antibodies are total antibodies measured by an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the antibodies are neutralizingantibodies measured by cellular assay. Tn some embodiments, the at least one immunomodulator comprises an IdeS or an FcRn blocker or inhibitor. In some embodiments, the at least one immunomodulator is an IdeS. In some embodiments, the at least one immunomodulator is an FcRNi. In some embodiments, the at least one immunomodulator is administered to the subject at least one day before the subsequent AAV-mediated gene therapy dose is administered. In some embodiments, the subsequent AAV-mediated gene therapy dose is administered after digested IgG products are cleared from the subject’s circulation. In some embodiments, the subsequent gene therapy comprises an AAV-SLB101 capsid and the first gene therapy comprises an AAV-rh74 capsid. In some embodiments, the subsequent gene therapy comprises an AAV-rh74 capsid and the first gene therapy dose comprises an AAV-SLB101 capsid.

[0014] Some embodiments provided herein relate to methods of treating a subject in need of a gene therapy, and uses thereof. In some embodiments, the methods include determining an antibody titer from the subject. In some embodiments, the methods include characterizing the subject as seronegative for the antibody titer. In some embodiments, the methods include administering a second gene therapy to the subject. In some embodiments, the subject was previously administered a first gene therapy different from the second gene therapy. In some embodiments, the antibody titer comprises an antibody against AAV1, AAV2, AAV3, AAV4, AAV5. AAV6, AAV7, AAV8, AAV9, AAVrhlO. AAVrh74, and / or AAV-SLB101. In some embodiments, the second gene therapy comprises a capsid specific to the antibody titer, and wherein the second gene therapy comprises a capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6. AAV7, AAV8, AAV9, AAVrhlO, AAVrh74, or AAV-SLB 101. In some embodiments, the first gene therapy comprises a capsid that differs from the second gene therapy, and wherein the first gene therapy comprises a capsid of AAV1, AAV2, AAV3. AAV4, AAV5. AAV6, AAV7. AAV8, AAV9, AAVrhlO. AAVrh74, and / or AAV-SLB101.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a non-limiting example of a heat map showing percent AAV9, AAV-SLB101. and AAVrh74 cross reactivity in donor samples from individuals with Duchenne muscular dystrophy (DMD).

[0016] FIG. 2 is a non-limiting example of total antibody (TAb) IgG titers in seropositive donor samples from individuals with Duchenne muscular dystrophy (DMD).

[0017] FIG. 3A is a non-limiting example of the distribution of anti-AAV IgG titers in seropositive donor samples from individuals with Duchenne muscular dystrophy (DMD).

[0018] FIG. 3B is a graph of total AAV-rh74 and AAV-SLB 101 binding antibodies in dosed mice measured using ELISA.

[0019] FIG 3C is a graph of total levels of AAV-rh74 and AAV-SLB 101 neutralizing antibodies in dosed mice measured using a cell-based assay.

[0020] FIG. 4 is a non-limiting example of microdystrophin (pDys) expression in skeletal muscle from mdx mice 30 days after dosing with AAV-SLBlOl-pDys as measured by Western blot intensity.

[0021] FIG. 5 is a non-limiting example of percent positive pDys5 positive myofibers in quadriceps, heart, and diaphragm four days after dosing with AAV-SLB 101-h-pDys5.

[0022] FIG. 6 is a non-limiting example of a longitudinal assessment of AAV-SLB 101 and AAVrh74 neutralizing antibody (NAb) titers over time in mice dosed with an AAVrh74 vector.

[0023] FIG. 7 is a non-limiting example of AAVrh74 and AAV-SLB 101 neutralizing antibody (NAb) titers in non-human primate (NHPs) 30 days post dosing with an AAVrh74 vector (n=7).

[0024] FIG. 8 is a non-limiting example of AAVrh74, AAV8, and AAV-SLB 101 neutralizing antibody (NAb) titers in individuals (Ind.) dosed with AAVrh74 (n=3).

[0025] FIG. 9 is a non-limiting example of a graph of total body luciferase imaging levels in mice following passive transfer of human sera and AAVSLB101-CK8-LUC transduction.

[0026] FIG. 10 is a non-limiting example of a correlation between total antibody (Tab) IgG titers and luciferase expression (total flux [photons / second (p / s)]).

[0027] FIG. 11 is a non-limiting example of tissue specific effects of increasing antibody titers on AAVSLB101-CK8-LUC transduction in quadriceps (top panel) and heart (bottom panel).

[0028] FIG. 12A is a non-limiting example of a longitudinal assessment of AAV-SLB101 and AAVrh74 neutralizing antibody (NAb) titers over time in mice dosed with an AAVrh74 vector.

[0029] FIG. 12B is a non-limiting example of a graph of biodistribution of the AAVrh74 vector across several tissues.

[0030] FIG. 12C is a non-limiting example of a graph of percentage of fibers positive for AAVrh74 transgene-derived pDys in quadriceps as detected by immunofluorescence using the MANHINGE2A antibody that recognizes the four-repeat pDys expressed from the AAVrh74 vector. The MANDYS106 antibody (negative control), recognizes the nNOS domain, which is not a component of the AAVrh74 transgene.

[0031] FIG. 13 is a non-limiting example of the effects of AAVrh74 dosing on protein expression from a subsequent dose of an AAV-SLB101 vector. The diagram is a timeline of the experiment. Western blot shows pDys5 expressed from the AAV-SLB101 vector, which encodes a five-repeat pDys sequence that includes the R16 / R17 neuronal nitric oxide synthase domain recognized by the MANDYS106 antibody. Expression is correlated to anti-AAVrh74 and anti-AAV-SLBlOl antibody titers measured at Day 30.

[0032] FIG. 14 is a non-limiting example of a correlation between AAV-SLB101 NAb Titers and AAV-SLB101 IgG Titers.DETAILED DESCRIPTION

[0033] Reference is made to particular features and / or non-limiting embodiments of the disclosure. It is to be understood that the disclosure in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment, or a particular claim, that feature may also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments, generally.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by those of skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0035] Pre-existing anti-AAV antibodies in serum arise from natural exposure to wild type AAV and vary by serotype, geography, and age. Total antibody (TAb) and neutralizing antibody (NAb) assays for anti-AAV antibodies may be useful for defining patient eligibility and optimizing dosing strategies. Anti-AAV antibodies include TAbs that recognize the AAV capsid of which NAbs, that block vector transduction, are a subset.

[0036] NAbs may be evaluated using in vitro cell-based transduction assays that measure functional inhibition of AAV-mediated gene transfer by patient antibodies. Such methods are more labor-intensive and variable than ELISA-based methods. They require live cells, transduction reagents and optimized conditions for each serotype. In vitro cell-based transduction assays enable determination of a transduction-inhibitory titer threshold. Titers may be correlated with reduced vector potency and may also be used to guide patient selection. For example, if a patient has a high NAb titer to a specific AAV serotype, that serotype may be unsuitable for dosing that patient. It may also be possible to determine NAb threshold levels under which transduction is efficacious.

[0037] TAbs may be evaluated using capture-based ELISA assays, which detect binding antibodies against AAV capsids without measuring functional neutralization. Interpretation of these assays may depend on assay sensitivity and defined thresholds. Thresholds are often empirically derived and may not directly correlate with impact on transduction. Such ELISA formatted assays for detecting TAbs may be operationally simpler for clinical trial integration as they may be more amenable to standardization and high-throughput implementation than cell-based assays.

[0038] Understanding the strengths and limitations of anti-AAV antibody assays is critical for informing patients selection for clinical trials , redosing feasibility, and interpretation of transduction. Holistic approaches that integrate assay type and titer threshold help select for patients likely to benefit from therapy while excluding those with truly inhibitory seropositivity. Strategies for redosing with different AAV serotypes may circumvent pre-existing immunity and enable transduction in patients previously treated with an AAV gene therapy.

[0039] “Humoral immunity” has its ordinary meaning as understood in light of the specification and may include a type of adaptive immunity that involves the production of antibodies by B lymphocytes. Humoral immunity involves the production of both neutralizingantibodies (NAbs) and non-neutralizing antibodies. NAbs are antibodies against viral surface antigens which can bind the free virus and prevent it from infecting a cell. NAbs may persist throughout a subject’s lifetime. Non-neutralizing antibodies are antibodies that bind to viruses but are unable to block viruses from infecting cells. Non-neutralizing antibodies may also be protective against viral infections by recruiting proteins or cells that destroy antibody-virus complexes in the extracellular space. Inside of cells, non-neutralizing antibodies may block the virus life cycle. Non-neutralizing antibodies may also impact viral infection through Fc-mediated capsid uptake into dendritic cells and macrophages and through increasing tissue inflammation. Total antibody (TAb) assays measure for all antibodies that bind to a viral vector, while NAb assays assess antibodies that prevent a viral vector from transducing cells.

[0040] “Immunogenicity” has its ordinary meaning as understood in light of the specification and may include the capacity to raise neutralizing antibodies (NAbs) which may result in infection inhibition. Anti- AAV antibodies, for example, antibodies resulting from environmental exposure or prior AAV treatment, can limit efficacy of AAV gene therapies. Neutralizing antibodies (NAbs) may prevent effective transduction of systemically delivered AAV gene therapies. A single dose of an AAV vector may elicit a robust humoral response, precluding redosing a subject with an AAV vector having the same, substantially the same, or a related AAV serotype, such as an AAV serotype derived from another (e.g., parental) serotype. Characterization of anti-AAV antibodies to therapeutic capsids may identify AAV capsids that can evade or mitigate immune response barriers and optimize treatment efficacy.

[0041] “Cross reactivity” has its ordinary meaning as understood in light of the specification and may include an antibody that can bind more than one an antigen. For example, an antibody that recognizes and binds an AAV capsid of one serotype and that also recognizes another AAV capsid having a different serotype has cross-reactivity for these two AAV serotypes. Subjects infected with an AAV serotype are often found to have antibodies in their sera that recognize AAV serotypes from viruses to which they were not exposed and are said to have cross-reacting antibodies.

[0042] “Seropositive” has its ordinary meaning as understood in light of the specification and may include the results of a laboratory test that shows the presence of a specific marker, such as an antibody, in the blood. A seropositive test result usually means that a subject has been exposed to or infected with a vims or other infectious agent and has madeantibodies against it. In some embodiments, a subject may be classified as seropositive because the antibodies in their blood cross react or recognize an antigen to which they have not been exposed. Determination of whether a subject is seropositive may require identification of a range or a threshold amount of antibody in the subject’s sera using laboratory tests. In some embodiments, seropositivity ranges and thresholds may be determined by testing the biological effects of a subject’ s antibody titers or levels. For example, having certain levels of an antibody in their serum may prevent or facilitate a biological event, such as the transduction of an AAV into a target tissue. In some embodiments, a subject that is seronegative for an AAV may be treated with that AAV, which transduces target tissues within the subject.

[0043] The term “intravenous immunoglobulin” or “IVIG” as used herein has its ordinary meaning as understood in light of the specification and may include IV-administered immunoglobulin. It is a control comprising pooled antibodies.

[0044] The term “IVIS BLI” as used herein has its ordinary meaning as understood in light of the specification and may include In Vivo Imaging System Bioluminescence Imaging. It is a technique that uses light-emitting enzymes to visualize biological processes within living animals. IVIS BLI typically involves injecting a substrate (like D-Luciferin) into an animal carrying a luciferase-expressing reporter gene, which then emits light. The emitted light is detected by the IVIS BLI instrument, creating images that can be used to study various biological phenomena. IVIS BLI may be done on different days after an animal has been administered an AAV vector that expresses a luciferase report gene. For example, imaging may be done between 1 and 14 or more days after dosing, e.g., 3, 5, 7, 9, 10, 11, 12 13, 14 or more days after dosing.

[0045] The term “immunomodulator” has its ordinary meaning as understood in light of the specification and may include a substance that changes the immune response of a subject by stimulating or suppressing the immune system. Immunomodulators may target different regulatory pathways and aspects of the immune system. Immunomodulators that decrease an immune response may be called immunosuppressants. Non-limiting examples of immunosuppressants include methylprednisolone, rapamycin, and rituximab. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) and neonatal Fc receptor (FcRn) inhibitors (FcRni) may also be considered immunomodulators that suppress the immune system. Various IdeS are known in the art, and include, for example, imilifidase, a cysteineproteinase secreted by Streptococcus pyogenes that interferes with phagocytic killing by specifically cleaving the heavy chain of all four immunoglobulin G subclasses in the lower hinge region. This rapid fragmentation disables IgG's ability to trigger complement or recruit immune cells for antibody-dependent cellular toxicity (ADCC), effectively removing pathogenic antibodies from circulation. As used herein, IdeS also refers to modifications, variants, or improvements to an IdeS, which may be administered to subjects to mediate degradation of existing NAbs to AAVs to improve and / or permit AAV-mediated transduction of target tissue. FcRn, also known as the Brambell receptor, is the major histocompatibility complex (MHC) I-related receptor encoded by the FCGRT gene. FcRnis inhibit the FcRn, which plays a role in the distribution, transport, and persistence of IgG. Preventing the binding of FcRn to IgG may shorten the half-life of IgG. Efgartigimod is an example of a FcRni.

[0046] The term “antibody” has its ordinary meaning as understood in light of the specification and may include proteins produced by the immune system that bind antigens. Examples of antigens include polypeptides, such as polypeptides on the surface of a microorganism (e.g., bacteria, fungi, parasites and viruses), chemicals, or any other molecule or fragment thereof that induces an immune response resulting in the production of antibodies. Antibodies are also known as immunoglobulins. There are five types of antibodies or immunoglobulins: IgM, IgD, IgG, IgA, and IgE. IgG is the most abundant immunoglobulin in the body and has a half-life of approximately 21 days in humans. High serum levels and long half-life of IgG are largely reliant on FcRn-mediated recapture and recirculation.

[0047] A “subject” has its ordinary meaning as understood in light of the specification and may include an animal, such as a mammal, that is the object of treatment using a method or composition as provided for herein. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and humans. In some embodiments, the subject is human. As used herein, the terms “patient.” and “human” may be used interchangeably.

[0048] The terms “treating,” “treatment,” “therapeutic,” or “therapy” have their ordinary meaning as understood in light of the specification, and do not necessarily mean total cure or abolition of the disease or condition. Any reduction, alleviation, improvement, or amelioration of any undesired signs or symptoms of a disease or condition, to any extent, may be considered treatment and / or therapy. To “treat” a disease as the term is used herein, meansto reduce the frequency of, or reduce, alleviate, or ameliorate, the severity of at least one sign or symptom of a disease or disorder experienced by a subject. As used herein, the term “ameliorate” means to improve, to make better or to become better. In some embodiments, a treatment is a dose of an AAV gene therapy vector. In some embodiments, amelioration or symptom improvement occurs when a patient receives an effective amount of a treatment. Treatment efficacy may be evaluated by determining if there has been a reversal of damage done to a tissue or tissues in the body or an improvement of a symptom or symptoms. Treatment may stimulate tissue regeneration, halt continued degeneration of a tissue and / or reverse damage. Treatment efficacy may be determined by conducting tests and comparing results to baselines measured before treatment was administered. In some cases, treatment may be determined by conducting tests and comparing results to results from individuals who have not been treated and may or may not be in need of treatment. Non-limiting examples of tests that may be administered to determine treatment efficacy include tests that measure molecules in blood or urine and physical tests that measure, for example, heart function such as arrhythmias, ejection fraction percentages, end-diastolic volumes (EDVs) and end-systolic volumes (ESVs). Tests may also be used to evaluate muscle size and / or muscle strength and range of motion to defect joint stiffness and contractures. Timed function tests may also be used to evaluate treatment efficacy including North Star Ambulatory Assessment (NSAA), which is a standard test for ambulant boys, assessing functional skills like getting up from the floor, walking, and climbing stairs. Timed function tests may also be used to measure ability to perform tasks in set times, such as the 30-Second Sit-to-Stand Test, which counts how many times a child can stand up from a chair in 30 seconds, the timed Up and Go (TUG) test, which measures time to stand, walk, turn, and sit, and the 6-Minute Walk Test (6MWT), which measures distance walked in 6 minutes. Tests may also measure expression levels and / or localization of the protein being expressed from the AAV vector. Tests may also measure changes in the levels of an endogenous wild type and / or mutated gene products, including proteins and RNAs, to determine treatment efficacy.

[0049] The term “administration,” as used herein, has its ordinary meaning as understood in light of the specification and may include the mechanism through which a treatment or a dose is conveyed to a subject or patient. As used herein, a treatment or a dose may comprise without limitation an AAV gene therapy vector or composition thereof, animmunomodulator, a drug, a pharmaceutical composition. A dose or treatment may be administered without limitation, orally, systemically including intravenously, intrathecally, intrasciatically, or interpleurally.

[0050] The term “effective amount,” as used herein, has its ordinary meaning as understood in light of the specification and may include an amount capable of preventing or treating a disease or condition or otherwise capable of producing an intended therapeutic effect, such as reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. In some embodiments, the treatment prevents the onset of disease symptoms. “Effective” may also be used to refer to the delivery of an AAV vector to a target tissue such that the gene of interest in the AAV vector is expressed at a level in the target tissue such that the symptoms or condition for which the subject is being treated subside, are ameliorated, or are reversed.

[0051] The term “polynucleotide” has its ordinary meaning as understood in light of the specification and may include a polymeric form of nucleotides of any length, including DNA, RNA, or analogs thereof. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Polynucleotide may be interchangeable to include double- and single-stranded molecules.

[0052] The term “recombinant” has its ordinary meaning as understood in light of the specification and may include a polynucleotide that is a product of various steps of alteration including but not limited to restriction enzyme digestion followed by ligation steps, alterations introduced by procedures such as polymerase chain reaction (PCR) (for example, introduction of restriction enzyme sites and nucleotide substitutions), and / or other procedures that result in a polynucleotide that is distinct from a polynucleotide found in nature and / or a combination of polynucleotides not found in nature. A recombinant virus refers to a viral particle that includes a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0053] The term “gene” has its ordinary meaning as understood in light of the specification and may include a polynucleotide containing at least one open reading frame capable of encoding a particular gene product. Any of the polynucleotide sequences describedherein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated.

[0054] The term “transgene” as used herein has its ordinary meaning as understood in light of the specification and may include a nucleic acid sequence to be positioned within a viral vector and encoding a polypeptide, protein, non-coding RNA, or other product of interest. In some embodiments, one recombinant AAV (rAAV) vector may comprise a sequence encoding one or more transgenes (which may be the same gene, or different genes). For example, one rAAV vector may comprise the coding sequence for 1, 2, 3, 4, 5, 6. 7, 8, 9, or 10 transgenes. Embodiments of the transgenes of the present disclosure relate to the treatment of one or more muscular condition including heart conditions, such as cardiomyopathies, and neuromuscular diseases or disorders, or other diseases or disorders as provided for herein.

[0055] The terms “gene transfer” or “gene delivery” have their ordinary meaning as understood in light of the specification and may include methods or systems for inserting DNA, such as a transgene, into host cells, such as those of a subject afflicted with a cardiomyopathy. In some embodiments, gene transfer yields transient expression of nonintegrated transferred DNA, extrachromosomal replication, and / or expression of transferred replicons (e.g., episomes). In some embodiments, gene transfer results in integration of transferred genetic material into the genomic DNA of host cells.

[0056] An “expression vector” has its ordinary meaning as understood in light of the specification and may include a vector comprising a region of nucleic acid (e.g., a transgene) that encodes a gene product (e.g., a polypeptide or protein) of interest. In some embodiments, vectors are used for achieving expression, e.g.. stable expression, of a nucleic acid or a protein in an intended target cell. An expression vector may also comprise control elements operatively linked to the transgene to facilitate expression of the encoded nucleic acid or protein in the target cell. In some embodiments, “expression construct” and “expression vector” may be used interchangeably.

[0057] An “expression cassette” as used herein has its ordinary meaning as understood in light of the specification and may include a gene or genes together with the combination of one or more regulatory elements to which the gene(s) are operably linked and which promote and / or regulate expression of the gene(s). Examples of regulatory elements that may be included in an expression cassette include but are not limited to promoters, introns,polyadenylation signals and 3’ UTRs. Expression cassettes may be components of plasmids. Expression cassettes may also be flanked by AAV inverted terminal repeats (ITRs) and be components of a viral vector such as an rAAV.

[0058] The term “AAV” is an abbreviation for adeno-associated virus and has its ordinary meaning as understood in light of the specification and may include a virus itself or derivatives thereof. AAV may include all subtypes, and both naturally occurring and recombinant forms, unless otherwise indicated. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”), which refers to AAV comprising a polynucleotide sequence not of AAV origin (e.g., a transgene). The term “AAV” includes AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3). AAV serotype 4 (AAV4), AAV serotype 5 (AAV5). AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), serotype rhlO AAV, serotype rh74 AAV, or any modifications or variants thereof. In some embodiments, the AAV is engineered to be myotropic and / or cardiotropic.

[0059] The terms “AAV,” “AAV particle,” and “rAAV vector particle” have their ordinary meaning as understood in light of the specification and may include a viral particle composed of at least an AAV capsid protein and an encapsidated polynucleotide, such as an AAV vector genome comprising an expression cassette.

[0060] The term “AAV-SLB101” refers to a myotropic viral construct comprising a modified AAV9 having an RGDLGLS (SEQ ID NO: 1) peptide inserted between positions 588 and 589 of VPl.

[0061] The term “heterologous” has its ordinary meaning as understood in light of the specification and may include genotypically distinct origins. For example, heterologous polynucleotides are derived from different species as compared to a reference species (for example a human gene inserted into a viral plasmid is a heterologous gene). A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. In some embodiments, a heterologous promoter may also be referred to as a recombinant promoter. A promoter that drives the expression of its native or natural gene is referred to as a native promoter. Other genetic elements that may also be referred to as native elements because they are used toregulate their endogenous genes, include but are not limited to, introns, 5’ UTRS, 3’ UTRS, and poly adenylation signals.

[0062] As used herein, the term “homologous” has its ordinary meaning as understood in light of the specification, and may include a nucleotide or amino acid sequence that shares a specified degree of sequence similarity or identity with a reference sequence and is derived from a common evolutionary origin or performs a substantially similar biological function. Homology may be determined using known sequence comparison algorithms and may be expressed as a percentage of sequence identity or similarity over a defined length of the sequence.

[0063] In some embodiments, regulatory elements, including promoters, may be homologous promoters, such as, for example, promoters that are homologous to a reference promoter and that retain the ability to direct or regulate transcription in a comparable manner. Such homologous promoters may differ in nucleotide sequence from the reference promoter while maintaining substantially similar regulatory activity, expression profile, or tissue specificity, and may be derived from the same species or a different species. The use of homologous promoters is expressly contemplated and encompassed by the present disclosure.

[0064] As used herein, the term “kit” has its ordinary meaning as understood in light of the specification and refers to variations of a portable, self-contained enclosure that includes at least one set of components to conduct one or more of the methods described herein.

[0065] The term “carrier” has its ordinary meaning as understood in light of the specification and may include a diluent, adjuvant, excipient, or vehicle with which the rAAV particle or preparation, and / or rAAV vectors is administered.

[0066] Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term “including” should be read to mean “including, without limitation,” “including but not limited to,” or the like; the term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term “having” should be interpreted as “having at least;” the term “includes” should be interpreted as “includes but is not limited to;” the term “example” is used to provide exemplary instances of the item in discussion, not an exhaustive or limitinglist thereof; and use of terms like “preferably,” “preferred,” “desired,” or “desirable,” and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function, but instead as merely intended to highlight alternative or additional features that may or may not be used in a particular embodiment. In addition, the term “comprising” is to be interpreted synonymously with the phrases “having at least” or “including at least.” When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and / or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should be read as “and / or” unless expressly stated otherwise.

[0067] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art may translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0068] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 90%” includes “90%.” In some embodiments, at least 95% homologous or identical includes 96%, 97%, 98%, 99%, and 100% homologous or identical to the reference sequence. In addition, when a sequence is disclosed as “comprising” a nucleotide or amino acid sequence, such a referenceshall also include, unless otherwise indicated, that the sequence “comprises,” “consists of’ or “consists essentially of’ the recited sequence.Vector

[0069] Provided herein are rAAV particles or rAAV preparations containing such particles. In several embodiments. rAAV particles comprise a viral capsid and one or more transgene as described herein, which is encapsidated by the viral capsid. Methods of producing rAAV particles are known in the art and are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing an rAAV vector may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP3 region as described herein), and transfected into a producer cell line such that the rAAV particle may be packaged and subsequently purified.

[0070] In some embodiments, the AAV is an AAV serotype 1 (AAV-1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), serotype rhlO AAV (AAVrhlO), serotype rh74 AAV (AAVrh74), or a pseudotyped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9). In some embodiments, the AAV vector comprises an AAV serotype 9, or is derived from an AAV serotype 9 vector. In some embodiments, the AAV vector comprises an AAV serotype 8, or is derived from an AAV serotype 8 vector. In some embodiments, the AAV vector comprises an AAV serotype rh74, or is derived from an AAV serotype rh74 vector. In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector comprising a capsid insertion. In some embodiments, the capsid insertion is between residues 588 and 589. In some embodiments, the insertion sequence comprises RGDLGLS (SEQ ID NO: 1). In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector comprising a capsid insertion having the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the AAV vector comprises or is derived from an AAV serotype 9 vector comprising a capsid comprisingthe amino acid sequence set forth in SEQ ID NO: 3. In some embodiments the AAV vector comprises or is derived from an AAV vector comprising a capsid having an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 2 or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID NO:2.

[0071] In some embodiments, a pseudotyped rAAV (e.g., AAV2 / 9. referring to an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9), is an AAV serotype 9 vector comprising a capsid insertion having the amino acid sequence set forth in SEQ ID NO: 1, or an AAV serotype 9 vector comprising a capsid comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments the AAV vector comprises or is derived from an AAV vector comprising a capsid having an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 2 or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more sequence identity to SEQ ID NO: 2.

[0072] In some embodiments, the AAV has about 70%, 75%, 80%, 85%. 90%, 95%, or 100% identity to any of the described AAV serotype vectors described herein, or a percent identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the AAV has between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 75%-100%, 75%-95%, 75%-90%, 75%-75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to an AAV serotype vector. In some embodiments, the AAV serotype vector comprises a capsid modification.

[0073] AAV serotypes and derivatives / pseudotypes, and methods of producing such derivatives / pseudotypes are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ.). Methods for producing and using pseudotyped rAAV vectors are known in the art (see. e.g., Duan et al, J. Virol.. 75:7662-7671, 2001; Halbert et al, J. Virol., 74:1524-1532, 2000; Zolotukhin et al, Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).

[0074] In some embodiments, an rAAV vector of the present disclosure comprises a nucleic acid sequence encoding, in order from 5’ to 3’, a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, a promoter operably linked to a transgene, apolyadenylation signal, and a second AAV inverted terminal repeat (ITR) sequence. In some embodiments, the rAAV vector comprises a nucleic acid sequence comprising an intron.

[0075] In some embodiments, the rAAV vector genome is circular. In some embodiments, the rAAV vector genome is linear. In some embodiments, the rAAV vector genome is single- stranded. In some embodiments, the rAAV vector genome is doublestranded. In some embodiments, the rAAV genome vector is a self-complementary rAAV vector.Recombinant Adeno- Associated Virus Vectors and Therapeutic Use Thereof

[0076] The therapeutic rAAV vectors, therapeutic rAAV particles, or the composition comprising the therapeutic rAAV particles of the present disclosure, may be used for gene therapy for treating neuromuscular or cardiac disorders. The therapeutic rAAV vectors, particles, and compositions comprising the therapeutic rAAV particles may be used for treatment of such neuromuscular or cardiac disorders when administered to a subject in need thereof, e.g., via intravenous delivery, vascular delivery into the coronary arteries and / or direct injection to the heart.Pharmaceutical Formulations and Administration

[0077] Compositions described herein may include a pharmaceutical excipient, buffer, or diluent, and may be formulated for administration to a subject. In some embodiments, the subject is a mammal, such as a mouse, rat, or primate. In some embodiments, the subject is a human. In some embodiments, the composition may be formulated for delivery to and uptake by a cell in a subject, for example, a human being. In some embodiments, compositions may further include a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof. Such compositions may be formulated for use in a variety of therapies, such as for example, in the prevention, and / or treatment of diseases or conditions such as peptide deficiency, polypeptide deficiency, peptide overexpression, polypeptide overexpression, including for example, conditions which result in diseases or disorders as described herein.

[0078] In some embodiments, formulations described herein include at least about 0.1% of the therapeutic agent (e.g., therapeutic rAAV particle or preparation) or more, although the percentage of the active ingredient(s) may be varied and may be between about 1% to about 90% or more of the weight or volume of the total formulation. An amount of therapeutic agent(s) in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art when preparing such pharmaceutical formulations. Additionally, a variety of dosages and treatment regimens may be desirable.

[0079] In some embodiments, the therapeutic rAAV particles or preparations may be delivered in suitably formulated pharmaceutical compositions disclosed herein, including, for example, subcutaneously, intracardially, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells (e.g., cardiomyocytes and / or other heart cells), tissues, or organs. In some embodiments, the therapeutic rAAV particles or the compositions including the therapeutic rAAV particles described herein may be delivered systemically via intravenous injection. In some embodiments, the therapeutic rAAV particles or the compositions including the therapeutic rAAV particles described herein may be injected directly into the heart of the subject. Direct injection to the heart may include injection into one or more of the myocardial tissues, the cardiac lining, or the skeletal muscle surrounding the heart, e.g.. using a needle catheter. In some embodiments, injection is directly into a muscular or cardiac tissue, for example, if delivery is performed concurrently with a surgical procedure or interventional procedure whereby access to the tissue is improved. In some embodiments, the interventional procedure includes any procedure wherein coronary or pulmonary perfusion is altered. In some embodiments, the interventional procedure includes one or more of percutaneous administration, catheterization, or coronary retroperfusion.

[0080] Some embodiments provided herein relate to methods and preparations for use with a subject, such as human or non-human subjects, a host cell in a subject, or a host cell derived from a subject. In some embodiments, the subject is a mammal. In some embodiments,the subject is a companion animal. “A companion animal,” as used herein, refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a human subject.

[0081] In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical compositions including a therapeutic, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a subject, such as a human.

[0082] In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical compositions including a therapeutic, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a subject, such as a human.

[0083] Some embodiments herein relate to the use of one or more excipients, for example, but not limited to. amino acids, salts, base buffers, detergents, and, optionally, stabilizers, in a formulation to promote recovery of a sample from the formulation following one or more stressors, for example, but not limited to, agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, as compared to the same formulation without the one or more excipients. In some embodiments, the one or more excipients in the formulation promote recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more of a sample from the formulation buffer following from agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, as compared to the same formulation without the one or more excipients.

[0084] As described herein, the inclusion of one or more amino acids may make a sample resistant to one or more stressors including, but not limited to. agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, as compared to the same formulation without the one or more amino acid. Some embodiments herein relate to the use of one or more amino acid in a formulation to promote recovery from and / or resistance to one or more stressor, for example, but not limited to, agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, as compared to the same formulation buffer without the one or more amino acid. In some embodiments, the one or more amino acid in theformulation promotes recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more of a sample from the formulation buffer following agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein, or an amount of sample that is in a range defined by any two of the preceding values.

[0085] In some embodiments, the one or more amino acid in the formulation promotes increased sample resistance to freeze-thaw (FT) cycling, as compared to the same formulation buffer without the one or more amino acids. In some embodiments, the one or more amino acids in the formulation promotes increased resistance of a sample in the formulation to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 freeze-thaw cycles, or increased resistance to a number of FT cycles that are in a range defined by any two of the preceding values, as compared to the same formulation without the one or more amino acids. In some embodiments, the formulation promotes recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%. 30%. 40%, 50%, 60%. 70%. 80%, 90%, 91%. 92%. 93%, 94%, 95%, 96%. 97%, 98%, 99%, or 100% more of a sample from the formulation buffer following 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 FT cycles, or an amount of sample that is in a range defined by any two of the preceding values.

[0086] In some embodiments, the one or more amino acids in the formulation promote increased sample resistance to agitation, as compared to the same formulation without the one or more amino acids. In some embodiments, the one or more amino acids in the formulation promote recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%. 40%. 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, 99%, or 100% more of a sample from the formulation buffer following agitation at about 1 rpm, 2 rpm, 3 rpm, 4 rpm, 5 rpm, 6 rpm, 7 rpm, 8 rpm, 9 rpm. 10 rpm, 20 rpm, 25 rpm, 30 rpm, 40 rpm, 50 rpm. 60 rpm, 70 rpm, 75 rpm, 80 rpm, 90 rpm, 100 rpm, 125 rpm, 150 rpm, 175 rpm, 200 rpm, 250 rpm, 300 rpm, 400 rpm, or 500 rpm, as compared to the same formulation buffer without the one or more amino acids.

[0087] In some embodiments, the one or more amino acid in the formulation buffer promote increased sample resistance to thermal stress as compared to the same formulation without the one or more amino acids. In some embodiments, the one or more amino acids in the formulation promote recovery of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more of a sample from the formulation following exposure to temperatures of about 1°C, 2°C, 3°C. 4°C, 5°C, 6°C, 7°C. 8°C, 9°C, 10°C, 20°C, 25°C, 30°C. 40°C, 45°C, 50°C, 55°C, or 60°C, as compared to the same formulation without the one or more amino acids.

[0088] In some embodiments, the formulations disclosed herein comprise one or more amino acid. In some embodiments, the one or more amino acid increase the stress resistance of the formulation as compared to the stress resistance of the same formulation without the one or more amino acid. For example, in some embodiments, the one or more amino acid increases resistance to agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein. In some embodiments, the one or more amino acid comprises alanine, arginine, glutamic acid, glycine, methionine, proline, and / or serine. In some embodiments, the formulation comprises about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM. 70 mM, 75 mM, 80 mM, 90 mM. 100 mM, 125 mM, 250 mM. 175 mM, or 200 mM alanine, arginine, glutamic acid, glycine, methionine, proline, and / or serine, or an amount of alanine, arginine, glutamic acid, glycine, methionine, proline, and / or serine that is in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 0.1-200 mM, 0.1-150 mM, 0.1-100 mM, 0.1-75 mM, 0.1-50 mM, 0.1-25 mM, 0.1-10 mM, 0.1-1 mM. 1-200 mM, 1-250 mM, 1-100 mM, 1-75 mM, 1-50 mM, 1-25 mM, 1-10 mM, 10-200 mM, 10-150 mM, 10-100 mM, 10-75 mM, 10-50 mM, 10-25 mM, 25-200 mM, 25-150 mM, 25-100 mM, 25-75 mM, 25-50 mM, 50-200 mM, 50-150 mM, 50-100 mM, 100-200 mM, 100-150 mM, or 150-200 mM, alanine, arginine, glutamic acid, glycine, methionine, proline, and / or serine. In some embodiments, the one or more amino acid comprises arginine. In some embodiments, the formulation comprises about 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, arginine, or an amount of arginine in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 20-70 mM, 20-60 mM, 20-50 mM, 20-40 mM, 20-30 mM, 30-70 mM, 30-60 mM, 30-50 mM, 30-40 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, 50-60 mM, or 60-70 mM arginine. In some embodiments, the one or more amino acid comprises proline. In some embodiments, the formulation comprises about 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM proline, or an amount of proline that is in a rangedefined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 20-70 mM, 20-60 mM, 20-50 mM, 20-40 mM, 20-30 mM, 30-70 mM. 30-60 mM, 30-50 mM, 30-40 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, 50-60 mM, or 60-70 mM proline. In some embodiments, the one or more amino acid comprises arginine and proline. In some embodiments, the formulation comprises about 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, arginine, or an amount of arginine that is in a range defined by any two of the preceding values and about 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, proline, or an amount of proline that is in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 20-70 mM, 20-60 mM, 20-50 mM, 20-40 mM, 20-30 mM, 30-70 mM, 30-60 mM, 30-50 mM, 30-40 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, 50-60 mM, or 60-70 mM arginine and about 20-70 mM, 20-60 mM, 20-50 mM, 20-40 mM, 20-30 mM, 30-70 mM, 30-60 mM, 30-50 mM, 30-40 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, 50-60 mM, or 60-70 mM proline.

[0089] In some embodiments, the formulation comprises one or more base buffer. In some embodiments, the one or more base buffer comprise acetate, citrate, histidine, phosphate, succinate, Tris, and / or Tris HC1. In some embodiments, the formulation comprises about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, of one or more of acetate, citrate, histidine, phosphate, succinate, and / or Tris, or an amount of acetate, citrate, histidine, phosphate, succinate, Tris, and / or Tris HC1, that is in a range defined by any of the preceding values. In some embodiments, the one or more base buffer comprises Tris. In some embodiments, the formulation comprises about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM. 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM. or 25 mM, Tris and / or Tris HC1 or an amount of Tris and / or Tris HC1 that is in a range defined by any of the preceding values. In some embodiments, the formulation comprises about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, or an amount of Tris or Tris HC1, that is in a range defined by any of the preceding values. In some embodiments, the formulation comprises about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, tris and Tris-HCl in combination or in an amount of Tris and Tris HCL, that is in a range defined by any of the preceding values. In several embodiments, the buffer comprises about 20 mM Tris. In some embodiments, the buffer comprises 20 mM Tris. In some embodiments, the buffer comprises about 20 mM of Tris and Tris-HCl in combination. In some embodiments, the buffer comprises 20 mM of Tris and Tris-HCl in combination.

[0090] In some embodiments, the formulation comprises one or more salts. In some embodiments, the one or more salt comprises MgC12 and / or NaCl. In some embodiments, the formulation comprises about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 57 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, or 250mM MgC12 and / or NaCl, or an amount of MgC12 and / or NaCl that is in a range defined by any two of the preceding values. In some embodiments, the one or more salt comprises MgC12. In some embodiments, the formulation comprises about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM MgC12, or an amount of MgC12 that is in a range defined by any two of the preceding values. For example, in some embodiments, the formulation comprises between about 0.1-5 mM, 0.1-3 mM, 0.1-1 mM, 0.1-0.5 mM, 0.5-5 mM, 0.5-3 mM, 0.5-1 mM, 1-5 mM, 1-3 mM, or 3-5 mM MgC12. In some embodiments, the formulation comprises about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 57 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 175 mM, 180mM, 190 mM, 200 mM, or 250 mM NaCl, or an amount of NaCl that is in a range defined by any two of the preceding values. In some embodiments, the one or more salt in the formulation promotes recovery of a sample from the formulation following exposure to one or more stressor, for example, but not limited to. agitation stress, shear stress, freeze-thaw stress, thermal stress, or any combination therein. In some embodiments, the stability of the formulation under forced degradation conditions due to storage at rapid temperature shits and multiple freeze-thaw cycles is improved by the addition of sodium chloride.

[0091] In some embodiments, the formulation comprises one or more detergent. In some embodiments, the formulation comprises one or more non-ionic detergent. In some embodiments, the formulation comprises one or more ionic, anionic, and / or cationic detergent.In some embodiments, the formulation comprises one or more polysorbate, for example, but not limited to, polysorbate 20, 40, 60, 65, and / or 80. In some embodiments, the formulation comprises one or more sorbitan, for example, but not limited to sorbitan stearate, sorbitan laurate, sorbitan sesquioleate, sorbitan oleate, sorbitan Tristearate, sorbitan palmitate and / or sorbitan trioleate. In some embodiments, the formulation comprises one or more poloxamer, for example, but not limited to, poloxamer 68. 88. 98, 108. 124. 188, 237, 338, and 407. In some embodiments, the detergent comprises sodium lauryl sulfate. In some embodiments, the formulation comprises benzalkonium chloride. In some embodiments, the formulation comprises centrimonium bromide. In some embodiments, the formulation comprises cocamidopropyl betaine. In some embodiments, the formulation comprises sodium cocoamphoacetate. In some embodiments, the one or more detergent comprises P188 and / or PS80. In some embodiments, the formulation comprises about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%. 0.08%, 0.09%, or 0.1 %. P188 and / or polysorbate 80 (PS80). or an amount of Pl 88 and / or PS 80 that is in a range defined by any two of the preceding values.

[0092] In some embodiments, the formulation comprises one or more stabilizer. In some embodiments, the one or more stabilizer comprise HSA, cyclodextrin, sucrose, and / or sorbitol. In some embodiments, the formulation comprises about 0.00%, 0.01%, 0.05%, 0.1%, 0.5%. 1%, 2%. 3%, 4%. 5%, 6%. 7%, 8%. 9%, 10%, 11%. 12%, 13%, 14%, 15%, 16%. 17%, 18%, 19%, or 20%, HSA, cyclodextrin, sucrose, and / or sorbitol, or an amount of HSA, cyclodextrin, sucrose, and / or sorbitol in a range defined by any two of the preceding values. In some embodiments, the formulation comprises about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM. 200 mM, 210 mM, 220 mM, 230 mM, 240 mM. 250 mM, 260 mM. 270 mM, 280 mM, 290 mM, or 300 mM, HSA, cyclodextrin, sucrose, and / or sorbitol, or an amount of HSA, cyclodextrin, sucrose, and / or sorbitol that is in a range defined by any two of the preceding values.

[0093] In some embodiments, the pH of the formulation is maintained. In some embodiments, the formulation comprises a pH between about 7 and 9. In some embodiments, the formulation comprises a pH of about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or a pH that is in a range defined by any two of the preceding values. In several embodiments, the formulation has a pH between 7.5 and 8.5. In several embodiments, the formulation has a pH between 7.7 and 8.3. In several embodiments, the formulation has a pH of about 8. In several embodiments, the formulation has a pH of 8.

[0094] The rAAVs or pharmaceutical compositions as described herein may be formulated for administration to host cell in an animal, and particularly a human being. In some embodiments, a plurality of injections, or other means of administration, are provided, for example 2, 3, 4, 5, 6, 7, 8, 9. 10 or more injections. Means of administration may be combined, if desired. In some embodiments, administration of the dosing is intramuscular.

[0095] In some embodiments, the number of rAAV particles administered to a subject may range from about 103to about 1016particles / mL, such as. for example, about 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, or 1016particles / mL. In some embodiments, the number of rAAV particles administered to a subject may range from about 1012to about 1016particles / mL. In some embodiments, the number of rAAV particles administered to a subject may range from about 1013to about 1015particles / mL. In some embodiments, the number of rAAV particles administered to a subject may range from about 103to about 1016vector genomes (vg)Zkg, such as for example, about 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, or 1016vg / kg. In some embodiments, the number of rAAV particles administered to a subject may range from about 1012to about 1016vg / kg. In some embodiments, the number of rAAV particles administered to a subject may range from about 1013to about 1015vg / kg. The rAAV particles may be administered as a single dose or divided into two or more doses as required to treat the particular disease or disorder.

[0096] As is apparent to a person skilled in the art in view of the teachings of this specification, a therapeutically effective dose of a viral vector may be empirically determined. A therapeutically effective dose may be administered in a single dose, a plurality of doses, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means of administration and dosage are well known to those of skill in the art and will vary depending on the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations may be carried out with the dose level and pattern being selected by the treating physician.

[0097] For administration of an injectable aqueous solution the solution may be suitably buffered, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions may be suitable for intravenous, intramuscular, intravitreal, subcutaneous and intraperitoneal administration. Sterile aqueous mediums that may be employed for such formulations will be known to those of skill in the art in light of the present disclosure. In some embodiments, the rAAV formulations comprise, consist of, or consist essentially of an active rAAV ingredient, a mono-basic buffer (e.g., sodium phosphate monobasic buffer, a di-basic salt (e.g., sodium phosphate di-basic), a sodium-based tonicifier (e.g., sodium chloride tonicifier), a non-sodium tonicifier (e.g., magnesium chloride hexahydrate tonicifier), a surfactant (e.g., poloxamer 188 surfactant), and / or water. In some embodiments, the rAAV formulations comprise, consist of, or consist essentially of active rAAV ingredient, sodium phosphate mono-basic buffer, sodium phosphate di-based, sodium chloride tonicifier, magnesium chloride hexahydrate tonicifier, poloxamer 188 surfactant, and / or water. In some embodiments, the active rAAV ingredient is present in the formulation in an amount described herein. In some embodiments, the mono-basic buffer (e.g., sodium phosphate mono-basic buffer) is present in the formulation at a concentration between about 0.2 mg / mL and about 0.5 mg / mL. In some embodiments, the di-basic salt (e.g., sodium phosphate di-basic) is present in the formulation at a concentration between about 1.5 mg / mL and about 4 mg / mL. In some embodiments, the sodium-based tonicifier (e.g., sodium chloride tonicifier) is present in the formulation at a concentration between about 8 mg / mL and about 12 mg / mL. In some embodiments, the non-sodium tonicifier (e.g., magnesium chloride hexahydrate tonicifier) is present in the formulation at a concentration between about 0.1 mg / mL and about 0.35 mg / mL. In some embodiments, the surfactant (e.g., poloxamer 188 surfactant) is present in the formulation at a concentration between about 0.05 mg / mL and about 0.8 mg / mL. In some embodiments, water is present to bring the volume of the formulation (e.g. a dosage unit) to 1 mL.

[0098] Some variation in dosage may occur depending on the condition of the subject being treated. The person responsible for administration may determine the appropriate dose for the individual subject. Moreover, for human administration, compositions may be prepared to meet sterility, pyrogenicity, and the general safety and purity standards as required by governing standard, e.g., FDA Office of Biologies standards.

[0099] Sterile injectable solutions may be prepared by incorporating the rAAV particles or preparations in the required amount in the appropriate solvent with one or more of the ingredients enumerated above, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the method of preparation may include vacuum-drying and / or freeze-drying techniques, which yield a powder of the active ingredient plus any additional ingredient from a previously sterile-filtered solution thereof.

[0100] An amount of rAAV particle and / or preparation and a time of administration of such particle and / or preparation will be within the purview of the skilled artisan having benefit of the present teachings. In some embodiments, administration of therapeutic ally-effective amounts of the rAAV particles or preparations of the present disclosure may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. In some embodiments, multiple or successive administrations of the rAAV particle or preparation may be performed, either over a relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions.

[0101] In some embodiments, rAAV particles may be administered in combination with additional agents, including, for example, proteins or polypeptides or various pharmaceutically active agents, including one or more administration of therapeutic polypeptides, biologically active fragments, or variants thereof. The rAAV particles or preparations may be delivered along with various other pharmaceutically acceptable agents as required or determined in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized.

[0102] In some embodiments, treatment of a subject with rAAV particles as described herein is capable of achieving or configured to achieve one or more of the following effects, including, for example: (i) reduction or amelioration of the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith;(iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the length of hospitalization; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; and / or (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.Methods of Treatment and Uses of the Compositions and Therapies

[0103] Some embodiments provided herein relate to methods of treating a subject with a subsequent AAV gene therapy dose after the subject was previously dosed with a first AAV gene therapy, and uses thereof. In some embodiments, the subject has antibodies that cross react with the subsequent gene therapy dose. In some embodiments, the subject does not have antibodies that cross react with the subsequent gene therapy dose. In some embodiments, total antibodies to the subsequent gene therapy dose are measured by ELISA. In some embodiments, the subject has neutralizing antibodies to the subsequent gene therapy dose. In some embodiments, the subject does not have neutralizing antibodies to the subsequent gene therapy dose. In some embodiments, the subject is seronegative for the subsequent gene therapy dose. In some embodiments, the subject is seropositive for the subsequent gene therapy dose. In some embodiments, neutralizing antibodies to the subsequent gene therapy dose are measured by a cell-based assay. In some embodiments, the first gene therapy dose is an AAV-SLB101 serotype. In some embodiments, the subsequent gene therapy dose is an AAV-SLB 101 serotype. In some embodiments, the first gene therapy dose is an AAV-rh74 serotype. In some embodiments, the subsequent gene therapy dose is an AAV-rh74 serotype.

[0104] Some embodiments provided herein relate to methods of treating a subject with a subsequent AAV gene therapy dose when the subject is seropositive for the subsequent AAV gene therapy, and uses thereof. In some embodiments, the subject was previously treated with a first gene therapy, and the first and the subsequent gene therapies are of different serotypes. In some embodiments, the subject in need of a subsequent AAV gene therapy dose has antibodies that specifically bind the capsid of the AAV viral particle of the subsequent dose. In some embodiments, the subject is first administered a therapeutically effective amountof at least one immunomodulator before being administered a therapeutically effective amount of a subsequent gene therapy dose. In some embodiments, the at least one immunomodulator comprises an IdeS or an FcRn blocker or inhibitor. In some embodiments, the at least one immunomodulator is an IdeS. In some embodiments, the at least one immunomodulator is an FcRNi. In some embodiments, the at least one immunomodulator is administered to the subject at least one day before the subsequent AAV-mediated gene therapy dose is administered. In some embodiments, the subsequent AAV-mediated gene therapy dose is administered after digested IgG products are cleared from the subject’s circulation. In some embodiments, intact IgG is rapidly cleaved within minutes of IdeS treatment. In some embodiments, peak reduction in intact IgG is within 2, 3, 4, 5 or 6 hours of IdeS treatment or in a time period that is within a range defined by any of the 2 preceding values. In some embodiments, cleaved IgG fragments (F(ab')2 and Fc) clear over 1, 2, 3, 4, 5, or 6 days or in a time period that is within a range defined by any of the 2 preceding values. In some embodiments, clearance of the cleaved IgG products is evaluated by ELISA. In some embodiments, the subject is measured an IdeS dose by IV infusion. In some embodiments, the subject is administered an IdeS dose of about 0.25 mg / kg BW. In several embodiments, the effective amount of Ides comprises mg / kg BW of about 0.01, 0.025, 0.05, 0.075, 0.1, 0.125. 0.15, 0.175. 0.2, 0.25. 0.5. 0.75, 1.0 or an amount of mg / kg BW that is within a range defined by any two of the preceding values.

[0105] Some embodiments provided herein relate to methods of increasing expression of a gene of interest (GOI) in a subject. In some embodiments, increasing expression of the GOI in a subject comprises administering a therapeutically effective amount of a polynucleotide encoding the GOI from an rAAV vector, a rAAV viral particle that includes an expression cassette encoding the GOI, or a composition including a viral particle including an expression cassette encoding the GOI. In some embodiments, the subject is seropositive for the AAV being used to dose the subject and an immunomodulator is administered before dosing. In some embodiments, the immunomodulator or immunomodulators are administered to the subject from between 1 day to 1 year before the subsequent AAV gene therapy is administered. In some embodiments, the immunomodulator is an IdeS. In some embodiments, the effectiveness of the immunomodulator is determined by evaluating biodistribution of the AAV vector and / or expression of the GOI. In some embodiments, the GOI is expressed in thetarget tissue before the subject’s TgG levels recover. Tn some embodiments, the GOT is a micro dystrophin.

[0106] In some embodiments, the effective amount of the AAV gene therapy is between about 1 x 1012to about 1 x 1016viral genomes (vg) / kg. In some embodiments, the therapeutically effective amount is about 5 x 1013to about 1 x 1014vg / kg or in a range defined by any two of the preceding values. In several embodiments, the effective amount comprises vg / kg of about 1.0 x 1013, 2.0 x 1013, 3.0 x 1013, 4.0 x 1013, 5.0 x 1013, 6.0 x 1013, 7.0 x 1013, 8.0 x 1013, 9.0 x 1013, 1.0 x 1014, or 2.0 x 1014or an amount of vg / kg that is within a range defined by any two of the preceding values. In some embodiments, the effective amount is about 2.0 x 1013vg / kg. In some embodiments, the effective amount is about 3.0 x 1013vg / kg. In some embodiments, the effective amount is about 4.0 x 1013vg / kg. In some embodiments, the effective amount is about 5.0 x 1013vg / kg. In some embodiments, the effective amount is about 6.0 x 1013vg / kg. In some embodiments, the effective amount is about 7.0 x 1013vg / kg. In some embodiments, the effective amount is about 8.0 x 1013vg / kg. In some embodiments, the effective amount is about 9.0 x 1013vg / kg. In some embodiments, the effective amount is about 1.0 x 1014vg / kg.

[0107] Any titles or subheadings used herein are for organizational purposes and should not be used to limit the scope of embodiments disclosed herein.EXAMPLES

[0108] The following non-limiting examples are illustrative only and are not intended to be a limitation on the scope of the disclosure.Example 1 : AAV antibody seroprevalence in individuals with Duchenne muscular dystrophy (DMD)

[0109] Neutralizing antibodies (NAbs) and total antibodies (TAbs) were measured in serum from a cohort of 58 individuals with DMD. Serum samples were from DMD donors aged > 2 to < 19 years with mean age of 10 (standard deviation = 4) years and obtained from CureDuchenne Link (cureduchenne.org / cureduchenne-link / ).

[0110] TAbs were measured using a sandwich ELISA that is AAV serotype and immunoglobulin serotype specific. Absorbance values were normalized to a human serumnegative control pool and evaluated using a statistical cut point. NAb titers were measured using a transduction inhibition assay that is AAV serotype specific. A human serum negative control pool was used, and the NAb titer was determined as the highest serum dilution that reduces expression by > 50% of a CMV-promoter driven reporter luciferase gene in an AAV vector.

[0111] Seroprevalence in donors was evaluated and compared for AAV-SLB101, AAV9, and AAVrh74 (FIG. 1). 98% of donors seropositive for anti- AAV-SLB 101 antibodies had antibodies that cross-reacted with AAVrh74, and 100% of donors seropositive for anti-AAV-SLB101 antibodies had antibodies that cross-reacted with AAV9. Cross-reactivity was assessed through kappa measure of agreement analysis (Sim and Wright, Phys Ther. 2005 Mar;85(3):257-268).

[0112] TAb IgG titers were measured in seropositive samples. Approximately 50% had equivalent titers across the three serotypes, while titers varied by 1 to 2 dilutions in the remaining 50% (FIG. 2).

[0113] Most seropositive donors had TAb IgG titers > 1:6400 (highest titer tested; towards AAVrh74 (58.33%), AAV9 (52.17%), and AAV-SLB101 (43.47%) (FIG. 3A).

[0114] Anti-AAV TAb IgG cross -reactivity for the three serotypes was between 98-100%, despite the vectors belonging to different clades, AAVrh74 (Clade E) and AAV9 (Clade F), and AAV-SLB 101 (derived from AAV9).

[0115] To determine the extent of antibody cross reactivity between AAVrh74 and AAV SLB 101, total binding antibodies (TAbs) were first quantified in mdx mice administered ascending doses of AAVrh74 or AAV-SLB 101 (FIG. 3B). One month post injection, ELISA measurements revealed robust anti AAVrh74 binding responses in AAVrh74 treated animals, whereas anti AAV SLB101 TAbs remained markedly lower across all dose levels (EIG. 3B). Conversely, mice treated with AAV SLB 101 generated high TAbs toward the SLB 101 capsid but exhibited minimal binding to AAVrh74, indicating limited serological cross reactivity between clade E (AAVrh74) and clade E-derived (AAV-SLB 101) vectors.

[0116] Neutralizing antibody (NAb) profiling using a cell-based assay mirrored these findings (EIG. 3C). Homologous NAbs to AAVrh74 were substantially elevated in AAVrh74 treated animals, while cross reactive NAbs toward AAV SLB 101 were consistently low or below detection (EIG. 3C). Together, these data demonstrate that AAVrh74 exposureinduces strong homologous responses but only weak cross-reactive responses toward AAV SLB101, suggesting antigenic distinction between the two capsids.Example 2: AAV-SLBlOl-mediated gene expression analysis

[0117] AAV-SLBlOl-mediated expression of a microdystrophin 5 (pDys5) coding sequence was compared to AAV9-mediated pDys5 expression (FIG. 4) and the timing of AAV-SLB 101-mediated microdystrophin 5 (pDys5) expression was evaluated in mouse heart, diaphragm, and quadriceps tissue (FIG. 5).

[0118] Higher levels of microdystrophin (pDys5) expression were observed in mdx mice (mouse model of Duchenne muscular dystrophy; Jackson Labs C5B16 / 10ScSn-DMDmdx / J #001801) after treatment with AAV-SLB 101 comprising a vector genome comprising a nucleic acid sequence encoding a pDys5 as measured by fold change in pDys5 expression versus AAV9 (FIG. 4).pDys5 was expressed in heart, diaphragm, and quadriceps within 4 days post AAV-SLB 101-pDys5 dosing as determined by the percentage of pDys5-positive myofibers (FIG.5).

[0119] Low cross -reactivity between AAVrh74 and AAV-SLB 101 in AAVrh74-dosed subjects combined with the ability of AAV-SLB 101 to induce rapid transduction and expression within days of dosing provides the opportunity to dose subjects previously dosed with AAVrh74 with AAV-SLB 101.Example 3: Cross Reactivity Profile Is Conserved in Nonhuman Primates and Duchenne Patients

[0120] Neutralizing antibodies (NAbs) to AAVrh74 and AAV-SLB 101 were measured in non-human primates (NHPs) (FIG. 7) and neutralizing antibodies (NAbs) to AAVrh74, AAV8 and AAV-SLB 101 were measured in human patients (FIG. 8) previously dosed with AAVrh74-gene therapy vectors.

[0121] To assess whether subjects previously dosed with AAVrh74 vectors develop cross-reacting antibodies to other AAV serotypes, archived serum samples from nonhuman primates (NHPs) (FIG. 7) and Duchenne patients with prior exposure to AAVrh74 (FIG. 8) were evaluated. In both species, NAb titers to AAVrh74 were substantially higherthan those measured against AAV-SLB101. NHPs previously dosed with an AAVrh74 vector had low AAV-SLB 101 neutralizing antibody (NAb) titers 30 days post dosing (FIG. 7). Human subjects dosed with AAVrh74 gene therapies also had lower titers of AAV-SLB 101 crossreacting NAbs compared to AAV8 cross-reacting NAbs and AAVrh74 NAbs (FIG. 8). Importantly, AAV SLB 101 NAb titers in previously dosed subjects remained low despite high anti-AAVrh74 titers (FIGs. 7 and 8).

[0122] These results unexpectedly demonstrate that AAV-SLB 101 NAb titers in AAVrh74-dosed subjects are unexpectedly low, providing an opportunity to subsequently dose AAVrh74-dosed subjects with AAV-SLB 101 gene therapy vectors. Notably, this finding highlights a key distinction between natural seroprevalence, where prior low-level environmental exposure to AAV generally does not elicit high neutralizing antibody titers, and high-dose systemic AAV administration, which is typically associated with robust anti-capsid antibody responses. In some cases, the AAV-SLB 101 gene therapies may be administered together with immunomodulators currently being used in the clinic.Example 4: Antibody cross reactivity and protein expression analysis following AAVrh74 dosing

[0123] In a longitudinal assessment of NAb titers following dosing with an AAVrh74 gene therapy vector(encoding a four-repeat pDys sequence under the control of the MHCK.7 promoter), AAV-SLB 101 NAb titers remained low out to day 60 (FIG. 6 and FIG.12 show the same data on different scales). In this experiment, a 3E13 vg / kg dose of an AAVrh74 gene therapy was administered to mice, and NAbs to both AAVrh74 and AAV-SLB 101 were measured over time. Mice had similar anti-AAVrh74 and anti-AAV-SLBlOl antibody titers 4 days post dosing (FIG. 12A). However, anti-AAVrh74 NAb titers increased over time and stayed persistently high, while anti- AV- SLB 101 NAb titers decreased and remained low (FIGs. 6 and 12A).

[0124] Biodistribution (FIG. 12B) and immunofluorescence (FIG. 12C) analyses confirmed robust AAVrh74 transduction in multiple muscle groups (FIG. 12B) and robust protein expression and localization in quadricep fibers (recognized by the MANHINGE2A antibody (DSHB)) (FIG. 12C). demonstrating that the low cross reactive anti- A AV- SLB 101 responses were not attributable to insufficient exposure to the AAVrh74 capsid (FIG. 12B).Therefore, lack of transduction cannot explain the low anti-AAV-SLBlOl antibody cross reactivity in mdx mice dosed with AAVrh74. Collectively, these findings indicate that even high dose AAVrh74 exposure does not generate appreciable anti AAV-SLB101 humoral immunity over time in the mdx mouse model.

[0125] The effects of AAVrh74 dosing on protein expression in mice subsequently dosed with an AAV-SLB101 vector were assessed. On Day 1, 4-week old mice were first administered 3.0E13 vg / kg of an AAVrh74-MHCK-pDys-H2 vector, which comprised a microdystrophin transgene with the H2 hinge domain expressed from a myosin heavy chain kinase (MHCK) promoter. (FIG. 13, timeline diagram). On Day 30, anti-AAVrh74 and anti-AAV-SLB 101 antibody titers were measured (FIG. 13, table) after which the mice were dosed with 3.0E13 vg / kg of an AAV-SLB101 vector comprising a microdystrophin-5 (pDys5) transgene expressed from a creatine kinase (CK8) promoter, which is recognized by the MANDYS106 antibody (Sigma which recognizes the nNOS domain). On Day 60, protein expression in mice dosed with the AAV-SLB 101-pDys5 vector was evaluated by Western blotting using an antibody that specifically recognizes the pDys5 (FIG. 13). On day 30, all mice had very high levels of NAbs against AAVrh74 (FIG. 13, table). On Day 60, transgene expression correlated with the anti- AAV-SLB 101 antibody titers measured on Day 30 (FIG.13). For example, mouse 2 had the lowest anti-AAV-SLBlOl antibody titer level (< 5) and the highest level of pDys5 protein expression (FIG. 13).Example 5: Assessing titer threshold at a clinically relevant dose of AAV-SLB 101 in a RAG2 knockout mouse model using passive transfer.

[0126] In this non-limiting example, the relationship between TAb titers and transduction neutralization is assessed using passive transfer studies in a RAG2 knock out mouse, which lacks T cell and B cells, thus making this model a clean system in which endogenous antibodies will not interfere.

[0127] On Day (-1), Rag2-RAGN12 mice were intravenously administered with human immunoglobulin (IVIG) that was confirmed to contain high amounts of anti- AAV-SLB 101 antibodies or plasma from AAV-SLB 101 seropositive human subjects that corresponded to a range of titers from low, mid, to high. On Day (0), mice were dosed (1E14 vg / kg) with an AAV-SLB 101 vector comprising a luciferase transgene operably linked to aCK8 promoter (AAVSLB101-CK8-LUC). On Days 3 and 7 or 14, whole body luciferase activity by in vivo imaging system bioluminescence imaging (IVIS-BLI) was conducted to measure luciferase expression as total flux (photons / second) in mice administered luciferin (FIG. 9). As total antibody (TAb) titers increased, the luciferase signal decreased, which is indicative of a reduction in transduction (FIG. 10). Similar results were obtained when the analysis was 14 days after AAV administration (data not shown).

[0128] AAV transduction into distinct target tissues of the Rag2-RAGN12 mice was differentially affected by increasing antibody titers. In quadriceps, transduction was decreased as antibody titers increased; however, low titer levels did not neutralize the capsid (FIG. 11). Thus, it is possible to establish a TAb titer cutoff for skeletal muscle. Transduction of heart tissue was relatively constant as antibody titers increased (FIG. 11). As such, titer thresholds may differ depending on the tissue being targeted with an AAV-gene therapy. For FIG. 11, the does level used was 1E14 vg / kg, and the x axis is the relative luminescence units in quadricep or heart, with the y axis the titer used for passive transfer (< 1:5 is a titer of less than 1:5; low is a titer of 1:40; low-mid is a titer of 1:80; mid is a titer of 1:160; mid-high is a titer of 1:320; and high is a titer of 1:640; IVIG is a 2 g / kg titer as a control).Example 6: Correlating AAV-SLB101 NAb titers with AAV-SLB101 IgG Titer Levels

[0129] In this non-limiting example, AAV-SLB101 NAbs titers were correlated with AAV-SLB101 IgG titers (FIG. 14). TAb and NAb assays for anti- AAV antibodies may be useful for defining patient eligibility and optimizing dosing strategies.Example 7: Administration of AAV-SLB 101 gene therapy vectors to subjects previously dosed with AAVrh74

[0130] An AAV-SLB 101 gene therapy vector is administered to a subject previously dosed with an AAVrh74 vector. The AAV-SLB 101 vector may be administered to subjects previously dosed with an AAVrh74 vector with or without first administering immunomodulators that reduce IgG antibody levels, for example, an IdeS and / or an FcRni.

[0131] An AAVrh74 gene therapy vector is administered to subjects seronegative for anti- AAV-SLB 101 antibodies. Anti-AAVrh74 TAb and NAb titers and anti- AAV-SLB 101 cross-reacting TAb and NAb titers are measured after administering the AAVrh74 vector andbefore administering the AAV-SLB101 vector (for example, 30 days post-administration of the AAVrh74 vector).

[0132] The subject is administered the AAV-SLB101 vector at least 30 days-post administration of the AAVrh74 vector. Expression of an AAV-SLBlOl-delivered gene of interest (GOI) is measured 30 days post-administration of the AAV-SLB101 vector and correlated with pre-AAV-SLBlOl administration levels of an ti- A AV- S LB 101 cross-reacting TAb and NAb titers to confirm titer levels that are permissive to AAV-SLB 101 administration.

[0133] For subjects that develop SLBlOl-cross reacting-TAb and NAb titers post AAVrh74 dosing, immunomodulators, (including, for example, an IdeS and / or an FcRni), are administered prior to AAV-SLB 101 dosing. Suitable subsequent dosing of AAVs include those which rapidly transduce prior to effects of an IdeS administration no longer remaining in effect.

[0134] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0135] Reference throughout the specification to “one example,” “another example,” “an example.” and so forth, means that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise. While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.

[0136] The use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have,” “has,” and “had,” is not limiting. As used in this specification, whether in atransitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0137] Features, materials, characteristics, or groups described in conjunction with a particular aspect, or example are to be understood to be applicable to any other aspect or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The protection is not restricted to the details of any foregoing examples. The protection extends to any one, or any combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any one, or any combination, of the steps of any method or process so disclosed.

[0138] Furthermore, certain features that are described in this disclosure in the context of separate implementations may also be combined in a single implementation. Additionally, various features described in the context of a single implementation may be implemented in multiple implementations separately or in a suitable sub-combination. Moreover, although features may be described above as acting in certain combinations, one or more feature from a claimed combination may be excised from the combination, and the combination may be claimed as a sub-combination or variation of a sub-combination.

[0139] Moreover, while operations may be depicted in the drawings or described in the specification in a particular order, such operations need not be performed in the particular order or in the sequential order shown, but all operations may be performed in the order needed to achieve desirable results. Other operations that are not depicted or described in the example methods and processes may also be incorporated. For example, one or more additional operation may be performed before, after, simultaneously, or between any of the describedoperations. Further, the operations may be rearranged or reordered in other implementations. Those skilled in the art will appreciate that in some examples, the actual steps taken in the processes illustrated and / or disclosed may differ from those shown in the figures. Depending on the example, certain of the steps described above may be removed or others may be added. Furthermore, the features and attributes of the specific examples disclosed above may be combined in different ways to form additional examples, all of which fall within the scope of the present disclosure.

[0140] For purposes of this disclosure, certain aspects, advantages, and features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular example. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

[0141] Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain examples include, while other examples do not include, certain features, elements, and / or steps. Thus, such conditional language is not generally intended to imply that features, elements, and / or steps are in any way required for one or more examples or that one or more examples necessarily include logic for deciding, with or without user input or prompting, whether these features, elements, and / or steps are included or are to be performed in any particular example.

[0142] Conjunctive language such as the phrase “at least one of X, Y, and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X. Y, or Z. Thus, such conjunctive language is not generally intended to imply that certain examples require the presence of at least one of X, at least one of Y, and at least one of Z.

[0143] Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result.

[0144] The scope of the present disclosure is not intended to be limited by the specific disclosures of preferred examples in this section or elsewhere in this specification, and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive. Although certain dependent claims may be described as depending from a single preceding claim, it is to be understood that each such dependent claim may alternatively be construed as depending from any one of the preceding claims, whether independent or dependent, unless expressly stated otherwise. All such alternative dependencies and combinations are expressly contemplated as part of the disclosure.

[0145] The described embodiments and examples of the present disclosure are intended to be illustrative rather than restrictive, and are not intended to represent every embodiment or example of the present disclosure, and thus, are not to be limited in scope by the specific embodiments and examples described herein. While the fundamental features of the disclosure as applied to various specific embodiments thereof have been shown, described, and pointed out, it will also be understood that various omissions, substitutions, and changes in the details of the compositions and methods that are disclosed, may become apparent and may be made by those skilled in the art without departing from the spirit of the disclosure. For example, it is expressly intended that all combinations of those method steps that perform substantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure. Moreover, it should be recognized that method steps shown and / or described in connection with any disclosed form or embodiment of the disclosure may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. Further, various modifications and variations may be made without departing from the spirit or scope of the disclosure as set forth in the following claims both literally and in equivalents recognized in law.

Claims

WHAT TS CLAIMED TS:

1. A method of treating a subject in need of a gene therapy, the method comprising:determining an an ti- A AV- S LB 101 antibody titer from the subject; characterizing the subject as seronegative for the anti- AAV-SLB 101 antibodies; andadministering a gene therapy comprising an AAV-SLB 101 capsid to the subject,wherein the subject was previously administered a gene therapy comprising an AAVrh74.

2. The method of claim 1, wherein the anti-AAV-SLBlOl antibodies are total antibodies (TAbs).

3. The method of claim 2, wherein the anti- AAV-SLB 101 antibodies are neutralizing antibodies (NAbs).

4. The method of claim 1, wherein the subject was previously administered the gene therapy comprising an AAVrh74 capsid between about 1 month and about 15 years before being administered the gene therapy comprising an AAV-SLB 101 capsid,5. The method of claim 1, wherein the subject was administered the gene therapy comprising an AAVrh74 capsid by intravenous administration.

6. The method of claim 1, wherein the subject was administered the AAV gene therapy comprising an AAVrh74 capsid intrathecally.

7. The method of claim 1, wherein the subject was administered the AAV gene therapy comprising an AAVrh74 capsid interpleurally.

8. The method of claim 1, wherein the subject has a neuromuscular or cardiac disease or disorder,9. The method of claim 8, wherein the neuromuscular disease or disorder is Duchenne muscular dystrophy or Friedrich’s ataxia.

10. A method of treating a subject in need of a gene therapy, the method comprising:determining an anti-AAVrh74 antibody titer from the subject; characterizing the subject as seronegative for the anti-AAVrh74 antibodies; andadministering a gene therapy comprising an AAVrh74 capsid to the subject, wherein the subject was previously administered a gene therapy comprising an SLB101 capsid.

11. The method of claim 10, wherein the anti-AAVrh74 antibodies are total antibodies (TAbs).

12. The method of claim 11, wherein the anti-AAVrh74 antibodies comprise neutralizing antibodies (NAbs).

13. The method of claim 10, wherein the subject was previously administered the gene therapy comprising an AAV-SLB101 capsid between about 1 month and about 15 years before being administered the gene therapy comprising an AAVrh74 capsid.

14. The method of claim 10, wherein the subject was administered the gene therapy comprising an AAV-SLB101 capsid by intravenous administration.

15. The method of claim 10, wherein the subject was administered the AAV gene therapy comprising an AAV-SLB101 capsid intrathecally.

16. The method of claim 10, wherein the subject was administered the AAV gene therapy comprising an AAV-SLB101 capsid interpleurally.

17. The method of claim 10, wherein the subject has a neuromuscular or cardiac disease or disorder.

18. The method of claim 17, wherein the neuromuscular disease or disorder is Duchenne muscular dystrophy or Friedrich’s ataxia.

19. A method of treating a subject in need of a subsequent gene therapy comprising an rAAV vector and having antibodies that specifically bind the capsid of the rAAV viral particle comprising the rAAV vector, the method comprising:administering to the subject a therapeutically effective amount of at least one immunomodulator; andadministering to the subject a therapeutically effective amount of the subsequent gene therapy,wherein the subject was previously treated with a first gene therapy, and wherein the first and the subsequent gene therapies are of different serotypes.

20. The method of claim 19, wherein the antibodies are total antibodies.

21. The method of claim 20, wherein the antibodies are neutralizing antibodies.

22. The method of claim 19, wherein the at least one immunomodulator comprises an IdeS or an FcRn blocker or inhibitor.

23. The method of claim 22, wherein the at least one immunomodulator is an IdeS.

24. The method of claim 22, wherein the at least one immunomodulator is an FcRNi.

25. The method of claim 22, wherein the at least one immunomodulator is administered to the subject at least one day before the subsequent AAV-mediated gene therapy dose is administered.

26. The method of claim 25, wherein the subsequent AAV-mediated gene therapy dose is administered after digested IgG products are cleared from the subject’s circulation.

27. The method of claim 19, wherein the subsequent gene therapy comprises an AAV-SLB101 capsid and the first gene therapy comprises an AAVrh74 capsid.

28. The method of claim 19, wherein the subsequent gene therapy comprises an AAVrh74 capsid and the first gene therapy dose comprises an AAV-SLB101 capsid.

29. A method of treating a subject in need of a gene therapy, the method comprising:determining an antibody titer from the subject;characterizing the subject as seronegative for the antibody titer; and administering a second gene therapy to the subject.wherein the subject was previously administered a first gene therapy different from the second gene therapy.

30. The method of claim 29, wherein the antibody titer comprises an antibody against AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrh74, and / or AAV-SLB101.

31. The method of claim 30, wherein the second gene therapy comprises a capsid specific to the antibody titer, and wherein the second gene therapy comprises a capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrh74, or AAV-SLB101,32. The method of claim 31, wherein the first gene therapy comprises a capsid that differs from the second gene therapy, and wherein the first gene therapy comprises a capsid ofAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrh74, and / or AAV-SLB101.

33. A method of treating a subject in need of a gene therapy, the method comprising:administering a gene therapy comprising an AAV-SLB101 capsid to the subject,wherein the subject was previously administered a gene therapy comprising an AAV-rh74 and is seronegative for anti-AAV-SLBlOl antibodies.

34. A method of treating a subject in need of a gene therapy, the method comprising:administering a gene therapy comprising an AAV-rh74 capsid to the subject, wherein the subject was previously administered a gene therapy comprising an AAV-SLB101 and is seronegative for anti-AAV-rh74 antibodies.

35. An immunomodulator for use in treating a subject having antibodies that specifically bind a capsid of a recombinant adeno-associated virus (rAAV) viral particle of a subsequent gene therapy comprising an rAAV vector, wherein the treatment enables administration of the subsequent gene therapy, wherein the subject was previously treated with a first gene therapy, and wherein the first gene therapy and the subsequent gene therapy are of different rAAV serotypes.

36. Use of a gene therapy comprising a recombinant viral vector in the treatment of a subject who is seronegative for antibodies against the gene therapy, wherein the subject was previously administered a first gene therapy different from the gene therapy used in the treatment.

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