Compositions and methods for avoiding humoral immunity

Abstract

The present disclosure provides, in part, compositions and methods for transiently removing neutralizing antibodies to AAV vectors, expanding the patient population suitable for gene therapy and, in addition, AAV re-medication / re-administration in patients previously treated with AAV vectors.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 914,682, filed Oct. 14, 2019, and U.S. Provisional Patent Application No. 62 / 797,495, filed Jan. 28, 2019. The entire disclosures of those applications are incorporated herein by reference in their entirety.

[0002] Description of Electronically Submitted Text Files The content of the text file submitted electronically with this application is incorporated by reference in its entirety: a computer - readable format copy of the sequence listing (file name: STRD_014_02WO_SeqList_ST25.txt, date of record: Jan. 28, 2020, file size: approximately 180 kilobytes).

[0003] Technical Field This application generally relates to the field of gene therapy, such as gene therapy using adeno - associated virus (AAV) vectors. More specifically, the present disclosure relates to compositions and methods for improving the efficacy of therapy with recombinant AAV using compositions and methods for reducing neutralizing antibodies against recombinant AAV.

[0004] Government Grants This invention was made with government support under Federal Grant No. R01HL089221 awarded by the National Heart, Lung, and Blood Institute (NIH / NHLBI), and Federal Grant No. R01GM127708 awarded by the National Institute of General Medical Sciences (NIH / NIGMS). The federal government has certain rights in this invention.

Background Art

[0005] Adeno-associated virus (AAV) is a helper-dependent parvovirus that can be used for human therapeutic gene delivery. Since the first gene therapy based on AAV1 was approved by the regulatory authorities in 2012, promising results of clinical trials involving recombinant AAV vectors for gene therapy in Leber congenital amaurosis, hemophilia, and other diseases have been reported.

[0006] These gene therapy trials use different natural AAV isolates but share the same exclusion criteria, requiring that the anti-AAV neutralizing antibody (NAb) titer be low or undetectable in the patient candidates to be enrolled. This eligibility criterion was established because of the high prevalence of pre-existing anti-AAV NAbs due to natural exposure in the human population. For example, the prevalence of anti-AAV1 NAb positivity (titer exceeding 1:2) in the entire population of human heart failure patients is approximately 60%. Also, most patients with high NAb titers against AAV serotype 2 also have measurable titers against AAV1, suggesting cross-reactivity between serotypes. NAbs can significantly reduce the gene transfer efficiency of AAV vectors by opsonization. Opsonization promotes clearance, alters biodistribution, blocks cell surface receptor binding, and / or adversely affects post-attachment steps essential for efficient transduction.

[0007] Efforts to develop strategies to overcome pre-existing anti-AAV Nab have focused on AAV capsid engineering and decoys, transient pharmacological immunosuppression, and plasmapheresis. These approaches have been found to be insufficient in enhancing AAV gene transfer by avoiding or reducing pre-existing NAbs in preclinical animal models and humans.

[0008] Therefore, there is a need in the art for compositions and methods that reduce, eliminate, or inactivate pre-existing anti-AAV NAbs and the generation of antibodies against AAV vectors after administration to a subject, in order to improve the effectiveness of gene delivery using AAV vectors. SUMMARY OF THE INVENTION

[0009] This specification provides compositions and methods for reducing neutralizing antibodies in a subject in need of a reduction in the amount of one or more neutralizing antibodies against a recombinant adeno-associated virus (AAV) vector. The compositions and methods described herein may improve the effectiveness of gene delivery, for example, by increasing the circulation time and / or infectivity of AAV in the subject's body. The compositions and methods described herein may also enable re-dosing of a therapeutic AAV to a subject who has previously received a therapeutic AAV. In some embodiments, administration of a wild-type or mutant antibody-degrading enzyme, such as IdeZ (or a fragment thereof), reduces neutralizing antibodies (NAbs) in a subject in need of a reduction in NAbs.

[0010] In some embodiments, the present disclosure provides a method for reducing neutralizing antibodies in a subject in need of a reduction in the amount of neutralizing antibodies against a recombinant adeno-associated virus (AAV) vector. The method includes administering to the subject an effective amount of a composition that promotes degradation of the neutralizing antibody.

[0011] Also provided is a method for preparing a subject in need of treatment for treatment with a recombinant adeno-associated virus (AAV) vector. The method includes administering to the subject an effective amount of a composition that (a) promotes degradation of neutralizing antibodies against the AAV vector and / or (b) reduces binding of the neutralizing antibody to an Fc receptor.

[0012] Also provided is a method for treating a subject in need of treatment with a recombinant adeno-associated virus (AAV) vector. The method includes (i) administering to the subject an effective amount of a composition that (a) promotes degradation of neutralizing antibodies against the AAV vector and / or (b) reduces binding of the neutralizing antibody to an Fc receptor, and (ii) administering to the subject an effective amount of the AAV vector.

[0013] Also provided is a method of treating a subject in need of treatment with a second recombinant adeno-associated virus (AAV) vector, who has been previously treated with a first recombinant AAV. The method includes (i) administering to the subject an effective amount of a composition that (a) promotes degradation of neutralizing antibodies against the first and / or second recombinant AAV vector and / or (b) reduces binding of the neutralizing antibodies to Fc receptors, and (ii) administering to the subject an effective amount of the second recombinant AAV vector.

[0014] Also provided is a method of reducing neutralizing antibodies in a subject in need of reducing neutralizing antibodies against an adeno-associated virus (AAV) vector containing a heterologous nucleic acid. The method includes administering to the subject an effective amount of an AAV vector and an effective amount of a composition that (a) promotes degradation of antibodies against the AAV vector or a recombinant protein encoded by the heterologous nucleic acid and / or (b) reduces binding of the antibodies to Fc receptors.

[0015] The compositions described herein may include, for example, an antibody-degrading enzyme or a fragment thereof. In some embodiments, the composition includes a vector containing a polynucleotide encoding an antibody-degrading enzyme or a fragment thereof. In some embodiments, the antibody-degrading enzyme or fragment thereof has cysteine protease activity. In some embodiments, the antibody-degrading enzyme specifically cleaves IgG. In some embodiments, the antibody-degrading enzyme has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0016] These and other embodiments are described in more detail below.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] Detailed Description of the Invention Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used in the detailed description herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0019] All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are incorporated herein by reference in their entirety.

[0020] All amino acid position designations of AAV capsid proteins in this disclosure and the appended claims are with respect to the numbering of the VP1 capsid subunit. Although it is obvious to those skilled in the art, the modifications described herein may result in modifications of the VP1, VP2 and / or VP3 capsid subunits when inserted into the AAV cap gene. Alternatively, the capsid subunits can be expressed independently to achieve modifications with only one or two of the capsid subunits (VP1, VP2, VP3, VP1 + VP2, VP1 + VP3, or VP2 + VP3).

[0021] Unless otherwise indicated by context, it is specifically intended that the various features described herein can be used in any combination.

[0022] Definitions In the description of this specification and the appended claims, the following terms are used.

[0023] The singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0024] Also, as used herein, the term "about", when referring to measurable values such as the length of a polynucleotide or polypeptide sequence, dosage, time, temperature, etc., is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of that particular amount.

[0025] Also, as used herein, "and / or" is to be construed to include any and all possible combinations of one or more of the associated listed items, as well as the case where the combinations are not applicable when interpreted as alternatives ("or").

[0026] The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. For example, if a concentration range is recited herein as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3% are explicitly recited herein. These are merely examples of specifically intended values, and all possible combinations of numerical values between and including the recited minimum and maximum values are to be considered as explicitly recited in this disclosure.

[0027] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAV rh32.33, AAV rh8, AAV rh10, AAV rh74, AAV hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, horned viper AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or later discovered. See, for example, BERNARD N. FIELDS et al., VIROLOGY Volume 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers). Several AAV serotypes and clades have been identified (see, for example, Gao et al, (2004) J. Virology 78:6381-6388; Moris et al, (2004) Virology 33-:375-383; and Table 2). In some embodiments, the AAV vector is selected from any of the AAV vectors disclosed in Table 1 of WO 2019 / 028306, which is incorporated herein by reference in its entirety.

[0028] As used herein, the term "chimeric AAV" means an AAV that includes a capsid protein having regions, domains, or individual amino acids derived from two or more different AAV serotypes. In some embodiments, the chimeric AAV includes a capsid protein comprising a first region derived from a first AAV serotype and a second region derived from a second AAV serotype. In some embodiments, the chimeric AAV includes a capsid protein comprising a first region derived from a first AAV serotype, a second region derived from a second AAV serotype, and a third region derived from a third AAV serotype. In some embodiments, the chimeric AAV may include regions, domains, or individual amino acids derived from two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. For example, the chimeric AAV may include regions, domains, and / or individual amino acids derived from the first and second AAV serotypes shown in Table 1 below. Here, AAVX+Y represents a chimeric AAV that includes sequences derived from AAVX and AAVY.

[0029] [Table 1]

[0030] By including individual amino acids or regions derived from multiple AAV serotypes in a single capsid protein, a capsid protein having multiple desired properties individually derived from multiple AAV serotypes can be obtained.

[0031] The genomic sequences of various serotypes of AAV and autonomous parvoviruses, as well as the sequences of native terminal repeats (TR), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example: GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579 (the disclosures of which are hereby incorporated by reference herein to teach parvovirus and AAV nucleic acid and amino acid sequences).For example, also see the following: Srivistava et al., (1983) J. Virology 45:555; Chiorini et al, (1998) J Virology 71:6823; Chiorini et al., (1999) J. Virology 73: 1309; Bantel-Schaal et al., (1999) J Virology 73:939; Xiao et al, (1999) J Virology 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al, (1986) J. Virol. 58:921; Gao et al, (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al, (2004) Virology 33:375-383; International Patent Publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303 (the disclosures of which are hereby incorporated by reference herein to teach parvovirus and AAV nucleic acid and amino acid sequences). Also see Table 2. The capsid structures of autonomous parvoviruses and AAVs are described in more detail by BERNARD N. FIELDS et al., VIROLOGY, Volume 2, Chapters 69·70 (4th Edition, Lippincott-Raven Publishers).See also the following descriptions of crystal structures: AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci. 99: 10405-10), AAV9 (DiMattia et al., (2012) J. Virol. 86:6947-6958), AAV8 (Nam et al, (2007) J. Virol. 81: 12260-12271), AAV6 (Ng et al., (2010) J. Virol. 84:12945-12957), AAV5 (Govindasamy et al. (2013) J. Virol. 87, 11187-11199), AAV4 (Govindasamy et al. (2006) J. Virol. 80:11556-11570), AAV3B (Lerch et al., (2010) Virology 403:26-36), BPV (Kailasan et al., (2015) J. Virol. 89:2603-2614), and CPV (Xie et al, (1996) J. Mol. Biol. 6:497-520 and Tsao et al, (1991) Science 251:1456-64).

[0032] [Table 2-1] [Table 2-2]

[0033] As used herein, the term "tropism" means that a virus preferentially infects a specific cell or tissue, followed by expression (e.g., transcription and optionally translation) of sequences carried by the viral genome within the cell (e.g., expression of a heterologous nucleic acid in the case of a recombinant virus).

[0034] As those skilled in the art will recognize, transcription of a heterologous nucleic acid sequence derived from a viral genome may not be initiated in the absence of a trans-acting factor, for example, in the case of a nucleic acid sequence regulated by an inducible promoter or other means. In the case of the rAAV genome, gene expression from the viral genome may be derived from stably integrated provirus, unintegrated episome, and any other form that the virus can take within the cell.

[0035] As used herein, "systemic tropism" and "systemic transduction" (and equivalent terms) indicate that the viral capsids or viral vectors of the present disclosure are respectively tropic for, or transduce, tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney, and / or pancreas). In embodiments, systemic transduction of muscle tissue (e.g., skeletal muscle, diaphragm muscle, and cardiac muscle) is observed. In other embodiments, systemic transduction of skeletal muscle tissue is achieved. For example, in certain embodiments, essentially all skeletal muscle throughout the body is transduced (although the efficiency of transduction may vary depending on the muscle type). In certain embodiments, systemic transduction of limb muscle, cardiac muscle, and diaphragm muscle is achieved. Optionally, the viral capsid or viral vector is administered via a systemic route (e.g., a systemic route such as intravenous, intra-articular, or intralymphatic). Alternatively, in other embodiments, the capsid or viral vector is delivered locally (e.g., plantar, intramuscular, intradermal, subcutaneous, topical).

[0036] Unless otherwise indicated, "efficient transduction" or "efficient tropism", or similar terms, may be determined with reference to an appropriate control (e.g., at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or more of the control transduction or tropism, respectively). In some embodiments, the viral vector efficiently transduces or has efficient tropism for skeletal muscle, cardiac muscle, diaphragm muscle, pancreas (including β-islet cells), spleen, gastrointestinal tract (e.g., epithelium and / or smooth muscle), cells of the central nervous system, lung, joint cells, and / or kidney. The appropriate control depends on various factors including the desired tropism characteristics. For example, AAV8 and AAV9 are very efficient in transducing skeletal muscle, cardiac muscle, and diaphragm muscle, but have the disadvantage of also transducing the liver with high efficiency. Thus, viral vectors can be identified that show efficient transduction of skeletal muscle, cardiac muscle, and / or diaphragm muscle by AAV8 or AAV9 but have very low transduction efficiency for the liver. Further, since the desired tropism characteristics may reflect tropism for multiple target tissues, the appropriate vector will of course sometimes show some compromise. By way of illustration, the viral vectors of the present disclosure may be less efficient than AAV8 or AAV9 in transducing skeletal muscle, cardiac muscle, and / or diaphragm muscle, but may still be highly desirable due to low levels of liver transduction.

[0037] Similarly, whether a virus "does not efficiently transduce" or "does not have efficient tropism" for a labeled tissue, or similar terms, may be determined with reference to an appropriate control. In certain embodiments, the viral vector does not efficiently transduce (i.e., does not have efficient tropism) for the liver, kidney, gonads, and / or germ cells. In certain embodiments, unwanted transduction of a tissue (e.g., the liver) is about 20% or less, about 10% or less, about 5% or less, about 1% or less, about 0.1% or less of the transduction level of the desired target tissue (e.g., skeletal muscle, diaphragm muscle, cardiac muscle, and / or cells of the central nervous system).

[0038] As used herein, the term "polypeptide" includes both peptides and proteins unless otherwise indicated.

[0039] A "polynucleotide" is a sequence of nucleotide bases and can be an RNA sequence, a DNA sequence, or a DNA-RNA hybrid sequence (including both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments is a single-stranded or double-stranded DNA sequence.

[0040] As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") means a polynucleotide that has been at least partially separated from at least some of the other components of a naturally occurring organism or virus (e.g., cellular or viral structural components that are generally found associated with that polynucleotide, or other polypeptides or nucleic acids). In representative embodiments, an "isolated" nucleotide is enriched by at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold, or more relative to the starting material.

[0041] Similarly, an "isolated" polypeptide means a polypeptide that has been at least partially separated from at least some of the other components of a naturally occurring organism or virus (e.g., cellular or viral structural components that are generally found associated with that polypeptide, or other polypeptides or nucleic acids). In representative embodiments, an "isolated" polypeptide is enriched by at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold, or more relative to the starting material.

[0042] As used herein, to "isolate" or "purify" (or a grammatically equivalent term) a polypeptide or viral vector means to separate the polypeptide or viral vector at least partially from at least some of the other components in the starting material. In representative embodiments, an "isolated" or "purified" polypeptide or viral vector is concentrated by at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold, or more relative to the starting material.

[0043] The compositions and methods disclosed herein find use in both veterinary and medical applications. Suitable subjects include both avians and mammals. As used herein, the term "avian" includes, but is not limited to, chickens, ducks, geese, quails, turkeys, pheasants, parrots, budgerigars, etc. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, cows, sheep, goats, horses, cats, dogs, rabbits, etc. Human subjects include neonates, infants, juveniles, adults, and geriatric subjects. In some embodiments, the human subject may be less than 6 months old, less than 2 years old, less than 5 years old, less than 10 years old, 10 - 18 years old, 19 - 29 years old, 30 - 35 years old, 36 - 40 years old, or over 40 years old. In representative embodiments, the subject "is in need of" the methods described herein. The terms "subject" and "patient" are used interchangeably herein with respect to human subjects.

[0044] A "therapeutic polypeptide" is a polypeptide that can alleviate, reduce, prevent, delay, and / or stabilize symptoms caused by the absence or deficiency of a protein in a cell or subject, and / or otherwise confer a benefit to the subject (e.g., an anti-cancer effect or improved transplant survival rate).

[0045] The terms "treat", "treating" or "treatment" (and grammatical variations thereof) mean that the severity of the condition of the subject is reduced, at least partially improved or stabilized, and / or that some alleviation, reduction, decrease, or stabilization of at least one clinical symptom is achieved, and / or that there is a delay in the progression of the disease or disorder.

[0046] The terms "prevent", "preventing" and "prevention" (and grammatical variations thereof) mean the prevention and / or delay of the onset of a disease, disorder, and / or clinical symptom in a subject, and / or a decrease in the severity of the onset of that disease, disorder, and / or clinical symptom as compared to what would occur in the absence of the methods of the present disclosure. The prevention may be complete (e.g., the disease, disorder, and / or clinical symptom is not present at all). The prevention may be partial such that the appearance and / or severity of onset of the disease, disorder, and / or clinical symptom in the subject is lower than what would occur in the absence of the present disclosure.

[0047] "Effective amount" means an amount sufficient to affect or reduce one or more symptoms of a disease, disorder, or condition when administered to a subject for treating that disease, disorder, or condition. The "effective amount" may vary depending, for example, on the disease, disorder, or condition, and / or its symptoms, the severity of the disease, disorder, condition, and / or its symptoms, the age, weight, and / or health status of the subject, and the judgment of the prescribing physician. The appropriate amount in any given instance may be ascertained by one of ordinary skill in the art or determined by routine experimentation. In some embodiments, the effective amount is a therapeutically effective amount.

[0048] As used herein, the terms "viral vector" or "gene delivery vector" mean viral (e.g., AAV) particles that function as nucleic acid delivery vehicles and contain a vector genome (e.g., viral DNA [vDNA]) encapsulated within the virion. Alternatively, depending on the context, the term "viral vector" may be used to refer to the vector genome / vDNA alone.

[0049] An "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that contains one or more heterologous nucleic acid sequences. rAAV vectors generally require only cis - type terminal repeats (TRs) to generate the virus. All other viral sequences are unnecessary and may be supplied in trans. Typically, the rAAV vector genome retains only one or more TR sequences, thus maximizing the size of the transgene that can be efficiently encapsulated by the vector. Sequences encoding structural and non - structural proteins may be provided in trans (e.g., from a vector such as a plasmid or by stably integrating the sequences into the packaging cells). In embodiments, the rAAV vector genome contains at least one TR sequence (e.g., an AAV TR sequence), optionally two TRs (e.g., two AAV TRs). The TR sequences are typically present at the 5' and 3' termini of the vector genome and flank the heterologous nucleic acids but need not be contiguous with them. The TRs may be the same as or different from each other.

[0050] The term "terminal repeat" or "TR" encompasses any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., is involved in desired functions such as replication, encapsidation, integration, and / or proviral rescue). The TR can be an AAV TR or a TR other than AAV. For example, TR sequences other than AAV, such as TR sequences of other parvoviruses (e.g., canine parvovirus (CPV), murine parvovirus (MVM), human parvovirus B-19), or any other suitable viral sequence (e.g., SV40 hairpin that functions as an SV40 origin of replication) can be used as the TR, and these can be further modified by cleavage, substitution, deletion, insertion, and / or addition. Further, the TR can be partially or fully synthetic.

[0051] The "AAV terminal repeat" or "AAV TR" can be derived from any AAV, and examples include, but are not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or any other AAV that is currently known or later discovered (see, for example, Table 2). As long as the terminal repeat is involved in desired functions such as replication, encapsidation, integration, and / or proviral rescue, the AAV terminal repeat does not have to have a native terminal repeat sequence (e.g., the native AAV TR sequence can be altered by insertion, deletion, cleavage, and / or missense mutations).

[0052] The viral vectors of the present disclosure can further be "targeted" viral vectors (e.g., having a directed tropism) and / or "hybrid" parvoviruses (i.e., those in which the viral TR and viral capsid are derived from different parvoviruses). The viral vectors of the present disclosure can further be double-stranded parvovirus particles. Thus, in some embodiments, a double-stranded (ds) genome can be encapsulated within the viral capsids of the present disclosure. Further, the viral capsid or genomic sequence can include other modifications such as insertions, deletions, and / or substitutions.

[0053] As used herein, the term "amino acid" encompasses any naturally occurring amino acid, its modified forms, and synthetic amino acids.

[0054] Naturally occurring left-handed (L-) amino acids are shown in Table 3.

[0055]

Table 3

[0056] Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples are shown in Table 4) and / or an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).

[0057]

Table 4-1

Table 4-2

[0058] Furthermore, amino acids that do not occur naturally may be "unnatural" amino acids (e.g., as described in Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006)). These unnatural amino acids can be advantageously used to chemically link the molecule of interest to the AAV capsid protein.

[0059] As used herein, the term "domain" is intended to include portions of protein sequences and structures that evolve, function, and exist independently of the remainder of the protein chain. A domain can form a small three-dimensional structure and is often independently stable and foldable. One domain may occur in various evolutionarily related proteins. Domains vary in length from about 25 amino acids to about 500 amino acids. "Domain" may also include domains derived from a wild-type protein in which one or more amino acid residues have been replaced by conservative substitutions. Since domains are self-stable in a protein environment, chimeric proteins can be created by "exchanging" domains between one protein and another by genetic manipulation.

[0060] As used herein, the terms "mutant", "mutants", "variant" or "variants" are intended to designate a protein or AAV in which one or more amino acids of the native protein or AAV have been replaced by another amino acid, and / or one or more amino acids of the parental AAV protein have been deleted, and / or one or more amino acids have been inserted into the protein or AAV, and / or one or more amino acids have been added to the parental protein or AAV. Such additions can occur at the N-terminus and / or C-terminus of the parental protein and also internally. In some embodiments, the amino acid sequence of the variant is at least 40%, at least 50%, at least 60%, or at least 70% identical to the amino acid sequence of the native protein.

[0061] As used herein, the term "vector" means any nucleic acid entity that can be amplified within a host cell. Thus, a vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication (e.g., a plasmid). Alternatively, a vector may be a vector that, when introduced into a host cell, integrates into the host cell genome and is replicated along with the chromosome into which it has integrated. The choice of vector often depends on the host cell into which it is introduced. Examples of vectors include, but are not limited to, plasmid vectors, phage vectors, viruses or cosmid vectors. A vector typically contains an origin of replication and at least one selectable gene (i.e., a gene that encodes an easily detectable product or whose presence is essential for cell growth).

[0062] The term "gene therapy" means a method of altering the expression of an endogenous gene by the exogenous administration of a gene. As used herein, "gene therapy" also means the replacement of a defective gene encoding a defective protein or the replacement of a missing gene by introducing a functional gene corresponding to the defective or missing gene into somatic or stem cells of an individual in need thereof. Gene therapy can be achieved by an ex vivo method in which differentiated or somatic stem cells are removed from an individual's body and a normal copy of the defective gene is introduced into cultured cells using a viral vector as a gene delivery vehicle. In addition, in vivo direct gene transfer techniques can be used to introduce genes in situ into cells in an individual's body using a wide range of viral vectors, liposomes, protein-DNA complexes or naked DNA to achieve a therapeutic result. The term "gene therapy" also means the replacement of a defective gene encoding a defective protein by introducing a polynucleotide that functions substantially similarly to the content that would function if the defective gene or protein were not defective into somatic or stem cells of an individual in need thereof.

[0063] The term "gene editing" means the insertion, deletion, or substitution of DNA at specific sites in the genome of an organism or cell. Gene editing may be performed using one or more targeted nucleotide systems (such as CRISPR / Cas systems, CRISPR / Cpf1 systems, zinc finger nucleases, TALENs, homing endonucleases, etc.).

[0064] As used herein, the term "Fc receptor" means the Fc gamma immunoglobulin receptor (FcγR) present on the cell surface. In humans, FcγR means one, several, or all of the receptor family including FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). As used herein, the term FcγR encompasses the naturally occurring polymorphisms of FcγRI (CD64), FcγyRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0065] As described herein, cysteine protease is an enzyme that degrades proteins. Cysteine proteases generally have a common catalytic mechanism that includes a catalytic triad or nucleophilic cysteine thiol of two residues. In some embodiments, the cysteine protease is an IgG cysteine protease that cleaves IgG such that the antigen-binding domain (Fab) and the constant domain (Fc) are separated from each other.

[0066] Composition for reducing neutralizing antibodies against biologic agents The present disclosure provides a composition capable of reducing neutralizing antibodies against a recombinant biologic agent or a drug entity in the body of a subject. In some embodiments, the recombinant biologic agent comprises a vector comprising a heterologous nucleic acid encoding one or more recombinant proteins. In some embodiments, the vector is a recombinant viral vector, such as a recombinant AAV vector.

[0067] In some embodiments, the composition reduces neutralizing antibodies against a recombinant biologic or drug entity in a subject by promoting the elimination or degradation of antibodies against the recombinant biologic or drug entity and / or reducing the binding of the antibodies against the recombinant biologic or drug entity to Fc receptors. In some embodiments, the composition reduces neutralizing antibodies against an AAV vector containing a heterologous nucleic acid in a subject by promoting the elimination or degradation of antibodies against one or more recombinant proteins encoded by the AAV vector or the heterologous nucleic acid and / or reducing the binding of the antibodies against the AAV vector to Fc receptors.

[0068] In some embodiments, the composition includes an antibody-degrading enzyme or a fragment thereof that degrades antibodies recognizing an adeno-associated virus (AAV) capsid protein or virion, or that can prevent neutralization of a recombinant AAV vector. In some embodiments, the composition includes a vector containing a polynucleotide encoding an antibody-degrading enzyme or a fragment thereof. In some embodiments, the antibodies include IgG (such as IgG1, IgG2a, IgG2b, and / or IgG3), IgM, IgE, and / or IgA. In an exemplary embodiment, the antibody includes IgG. Thus, in some embodiments, the composition includes an IgG-degrading enzyme or a fragment thereof. In some embodiments, the IgG-degrading enzyme or a fragment thereof has cysteine protease activity.

[0069] In some embodiments, the IgG-degrading enzyme or a fragment thereof is isolated or derived from a bacterium (e.g., derived from a bacterium of the genus Streptococcus). In some embodiments, the IgG-degrading enzyme includes an amino acid sequence having at least about 50% (e.g., about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or 100%, including all values and subranges therebetween) identity to the following amino acid sequence of Streptococcus equi shown in SEQ ID NO: 1 below.

[0070]

Table 5

[0071] The sequence of SEQ ID NO: 1 corresponds to the IdeZ protein. A exemplary crystal structure of the IdeZ protein is shown in Figure 5 (each figure shows a view at a different angle).

[0072] As disclosed herein, the IdeZ protein is a cysteine protease that recognizes group A streptococcus and inactivates IgG antibodies by cleaving IgG in the lower hinge region of the heavy chain to yield one F(ab’)2 fragment and one homodimeric Fc fragment. This IgG-degrading enzyme has a short half-life and is very efficient but rapidly eliminated from circulation with transient IgG removal.

[0073] In some embodiments, the IgG-degrading enzyme or a fragment thereof is isolated from or derived from Streptococcus pyogenes. In some embodiments, the IgG-degrading enzyme comprises an amino acid sequence having at least about 50% (e.g., about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or 100%, including all values and subranges therebetween) identity to SEQ ID NO: 13 or SEQ ID NO: 14.

[0074] In some embodiments, the IgG-degrading enzyme or a fragment thereof is a synthetic enzyme. In some embodiments, the IgG-degrading enzyme comprises a sequence having at least about 50% (e.g., about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or 100%, including all values and subranges therebetween) identity to any one of SEQ ID NOs: 15-52.

[0075] The composition for reducing the neutralizing antibody described in this specification may contain a fusion protein comprising an antibody-degrading enzyme or a fragment thereof and a second protein. In some embodiments, the second protein is an IgG protease.

[0076] In some embodiments, the composition for reducing the neutralizing antibody reduces the binding of the antibody to the Fc receptor for the AAV vector. In some embodiments, the composition promotes the rapid elimination of the antibody against the AAV vector by binding to the Fc receptor of the antibody. For example, the composition may promote the rapid elimination of IgG against the AAV vector by binding to the receptor of IgG (e.g., FcRN), thereby also promoting the internalization and degradation of the IgG receptor. In some embodiments, the composition contains a therapeutic antibody. In some embodiments, the therapeutic antibody is IgG. In some embodiments, the therapeutic antibody is rozanolixizumab. In some embodiments, the dose of the therapeutic antibody is from 0.05 mg / kg to about 150 mg / kg, such as about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, or about 150 mg / kg (including all values and subranges therebetween).

[0077] In some embodiments, a composition that reduces neutralizing antibodies reduces and / or inhibits complement activation. For example, the composition may prevent C1q from binding to an antigen-antibody complex (e.g., an AAV-antibody complex) by cleaving the neutralizing antibody. By reducing and / or inhibiting complement activation, downstream steps of the complement cascade are prevented, such as the recruitment of immune cells and opsonization (e.g., of AAV). In some embodiments, treating a subject with a composition that reduces neutralizing antibodies prior to AAV-based therapy prevents complement activation in the subject's body upon administration of AAV. In some embodiments, complement activation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% upon administration of AAV. In some embodiments, treating a patient with a composition that reduces neutralizing antibodies concurrently with AAV-based therapy prevents complement activation in the patient's body due to the AAV therapy. In some embodiments, complement activation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, treating a patient with one of the compositions that reduces neutralizing antibodies after AAV-based therapy reduces complement activation in the patient's body. In some embodiments, complement activation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0078] Depending on the embodiment, the compositions disclosed herein may further comprise at least one pharmaceutically acceptable carrier, excipient, and / or solvent, such as a solvent, buffer, solution, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Depending on the embodiment, the pharmaceutically acceptable carrier, excipient, and / or solvent may include physiological saline, buffered physiological saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, phosphate buffer solution, amino acid-based buffer, bicarbonate buffer solution, and combinations thereof. Depending on the embodiment, the pharmaceutically acceptable carrier, excipient, and / or solvent includes phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) or suitable mixtures thereof. Depending on the embodiment, the compositions disclosed herein may further comprise trace amounts of an emulsifying or wetting agent, or a pH buffering agent.The formulations of the compositions disclosed herein may be prepared for storage in the form of, for example, lyophilized powders, pastes, aqueous solutions or suspensions, by mixing with a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[0079] Depending on the embodiment, the compositions disclosed herein further comprise other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers (such as chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, or albumin, etc.). Depending on the embodiment, the compositions disclosed herein may further comprise antibacterial and antifungal agents (parabens, chlorobutanol, phenol, sorbic acid or thimerosal), isotonic agents (sugars or sodium chloride), and / or absorption delaying agents (such as aluminum monostearate and gelatin).

[0080] Depending on the embodiment, the compositions of the present disclosure are formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts derived from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.) (formed with the free amino groups of proteins). Depending on the embodiment, salts formed with the free carboxyl groups of proteins may be derived from inorganic bases (such as sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (such as isopropylamine, trimethylamine, histidine, procaine), etc.

[0081] Depending on the embodiment, the composition is in the form of a solid (such as a lyophilized powder suitable for reconstitution), solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Depending on the embodiment, the composition may be formulated for delivery using liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, liposomes, nanospheres, nanoparticles, etc.

[0082] Dosage and administration method of the composition for reducing neutralizing antibodies Administration of any one of the compositions for reducing neutralizing antibodies against the biologic agents disclosed herein may be by injection, infusion, or a combination thereof. A pharmaceutical composition comprising any one of the compositions described herein may be administered at a dosage of about 0.05 mg / kg to about 150 mg / kg, such as, for example, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, or about 150 mg / kg (including all values and subranges therebetween).

[0083] A therapeutically effective amount of any one of the compositions disclosed herein may be administered in a single dose, but is not limited to a single dose. Thus, administration may be in 1 to 50 doses, such as, for example, 2 doses, 5 doses, 10 doses, 15 doses, 20 doses, 25 doses, 30 doses, 35 doses, 40 doses, 45 doses, or 50 doses (including all values and subranges therebetween). If there are more than one administration in the method, the interval between administrations may be from about 1 minute to about 1 month, such as, for example, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 40 minutes, about 1 hour, about 2 hours, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 24 hours, about 2 days, about 5 days, about 10 days, about 15 days, about 20 days (including all subranges and values therebetween). The present invention is not limited to a temporally equidistant dosing interval and includes non-equidistant dosing, such as, by way of non-limiting example only, a priming schedule consisting of administrations on day 1, day 4, day 7, and day 25.

[0084] For example, dosing schedules such as once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, etc. can be utilized in the present invention. The dosing schedule encompasses dosing for a total period of about 1 day to about 1 year, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months (including all values and sub-ranges therebetween).

[0085] Examples of the periods of the above dosing schedules are shown. The period may be repeated, for example, approximately every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, every 42 days, every 49 days, every 56 days, every 63 days, every 70 days, etc. There may be intervals without dosing during the period, and the intervals are approximately, for example, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 70 days, etc.

[0086] The compositions disclosed herein may be administered in combination with one or more additional therapeutic agents. Methods of co-administration with additional therapeutic agents are well known in the art (Hardman, et al. (eds.) (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.).

[0087] In this specification, subjects to be treated include mammals, such as humans and non-human primates. In some embodiments, the subject may be selected from humans, non-human primates, cows, sheep, goats, horses, cats, dogs, and rabbits.

[0088] Method for reducing neutralizing antibodies against a biologic The present disclosure provides a method for reducing neutralizing antibodies against a recombinant biologic or a drug entity in a subject's body. This method includes administering to the subject a therapeutically effective amount of a recombinant biologic or a drug entity and any one of (a) a therapeutically effective amount of a composition that promotes the degradation of antibodies against the recombinant biologic or the drug entity and / or (b) a therapeutically effective amount of a composition that reduces the binding of the antibody to the Fc receptor, as disclosed herein.

[0089] In some embodiments, a method for reducing the amount of neutralizing antibodies against a recombinant adeno-associated virus (AAV) vector in a subject's body includes administering to the subject a therapeutically effective amount of a composition that promotes the degradation of the neutralizing antibodies.

[0090] In some embodiments, a method for preparing a subject for treatment with a recombinant adeno-associated virus (AAV) vector includes administering to the subject a therapeutically effective amount of a composition that (a) promotes the degradation of neutralizing antibodies against the AAV vector and / or (b) reduces the binding of the neutralizing antibodies to the Fc receptor.

[0091] In some embodiments, a method for treating a subject in need of treatment with a recombinant adeno-associated virus (AAV) vector includes (i) administering to the subject a therapeutically effective amount of a composition that (a) promotes the degradation of neutralizing antibodies against the AAV vector and / or (b) reduces the binding of the neutralizing antibodies to the Fc receptor, and (ii) administering to the subject a therapeutically effective amount of the AAV vector.

[0092] In some embodiments, a method of treating a subject previously treated with a first recombinant adeno-associated virus (AAV) with a second recombinant AAV vector comprises (i) administering to the subject a therapeutically effective amount of a composition that (a) promotes degradation of neutralizing antibodies against the first and / or second recombinant AAV vector and / or (b) reduces binding of the neutralizing antibodies to Fc receptors, and (ii) administering to the subject a therapeutically effective amount of the second recombinant AAV vector.

[0093] In some embodiments, a method of reducing neutralizing antibodies against an adeno-associated virus (AAV) vector comprising a heterologous nucleic acid in a subject's body comprises administering to the subject a therapeutically effective amount of an AAV vector and a therapeutically effective amount of a composition that (a) promotes degradation of antibodies against the AAV vector or a recombinant protein encoded by the heterologous nucleic acid and / or (b) reduces binding of the antibodies to Fc receptors.

[0094] In some embodiments, a method of reducing neutralizing antibodies against any one of the adeno-associated virus (AAV) vectors disclosed herein in a subject's body comprises administering to the subject a therapeutically effective amount of an AAV vector and any one of a therapeutically effective amount of the compositions disclosed herein that (a) promotes degradation of antibodies against the AAV vector or a recombinant protein encoded by the heterologous nucleic acid and / or (b) reduces binding of the antibodies to Fc receptors.

[0095] (a) A composition that promotes the degradation of neutralizing antibodies against the first and / or second recombinant AAV vector and / or (b) reduces the binding of neutralizing antibodies to Fc receptors may contain an antibody-degrading enzyme or a fragment thereof. In some embodiments, the composition comprises a vector containing a polynucleotide encoding an antibody-degrading enzyme or a fragment thereof. In some embodiments, the antibody-degrading enzyme or a fragment thereof may have cysteine protease activity. In some embodiments, the antibody-degrading enzyme specifically cleaves IgG. In some embodiments, the antibody-degrading enzyme or a fragment thereof is derived from the genus Streptococcus. In some embodiments, the antibody-degrading enzyme comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

[0096] In some embodiments, the composition may comprise a fusion protein comprising a first protein and a second protein, and the first protein may be an antibody-degrading enzyme or a fragment thereof. In some embodiments, the first protein and the second protein are separated by a linker. In some embodiments, the second protein is an IgG protease.

[0097] The composition may be administered via a systemic (e.g., intravenous, intra-articular or intralymphatic) route. In some embodiments, the composition is delivered locally (e.g., intramuscular, intradermal, subcutaneous, topical). In some embodiments, the composition is administered directly to a location known to contain neutralizing antibodies, such as cerebrospinal fluid (CSF). The composition may contain a pharmaceutically acceptable carrier and / or diluent.

[0098] In some embodiments, an antibody-degrading enzyme or a fragment thereof at about 0.1 mg / kg to about 100 mg / kg is administered to a subject. In some embodiments, a fusion protein at about 0.1 mg / kg to about 100 mg / kg is administered to a subject. In some embodiments, an antibody-degrading enzyme or a fragment thereof or a fusion protein at about 0.05 mg / kg to about 150 mg / kg, such as about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, or about 150 mg / kg (including all values and subranges therebetween) is administered to a subject.

[0099] In some embodiments, the neutralizing antibodies to be decreased and / or degraded include IgG, IgM, IgE, and / or IgA. In some embodiments, the neutralizing antibody is IgG. In some embodiments, the antibody is a neutralizing antibody against an AAV vector containing a transgene. In some embodiments, the antibody binds to a recombinant protein encoded by the transgene. In some embodiments, the antibody binds to an adeno-associated virus capsid protein or its virion.

[0100] In some embodiments, the recombinant AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV vector. In some embodiments, the recombinant AAV vector comprises a capsid protein having a sequence of any one of SEQ ID NOs: 2-12, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the AAV vector is a wild-type AAV vector. In some embodiments, the AAV vector is a mutant AAV vector. In some embodiments, the AAV vector is a wild-type AAV1 vector. In some embodiments, the AAV vector is a wild-type AAV2 vector. In some embodiments, the AAV vector is a wild-type AAV4 vector. In some embodiments, the AAV vector is a wild-type AAV8 vector. In some embodiments, the AAV vector is a wild-type AAV9 vector. In some embodiments, the AAV vector is a mutant AAV1 vector. In some embodiments, the AAV vector is a mutant AAV2 vector. In some embodiments, the AAV vector is a mutant AAV4 vector. In some embodiments, the AAV vector is a mutant AAV8 vector. In some embodiments, the AAV vector is a mutant AAV9 vector. In some embodiments, the recombinant AAV vector comprises a heterologous nucleic acid encoding a therapeutic protein or a therapeutic RNA.

[0101] In some embodiments, the methods described herein include reducing the interaction of an antibody with its cognate cell surface receptor. Such methods may expand the patient population suitable for gene therapy and may further enable AAV re-dosing / readministration to patients previously treated with an AAV vector.

[0102] In some embodiments of the methods of the disclosure, the AAV vector is administered to the subject concomitantly with the composition. In some embodiments, the AAV vector is administered to the subject after administration of the composition. In some embodiments, the AAV vector is administered to the subject before administration of the composition. In some embodiments, the method further comprises administering one or more additional doses or a second dose of a second AAV vector comprising a second heterologous nucleic acid. In some embodiments, the first AAV vector and the second AAV vector comprise the same AAV capsid protein. In some embodiments, the first AAV vector and the second AAV vector comprise different AAV capsid proteins.

[0103] In some embodiments, the method promotes degradation of antibodies against the AAV vector. In some embodiments, the level of the antibody is reduced to a level within the range of about 95% to about 0.01% (e.g., about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, about 0.1%, or about 0.01%, including all values and subranges therebetween) relative to the level of the antibody in the body of a control subject. As used herein, a control subject is administered a recombinant biologic, such as an AAV vector, but not any one of the compositions disclosed herein. In some embodiments, the method results in degradation of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the antibody after administration of the composition.

[0104] In some embodiments, the subject has been previously administered a recombinant protein. Thus, in some embodiments, administration of any one of the compositions disclosed herein results in a decrease in the circulating concentration of antibodies produced against a previous dose of recombinant protein in the body of the subject.

[0105] Importantly, the compositions and methods of the present disclosure can be used in combination with other pharmacological or interventional techniques that can reduce antibodies.

[0106] Recombinant viral vector In some embodiments, the vectors disclosed herein are useful for in vitro, ex vivo, and in vivo delivery of heterologous nucleic acids to cells. In some embodiments, the vector is a viral vector, such as an AAV vector. In particular, viral vectors can be advantageously employed to deliver or transfer nucleic acids to animal cells, such as mammalian cells. In some embodiments, the viral vector comprises a recombinant viral capsid, such as an AAV capsid, that encapsulates the heterologous nucleic acid. In some embodiments, the recombinant viral capsid comprises a recombinant capsid protein, such as a recombinant AAV capsid protein. Further details regarding viral vectors, viral capsids, and / or capsid proteins that may be used in accordance with the present disclosure are provided in International Applications PCT / US2019 / 025617, PCT / US2019 / 025584, and PCT / US2019 / 025610 (the contents of each application are incorporated herein by reference in their entirety for all purposes).

[0107] In some embodiments, the AAV vector comprises a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh.32.33, AAVrh74, bovine AAV, avian AAV, or any other AAV that is currently known or later recognized. In some embodiments, the AAV capsid protein is chimeric.

[0108] In some embodiments, the capsid protein is an AAV capsid protein (VP1, VP2, and / or VP3) that contains modifications (e.g., substitutions) in the amino acid sequence, and the viral capsid and viral vector contain the modified AAV capsid protein. In some embodiments, the modifications described herein can confer one or more desired properties (including, but not limited to, the ability to avoid neutralizing antibodies) to the viral vector containing the modified AAV capsid protein.

[0109] In some embodiments, the AAV capsid protein contains one or more amino acid substitutions, and the one or more substitutions modify one or more antigenic sites of the AAV capsid protein. As a result of the modification of the one or more antigenic sites, binding of an antibody to the one or more antigenic sites is inhibited, and / or neutralization of the infectivity of the viral particles containing the AAV capsid protein is inhibited. In some embodiments, as a result of the modification of the one or more antigenic sites, binding of an antibody to the one or more antigenic sites is inhibited. In some embodiments, the modified antigenic site can prevent an antibody from binding to, recognizing, or neutralizing the AAV capsid. Here, the antibody is IgG (such as IgG1, IgG2a, IgG2b, IgG3), IgM, IgE, or IgA. In some embodiments, as a result of the modification of the one or more antigenic sites, the infectivity of the viral particles containing the AAV capsid protein is neutralized.

[0110] One or more amino acid substitutions may be present in one or more antigen footprints identified by peptide epitope mapping and / or cryo-electron microscopy studies of the AAV-antigen complex comprising the AAV capsid protein. In some embodiments, one or more antigenic sites are common antigenic motifs or CAMs as described in WO 2017 / 058892 (incorporated herein by reference in its entirety). In some embodiments, the antigenic site is present in a variable region (VR) of the AAV capsid protein, such as VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, VR-IX. In some embodiments, one or more antigenic sites are present in the HI loop of the AAV capsid protein.

[0111] In some embodiments, one or more amino acid substitutions replace any 6, 7, or 8 amino acids of the AAV capsid protein derived from any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAV10, AAV11, AAV12, AAVrh32.22, bovine AAV, or avian AAV. In some embodiments, the substitution introduces a deletion into the AAV capsid sequence. For example, a sequence of 6, 7, 8, or 9 amino acids is substituted to replace 7, 8, 9, or 10 amino acids, respectively, of the native amino acid capsid sequence. In some embodiments, the substitution introduces an insertion into the AAV capsid sequence. For example, a sequence of 6, 7, 8, or 9 amino acids is substituted to replace 5, 6, 7, or 8 amino acids, respectively, of the native amino acid capsid sequence.

[0112] In some embodiments, one or more substitutions at one or more antigenic sites can introduce one or more antigenic sites derived from the capsid protein of a first AAV serotype into the capsid protein of a second AAV serotype different from the first AAV serotype.

[0113] As used herein, "substitution" may refer to a single amino acid substitution or may refer to substitutions of two or more amino acids. For example, in some embodiments, the capsid proteins of the present disclosure may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. single amino acid substitutions. In some embodiments, the capsid proteins of the present disclosure may include one or more substitutions of a plurality of consecutive amino acids, such as one or more substitutions of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive amino acids.

[0114] Furthermore, in embodiments described herein where an amino acid residue is substituted with any other amino acid residue other than that amino acid residue present in the wild-type or native amino acid sequence, the any other amino acid residue is any natural or non-natural amino acid residue known in the art (see, for example, Tables 3 and 4). In some embodiments, the substitution may be a conservative substitution, and in some embodiments, the substitution may be a non-conservative substitution.

[0115] In some embodiments, the AAV capsid protein includes a first amino acid substitution and a second amino acid substitution, and the first amino acid substitution and the second amino acid substitution each modify a separate antigenic site of the AAV capsid protein. In some embodiments, the AAV capsid protein includes a first amino acid substitution, a second amino acid substitution, and a third amino acid substitution, and the first amino acid substitution, the second amino acid substitution, and the third amino acid substitution each modify a separate antigenic site of the AAV capsid protein.

[0116] Any one of the AAV capsids described herein may further include a modification (e.g., substitution or deletion) within the HI loop. The HI loop is a protruding domain on the AAV capsid surface that exists between β-strands βH and βI and extends from each VP subunit that overlaps with the viral protein (VP) consisting of five adjacent parts. In some embodiments, the AAV capsid includes 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions within the HI loop. In some embodiments, the AAV capsid protein includes 1, 2, 3, or 4 amino acid substitutions, each substitution modifying a separate antigenic site of the AAV capsid protein, and at least one of the amino acid substitutions modifies the HI loop of the capsid protein. In some embodiments, the AAV capsid protein includes first, second, third, and fourth amino acid substitutions.

[0117] In some embodiments, the AAV capsid proteins disclosed herein are encoded and expressed by a nucleotide sequence or an expression vector containing the same. The nucleotide sequence may be a DNA sequence or an RNA sequence.

[0118] In some embodiments, a modified capsid protein is produced by modifying any AAV capsid protein known currently or discovered later. Further, the AAV capsid protein to be modified may be a naturally occurring AAV capsid protein (for example, AAV2, AAV3a or 3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein or any of the AAVs shown in Table 2), but is not limited thereto. As will be apparent to those skilled in the art, various manipulations of AAV capsid proteins are known in the art, and the present disclosure is not limited to the modification of naturally occurring AAV capsid proteins. For example, the capsid protein to be modified may already be modified in comparison to a naturally occurring AAV (for example, a naturally occurring AAV capsid protein, such as AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any other AAV known currently or discovered later). In some embodiments, the capsid protein may be a chimeric capsid protein. In some embodiments, the capsid protein may be a manipulated AAV, such as AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B. Such AAV capsid proteins are also included within the scope of the present disclosure.

[0119] Thus, depending on the embodiment, the AAV capsid protein to be modified may be derived from naturally occurring AAV, but further includes one or more foreign sequences inserted and / or substituted with respect to the capsid protein (e.g., those that are exogenous to the wild-type virus) and / or is modified by deletion of one or more amino acids. In some embodiments, the modification to the AAV capsid protein is a “selective” modification. This approach is in contrast to past studies that have exchanged entire subunits or large domains between AAV serotypes (see, e.g., International Patent Publication WO 00 / 28004 and Hauck et al., (2003) J. Virology 77:2768-2774). In certain embodiments, as a result of the “selective” modification, there occur insertions and / or substitutions and / or deletions of about 20 or fewer, about 18 or fewer, about 15 or fewer, about 12 or fewer, about 10 or fewer, about 9 or fewer, about 8 or fewer, about 7 or fewer, about 6 or fewer, about 5 or fewer, about 4 or fewer, or about 3 or fewer contiguous amino acids. The modified capsid proteins and capsids of the present disclosure may further include any other modifications that are currently known or later recognized.

[0120] Accordingly, when referring in this specification to a particular AAV capsid protein (e.g., an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein, or a capsid protein derived from any of the AAVs shown in Table 2, etc.), it is intended to encompass the native capsid protein and capsid proteins having modifications other than those of the present disclosure. Such modifications include substitutions, insertions, and / or deletions. In certain embodiments, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 (other than the insertions of the present disclosure) amino acids inserted as compared to the native AAV capsid protein sequence. In an embodiment, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 (other than the amino acid substitutions of the present disclosure) amino acid substitutions as compared to the native AAV capsid protein sequence. In embodiments of the present disclosure, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 (other than the amino acid deletions of the present disclosure) amino acid deletions as compared to the native AAV capsid protein sequence.

[0121] Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. One may determine sequence similarity or identity using standard techniques known in the art, for example, by the method of local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the method of sequence identity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48, 443 (1970), by the method of similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computer execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA of Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), by the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or by inspection, etc., but not limited to these.

[0122] Another suitable algorithm is the BLAST algorithm described in Altschul et al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); http: / / blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which may be set to their default values. The parameters are dynamic values and are established by the program itself according to the composition of the specific sequence to search for the target sequence and the composition of the specific database. However, the values may be adjusted to increase sensitivity.

[0123] Another useful algorithm is the gapped BLAST reported in Altschul et al, (1997) Nucleic Acids Res. 25, 3389-3402.

[0124] In some embodiments, the AAV vector includes an AAV capsid and an AAV genome and has a phenotype that avoids neutralizing antibodies. Also, the AAV virus particles or vectors disclosed herein may have, in addition to a phenotype that avoids neutralizing antibodies, a phenotype that enhances or maintains transduction efficiency.

[0125] The viral vectors according to the present disclosure may be produced by expression using any method known in the art, such as the baculovirus system.

[0126] According to embodiments of the present disclosure, the viral capsid may be a targeted viral capsid that includes a targeting sequence (e.g., substituted or inserted with respect to the viral capsid) that directs the viral capsid to interact with a cell surface molecule present in a desired target tissue. For example, the viral capsids of the present disclosure may have relatively low tropism for a particular target tissue of interest (e.g., liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestine, skin, endothelial cells, and / or lung). By advantageously incorporating the targeting sequence into these poorly transducing vectors, the viral capsid can be imparted with the desired tropism, optionally a selective tropism for a particular tissue. AAV capsid proteins, capsids, and vectors containing the targeting sequence are described, for example, in International Patent Publication WO 00 / 28004. As another example, one or more non-natural amino acids (such as those described in Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006)) can be incorporated into the orthogonal sites of the AAV capsid subunits of the present disclosure as a means of redirecting poorly transducing vectors to a desired target tissue. These non-natural amino acids can be advantageously used to chemically link a molecule of interest to the AAV capsid protein. Examples of AAV capsid proteins include glycans (mannose: dendritic cell targeting); RGD, bombesin, or neuropeptides for targeted delivery to specific cancer cell types; RNA aptamers or peptides selected from phage display that are targeted to specific cell surface receptors, such as growth factor receptors, integrins, etc., but are not limited thereto. Methods for chemically modifying amino acids are known in the art.

[0127] According to embodiments of the present disclosure, the capsid protein, viral capsid or vector of the present disclosure may have a transduction efficiency equal to or higher than that of the AAV serotype from which the capsid protein, viral capsid or vector of the present disclosure is derived. According to embodiments of the present disclosure, the capsid protein, viral capsid or vector of the present disclosure may have a transduction efficiency lower than that of the AAV serotype from which the capsid protein, viral capsid or vector of the present disclosure is derived. According to embodiments of the present disclosure, the capsid protein, viral capsid or vector of the present disclosure may have a tropism equal to or higher than that of the AAV serotype from which the capsid protein, viral capsid or vector of the present disclosure is derived. According to embodiments of the present disclosure, the capsid protein, viral capsid or vector of the present disclosure may have an altered or different tropism compared to the tropism of the AAV serotype from which the capsid protein, viral capsid or vector of the present disclosure is derived. According to embodiments of the present disclosure, the capsid protein, viral capsid or vector of the present disclosure may be targeted to or engineered to have tropism for brain tissue. According to embodiments of the present disclosure, the capsid protein, viral capsid or vector of the present disclosure may be targeted to or engineered to have tropism for liver tissue.

[0128] As those skilled in the art will recognize, depending on whether the corresponding amino acid position is partially present, fully present, or not present at all in the virus for a given AAV capsid protein, the corresponding modification is an insertion and / or substitution. As described elsewhere herein, the corresponding amino acid position will be apparent to those skilled in the art using well-known techniques.

[0129] AAV viral vector In some embodiments, the viral vector comprises a modified AAV capsid comprising the modified capsid subunit of the present disclosure and a vector genome. For example, in some embodiments, the viral vector comprises (a) a modified viral capsid (e.g., a modified AAV capsid) comprising the modified capsid protein of the present disclosure, and (b) a heterologous nucleic acid comprising terminal repeats (e.g., AAV TR), wherein the heterologous nucleic acid comprising terminal repeats is encapsidated by the modified viral capsid. The nucleic acid may comprise two terminal repeats (e.g., two AAV TR).

[0130] In some embodiments, the viral vector of the present disclosure has (i) lower liver transduction compared to the transduction level by the viral vector in the absence of the modified capsid protein, (ii) exhibits higher systemic transduction in the body of an animal subject compared to the level observed with the viral vector in the absence of the modified capsid protein, (iii) shows higher motility in endothelial cells compared to the motility level by the viral vector in the absence of the modified capsid protein, and / or (iv) shows selective enhancement of transduction in muscle tissues (e.g., skeletal muscle, cardiac muscle, and / or diaphragm muscle), (v) shows selective enhancement of transduction in liver tissue, and / or (vi) reduces transduction in brain tissue (e.g., neurons) compared to the transduction level by the virus in the absence of the modified capsid protein. In certain embodiments, the viral vector has systemic transduction in the liver.

[0131] In some embodiments, the viral vector is a recombinant viral vector comprising a heterologous nucleic acid encoding a polypeptide or functional RNA of interest. In some embodiments, the nucleic acid is a nucleic acid encoding a polypeptide such as a therapeutic polypeptide (e.g., for medical or veterinary use) or an immunogenic polypeptide (e.g., for a vaccine) or an RNA.

[0132] Alternatively, the immunogenic polypeptide may be any tumor or cancer cell antigen. The tumor or cancer cell antigen may be expressed on the surface of cancer cells.

[0133] Although it is obvious to those skilled in the art, heterologous nucleic acids can be operably linked to appropriate control sequences. For example, heterologous nucleic acids can be operably linked to expression control sequences such as transcription / translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES) within the sequence, promoters, and / or enhancers. Further, controlled expression of the heterologous nucleic acid of interest can be achieved at the post-transcriptional level, for example, by regulating the alternative splicing of various introns by the presence or absence of oligonucleotides, small molecules, and / or other compounds that selectively block splicing activity at specific sites.

[0134] As recognized by those skilled in the art, various promoter / enhancer sequences can be used depending on the desired level and tissue-specific expression. The promoter / enhancer sequences can be constitutive or inducible depending on the desired expression pattern. The promoter / enhancer sequences can be endogenous or exogenous, and can be natural sequences or synthetic sequences. "Exogenous" means that the transcription start region is not found in the wild-type host into which the transcription start region is introduced.

[0135] In certain embodiments, the promoter / enhancer sequence may be specific to the target cell or the subject to be treated. In representative embodiments, the promoter / enhancer sequence may be specific to the heterologous nucleic acid sequence. The promoter / enhancer sequence is generally selected to function within the target cell of interest. Further, in certain embodiments, the promoter / enhancer sequence is a mammalian promoter / enhancer sequence. The promoter / enhancer sequence can be constitutive or inducible.

[0136] Inducible expression control sequences are typically advantageous in applications where it is desirable to control the expression of heterologous nucleic acid sequences. Inducible promoter / enhancer sequences for gene delivery may be tissue-specific or preferential promoter / enhancer sequences, such as muscle-specific or preferential (e.g., specific or preferential for cardiac muscle, skeletal muscle, and / or smooth muscle), nerve tissue-specific or preferential (e.g., specific or preferential for the brain), eye-specific or preferential (e.g., specific for the retina and specific for the cornea), liver-specific or preferential, bone marrow-specific or preferential, pancreas-specific or preferential, spleen-specific or preferential, and lung-specific or preferential promoter / enhancer sequences. Other inducible promoter / enhancer sequences include hormone-inducible and metal-inducible sequences. Exemplary inducible promoter / enhancer sequences include, but are not limited to, Tet on / off sequences, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters.

[0137] Viral vectors according to the present disclosure provide a means for delivering heterologous nucleic acids to a wide range of cells, including dividing and non-dividing cells. For example, viral vectors can be employed to deliver nucleic acids of interest to cells in vitro for producing polypeptides in vitro or for ex vivo gene therapy. Viral vectors are further useful in methods of delivering nucleic acids to a subject in need thereof to express, for example, immunogenic or therapeutic polypeptides or functional RNAs. In this way, polypeptides or functional RNAs can be produced in vivo in a subject. The subject may be one that requires the polypeptide due to a deficiency thereof. Further, the method may be carried out for reasons that the production of a polypeptide or functional RNA in the subject's body may have some beneficial effect compromised.

[0138] The viral vectors of the present disclosure may be employed to treat and / or prevent any medical condition in which it is beneficial to deliver a therapeutic polypeptide or functional RNA by delivering a heterologous nucleic acid encoding the polypeptide or functional RNA. Gene transfer is quite useful for understanding medical conditions and providing treatment. There are many genetic diseases in which the defective gene is known and cloned. Generally, the above medical conditions are classified into two types. Generally, a recessively inherited (usually enzymatic) deficiency state and a disequilibrium state in which regulatory or structural proteins may be involved and which typically inherit dominantly. In the case of deficiency state diseases, gene transfer can be used to introduce a normal gene into the affected tissue for replacement therapy, and furthermore, an antisense mutation can be used to create an animal model of the disease. In the case of disequilibrium conditions, gene transfer can be used to cause the condition in a model system and used in attempts to counteract the condition. Thus, the viral vectors according to the present disclosure enable the treatment and / or prevention of genetic diseases.

[0139] The viral vectors according to the present disclosure may be employed to provide functional RNA to cells in vitro or in vivo. The functional RNA may be, for example, non-translated RNA. According to embodiments, the expression of a specific target protein by the cell may be reduced by the expression of the functional RNA in the cell. Thus, the functional RNA can be administered to reduce the expression of a specific protein in the body of a subject in need thereof. According to embodiments, the expression of a specific target protein by the cell may be increased by the expression of the functional RNA in the cell. Thus, the functional RNA can be administered to increase the expression of a specific protein in the body of a subject in need thereof. According to embodiments, the splicing of a specific target RNA in the cell may be regulated by the expression of the functional RNA. Thus, the functional RNA can be administered to regulate the splicing of a specific RNA in the body of a subject in need thereof. According to embodiments, the function of a specific target protein by the cell may be regulated by the expression of the functional RNA in the cell. Thus, the functional RNA can be administered to regulate the function of a specific protein in the body of a subject in need thereof. Further, the functional RNA can be administered to cells in vitro to regulate gene expression and / or cell physiology (e.g., to optimize a cell or tissue culture system or in a screening method).

[0140] Alternatively, the viral vector may be administered to cells ex vivo, and the modified cells may be administered to a subject. A viral vector containing a heterologous nucleic acid can be introduced into a cell to express a heterologous nucleic acid encoding an immunogen in the cell, and an immune response against the immunogen can be induced in the body of the subject. In certain embodiments, the cell is an antigen-presenting cell (e.g., a dendritic cell).

[0141] Kit Another aspect of the present disclosure provides a kit for reducing and / or removing neutralizing antibodies and / or immunoglobulins against recombinant biologics and / or drug entities in a subject's body. The kit comprises, consists of, or consists essentially of any one of the compositions described herein, a means for administering the composition, and instructions for use. In some embodiments, the reagent comprises an immunoglobulin G (IgG) degrading enzyme.

[0142] Numbered Embodiments The following numbered embodiments are included within the scope of the present disclosure.

[0143] 1. A method of reducing neutralizing antibodies in a subject's body that requires a reduction in the amount of neutralizing antibodies against a recombinant adeno-associated virus (AAV) vector, the method comprising administering to the subject a therapeutically effective amount of a composition that promotes degradation of the neutralizing antibodies.

[0144] 2. The method of embodiment 1, wherein the neutralizing antibody is IgG, IgM, IgE, or IgA.

[0145] 3. The method of embodiment 2, wherein the neutralizing antibody is IgG.

[0146] 4. The method of any one of embodiments 1-3, wherein the recombinant AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV vector.

[0147] 5. The method of embodiment 4, wherein the AAV vector is a wild-type AAV vector.

[0148] 6. The method of embodiment 4, wherein the AAV vector is a mutant AAV vector.

[0149] 7. The method according to any one of embodiments 1 to 6, wherein the recombinant AAV vector comprises a heterologous nucleic acid encoding a therapeutic protein or a therapeutic RNA.

[0150] 8. The method according to any one of embodiments 1 to 7, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the antibodies in the body of the subject are degraded after administration of the composition.

[0151] 9. The method according to any one of embodiments 1 to 8, wherein the composition comprises an antibody-degrading enzyme or a fragment thereof.

[0152] 10. The method according to any one of embodiments 1 to 8, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or a fragment thereof.

[0153] 11. The method according to embodiment 9 or 10, wherein the antibody-degrading enzyme or a fragment thereof has cysteine protease activity.

[0154] 12. The method according to any one of embodiments 9 to 11, wherein the antibody-degrading enzyme specifically cleaves IgG.

[0155] 13. The method according to any one of embodiments 9 to 12, wherein the antibody-degrading enzyme or a fragment thereof is derived from the genus Streptococcus.

[0156] 14. The method according to any one of embodiments 9 to 13, wherein the antibody-degrading enzyme comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

[0157] 15. The method according to embodiment 14, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

[0158] 16. The method according to any one of embodiments 1 to 15, wherein the composition comprises a fusion protein comprising a first protein and a second protein, and the first protein is an antibody-degrading enzyme or a fragment thereof.

[0159] 17. The method according to embodiment 16, wherein the first protein and the second protein are separated by a linker.

[0160] 18. The method according to embodiment 16 or 17, wherein the second protein is an IgG protease.

[0161] 19. The method according to any one of embodiments 9 to 15, wherein about 0.1 mg / kg to about 100 mg / kg of the antibody-degrading enzyme or a fragment thereof is administered to the subject.

[0162] 20. The method according to any one of embodiments 1 to 19, wherein the administration reduces the binding of the antibody to the Fc receptor.

[0163] 21. The method according to any one of embodiments 1 to 20, wherein the composition is administered intravenously.

[0164] 22. The method according to any one of embodiments 1 to 21, wherein the composition comprises a pharmaceutically acceptable carrier and / or diluent.

[0165] 23. The method according to any one of embodiments 1 to 22, wherein the subject is a human.

[0166] 24. The method according to any one of embodiments 1 to 23, wherein the subject is treated with the recombinant adeno-associated virus (AAV) vector prior to administration of the composition.

[0167] 25. The method according to any one of embodiments 1 to 23, wherein the subject is not treated with the recombinant AAV prior to administration of the composition.

[0168] 26. A method of preparing a subject for treatment with a recombinant adeno-associated virus (AAV) vector, the method comprising administering to the subject an effective amount of a composition that (a) promotes degradation of neutralizing antibodies against the AAV vector and / or (b) reduces binding of the neutralizing antibodies to Fc receptors.

[0169] 27. The method of embodiment 26, wherein the neutralizing antibody is IgG, IgM, IgE, or IgA.

[0170] 28. The method of embodiment 27, wherein the neutralizing antibody is IgG.

[0171] 29. The method of any one of embodiments 26-28, wherein the recombinant AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV vector.

[0172] 30. The method of embodiment 29, wherein the AAV vector is a wild-type AAV vector.

[0173] 31. The method of embodiment 29, wherein the AAV vector is a mutant AAV vector.

[0174] 32. The method of any one of embodiments 26-31, wherein the recombinant AAV comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA.

[0175] 33. The method of any one of embodiments 26-32, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the antibodies are degraded in the subject's body after administration of the composition.

[0176] 34. The method of any one of embodiments 26-33, wherein the composition comprises an antibody-degrading enzyme or a fragment thereof.

[0177] 35. The method according to any one of embodiments 26 to 33, wherein the composition comprises a vector containing a polynucleotide encoding an antibody-degrading enzyme or a fragment thereof.

[0178] 36. The method according to embodiment 34 or 35, wherein the antibody-degrading enzyme or a fragment thereof has cysteine protease activity.

[0179] 37. The method according to any one of embodiments 26 to 36, wherein the antibody-degrading enzyme specifically cleaves IgG.

[0180] 38. The method according to any one of embodiments 26 to 37, wherein the antibody-degrading enzyme or a fragment thereof is derived from the genus Streptococcus.

[0181] 39. The method according to any one of embodiments 26 to 38, wherein the antibody-degrading enzyme comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

[0182] 40. The method according to embodiment 39, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

[0183] 41. The method according to any one of embodiments 26 to 40, wherein the composition comprises a fusion protein comprising a first protein and a second protein, and the first protein is an antibody-degrading enzyme or a fragment thereof.

[0184] 42. The method according to embodiment 41, wherein the first protein and the second protein are separated by a linker.

[0185] 43. The method according to embodiment 41 or 42, wherein the second protein is an IgG protease.

[0186] 44. The method according to any one of embodiments 34 to 43, wherein about 0.1 mg / kg to about 100 mg / kg of the antibody-degrading enzyme or a fragment thereof is administered to the subject.

[0187] 45. The administration is by any one of the methods of Embodiments 28 to 44 that reduces the binding of the antibody to the Fc receptor.

[0188] 46. The composition is administered intravenously, by any one of the methods of Embodiments 26 to 45.

[0189] 47. The composition comprises a pharmaceutically acceptable carrier and / or diluent, by any one of the methods of Embodiments 26 to 46.

[0190] 48. The subject is human, by any one of the methods of Embodiments 26 to 47.

[0191] 49. A method for treating a subject in need of treatment with a recombinant adeno-associated virus (AAV) vector, (i) (a) administering to the subject an effective amount of a composition that promotes the degradation of neutralizing antibodies against the AAV vector and / or (b) reduces the binding of the neutralizing antibodies to the Fc receptor, and (ii) administering to the subject an effective amount of the AAV vector comprising the method.

[0192] 50. The AAV vector is administered simultaneously with the composition, by the method of Embodiment 49.

[0193] 51. The AAV vector is administered after the administration of the composition, by the method of Embodiment 49.

[0194] 52. The AAV vector is administered before the administration of the composition, by the method of Embodiment 49.

[0195] 53. Further comprising administering a second AAV vector to the subject, by any one of the methods of Embodiments 49 to 52.

[0196] 54. The method of embodiment 53, wherein the AAV vector and the second AAV vector comprise AAV capsid proteins having the same serotype.

[0197] 55. The method of embodiment 53, wherein the AAV vector and the second AAV vector comprise AAV capsid proteins having different serotypes.

[0198] 56. The method of any one of embodiments 45 - 55, wherein the neutralizing antibody is IgG, IgM, IgE, or IgA.

[0199] 57. The method of embodiment 56, wherein the neutralizing antibody is IgG.

[0200] 58. The method of any one of embodiments 49 - 57, wherein the recombinant AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV vector.

[0201] 59. The method of embodiment 58, wherein the AAV vector is a wild - type AAV vector.

[0202] 60. The method of embodiment 58, wherein the AAV vector is a mutant AAV vector.

[0203] 61. The method of any one of embodiments 49 - 60, wherein the recombinant AAV vector comprises a heterologous nucleic acid encoding a therapeutic protein or a therapeutic RNA.

[0204] 62. The method of any one of embodiments 49 - 61, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the antibodies are degraded after administration of the composition.

[0205] 63. The method according to any one of embodiments 49 to 62, wherein the composition comprises an antibody-degrading enzyme or a fragment thereof.

[0206] 64. The method according to any one of embodiments 49 to 62, wherein the composition comprises a vector containing a polynucleotide encoding an antibody-degrading enzyme or a fragment thereof.

[0207] 65. The method according to embodiment 63 or 64, wherein the antibody-degrading enzyme or a fragment thereof has cysteine protease activity.

[0208] 66. The method according to any one of embodiments 63 to 65, wherein the antibody-degrading enzyme specifically cleaves IgG.

[0209] 67. The method according to any one of embodiments 63 to 66, wherein the antibody-degrading enzyme or a fragment thereof is derived from the genus Streptococcus.

[0210] 68. The method according to any one of embodiments 63 to 67, wherein the antibody-degrading enzyme comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

[0211] 69. The method according to embodiment 68, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

[0212] 70. The method according to any one of embodiments 49 to 69, wherein the composition comprises a fusion protein comprising a first protein and a second protein, and the first protein is an antibody-degrading enzyme or a fragment thereof.

[0213] 71. The method according to embodiment 70, wherein the first protein and the second protein are separated by a linker.

[0214] 72. The method according to embodiment 70 or 71, wherein the second protein is an IgG protease.

[0215] 73. The method according to any one of embodiments 63 to 72, wherein the antibody degrading enzyme or a fragment thereof at about 0.1 mg / kg to about 100 mg / kg is administered to the subject.

[0216] 74. The method according to any one of embodiments 49 to 73, wherein the administration reduces the binding of the antibody to the Fc receptor.

[0217] 75. The method according to any one of embodiments 49 to 74, wherein the composition is administered intravenously.

[0218] 76. The method according to any one of embodiments 49 to 75, wherein the composition comprises a pharmaceutically acceptable carrier and / or diluent.

[0219] 77. The method according to any one of embodiments 49 to 76, wherein the subject is human.

[0220] 78. A method of treating a subject in need of treatment with a second AAV vector, the subject having been previously treated with a first recombinant adeno-associated virus (AAV), (i) administering to the subject an effective amount of a composition that (a) promotes degradation of neutralizing antibodies against the first and / or second recombinant AAV vectors and / or (b) reduces the binding of the neutralizing antibodies to the Fc receptor, and (ii) administering to the subject an effective amount of the second recombinant AAV vector comprising the method.

[0221] 79. The method according to embodiment 78, wherein the first recombinant AAV and the second recombinant AAV have the same serotype.

[0222] 80. The method according to embodiment 78, wherein the first recombinant AAV and the second recombinant AAV have different serotypes.

[0223] 81. The method according to any one of embodiments 78 to 80, wherein the neutralizing antibody is IgG, IgM, IgE, or IgA.

[0224] The method of embodiment 81, wherein the neutralizing antibody is IgG.

[0225] The method of any one of embodiments 76 - 82, wherein the recombinant AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV vector.

[0226] The method of embodiment 83, wherein the AAV vector is a wild - type AAV vector.

[0227] The method of embodiment 83, wherein the AAV vector is a mutant AAV vector.

[0228] The method of any one of embodiments 78 - 85, wherein the recombinant AAV contains a heterologous nucleic acid encoding a therapeutic protein or a therapeutic RNA.

[0229] The method of any one of embodiments 78 - 86, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the antibodies are degraded after administration of the composition.

[0230] The method of any one of embodiments 78 - 87, wherein the composition contains an antibody - degrading enzyme or a fragment thereof.

[0231] The method of any one of embodiments 78 - 87, wherein the composition contains a vector containing a polynucleotide encoding an antibody - degrading enzyme or a fragment thereof.

[0232] The method of embodiment 88 or 89, wherein the antibody - degrading enzyme or a fragment thereof has cysteine protease activity.

[0233] 91. The method according to any one of embodiments 78 to 90, wherein the antibody-degrading enzyme specifically cleaves IgG.

[0234] 92. The method according to any one of embodiments 78 to 91, wherein the antibody-degrading enzyme or a fragment thereof is derived from the genus Streptococcus.

[0235] 93. The method according to any one of embodiments 78 to 92, wherein the antibody-degrading enzyme comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

[0236] 94. The method according to embodiment 93, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

[0237] 95. The method according to any one of embodiments 87 to 94, wherein the composition comprises a fusion protein comprising a first protein and a second protein, and the first protein is an antibody-degrading enzyme or a fragment thereof.

[0238] 96. The method according to embodiment 95, wherein the first protein and the second protein are separated by a linker.

[0239] 97. The method according to embodiment 95 or 96, wherein the second protein is an IgG protease.

[0240] 98. The method according to any one of embodiments 78 to 97, wherein about 0.1 mg / kg to about 100 mg / kg of the antibody-degrading enzyme or a fragment thereof is administered to the subject.

[0241] 99. The method according to any one of embodiments 78 to 98, wherein the administration reduces the binding of the antibody to its Fc receptor.

[0242] 100. The method according to any one of embodiments 78 to 99, wherein the composition is administered intravenously.

[0243] 101. The method of any one of embodiments 78 to 100, wherein the composition comprises a pharmaceutically acceptable carrier and / or diluent.

[0244] 102. The method of any one of embodiments 78 to 101, wherein the subject is human.

[0245] 103. A method for reducing neutralizing antibodies in a subject in need of reducing neutralizing antibodies against an adeno-associated virus (AAV) vector comprising a heterologous nucleic acid, the method comprising administering to the subject an effective amount of the AAV vector and (a) an effective amount of a composition that promotes degradation of antibodies against the AAV vector or a recombinant protein encoded by the heterologous nucleic acid and / or (b) reduces binding of the antibody to an Fc receptor.

[0246] 104. The method of embodiment 103, wherein the antibody is IgG.

[0247] 105. The method of embodiment 103 or 104, wherein the subject is administered the AAV vector simultaneously with the composition.

[0248] 106. The method of embodiment 103 or 104, wherein the subject is administered the AAV vector after administration of the composition.

[0249] 107. The method of embodiment 103 or 104, wherein the subject is administered the AAV vector before administration of the composition.

[0250] 108. The method of embodiment 107, further comprising administering a single dose or multiple doses of a second AAV vector comprising a second heterologous nucleic acid.

[0251] 109. The method of embodiment 108, wherein the AAV vector and the second AAV vector comprise an AAV capsid protein having the same serotype.

[0252] 110. The method of embodiment 108, wherein the AAV vector and the second AAV vector comprise AAV capsid proteins having different serotypes.

[0253] 111. The method of any one of embodiments 102 - 110, wherein the composition further comprises a pharmaceutically acceptable carrier and / or diluent.

[0254] 112. The method of any one of embodiments 102 - 111, wherein the composition promotes degradation of the antibody.

[0255] 113. The method of embodiment 112, wherein the level of the antibody in the body of the subject is reduced to a level within the range of about 95% to about 0.01% of the level of the antibody in the body of a control subject, and the control subject is administered the AAV vector but not the composition.

[0256] 114. The method of embodiment 112 or 113, wherein the composition comprises an antibody-degrading enzyme or a fragment thereof.

[0257] 115. The method of embodiment 112 or 113, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or a fragment thereof.

[0258] 116. The method of embodiment 114 or 115, wherein the antibody-degrading enzyme or a fragment thereof has IgG cysteine protease activity.

[0259] 117. The method of any one of embodiments 114 - 116, wherein the antibody-degrading enzyme or a fragment thereof is derived from the genus Streptococcus.

[0260] 118. The method of any one of embodiments 114 - 117, wherein the antibody-degrading enzyme comprises an amino acid sequence having at least 50% identity to the amino acid sequence of SEQ ID NO: 1.

[0261] 119. The method of any one of embodiments 114 - 118, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

[0262] 120. The method according to any one of embodiments 114 to 119, wherein the composition comprises a fusion protein comprising the antibody-degrading enzyme or a fragment thereof and a second protein.

[0263] 121. The method according to embodiment 120, wherein the second protein is an IgG protease.

[0264] 122. The method according to any one of embodiments 114 to 121, wherein the subject is administered from about 0.1 mg / kg to about 100 mg / kg of the antibody-degrading enzyme or a fragment thereof.

[0265] 123. The method according to any one of embodiments 108 to 122, wherein the subject is human.

[0266] 124. The method according to embodiment 102, wherein the composition reduces the binding of the antibody to its Fc receptor.

[0267] Note that the above description and the following examples are intended to be illustrative and not to limit the scope of the present invention. Other aspects, advantages, and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention pertains.

Example

[0268] Example 1: Cloning, Expression, and Purification of Recombinant IdeZ (rIdeZ)

[0269] The IdeZ coding sequence was cloned into the pGEX-6P-3 vector using BamHI and SalI restriction sites to generate an IdeZ fusion with an N-terminal GST tag (GST-IdeZ) (Figure 1A). Expression of GST-IdeZ was controlled under the lac operon and production was induced by adding IPTG. The IdeZ protein was purified using glutathione sepharose and eluted with excess glutathione. Expression and purification were monitored using SDS-PAGE (Figure 1B). Recombinant IdeZ was quantified using BSA as a standard with Biorad Imagelab™ software.

[0270] Example 2: IdeZ cleaves recombinant mouse IgG and serum IgG of multiple species

[0271] To determine whether rIdeZ was active in vitro, mouse, primate, and human serum samples were treated with recombinant GST-IdeZ (1 μg) at 37°C for 3 hours. As shown in Figure 6, GST-IdeZ cleaved IgG present in human and primate sera.

[0272] In another experiment, recombinant mouse IgG (40 μg) was incubated with rIdeZ (NEB P0770S, 160 units) at 37°C for 2 hours. The reaction was analyzed by SDS-PAGE under non-reducing conditions and stained with Coomassie blue (Figure 2A). In the presence of IdeZ, recombinant mouse IgG was cleaved into multiple bands as indicated by the * marks. The arrow indicates the IdeZ protein.

[0273] In further experiments, mouse, primate, and human serum samples were treated with untreated (-) or recombinant IdeZ (+) (NEB P0770S, 320 units) at 37 °C for 3 hours. The reactions were diluted 1:10 and analyzed by SDS-PAGE under reducing conditions. The gels were then stained with Coomassie blue. As shown in Figure 2B, the IgG present in the serum was cleaved by IdeZ. In Figure 2B, the lower gel represents an overexposure of the portion of the gel containing the IgG heavy chain cleavage product (approx. 31 kDa). IdeZ also cleaved IgG in serum samples from additional human patients (Donors 1 - 5) (Figure 2D). In a similar experiment, it was also observed that IdeZ was able to cleave canine serum IgG (Figure 2C). In summary, these data demonstrate that IdeZ can cleave IgG in the sera of multiple species.

[0274] Example 3: IdeZ cleaves human IVIG in vitro and in vivo

[0275] Human intravenous immunoglobulin (IVIG) was incubated with GST-IdeZ (1 μg) or IdeZ (NEB P0770S or Genscript) at 37 °C for 2 hours. The reactions were analyzed by SDS-PAGE under reducing conditions and stained with Coomassie blue. As shown in Figure 3B, IdeZ cleaved human IVIG in vitro.

[0276] Mice were injected intraperitoneally with 8 mg of human IVIG. The same mice were injected intravenously with PBS (-) or recombinant IdeZ (2.5 mg / kg) (+) 24 hours later. 72 hours after the IVIG injection, blood samples were collected and analyzed by SDS-PAGE under reducing conditions using immunoblotting. IVIG was probed with goat anti-human IgG Alexa Fluor 647 (1:10,000). In the presence of IdeZ, as indicated by the * marks, human IVIG was digested into multiple smaller cleavage products (Figure 3A). These data show that IdeZ cleaves human IVIG in vivo.

[0277] In a similar experiment, mice were intraperitoneally injected with 8 mg of human IVIG. Twenty-four hours later, the same mice were intravenously injected with PBS (-) or recombinant GST-IdeZ (2.5 mg / kg) (+). Blood samples were collected before injection, 24 hours, 48 hours, and 72 hours after IVIG injection. As shown in Figure 7, IdeZ cleaved human IVIG within 24 hours, and the level of cleavage continued to increase until the 72-hour (day 3) time point.

[0278] Example 4: Dose analysis of IVIG cleavage by GST-IdeZ

[0279] Dose analysis of IVIG cleavage by GST-IdeZ was also performed. In this experiment, mice were intraperitoneally injected with 8 mg of human IVIG. Twenty-four hours later, the same mice were intravenously injected with PBS (-) or recombinant GST-IdeZ (0.25 mg / kg) (+). As shown in Figures 8A-8B, cleavage was dose-dependent. At the maximum dose (2.5 mg / kg), almost all of the IVIG in each sample was cleaved, as evidenced by the change in band size.

[0280] The cleavage site of IdeZ is within the hinge region of human immunoglobulin. To confirm that IdeZ was cleaving IVIG, serum samples from PBS (-) or 1 mg / kG IdeZ (+) mice were run on an SDS-PAGE gel and probed with anti-Fab or anti-Fc antibodies. As shown in Figure 9, the Fab band changed in size (from approximately 250 kDa to approximately 150 kDa) after IdeZ treatment, indicating that cleavage had occurred. The Fc band appeared at approximately 50 kDa in the IdeZ-treated sample, indicating that this domain had been separated from the Fab.

[0281] The analysis results of the neutralization of AAV8-Luc by human IVIG were also prepared. GST-IdeZ (1 μg) untreated and treated human IVIG were serially diluted 2-fold from 1:1000 to 1:102,400, then co-incubated with AAV8-Luc and administered to the cells in the culture medium (100,000 vg / cell). As demonstrated by the curve in Figure 10, the neutralization of AAV8-Luc was reduced in the presence of GST-IdeZ.

[0282] Example 5: IdeZ restores hepatic transduction of AAV8-Luc in IVIG-treated mice

[0283] Mice were intraperitoneally injected with 8 mg of human IVIG. Twenty-four hours later, the same mice were intravenously injected with PBS or recombinant IdeZ (2.5 mg / kg) and AAV8-Luc (5 x 10 12 vg / kg). The luciferase transgene expression levels in the liver were analyzed 4 weeks after injection. The luciferase expression levels were normalized to the total tissue protein concentration and represented as relative light units (RLU) per gram of liver tissue. All experiments were performed 3 times. *: p value < 0.05, L.O.D = limit of detection.

[0284] As shown in Figure 4, IVIG-treated mice showed lower levels of AAV8-Luc transduction in the liver. However, IVIG-treated mice co-injected with IdeZ showed levels of AAV8-Luc hepatic transduction similar to those of PBS-treated mice.

[0285] The copy number of AAV8-Luc in mouse liver samples was calculated. As shown in Figure 11, the copy number of AAV8-Luc per cell was higher in the samples from IVIG-treated mice co-injected with AAV8-Luc and IdeZ. Hepatic transduction was very low in IdeZ-untreated mice.

[0286] In summary, these data indicate that IdeZ reduces the neutralization of AAV by IVIG and promotes the hepatic transduction of AAV8-Luc.

[0287] Example 6: IdeZ restores AAV9-Luc hepatic and cardiac transduction in IVIG-treated mice

[0288] Mice were intraperitoneally injected with 8 mg of human IVIG. Seventy-two hours later, the same mice were intravenously injected with PBS or recombinant GST-IdeZ (2.5 mg / kg). Then, 72 hours after IdeZ treatment, the mice were intravenously injected with AAV9-Luc (2×10 11 vg / mouse). Luciferase transgene expression levels were analyzed in the liver and heart 4 weeks after injection. Luciferase expression levels were normalized to the total tissue protein concentration and represented as relative light units (RLU) per gram of liver tissue.

[0289] IVIG-treated male and female mice showed lower AAV9-Luc transduction levels in the liver (Figure 12A, Figure 12C) and heart (Figure 12B, Figure 12D). However, IVIG-treated mice co-injected with GST-IdeZ showed AAV9-Luc hepatic and cardiac transduction levels similar to those of PBS-treated mice.

[0290] In summary, these data indicate that IdeZ abrogates the neutralization of AAV by IVIG and promotes AAV9-Luc hepatic and cardiac transduction (L.O.D = limit of detection).

[0291] Example 7: IdeZ improves AAV9-Luc hepatic and cardiac transduction levels in mice treated with patient sera

[0292] Serum samples obtained from 18 human patients were tested for their ability to neutralize AAV9 transduction in the liver and heart.

[0293] Two mice per human serum sample were used in the study, and both mice were intraperitoneally injected with 100 μl of human patient serum. Then, 72 hours later, the mice were intravenously injected with PBS or recombinant GST-IdeZ (2.5 mg / kg). Subsequently, 72 hours after IdeZ treatment, the mice were intravenously injected with AAV9-Luc (2 x 10 11(vg / mouse) was intravenously injected.

[0294] The transduction levels in the liver and heart were analyzed 4 weeks after injection. AAV9-Luc (2×10 11 (vg / mouse) was injected and the transduction levels were normalized against control mice that were not serum-treated.

[0295] As shown in Figures 13A and 13B, mice treated with human patient serum showed different transduction levels. However, mice treated with strongly neutralizing patient serum showed higher liver (Figure 13A) and heart (Figure 13B) transduction when co-injected with GST-IdeZ.

[0296] In summary, these data indicate that IdeZ antagonizes the neutralization of AAV by patient serum and promotes the liver and heart transduction of AAV9-Luc.

[0297] As will be readily appreciated by those skilled in the art, the present disclosure is capable of carrying out the objectives and is well adapted to obtain the mentioned objectives and advantages and those inherent therein. The present disclosure described herein is presently representative of the preferred embodiments, exemplary, and is not intended as a limitation on the scope of the present disclosure. Those changes and other uses that fall within the spirit of the scope of the present disclosure as defined by the claims will occur to those skilled in the art.

Claims

**Claim 1** A composition for reducing the amount of a neutralizing antibody in a subject in need of such reduction, the composition comprising a therapeutically effective amount of an antibody-degrading enzyme having the sequence of SEQ ID NO:1, wherein the neutralizing antibody is a neutralizing antibody against a recombinant AAV vector and / or a neutralizing antibody against a therapeutic protein or therapeutic RNA encoded by the recombinant AAV vector. **Claim 2** A composition for treating a subject in need of treatment with a second recombinant adeno-associated virus (AAV) vector, the subject having been previously treated with a first recombinant AAV vector, the composition comprising a therapeutically effective amount of an antibody-degrading enzyme having the sequence of SEQ ID NO:1, wherein the composition (a) promotes degradation of the neutralizing antibody and / or (b) reduces binding of the neutralizing antibody to an Fc receptor, and the neutralizing antibody is a neutralizing antibody against the first recombinant AAV vector, the second recombinant AAV vector, and / or a therapeutic protein or therapeutic RNA encoded by these AAV vectors. **Claim 3** The composition according to claim 1, wherein the subject in need of reduction of the amount of the neutralizing antibody is treated with the recombinant AAV vector (i) before administration of the composition or (ii) after administration of the composition. **Claim 4** The composition according to claim 1 or 2, wherein the therapeutically effective amount of the antibody-degrading enzyme comprises from about 0.1 mg / kg to about 100 mg / kg. **Claim 5** The composition according to any one of claims 1-4, wherein the neutralizing antibody is an IgG, IgM, IgE, or IgA antibody. **Claim 6** The composition according to claim 1, wherein the recombinant AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV vector. **Claim 7** The composition according to any one of claims 1-6, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the neutralizing antibody in the subject is degraded after administration of the composition. **Claim 8** The composition according to any one of claims 1 to 7, further comprising a pharmaceutically acceptable carrier and / or diluent.

9. The composition according to claim 1, wherein the composition promotes degradation of the neutralizing antibody and / or reduces binding of the neutralizing antibody to an Fc receptor.

10. The antibody-degrading enzyme is part of a fusion protein, the fusion protein comprises a second protein, the second protein is an IgG protease, and the antibody-degrading enzyme and the second protein are separated by a linker. The composition according to any one of claims 1 to 9.

11. The composition according to any one of claims 1 to 10, which is administered intravenously to the subject.

12. The composition according to claim 2, wherein the first recombinant AAV vector and the second recombinant AAV vector have the same serotype, or the first recombinant AAV vector and the second recombinant AAV vector have different serotypes.

13. The composition according to claim 2, wherein the first recombinant AAV vector encodes a first therapeutic protein or therapeutic RNA, and the second recombinant AAV encodes a second therapeutic protein or therapeutic RNA.

14. The composition according to any one of claims 1 to 13, which is administered to the subject once or multiple times.

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