Compositions and methods for increasing or enhancing transformation of gene therapy vectors and for removing or reducing immunoglobulins

Abstract

Disclosed herein are methods of treating gene therapy neutralizing antibodies that may develop or have pre-presence by administering a protease that cleaves a peptide bond present in an immunoglobulin or by administering a glycosidase that cleaves a carbohydrate residue present in an immunoglobulin or other similar enzymatic cleavage of an in vivo immunoglobulin and a method for treating a patient. Also disclosed are methods of gene therapy treatment of disease in a patient in need thereof using IdeS and other immunoglobulin G degrading enzyme polypeptides.

Description

[0001] This application is a divisional application of an invention patent application with an application date of July 17, 2019, application number 201980048115.9, and invention name “Compositions and methods for increasing or enhancing the transformation of gene therapy vectors and for removing or reducing immunoglobulins”.

[0002] Related Applications

[0003] This application claims priority to European Patent Application No. EP18305971.6 filed on July 17, 2018 and U.S. Provisional Patent Application No. 62 / 768,731 filed on November 16, 2018. The entire contents of the above applications are incorporated herein by reference, including all texts, tables, sequence listings and drawings. Background Art

[0004] The adaptive immune system can target adeno-associated virus (AAV) and other viral vectors as well as lipid-, polymer-, and protein-based nanoparticle gene therapy approaches, resulting in reduced efficacy and the possibility that patients become completely refractory to therapeutic interventions. The adaptive immune system relies on the development of antigen-specific immunoglobulin (e.g., IgG) antibodies, which lead to inhibition or clearance of target molecules. Because humans are naturally exposed to wild-type AAV, pre-existing anti-AAV antibodies can hinder AAV gene therapy. In addition, the development of anti-AAV antibodies after AAV gene transfer can prevent re-administration of the same or cross-reactive vectors.

[0005] Postnatal exposure to wild-type AAV induces antibodies against the viral capsid within the first two years of life (Calcedo et al. (2011) Clin Vaccine Immunol. 18, 1586-8; Erles et al. (1999) J Med Virol. 59, 406-11; Li et al. (2012) Gene Ther. 19, 288-94). Depending on the AAV serotype and the age of the individual, the proportion of subjects who are positive for anti-AAV antibodies can reach 60%. Even when present at relatively low titers, some anti-AAV antibodies target viral epitopes that are critical for cell entry and neutralize viral infectivity. Importantly, anti-AAV antibodies are highly cross-reactive with different AAV serotypes (Boutin et al. (2010) Hum Gene Ther. 21, 704-12). As demonstrated by several preclinical and clinical studies, such neutralizing antibodies greatly reduce transduction efficiency, especially when the vector is delivered directly into the blood (Manno et al. (2006) Nat Med. 12, 342-7; Masat et al. (2013) Discov Med. 15, 379-89; Arruda et al. (2010) Blood. 115, 4678-88; Haurigot et al. (2010) Mol Ther. 18, 1318-29; Jiang et al. (2006) Blood. 108, 3321-8; Scallan et al. (2006) Blood. 107, 1810-7).

[0006] The high prevalence of neutralizing anti-AAV antibodies prevents some subjects from participating in gene transfer trials of AAV vectors and will prevent some patients from receiving approved AAV gene therapy, making it impossible for some patients to obtain potentially life-saving therapies. In addition, neutralizing anti-AAV antibodies are induced after AAV gene transfer, which can hinder transduction efficiency when the same individual is re-administered. For example, hemophilia B is a rare X-linked bleeding disease caused by deficiency or dysfunction of coagulation factor IX (FIX), and when it is severe (circulating FIX levels are less than 1% of normal), it leads to frequent bleeding episodes, the risk of arthritis development and early death (Mannucci et al. (2001) N Engl J Med. 344, 1773-9). Current treatments for the prevention or treatment of bleeding with exogenous FIX administration are not ideal. The short half-life of the molecule means frequent intravenous injections, which are associated with high costs and the risk of producing inhibitory antibodies. The monogenic nature of hemophilia B makes gene therapy an attractive option for restoring continuous endogenous FIX expression and circumventing the limitations of current therapies. In this regard, although AAV-mediated gene transfer has shown great promise in long-term correction of hemophilia B in severely ill patients (Nathwani et al. (2014) N Engl J Med. 371, 1994-2004; Nathwani et al. (2011) N Engl J Med. 365, 2357-65; George et al. (2017) N Engl J Med. 377, 2215-2227), the presence of pre-existing anti-AAV antibodies has created an obstacle to the use of AAV-mediated gene therapy to treat certain patients with hemophilia B. Similarly, although AAV-mediated gene transfer has shown great promise for the treatment of hemophilia A, spinal muscular atrophy (Mendell et al., 2017, N. Engl. J. Med., 377: 1713-1722), and many other diseases, the presence of pre-existing anti-AAV antibodies has likewise created an obstacle to the treatment of certain patients with these diseases and disorders.

[0007] Proteolysis of immunoglobulins is a common mechanism used by pathogens to circumvent host defense systems (Travis et al. (2000) Biochim Biophys Acta. 1477, 35-50; Travis et al. (1995) Trends Microbiol. 3, 405-7). For example, IdeS is a naturally occurring 35 kDa cysteine ​​protease (particularly an endopeptidase) expressed by pathogenic Streptococcus pyogenes that is specific for target sequences found in human IgG, in addition to several other species. IdeS is able to cleave IgG below the hinge region (von Pawel-Rammingen et al. (2003) Curr Opin Microbiol. 6, 50-5), resulting in the production of F(ab')2 and Fc / 2 fragments (Ryan et al. (2008) Mol Immunol. 45, 1837-46).

[0008] As a further example, EndoS is a naturally occurring glycosidase (particularly an endoglycosidase) from Streptococcus pyogenes that specifically hydrolyzes glycans from human IgG and alters antibody effector functions, including Fc receptor binding.

[0009] Methods of treating patients who may develop or already have pre-existing neutralizing antibodies to a gene therapy vector by administering a protease that cleaves peptide bonds present in an immunoglobulin or by administering a glycosidase that cleaves carbohydrate residues present on an immunoglobulin or other similar enzymatic cleavages of an immunoglobulin in vivo are particularly described herein. Methods of treating patients who may develop or already have pre-existing antibodies that bind to a heterologous polynucleotide or a protein or peptide encoded by a heterologous polynucleotide encapsidated by a gene therapy vector are also particularly described herein by administering a protease that cleaves peptide bonds present in an immunoglobulin or by administering a glycosidase that cleaves carbohydrate residues present on an immunoglobulin or other similar enzymatic cleavages of an immunoglobulin in vivo are also particularly described herein. SUMMARY OF THE INVENTION

[0011] Disclosed herein are methods for reducing antibody (e.g., IgG) levels in human plasma using proteases such as IdeS. The methods according to the invention are particularly useful for treating patients with pre-existing neutralizing antibodies to gene therapy vectors, as well as patients who have been previously treated with gene therapy vectors.

[0012] In certain embodiments, a method for treating a subject in need of treatment for a disease caused by loss of protein function or activity comprises: (a) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide encoding a protein or peptide that provides or supplements the protein function or activity; and (b) administering to the subject an amount of a protease or glycosidase that effectively degrades or digests and / or inhibits or reduces the effector function of antibodies that bind to the recombinant viral vector and / or the protein or peptide encoded by the heterologous polynucleotide.

[0013] In certain embodiments, a method for treating a subject in need of treatment for a disease caused by increased protein function activity or expression comprises: (a) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, decreases or reduces the increase in protein function, activity or expression; and (b) administering to the subject a protease or glycosidase that effectively degrades or digests and / or inhibits or reduces the effector function of an antibody that binds to the recombinant viral vector.

[0014] In certain embodiments, a method for treating a subject in need of treatment for a disease caused by loss of protein function or activity comprises: (a) administering to the subject a protease or glycosidase that effectively degrades or digests and / or inhibits or reduces the effector function of a viral vector-bound antibody; and (b) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide encoding a protein or peptide that provides or supplements the protein function or activity.

[0015] In certain embodiments, a method for treating a subject in need of treatment for a disease caused by increased protein function, activity or expression comprises: (a) administering to the subject a protease or glycosidase that effectively degrades or digests and / or inhibits or reduces the effector function of the viral vector-bound antibody; and (b) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, decreases or reduces the increase in protein function, activity or expression.

[0016] In certain embodiments, in the method for treating a subject, step (b) is performed within about 90 days after performing step (a). In certain embodiments, step (b) is performed within about 60 days before or after step (a). In certain embodiments, step (b) is performed within about 45 days before or after step (a). In certain embodiments, step (b) is performed within about 30 days before or after step (a). In certain embodiments, step (b) is performed within about 21 days before or after step (a). In certain embodiments, step (b) is performed within about 14 days before or after step (a). In certain embodiments, step (b) is performed within about 7 days before or after step (a). In certain embodiments, step (b) is performed within about 72 hours before or after step (a). In certain embodiments, step (b) is performed within about 48 hours before or after step (a). In certain embodiments, step (b) is performed within about 24 hours before or after step (a). In certain embodiments, step (b) is performed within about 12 hours before or after step (a). In certain embodiments, step (b) is performed within about 6 hours before or after step (a).

[0017] In certain embodiments, the protease comprises a cysteine ​​protease or a thiol protease. In certain embodiments, the protease comprises a protease from Streptococcus pyogenes, Streptococcus equi, or Mycoplasma canis. In certain embodiments, the protease comprises IdeS or a modified variant thereof as set forth in any one of SEQ ID NOs: 3-18, 23, or 48.

[0018] In certain embodiments, the glycosidase comprises an endoglycosidase. In certain embodiments, the endoglycosidase comprises the sequence set forth in any one of SEQ ID NOs: 44-47.

[0019] In certain embodiments, the protease or glycosidase degrades or digests and / or inhibits or reduces the effector function of the human antibody.

[0020] In certain embodiments, the viral vector comprises a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

[0021] In certain embodiments, the lentiviral vector comprises an envelope protein to which the antibody binds.

[0022] In certain embodiments, the AAV vector comprises a capsid protein to which the antibody binds.

[0023] In certain embodiments, the AAV vector comprises antibody-bound VP1, VP2, and / or VP3 capsid proteins.

[0024] In certain embodiments, the AAV vector comprises a VP1, VP2 and / or VP3 capsid protein having 60% or greater sequence identity with a VP1, VP2 and / or VP3 capsid protein selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, Rh10, Rh74, SEQ ID NO: 1 and SEQ ID NO: 2 VP1, VP2 and / or VP3 capsid proteins.

[0025] In certain embodiments, the AAV vector comprises VP1, VP2 and / or VP3 capsid proteins having 100% sequence identity with VP1, VP2 and / or VP3 capsid proteins selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, Rh10, Rh74, SEQ ID NO: 1 and SEQ ID NO: 2 VP1, VP2 and / or VP3 capsid proteins.

[0026] In certain embodiments, the subject has antibodies that bind to the viral vector.

[0027] In certain embodiments, the subject does not have antibodies that bind to the viral vector.

[0028] In certain embodiments, the subject has antibodies that bind to the protein or peptide encoded by the heterologous polynucleotide.

[0029] In certain embodiments, the antibodies comprise IgG, IgM, IgA, IgD and / or IgE.

[0030] In certain embodiments, the method comprises determining the presence, quantifying the amount, or effector function of viral vector binding antibodies present in the subject before performing step (a), after performing step (a) but before performing step (b), and / or after performing steps (a) and (b).

[0031] In certain embodiments, the method comprises analyzing a biological sample from the subject for the presence, amount, or effector function of viral vector binding antibodies present in the sample before performing step (a), after performing step (a) but before performing step (b), and / or after performing steps (a) and (b).

[0032] In certain embodiments, the biological sample from the subject is a blood product. In certain embodiments, the blood product comprises plasma or serum.

[0033] In certain embodiments, the methods result in a reduction of the viral vector binding antibodies by 20-50%, 50-75%, 75-90%, 90-95%, or 95% or more.

[0034] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:100,000, wherein 1 part of the biological sample or blood product diluted in 100,000 parts of buffer results in 50% neutralization of the viral vector.

[0035] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:50,000, wherein 1 part of the biological sample or blood product diluted in 50,000 parts buffer results in 50% neutralization of the viral vector.

[0036] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:10,000, wherein 1 part of the biological sample or blood product diluted in 10,000 parts buffer results in 50% neutralization of the viral vector.

[0037] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:1,000, wherein 1 part of the biological sample or blood product diluted in 1,000 parts of buffer results in 50% neutralization of the viral vector.

[0038] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:100, wherein 1 part of the biological sample or blood product diluted in 100 parts of buffer results in 50% neutralization of the viral vector.

[0039] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:10, wherein 1 part of the biological sample or blood product diluted in 10 parts buffer results in 50% neutralization of the viral vector.

[0040] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:5, wherein 1 part of the biological sample or blood product diluted in 5 parts buffer results in 50% neutralization of the viral vector.

[0041] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:4, wherein 1 part of the biological sample or blood product diluted in 4 parts buffer results in 50% neutralization of the viral vector.

[0042] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:3, wherein 1 part of the biological sample or blood product diluted in 3 parts buffer results in 50% neutralization of the viral vector.

[0043] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:2, wherein 1 part of the biological sample or blood product diluted in 2 parts buffer results in 50% neutralization of the viral vector.

[0044] In certain embodiments, the viral vector binding antibodies present in a biological sample or blood product from a subject are less than about 1:1, wherein 1 part of the biological sample or blood product diluted in 1 part buffer results in 50% neutralization of the viral vector.

[0045] In certain embodiments, the method comprises determining the presence or quantifying the amount of antibodies that bind to the polypeptide or peptide encoded by the heterologous polynucleotide after performing step (a) but before performing step (b) and / or after performing steps (a) and (b).

[0046] In certain embodiments, the method comprises determining the presence or quantifying the amount of nucleic acid-binding antibodies after performing step (a) but before performing step (b) and / or after performing steps (a) and (b).

[0047] In certain embodiments, the subject has a pulmonary disease (e.g., cystic fibrosis), a bleeding disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disorder (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, a lysosomal storage disease (e.g., aspartylglucosuria, Batten disease, late infantile neuronal lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2-ganglioneuropathies type I (Tay et al., Sachs disease), GM2-ganglioneuroses type II (Sandhoff disease), seborrheic lesions type I (sialidosis type I and II), type II (I cell disease), type III (pseudo-Hurler disease) and type IV, mucopolysaccharidoses (Hurler disease and variants, Hunter, Sanfilippo types A, B, C, D, Morquio types A and B, Maroteaux-Lamy, and Sly diseases), Niemann-Pick disease types A / B, C1, and C2, and Schindler disease types I and II), hereditary angioedema (HAE), copper or iron storage diseases (e.g., Wilson's disease or Menkes' disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), solid organ (e.g., brain, liver, kidney, heart) diseases, or infectious viral (e.g., hepatitis B and C, HIV, etc.), bacterial, or fungal diseases. In certain embodiments, the subject has a coagulation disorder. In certain embodiments, the subject has hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any coagulation factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency.

[0048] In certain embodiments, the subject suffers from anemia; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharides, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors), or a thrombocytopathy (e.g., Bernard Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency).

[0049] In certain embodiments, the subject suffers from a disease that affects or originates from the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. In certain embodiments, CNS or neurodegenerative diseases are Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, polyglutamine repeat disease or Parkinson's disease. In certain embodiments, CNS or neurodegenerative diseases are polyglutamine repeat disease. In certain embodiments, polyglutamine repeat disease is spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7 or SCA17).

[0050] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiogenin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF) , transforming growth factor alpha (TGFα), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGFβ, activins, inhibins, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophins, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0051] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor alpha and beta, interferon alpha, beta and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

[0052] In certain embodiments, the heterologous polynucleotide encodes CFTR (cystic fibrosis transmembrane regulator), blood coagulation (coagulation) factors (factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), an increase in functional coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globulin, α-globulin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, β-25 glucocorticoid glycosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial cell-derived growth factor, transforming growth factor alpha and beta, cytokines, interferon alpha, interferon beta, interferon gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid aminotransferase (Gyrate Atrophy), retinoic acid relaxin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin A, GM2-AP, NPC1, VPC2, saposin, one or more zinc finger nucleases for genome editing, and one or more donor sequences for use as repair templates for genome editing.

[0053] In certain embodiments, the heterologous polynucleotide encodes an inhibitory nucleic acid. In certain embodiments, the inhibitory nucleic acid is selected from the group consisting of siRNA, antisense molecules, miRNA, RNAi, ribozymes, and shRNA. In certain embodiments, the inhibitory nucleic acid binds to a gene, a transcript of a gene, or a transcript of a gene associated with a polynucleotide repeat disease, the gene being selected from the group consisting of the huntingtin protein (HTT) gene, a gene associated with atrophy of the dentate nucleus, the globus pallidus, and the androgen receptor on chromosome X in spinal cord muscular atrophy, human Ataxin-1, -2, -3, and -7, Ca v2.1 P / Q voltage-dependent calcium channel (CACNA1A), TATA-binding protein, Ataxin 8 reverse chain (ATXN8OS), serine / threonine protein phosphatase 2A55 in spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, 17) kDa regulatory subunit Bβ isoform, FMR1 (fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMR1 (fragile X mental retardation 2) in fragile XE mental retardation, or AF4 / FMR2 family member 2; troponin kinase (MT-PK) in myotonic dystrophy; Frataxin in Friedreich's ataxia; mutants of the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transcriptional gene transactivator in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene in HIV infection; CC chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute renal failure in renal transplantation or renal injury with delayed graft function; protein kinase N3 (PKN3) in late recurrent or metastatic solid malignancies; LMP2, LMP2 also known as proteasome subunit beta 9 type (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta 8 type (PSMB 8), metastatic melanoma; MECL1 also known as proteasome subunit beta 10 type (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis inhibitory B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2 adrenergic receptor, glaucoma; RTP801 / Redd1, also known as DNA damage-inducible transcript 4 protein in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6A N17K mutant protein in congenital hyperthyroidism;Influenza A virus genome / gene sequence in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence in SARS infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola virus virus genome / gene sequence in Ebola virus infection; hepatitis B and hepatitis C virus genome / gene sequence in hepatitis B and hepatitis C infection; herpes simplex virus (HSV) genome / gene sequence in HSV infection; coxsackie virus B3 genome / gene sequence in coxsackie virus B3 infection; pathogenic allele silencing (allele-specific silencing) of genes, such as trunk protein A (TOR1A) in primary dystonia, specific pan I class and HLA alleles in transplantation; and mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP). ;

[0054] In certain embodiments, the protein encoded by the heterologous polynucleotide comprises a gene editing nuclease. In certain embodiments, the gene editing nuclease comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). In certain embodiments, the gene editing nuclease comprises a functional type II CRISPR-Cas9.

[0055] In certain embodiments, step (a) and / or step (b) of the method according to the present invention is performed two or more times.

[0056] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0057] Also disclosed herein are compositions, such as, but not limited to, kits and kits, having components useful for practicing the methods according to the present invention.

[0058] In certain embodiments, the kit or test kit is provided with: (a) a recombinant viral vector comprising a heterologous polynucleotide encoding a protein or peptide; (b) a protease or glycosidase that degrades or digests the antibody; and (c) a label with instructions for performing the methods disclosed herein. In certain embodiments, (a) and (b) are in separate or identical containers.

[0059] In certain embodiments, the kit or test kit is provided with: (a) a recombinant viral vector comprising a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, reduces or decreases protein expression; (b) a protease or glycosidase that degrades or digests the antibody; and (c) a label with instructions for performing the methods disclosed herein. In certain embodiments, (a) and (b) are in separate or identical containers.

[0060] In certain embodiments, a method of treating a disease that is treatable by gene therapy using a vector comprises administering to a subject in need thereof a therapeutically effective amount of an immunoglobulin G degrading enzyme polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 : is a graph showing the hydrolysis of human IgG in mice reconstituted with IVIg. Wild-type C57BL / 6 mice were reconstituted with human IgG (IVIg, 9 mg / mouse) and injected with IdeS (250 IU / mouse, 3 mice) or PBS (2 mice) 30 minutes later. Blood was collected before and 1, 6, and 10 hours after IdeS injection. Intact human IgG was measured in mouse serum by ELISA and expressed as μg / mL using IVIg as a standard.

[0062] Figure 2 Schematic diagram of the experimental protocol for neutralizing anti-AAV IgG in vivo. Wild-type C57BL / 6 mice (6 mice per group) or hemophilia B (HB) mice (4 mice per group) were reconstituted with human IgG (IVIg, 9 mg / mouse, day (D) minus 1 (D-1)) and injected with IdeS (250, 500 or 1250 IU / mouse, D-1+30 minutes) 30 minutes (min, mn) later (D0), mice were injected with AAV8 vectors (2×1010 vg / mouse). Blood was collected 15 minutes after IVIg injection, 24 hours after IdeS injection, and on D-7, D7, D14 and D28. At euthanasia (D28), livers were collected for vector genome copy number (VGCN) assessment. Control mice received IVIg alone, Ides alone, or PBS only before injection of AAV vectors.

[0063] Figure 3A , 3B , 3C, 3D, 3E, and 3F are graphs showing the effects of IdeS on human anti-AAV8 IgG, anti-AAV8 neutralizing antibodies (NAbs), anti-AAV2 IgG, anti-AAV2 Nabs, anti-AAV9 IgG, and anti-AAV9 Nabs, respectively, in passively immunized mice. Blood was collected from mice (6 mice / group) injected with IVIg and IdeS (IVIg / IdeS) or IVIg alone (IVIg / PBS) at D-1+15 minutes and D-1+24 (see Figure 2 ). Figure 3A , Figure 3B , Figure 3C and Figure 3D In the experiment, the dose of IdeS was 250 IU / mouse, and in Figure 3E and 3FThe dose of IdeS was 500 IU / mouse. The level of anti-AAV IgG was measured by ELISA, and the level of AAV NAb was measured by neutralization assay. Figure 3A , 3C The levels of anti-AAV8, anti-AAV2, and anti-AAV9 IgG in 3E and 3E are expressed in μg / mL, respectively. Figure 3B , 3D The levels of anti-AAV8, anti-AAV2, and anti-AAV9 NAbs in 3F and 4F were expressed as titers (1 / x), respectively. Data are expressed as mean ± SD. Statistical differences were evaluated using the nonparametric two-sided Mann-Whitney test (ns: not significant).

[0064] Figure 4A , 4B 4C and 4D are graphs showing that IdeS treatment rescues transgene expression using AAV8 vectors in passively immunized mice. Figure 4A and 4B ) or 1250 IU / mouse ( Figure 4C and 4D )) or PBS mice (6 mice / group) were treated with a gene encoding human factor IX (hFIX) ( Figure 4A and 4B ) or Gaussia luciferase (GLuc) ( Figure 4C and 4D ) were treated with 2x1010 vg / mouse AAV8 vector. Figure 4A : hFIX plasma levels were measured using a specific anti-hFIX ELISA. hFIX levels were expressed as μg / mL using purified hFIX as a standard. Figure 4C : Gaussia luciferase activity in plasma was measured using a luminescence assay. GLuc activity was expressed as RLU (Relative Light Units). Figure 4B and 4D : Number of vector genome copies per diploid genome in mouse liver by qPCR at sacrifice. Data are presented as mean ± SD. Statistical analysis was performed by two-way ANOVA with Dunnett's test.

[0065] Figure 5A and 5B is a graph showing an assay for in vitro digestion of human and non-human primate anti-AAV8 IgG by IdeS. IVIg (200 μg), serum from AAV8 seropositive humans, and serum from AAV8 seropositive monkeys were incubated with IdeS (100 IU) or PBS at 37°C for 22 hours. Figure 5A Depicted are the levels of anti-AAV8 IgG measured by ELISA. Figure 5BDepicted are the results of the AAV neutralization assay (left Y-axis) and the % neutralization activity observed at dilution (right Y-axis, black stars). Data are presented as mean ± SD.

[0066] Fig. 6A and 6B is a graph showing that IdeS treatment allows effective gene therapy with AAV8 vectors in passively immunized hemophilia B (HB) mice. HB mice (4 mice per group) were reconstituted with human IgG (IVIg, 9 mg / mouse, D-1) and injected with IdeS (1250 IU / mouse, D-1+30 minutes) 30 minutes later. 24 hours later (D0), mice were injected with AAV8 vectors carrying the hFIX transgene (2×1010 vg / mouse). Blood was collected 24 hours after IdeS injection and at D7 and D14. The levels of hFIX in plasma were measured using a dedicated ELISA ( Fig. 6A At D21, the efficacy of gene therapy in HB mice was assessed using a tail clip bleeding assay, in which blood loss over 20 min was estimated ( Figure 6B ). Data are expressed as mean ± SD. Statistical analysis was performed by two-way ANOVA with Dunnett's test for hFIX ELISA and one-way ANOVA with Dunnett's test for tail clip assay (*: p<0.05; ***: p<0.001).

[0067] Figure 7 is a schematic diagram of the experimental scheme in NHP. Two male cynomolgus monkeys seropositive for AAV8 (NAb titer of 1:17.2) were infused with 2x1012vg / kg of AAV8-hFIX vector (D0). Before injection of the vector, NHP2 received two injections (iv) of IdeS (500μg / kg) on ​​D-2 and D-1 (NHP treatment). NHP1 did not receive prior treatment (control NHP). Blood samples were collected before and after injection of IdeS and at D0, D1, D4, D7, D13, D21, D28, D35 and D50. At euthanasia (D50), liver biopsies were collected from 4 liver lobes for VGCN evaluation.

[0068] Fig. 8A , 8B 8A and 8C are graphs showing the effect of IdeS treatment on IgG in AAV8 seropositive monkeys. Fig. 8A : Intact IgG levels in serum were measured by ELISA using purified monkey IgG as a standard and expressed as mg / mL. Figure 8B: Anti-AAV8 IgG levels in serum were measured before and after IdeS treatment by ELISA. In treated monkeys (NHP2), the level of anti-AAV8 IgG decreased 2.7-fold after IdeS injection (from 2.86 to 1.05 μg / mL). Figure 8C : Anti-AAV8 NAb levels were measured before and after Ides treatment using a neutralization assay. Anti-AAV8 NAb titers were reduced 3-fold in treated monkeys (NHP2) (titer decreased from 17.2 to 5.4). Data are expressed as mean ± SD. Statistical analysis was performed by two-way ANOVA with Tukey's test (ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0069] Fig.9A and 9B is a graph showing higher transgene expression in monkeys seropositive to AAV8 treated with IdeS. Fig.9A : hFIX plasma levels were measured using a specific anti-hFIX ELISA. hFIX levels were expressed as ng / mL using purified hFIX as a standard. Fig. 9B : Vector genome copy number (VGCN) per diploid genome in the liver by qPCR at the time of sacrifice. VGCN assessment was performed in 4 wedge biopsies taken from different parts of the 4 liver lobes (right, left, caud and quad). Data are expressed as mean ± SD. Statistical analysis was performed by two-way SNOVA with Sidak's multiple comparison test (***: p < 0.001; ****: p < 0.0001). Compared with control monkeys (NHP1), monkeys treated with IdeS (NHP2) had higher levels of hFIX in plasma and higher AAV liver transduction efficiency.

[0070] Fig. 10A and 10B Graphs showing the levels of anti-AAV8 IgG and anti-AAV8 NAb, respectively, in AAV8 seropositive monkeys treated with IdeS. The levels of anti-AAV8 IgG were measured by ELISA and expressed in μg / mL ( Fig. 10A ). Anti-AAV8 NAbs were measured in the neutralization assay and expressed as titers (1:x) ( Fig. 10B ). Data are expressed as mean ± SD. Statistical analysis was performed by two-way ANOVA with Tukey's multiple comparison test (****: p < 0.0001).

[0071] Fig.11Schematic diagram of the experimental scheme for AAV8 re-administration in NHP. Two male cynomolgus monkeys (Nab titer 1:31.6) seropositive for AAV8 were infused with 5x1012vg / kg of AAV8-hFIX vector (D0). Prior to the injection of the vector, NHP4 received a single intravenous (IV) injection of IdeS (500μg / kg) on ​​D-1 day (NHP treatment). NHP3 did not receive prior treatment (control NHP). Subsequently, the two monkeys received two IV injections of IdeS (500μg / kg) on ​​days 80 and 81, and received an injection of 5x1012vg / kg of AAV8-hFIX vector on day 82. Blood samples were collected at different time points during the experiment. At euthanasia (D105), liver biopsies were collected from 4 liver lobes for VGCN evaluation.

[0072] Fig. 12A and Fig. 12B is a graph showing that IdeS treatment allows efficient re-administration of AAV vectors in AAV8 seropositive monkeys, resulting in increased transgene expression. Fig. 12A : hFIX plasma levels were measured using a specific anti-hFIX ELISA. hFIX levels were expressed as ng / mL using purified hFIX as a standard. Fig. 12B : The vector genome copy number (VGCN) per diploid genome in the liver was determined by qPCR at the time of sacrifice. VGCN assessment was performed in 4 wedge biopsies taken from different parts of the 4 liver lobes (left, right, caud and quad). Data are expressed as mean ± SD. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparison test (*: p < 0.05; **: p < 0.01; ****: p < 0.0001).

[0073] Fig.13A and 13B are graphs showing the levels of anti-AAV8 IgG and anti-AAV8 NAb, respectively, in AAV8 seropositive monkeys treated with IdeS after re-administration of AAV8 vectors. Fig.13A : Anti-AAV8 IgG levels were measured by ELISA. Fig. 13B : Anti-AAV8 Nab titers were measured using a neutralization assay. Data are presented as mean ± SD.

[0074] Fig.14A , 14B and 14C showing human patient sera incubated with increasing amounts of IdeS ( Fig.14A ), non-human primate (rhesus monkey) plasma ( Fig. 14B ) and hamster plasma ( Fig. 14C) by IdeS cleavage of IgG. The samples were incubated at 37°C for 1 hour in the absence of IdeS or in the presence of increasing concentrations of IdeS. The reaction was terminated by adding sample buffer. The samples were analyzed by non-reducing SDS-PAGE and Coomassie staining.

[0075] Fig.15 is a graph showing the level of GAA activity in mouse plasma after infusion of AAV-Spk1-GAA vector in animals immunized with different amounts of IVIg (the IVIg was pretreated with or without IdeZ). AAV-Spk1-GAA vector was infused one day after IVIg immunization. Transgenic activity was assessed by GAA activity assay 2 weeks after vector administration. GAA activity for each mouse in each group was plotted in nmol / hr / mL. Vectors were administered only to control mice in the absence of IVIg.

[0076] Fig.16 The titer levels of anti-Spk1 neutralizing antibodies (NAbs) in mouse plasma before and after IdeS infusion are shown. The relative NAb titer levels in this study are designated as low titer (<1:1, 1:1-1:2.5) (bold), mid-range (1:2.5-1:5) (bold italics), and high (>1:5-1:10) (italics).

[0077] Fig.17 The levels of anti-Spk1 IgG NAb in mouse plasma before and after IdeS infusion are shown (ng / mL). Negative control animals were not treated with IVIg or IdeS. IdeS low used in the study refers to 0.4 mg / kg IdeS, and IdeS high refers to 4 mg / kg IdeS.

[0078] Fig.18 Shown is the level of GAA activity (nmol / hr / mL) in mouse plasma after infusion of the AAV-Spk1-GAA vector in animals immunized with IVIg and then treated with IdeS. All animals received 2x10 12 vg / kg AAV-Spk1-GAA vector. Transgene activity was measured in plasma samples of mice immunized with IVIg, then treated with or without IdeS, and finally administered with vector. Transgene activity was assessed by GAA activity assay 1 week after vector administration. GAA activity for each mouse in each group was plotted in nmol / hr / mL.

[0079] Fig.19 Animals previously treated with IdeS (0, 0.4, 1.0, or 2.0 mg / kg) and infused with AAV-Spk1-GAA vector (2x1012 vg / kg) after two weeks of GAA activity levels (nmol / hr / mL). The GAA activity of each mouse in each group was plotted. Mice in the IVIg-administered group that did not develop a corresponding anti-Spk1 Nab titer (i.e., Nab titer <1:1 or 1:1-1:2.5 before IdeS treatment) were excluded.

[0080] Fig. 20 Shown are anti-Spk1 Nab titer levels in C57BL / 6 mouse plasma with artificial titers of human anti-capsid neutralizing IgG measured before and after infusion with different IdeS formulations (Batch #1 and Batch #2).

[0081] Fig.21 is a graph showing human Factor VIII levels in plasma from C57BL / 6 mice with artificial titers of human anti-capsid neutralizing IgG, measured before and 1 and 2 weeks after dosing with AAV-Spk1-hFVIII.

[0082] Fig. 22 is a graph showing anti-Spk1 capsid IgG levels (ng / mL) in mouse plasma before and after IdeS infusion. IVIg was provided to C57BL / 6 mice to induce artificial titers of human anti-capsid neutralizing IgG. Negative control animals were not treated with IVIg or IdeS. The low IVIg used in the study refers to 300 mg / kg IVIg, the medium IVIg refers to 800 mg / kg, and the high IVIg refers to 1600 mg / kg. In each IVIg group, animals were treated with increasing doses of IdeS (0, 0.4, 1.0, 2.0 mg / kg IdeS). Animals that were treated with IVIg but did not show anti-capsid IgG responses were excluded from the figure. DETAILED DESCRIPTION OF THE INVENTION

[0084] Provided herein are methods for improving the benefits or effectiveness of gene therapy, including administering agents that degrade or digest antibodies and / or inhibit or reduce the effector functions of antibodies. Also provided herein are methods for degrading or digesting antibodies that bind to viral vectors (e.g., recombinant viral vectors) and / or antibodies that degrade or digest proteins or peptides encoded by heterologous polynucleotides encapsidated with nucleic acids or recombinant viral vectors and / or inhibit or reduce the effector functions of antibodies bound to recombinant viral vectors, and / or nucleic acids or proteins or peptides encoded by heterologous polynucleotides encapsidated with recombinant viral vectors. Also provided herein are methods for re-administering or re-administering gene therapy vectors to subjects who have previously administered gene therapy vectors, and wherein the subject has produced antibodies that bind to and / or neutralize the gene therapy vectors.

[0085] In certain embodiments, the method comprises administering to the subject an amount of a protease that is effective in degrading or digesting the antibody, or a glycosidase that is effective in inhibiting or reducing the effector function of the antibody that binds to the recombinant viral vector, and / or nucleic acid, and / or a protein or peptide encoded by a heterologous polynucleotide. In certain embodiments, the method comprises administering to the subject an amount of an endopeptidase that is effective in degrading or digesting the antibody, or an endoglycosidase that is effective in inhibiting or reducing the effector function of the antibody that binds to the recombinant viral vector, and / or nucleic acid, and / or a protein or peptide encoded by a heterologous polynucleotide.

[0086] In certain embodiments, the method comprises administering to a subject an amount of a glycosidase effective to reduce Fc receptor binding of an antibody that binds to a recombinant viral vector, and / or a nucleic acid and / or a protein or peptide encoded by a heterologous polynucleotide. In certain embodiments, the method comprises administering to a subject an amount of an endoglycosidase effective to reduce Fc receptor binding of an antibody that binds to a recombinant viral vector, and / or a nucleic acid and / or a protein or peptide encoded by a heterologous polynucleotide.

[0087] In certain embodiments, the present invention relates to immunoglobulin G degrading enzyme peptides for use in treating a disease in a patient in need thereof by gene therapy using a vector.

[0088] In certain embodiments, the present invention relates to i) an immunoglobulin G degrading enzyme polypeptide, and ii) a vector as a combined preparation for simultaneously, separately or sequentially treating a disease treatable by gene therapy using the vector in a patient in need thereof.

[0089] In certain embodiments, the present invention relates to a combination of an immunoglobulin G degrading enzyme polypeptide and a vector for use in treating a disease in a patient in need thereof by gene therapy using the vector.

[0090] In certain embodiments, the immunoglobulin G degrading enzyme polypeptide is administered before, simultaneously (concomitantly) or after the vector.

[0091] As used herein, the term "disease treated by gene therapy using a vector" refers to a disease in which a polynucleotide (encoding at least one polypeptide or inhibitory nucleic acid) is delivered to a patient's cells as a drug to treat the disease (gene therapy).

[0092] In certain embodiments, the vector encodes at least one specific polypeptide or inhibitory nucleic acid that can be used to treat a disease. In certain embodiments, the vector encodes / contains a therapeutic polynucleotide suitable for treating a disease.

[0093] In certain embodiments, the vector encodes / comprising a therapeutic polynucleotide suitable for treating a disease using gene therapy.

[0094] In certain embodiments, the patient is a vector seropositive patient.

[0095] In certain embodiments, the patient is a vector seronegative patient.

[0096] As used herein, the term "immunoglobulin G degrading enzyme polypeptide" refers to a polypeptide that is a protease that degrades immunoglobulin G. In certain embodiments, the immunoglobulin G degrading enzyme (IdeS) polypeptide is a papain, pepsin or immunoglobulin G degrading enzyme polypeptide of Streptococcus pyogenes, or a variant from other bacteria or microorganisms, or an engineered form thereof. In certain embodiments, the immunoglobulin G degrading enzyme polypeptide specifically degrades immunoglobulin G. In certain embodiments, the immunoglobulin G degrading enzyme polypeptide comprises any one of the sequences selected from SEQ ID NO: 3-43 or 48.

[0097] As used herein, the terms "patient" and "subject" refer interchangeably to animals, typically mammals, such as rodents, felines, canines, and primates. In particular, the subject or patient according to the present invention is a human.

[0098] As used herein, the term "treatment" refers to prophylactic or preventative treatment, such as gene therapy, and curative or disease-modifying treatment, including treatment of subjects at risk of or suspected of having a disease and subjects who are ill or diagnosed with a disease or medical condition, and includes inhibition of clinical relapse. Treatment can be administered to a subject who has a medical condition or may eventually have a condition to prevent, cure, delay the onset of the condition or recurrent condition, reduce severity or alleviate one or more symptoms, or to extend the subject's survival beyond what would be expected in the absence of such treatment.

[0099] Further provided herein are compositions, for example, provided in the form of a kit or a test kit, having (a) a recombinant viral vector comprising a heterologous polynucleotide encoding a protein or peptide; (b) a protease or glycosidase that degrades or digests the antibody; and (c) a label with instructions for performing the methods disclosed herein, wherein (a) and (b) are in separate or identical containers. In certain embodiments, viral vectors useful in the present invention include, for example, but not limited to, adeno-associated virus (AAV) vectors. In certain embodiments, viral vectors useful in the present invention include, for example, but not limited to, retroviruses, adenoviruses, helper-dependent adenoviruses, hybrid adenoviruses, herpes simplex viruses, lentiviruses, poxviruses, Epstein-Barr viruses, vaccinia viruses, and human cytomegalovirus vectors, including recombinant forms thereof.

[0100] As used herein, but not limited to, the term "recombinant" as a modifier of viral vectors (e.g., recombinant AAV (rAAV) vectors) and sequences (e.g., recombinant polynucleotides and polypeptides) refers to compositions that have been manipulated (i.e., engineered) in a manner that does not normally occur in nature. A specific example of a recombinant AAV vector is the insertion of a nucleic acid (heterologous polynucleotide) that is not normally present in the wild-type AAV genome into the viral genome. An example is the cloning of a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence into a vector with or without the 5', 3', and / or intron regions of the gene that are normally associated with the AAV genome. Although the term "recombinant" is not always used herein for AAV vectors and sequences (e.g., polynucleotides), recombinant forms comprising AAV vectors, polynucleotides, etc. are expressly included despite any such omissions.

[0101] For example, a "rAAV vector" is derived from the wild-type genome of AAV by removing all or part of the wild-type AAV genome using molecular methods and replacing it with a non-natural (heterologous) nucleic acid (e.g., a nucleic acid encoding a therapeutic protein or polynucleotide sequence). Typically, for rAAV vectors, one or two inverted terminal repeat (ITR) sequences of the AAV genome are retained. rAAV is different from the AAV genome because all or part of the AAV genome has been replaced by a non-natural sequence, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence, relative to the AAV genomic nucleic acid. Therefore, the incorporation of non-natural (heterologous) sequences defines AAV as a "recombinant" AAV vector, which can be referred to as a "rAAV vector."

[0102] Recombinant AAV vector sequences can be packaged - referred to herein as "particles" for subsequent infection (transduction) of cells ex vivo, in vitro or in vivo. When the recombinant vector sequences are encapsidated or packaged into AAV particles, the particles may also be referred to as "rAAV", "rAAV particles" and / or "rAAV virions". Such rAAV, rAAV particles and rAAV virions include proteins that encapsidate or package the vector genome. In the case of AAV, specific examples include capsid proteins.

[0103] "Vector genome", which may be abbreviated as "vg", refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form rAAV particles. In the case where the recombinant plasmid is used to construct or prepare a recombinant AAV vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone", which is very important for cloning and amplifying plasmids (processes necessary for propagation and recombinant AAV vector production), but it itself is not packaged or encapsidated into rAAV particles. Therefore, "vector genome" refers to the nucleic acid packaged or encapsidated by rAAV.

[0104] As used herein, the term "serotype" with respect to AAV vectors refers to a capsid that is serologically distinct from other AAV serotypes. Serological uniqueness is determined based on the lack of cross-reactivity between antibodies against one AAV compared to another AAV. Differences in cross-reactivity are generally attributed to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Due to the homology of capsid protein sequences, antibodies against one AAV may cross-react with one or more other AAV serotypes.

[0105] Under the traditional definition, serotype refers to the neutralization activity of the target virus tested for serum of all existing and characteristic serotypes, and no antibodies to neutralize the target virus are found. With the discovery of more naturally occurring virus isolates and / or the production of capsid mutants, there may be serological differences or no serological differences with any currently existing serotype. Therefore, in the case where a new virus (e.g., AAV) does not have serological differences, the new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, mutant viruses with modified capsid sequences have not yet been subjected to serological tests for neutralization activity to determine whether they belong to another serotype according to traditional serotype definitions. Therefore, for convenience and to avoid repetition, the term "serotype" refers broadly to serologically different viruses (e.g., AAV) and serologically different viruses (e.g., AAV) within a subgroup or variant of a given serotype.

[0106] rAAV vectors include any strain or serotype. For example, but not limited to, rAAV vector genomes or particles (capsids, such as VP1, VP2 and / or VP3) can be based on any AAV serotype, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10 or AAV-2i8. The vectors can be based on the same strain or serotype (or subgroup or variant), or different from each other. For example, but not limited to, rAAV plasmids or vector genomes or particles (capsids) based on a serotype genome can be the same as one or more capsid proteins that package the vector. In addition, the rAAV plasmid or vector genome can be based on an AAV serotype genome that has one or more capsid proteins that are different from the packaging vector genome, in which case at least one of the three capsid proteins can be a different AAV serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2), or a variant thereof. More specifically, the rAAV2 vector genome may comprise the AAV2 ITRs, but a capsid from a different serotype, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2), or variants thereof. Thus, the rAAV vector includes gene / protein sequences that are identical to those characteristic of a particular serotype as well as "mixed" serotypes (which may also be referred to as "pseudotypes").

[0107] In certain embodiments, the vector is an AAV vector, a lentiviral vector, or an adenoviral vector.

[0108] In certain embodiments, the term vector also includes recombinant vectors that induce neutralizing immunoglobulin G after the first administration.

[0109] In certain embodiments, the vector is an AAV vector. In certain embodiments, the vector is an AAV8 vector encoding human FIX (comprising a therapeutic polynucleotide encoding human FIX). In certain embodiments, the vector is an AAV8 vector encoding human FIX, which can be used to treat hemophilia.

[0110] In certain embodiments, the rAAV plasmid or vector genome or particle is based on a reptile or invertebrate AAV variant, such as a snake and lizard parvovirus (Pénzes et al., 2015, J. Gen. Virol., 96:2769–2779) or an insect and shrimp parvovirus (Roekring et al., 2002, Virus Res., 87:79–87).

[0111] In certain embodiments, the recombinant plasmid or vector genome or particle is based on a bocavirus variant. For example, human bocavirus variants are described in Guido et al., 2016, World J. Gastroenterol., 22:8684–8697.

[0112] In certain embodiments, the rAAV vector comprises or consists of a capsid protein that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2) capsid proteins (VP1, VP2 and / or VP3 sequences). In certain embodiments, the rAAV vector comprises or consists of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, or AAV-2i8, ITRs.

[0113] In certain embodiments, rAAV vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, and AAV-2i8 variants thereof (e.g., ITR and capsid variants, such as amino acid insertions, additions, substitutions, and deletions), for example, as described in WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO 2015 / 013313 (International Application PCT / US2014 / 047670), and US2013 / 0059732 (U.S. Patent Application No. 13 / 594,773).

[0114] rAAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2) and variants, hybrids and chimeric sequences thereof can be constructed using recombinant techniques known to the skilled artisan to include one or more heterologous polynucleotide sequences (transgenes) flanked by one or more functional AAVITR sequences. The AAV vector typically retains at least one functional flanking ITR sequence, which is necessary for the rescue, replication and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome will include sequences required for replication and packaging in cis (e.g., functional ITR sequences). In certain embodiments, the lentivirus used in the present invention can be human immunodeficiency-1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV) or caprine arthritis encephalitis virus (CAEV). Lentiviral vectors are capable of efficiently delivering, integrating and long-term expressing heterologous polynucleotide sequences into non-dividing cells in vitro and in vivo. A variety of lentiviral vectors are known in the art, see Naldini et al. (Proc. Natl. Acad. Sci. USA, 93: 11382-11388 (1996); Science, 272: 263-267 (1996)), Zufferey et al., (Nat. Biotechnol., 15: 871-875, 1997), Dull et al., (J Virol. 1998 Nov; 72 (11): 8463-71, 1998), U.S. Pat. Nos. 6,013,516 and 5,994,136, any of which may be suitable viral vectors for use in the present invention. An immune response, such as humoral immunity, may develop against a wild-type virus in a subject exposed to the wild-type virus. Such exposure may result in pre-existing antibodies in the subject binding to a viral vector based on the wild-type virus, even before treatment with a gene therapy method using a viral vector.

[0115] As used herein, the term "AAV vector" has its ordinary meaning in the art and refers to vectors derived from adeno-associated virus serotypes, including but not limited to AAV1, AAV2, variant AAV2, AAV3, variant AAV3, AAV3B, variant AAV3B, AAV4, AAV5, AAV6, variant AAV6, AAV7, AAV8, AAV9, AAV10 (such as AAVcy10 and AAVrh10), AAVrh74, AAVDJ, AAV-Anc80; AAV-LK03, AAV2i8 and porcine AAV (such as AAVpo4 and AAVpo6 capsids), or any other naturally occurring AAV serotype with a chimeric capsid or that can infect humans, monkeys or other species, as well as all other serotypes engineered by shuffling, peptide insertion, error-prone PCR, rational design or using machine learning and other techniques, such as AAVLK03, AAVDJ, Anc80, AAV2i8, AAV-PHP-B. AAV vectors may have one or more AAV wild-type genes that are completely or partially deleted, preferably rep and / or cap genes, but retain functional flanking reverse terminal repeat (ITR) sequences. Functional ITR sequences are essential for the relief, replication and packaging of AAV virions. Therefore, AAV vectors are defined herein as including at least those sequences (e.g., functional ITRs) required for cis replication and packaging viruses. ITRs do not have to be wild-type nucleotide sequences, and can be changed, for example, by insertion, deletion or substitution of nucleotides, as long as the sequence provides functional relief, replication and packaging. AAV expression vectors are constructed using known techniques to provide at least operably connected components, control elements (including transcription initiation regions), nucleic acid molecules of the present invention, and transcription termination regions in the transcription direction. Control elements are selected to work in mammalian cells. The resulting construct containing operably connected components is combined with functional AAV ITR sequences (5' and 3').

[0116] "Adeno-associated virus inverted terminal repeats" or "AAV ITRs" refer to regions recognized in the art found at each end of the AAV genome that function together in cis as an origin of DNA replication and a packaging signal for the virus. Together with the AAV rep coding region, the AAV ITRs provide for efficient excision and rescue of nucleotide sequences inserted between the two flanking ITRs and their integration into the mammalian cell genome. The nucleotide sequences of the AAV ITR region are known. For example, for AAV-2 sequences, see Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (BN Fields and DM Knipe, eds.). As used herein, "AAV ITR" does not necessarily comprise a wild-type nucleotide sequence, but may be altered, for example, by insertion, deletion or substitution of nucleotides. In addition, AAV ITRs may be derived from any of several AAV serotypes, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, and the like. In addition, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV vector are not necessarily the same or derived from the same AAV serotype or isolate, as long as they can function as expected, that is, allow the target sequence to be excised and rescued from the host cell genome or vector, and allow the heterologous sequence to be integrated into the recipient cell genome when the AAV Rep gene product is present in the cell. In certain embodiments, the AAV vector of the present invention is selected from vectors of AAV serotypes with tropism and high transduction efficiency in cells of the mammalian central and peripheral nervous systems, particularly neurons, neuronal progenitor cells, astrocytes, oligodendrocytes and glial cells. In certain embodiments, the AAV vector is AAV4, AAV9 or AAVrh10, which have been described as well transducing brain cells (especially neurons). In certain embodiments, the AAV vector of the present invention is a double-stranded self-complementary AAV (scAAV) vector. As an alternative to using a single-stranded AAV vector, a self-complementary vector can be used. The efficacy of AAV vectors in terms of the number of genome-containing particles required for transduction is hampered by the need to convert the single-stranded DNA (ssDNA) genome to double-stranded DNA (dsDNA) prior to expression. This step can be circumvented by using self-complementary vectors that package inverted repeat genomes that can fold into dsDNA without the need for DNA synthesis or base pairing between multiple vector genomes. The resulting self-complementary AAV (scAAV) vectors increase the resulting expression of the transgene.For a review of AAV biology, ITR function and scAAV constructs, see McCarty DM. Self-complementary AAV vectors; advances and applications. Mol. Ther. 2008 October; 16 (10): 1648-51, first paragraph in its entirety, the disclosure of AAV and scAAV constructs, ITR function and the role of ΔTRS ITR in scAAV constructs is incorporated herein by reference. rAAV vectors containing ΔTRS ITRs cannot be properly nicked during the replication cycle, thus generating a self-complementary double-stranded AAV (scAAV) genome that can be efficiently packaged into infectious AAV particles. The various rAAV, ssAAV, and scAAV vectors, as well as the advantages and disadvantages of each type of vector for specific applications and methods of using such vectors in gene transfer applications are well known to those of skill in the art (see, e.g., Choi et al., 2005, J. Virol., 79(11):6801-7; McCarty et al., 2004, Annu Rev Genet., 38:819-45; McCarty et al., 2001, Gene Ther., 8(16):1248-54; and McCarty 2008, Mol. Ther., 16(10):1648-56; all cited references are incorporated herein by reference for their disclosures of AAV, rAAV, and scAAV vectors).

[0117] The AAV vector of the present invention can be constructed by directly inserting the selected sequence into the AAV genome from which the major AAV open reading frame ("ORF") has been excised. Other parts of the AAV genome can also be deleted, as long as enough ITR portions are retained to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 and WO 93 / 03769. Alternatively, the AAV ITR can be excised from the viral genome or from the same and fused 5' and 3' AAV vector containing the selected nucleic acid construct present in another vector using standard ligation techniques. AAV vectors containing ITRs have been described in, for example, U.S. Patent No. 5,139,941. In particular, several AAV vectors are described therein, which are available from the American Type Culture Collection ("ATCC") with accession numbers 53222, 53223, 53224, 53225 and 53226. In addition, chimeric genes can be synthetically produced to include AAV ITR sequences arranged at 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expressing chimeric gene sequences in mammalian CNS and PNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. In order to produce AAV virions, the AAV expression vector is introduced into a suitable host cell using known techniques (e.g., by transfection). Many transfection techniques are well known in the art. For example, see Graham et al., 1973; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981. Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome-mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Felgner et al., 1987), and nucleic acid delivery using high-speed microprojectiles (Klein et al., 1987).

[0118] Typically, the vector of the present invention comprises an expression cassette. As used herein, the term "expression cassette" refers to a nucleic acid construct comprising a nucleic acid element sufficient to express a nucleic acid molecule of the present invention. Typically, an expression cassette comprises a nucleic acid molecule of the present invention operably connected to a promoter sequence. The term "operably connected" refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment, so that the function of one is affected by another. For example, when a promoter is able to affect the expression of the coding sequence (for example, the coding sequence is under the transcriptional control of the promoter), the promoter is operably connected to the coding sequence. The coding sequence can be operably connected to a regulatory sequence in a sense or antisense direction. In certain embodiments, the promoter is a heterologous promoter. As used herein, the term "heterologous promoter" refers to a promoter that is not found to be operably connected to a given coding sequence in nature. In certain embodiments, the expression cassette may include other elements, such as introns, enhancers, polyadenylation sites, woodchuck response elements (WREs) and / or other elements known to affect the expression level of the coding sequence. As used herein, the term "promoter" refers to a nucleotide sequence that can control the expression of a coding sequence or functional RNA. Typically, the nucleic acid molecule of the present invention is located at the 3' of the promoter sequence. In certain embodiments, the promoter sequence is composed of proximal and more distal upstream elements, and may include an enhancer element. "Enhancer" is a nucleotide sequence that can stimulate promoter activity, and may be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. In certain embodiments, the promoter is derived from a natural gene in its entirety. In certain embodiments, the promoter is composed of different elements derived from different naturally occurring promoters. In certain embodiments, the promoter comprises a synthetic nucleotide sequence. It will be appreciated by those skilled in the art that different promoters will direct the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions or the presence or absence of drugs or transcriptional cofactors. Ubiquitous, cell type-specific, tissue-specific, developmental stage-specific and conditional promoters, such as drug-responsive promoters (e.g., tetracycline-responsive promoters) are well known to those skilled in the art. Examples of promoters include, but are not limited to, phosphoglycerate kinase (PKG) promoter; CAG (a complex of CMV enhancer, chicken beta actin promoter (CBA) and rabbit beta globin intron); NSE (neuron-specific enolase); synapsin or NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoter (such as CMV) immediate early promoter region (CMVIE); SFFV promoter; Rous sarcoma virus (RSV) promoter; synthetic promoter; hybrid promoter, etc. Other promoters may be of human origin or from other species, including mice.Common promoters include, for example, human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat, β-actin, rat insulin promoter, phosphoglycerate kinase promoter, human α-1 antitrypsin (hAAT) promoter, transthyretin promoter, TBG promoter and other liver-specific promoters, desmin promoter and similar muscle-specific promoters, EF1-α promoter, CAG promoter and other constitutive promoters, hybrid promoters with multiple tissue specificities, promoters specific to neurons (such as synaptophysin and glyceraldehyde-3-phosphate dehydrogenase promoters), all of which are well-known and easily available promoters to those skilled in the art, which can be used to obtain high-level expression of the encoding target sequence. In addition, sequences derived from non-viral genes (such as mouse metallothionein genes) can also be used herein. For example, the promoter sequence is commercially available from Stratagene (San Diego, CA).

[0119] In certain embodiments, the expression cassette comprises a suitable secretion signal sequence, which will allow secretion of the polypeptide encoded by the nucleic acid molecule of the present invention. As used herein, the term "secretion signal sequence" or a variant thereof refers to an amino acid sequence having the function of enhancing (as defined above) secretion of an operably connected polypeptide from a cell compared to the secretion level seen with a natural polypeptide. As defined above, "enhanced" secretion refers to an increase in the relative proportion of a polypeptide synthesized by a cell secreted by a cell; the absolute amount of secreted protein does not necessarily increase. In certain embodiments, substantially all (i.e., at least 95%, 97%, 98%, 99% or more) polypeptides are secreted. However, as long as the secretion level is increased compared to a natural polypeptide, substantially all or even most of the polypeptides are not necessarily secreted. Typically, the secretion signal sequence is cut in the endoplasmic reticulum, and in certain embodiments, the secretion signal sequence is cut before secretion. However, as long as the secretion of the polypeptide from the cell is enhanced and the polypeptide is functional, the secretion signal sequence is not necessarily cut. Therefore, in certain embodiments, the secretion signal sequence is partially or completely retained. The secretion signal sequence may be derived in whole or in part from the secretion signal of the secreted polypeptide (i.e., from a precursor) and / or may be synthesized in whole or in part. The length of the secretory signal sequence is not critical; generally, known secretory signal sequences are about 10-15 to 50-60 amino acids in length. In addition, known secretory signals from secreted polypeptides can be altered or modified (e.g., by substitution, deletion, truncation or insertion of amino acids), as long as the resulting secretory signal sequence acts to enhance the secretion of the operably linked polypeptide. The secretory signal sequence of the present invention may comprise, consist essentially of, or consist of a naturally occurring secretory signal sequence or a modification thereof (as described above). Numerous secretory proteins and sequences that direct secretion from cells are known in the art. The secretory signal sequence of the present invention may further be wholly or partially synthetic or artificial. Synthetic or artificial secretory signal peptides are known in the art, for example, see Barash et al., Biochem.Biophys.Res.Comm.294:835-42 (2002).

[0120] In certain embodiments, the present invention includes a method of treating a disease by gene therapy using a vector, comprising administering a therapeutically effective amount of an immunoglobulin G degrading enzyme polypeptide to a subject in need thereof.

[0121] Nucleic acid, vector, recombinant host cell and use thereof

[0122] In certain embodiments, the present invention relates to a nucleic acid sequence encoding an immunoglobulin G degrading enzyme polypeptide for use in treating a disease in a patient in need thereof by gene therapy using a vector.

[0123] In certain embodiments, the present invention comprises: i) a nucleic acid sequence encoding an immunoglobulin G degrading enzyme polypeptide, and ii) a vector as a combined preparation for simultaneously, separately or sequentially treating a disease treatable by gene therapy using the vector in a patient in need thereof.

[0124] In certain embodiments, the present invention includes a combination of a nucleic acid sequence encoding an immunoglobulin G degrading enzyme polypeptide and a vector for use in treating a disease treatable by gene therapy using the vector in a patient in need thereof.

[0125] In certain embodiments, the present invention includes a nucleic acid sequence encoding the IdeS polypeptide of SEQ ID NO: 4 as described herein or a functional conservative variant thereof.

[0126] In certain embodiments, the nucleic acid sequence encoding the IdeS polypeptide of SEQ ID NO:4 comprises or consists of SEQ ID NO:52.

[0127] In certain embodiments, the nucleic acid sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the sequence of SEQ ID NO:52.

[0128] In certain embodiments, mRNA of the nucleic acids of the invention may be used.

[0129] The nucleic acids of the invention can be produced by any technique known per se in the art, such as but not limited to any chemical, biological, genetic or enzymatic technique, alone or in combination.

[0130] In certain embodiments, the present invention includes an expression vector comprising a nucleic acid sequence encoding an immunoglobulin G degrading enzyme polypeptide for use in treating a disease treatable by gene therapy using the vector in a patient in need thereof.

[0131] In certain embodiments, the expression vector comprises a nucleic acid sequence encoding an amino acid sequence comprising SEQ ID NO: 52 as described above or a functional conservative variant thereof.

[0132] In certain embodiments, the vector comprising SEQ ID NO: 52 or a functional conservative variant thereof is not an AAV vector.

[0133] The expression vector that can be used for the present invention may include at least one expression control element that is operably connected to the nucleic acid sequence. The expression control element is inserted into the vector to control and regulate the expression of the nucleic acid sequence. Examples of expression control elements include, but are not limited to, the lac system, the operator and promoter region of bacteriophage λ, yeast promoters, and promoters derived from polyoma virus, adenovirus, retrovirus, slow virus, or SV40. Other preferred or required operating elements include, but are not limited to, leader sequences, stop codons, polyadenylation signals, and any other sequences necessary or preferred for the appropriate transcription and subsequent translation of the nucleic acid sequence in the host system. Those skilled in the art will appreciate that the correct combination of the required or preferred expression control elements will depend on the selected host system. It will also be appreciated that the expression vector should include other elements necessary for transferring and subsequently replicating the expression vector containing the nucleic acid sequence in the host system. Examples of the element include, but are not limited to, an origin of replication and a selectable marker. Those skilled in the art will further appreciate that such vectors can be easily constructed using conventional methods or commercially available.

[0134] In certain embodiments, the invention includes a host cell comprising an expression vector as described above.

[0135] In certain embodiments, the host cell can be, for example, but not limited to, eukaryotic cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as Escherichia coli. The means by which the vector carrying the gene can be introduced into the cell include, but are not limited to, microinjection, electroporation, transduction or transfection using DEAE-dextran, lipofection, calcium phosphate or other methods known to those skilled in the art.

[0136] In certain embodiments, a eukaryotic expression vector that functions in eukaryotic cells is used. Examples of such vectors include, but are not limited to, viral vectors, such as retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, poxviruses, polioviruses; lentiviruses, bacterial expression vectors, plasmids (such as pcDNA3), or baculovirus transfer vectors. Preferred eukaryotic cell lines include, but are not limited to, COS cells, CHO cells, HeLa cells, NIH / 3T3 cells, 293 cells (ATCC#CRL1573), T2 cells, dendritic cells, or monocytes.

[0137] An immune response, such as humoral immunity, can also be generated against the recombinant viral vector and / or the heterologous polynucleotide or the protein or peptide encoded by the heterologous polynucleotide encapsidated by the viral vector, resulting in inhibition or reduction of transduction of viral vector cells, expression or function of the heterologous polynucleotide, or function or activity of the protein or peptide encoded by the heterologous polynucleotide in a subject to which the viral vector is administered.

[0138] Antibodies that bind to viral vectors (such as recombinant viral vectors) used in the present invention, which may be referred to as "neutralizing" antibodies, may reduce or inhibit cell transduction by viral vectors that may be used for gene therapy. As a result, while not being bound by theory, cell transduction is reduced or inhibited, thereby reducing the introduction of virally packaged heterologous polynucleotides into cells and subsequent expression, and, where appropriate, subsequent translation into proteins or peptides. In addition, antibodies that bind to heterologous polynucleotides or proteins or peptides encoded by heterologous polynucleotides encapsidated by viral vectors may inhibit the expression of heterologous polynucleotides, the function or activity of heterologous polynucleotides, or the function or activity of proteins or peptides encoded by heterologous polynucleotides.

[0139] Therefore, there may be antibodies that bind to a recombinant viral vector (e.g., AAV) in a subject and / or there may be antibodies that bind to a protein or peptide encoded by a heterologous polynucleotide. In addition, there may be antibodies that bind to a heterologous polynucleotide encapsidated by a recombinant viral vector.

[0140] Antibodies that bind to recombinant viral vectors (e.g., AAV) or to proteins or peptides encoded by heterologous polynucleotides, if produced, can be degraded or digested by proteases as described herein. Antibodies that bind to recombinant viral vectors (e.g., AAV) or to proteins or peptides encoded by heterologous polynucleotides, if produced, can also have their effector functions reduced or inhibited, as described herein.

[0141] As used herein, "effector function" with respect to an antibody refers to the normal functional attributes of an antibody. For example, non-limiting examples of antibody functional attributes include binding to antigens; activating the complement cascade (called complement-dependent cytotoxicity); binding to Fc receptors on effector cells (e.g., macrophages, monocytes, natural killer cells, and eosinophils), thereby causing antibody-dependent cellular cytotoxicity (ADCC); and as a signal for the ingestion of bound antigens / pathogens by immune cells (e.g., phagocytes and dendritic cells). Therefore, the reduction or inhibition of antibody effector functions may refer to any one or more of the aforementioned non-limiting functional attributes. Effector function assays are known in the art and described, for example, in WO2016012285.

[0142] "Fc receptor" refers to any Fc receptor. Specific non-limiting examples of Fc receptors include Fcγ immunoglobulin receptors (FcγRs) present on cells. In humans, FcγR refers to one, some or all of the Fc receptor family comprising FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) and FcγRIIIB (CD16b). FcγR includes naturally occurring polymorphisms of FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) and FcγRIIIB (CD16b).

[0143] In certain embodiments, antibody binding to the viral vector is reduced or inhibited by protease means.

[0144] In certain embodiments, antibodies that bind to Fc receptors on effector cells (such as macrophages, monocytes, natural killer cells, or eosinophils) are reduced or inhibited by glycosidase means. In certain embodiments, endoglycosidases hydrolyze glycan structures on the Fc interaction domain of antibodies. In certain embodiments, endoglycosidases hydrolyze glycan structures on the Fc interaction domain of IgG, such as the N-linked biantennary glycans at position Asn-297 (Kabat numbering).

[0145] In certain embodiments, antibody activation of the complement cascade is reduced or inhibited by protease means.

[0146] In certain embodiments, stimulation or uptake of the antibody by an immune cell (eg, a phagocyte or dendritic cell) is reduced or inhibited by protease or glycosidase means.

[0147] In certain embodiments, prior to administering the recombinant viral (e.g., AAV) vector, a protease and / or glycosidase is administered to the subject. In certain embodiments, after administering the recombinant viral (e.g., AAV) vector, a protease and / or glycosidase is administered to the subject. In certain embodiments, the recombinant viral (e.g., AAV) vector and the protease and / or glycosidase are administered substantially simultaneously or approximately simultaneously.

[0148] Antibodies include any of IgG, IgM, IgA, IgD and / or IgE. Therefore, the present invention relates to digesting, degrading or reducing or inhibiting the effector functions of any one of these five classes of antibodies, any two of these five classes of antibodies, any three of these five classes of antibodies, any four of these five classes of antibodies, or all five of these five classes of antibodies.

[0149] The level of antibodies in the subject can be analyzed, measured or determined before and / or after the administration of the recombinant viral vector. The level of antibodies in the subject can also be analyzed, measured or determined before and / or after the administration of a protease or glycosidase. The level of antibodies in the subject can also be analyzed or measured multiple times before and / or after the administration of the recombinant viral vector and before and / or after the administration of a protease or glycosidase.

[0150] The effector function of the antibody in the subject can be analyzed, measured or determined before and / or after administration of the recombinant viral vector. The effector function of the antibody in the subject can also be analyzed, measured or determined before and / or after administration of a protease or glycosidase. The effector function of the antibody in the subject can also be analyzed or measured multiple times before and / or after administration of the recombinant viral vector and before and / or after administration of a protease or glycosidase.

[0151] An increase in the equilibrium binding constant corresponds to a decrease in binding between IgG and Fc receptors. Thus, a decrease in Fc receptor binding of an antibody due to glycosidase activity can result in an increase in the equilibrium binding constant of the IgG:FcR interaction. A decrease in Fc receptor binding of an antibody can result in an increase in the equilibrium binding constant of the IgG:FcR interaction by at least 1-fold, at least 2-fold, at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 7-fold, or at least 8-fold.

[0152] In certain embodiments, prior to administering the recombinant viral vector to the subject, the subject's immune response (e.g., humoral immune response) caused by exposure to the wild-type virus is treated by administering an amount of a protease and / or glycosidase that effectively degrades or digests antibodies bound to the wild-type virus-based recombinant viral vector.

[0153] In certain embodiments, an immune response (e.g., a humoral immune response) resulting from administration of a recombinant viral vector (e.g., AAV) is treated by administering an amount of a protease and / or glycosidase that is effective to degrade or digest an antibody bound to the recombinant viral vector or an antibody bound to a heterologous polynucleotide or a protein or peptide encoded by a heterologous polynucleotide encapsidated by the viral vector.

[0154] In certain embodiments, prior to administering the recombinant viral vector to a subject, a protease and / or glycosidase is administered to inhibit or prevent an immune response (e.g., a humoral immune response) against the recombinant viral vector or antibodies that bind to a heterologous polynucleotide or a protein or peptide encoded by a heterologous polynucleotide encapsidated by the viral vector.

[0155] In certain embodiments, a protease and / or glycosidase is administered to a subject prior to an immune response (e.g., a humoral immune response), such as the production of neutralizing antibodies or antibodies that bind to a protein or peptide encoded by a heterologous polynucleotide or a heterologous polynucleotide encapsidated by a viral vector.

[0156] Proteases are enzymes that degrade or digest proteins. As used herein, proteases are also known as peptidases, proteases, peptide hydrolases or proteolytic enzymes.

[0157] The proteases useful in the present invention can be divided into two major categories according to their substrate specificity. Proteases can be exo-types (exoproteinases or exopeptidases) that hydrolyze the peptide bonds located at the N-terminus or C-terminus. For example, non-limiting examples of exoproteinases or exopeptidases include flavoenzymes (Novozymes), ProteaAX (Amano) and pancreatin from porcine pancreas.

[0158] The protease that can be used in the present invention can be an endo-type (endoprotease or endopeptidase) that hydrolyzes peptide bonds inside the polypeptide chain. For example, examples of endoproteases include IdeS, IdeZ, IgdE, IdeMC, trypsin, chymotrypsin, papain and pepsin.

[0159] Examples of proteases useful in the present invention include, for example but not limited to, cysteine ​​proteases from Streptococcus pyogenes, Streptococcus equi, Mycoplasma canis, Streptococcus agalactiae, Streptococcus pseudosuis, or Pseudomonas. In certain embodiments, the protease comprises an endopeptidase IdeS from Streptococcus pyogenes as shown in any one of SEQ ID NOs: 3-18, or a modified variant thereof. In certain embodiments, the protease comprises a protease as shown in SEQ ID NO: 19 or SEQ ID NO: 20, or a modified variant thereof. In certain embodiments, the protease comprises an endopeptidase IdeZ from Streptococcus equi as shown in any one of SEQ ID NOs: 21-43, or a modified variant thereof.

[0160] Other proteases useful in the present invention include, for example, but not limited to, IgdE enzymes from S.suis, S.porcinus, and S.equi described in WO 2017 / 134274. Other proteases useful in the present invention include, for example, but not limited to, IdeMC and homologues described in WO 2018 / 093868. Other endopeptidases useful in the present invention include, for example, but not limited to, IdeZ with and without N-terminal methionine and signal peptide and IdeS / IdeZ hybrid proteins described in WO 2016 / 128559. Other proteases that can be used in the present invention include, but are not limited to, those described in Jordan et al. (N Engl. J Med. 377; 5, 2017), Lannergard and Guss (FEMS Microbiol Lett., 262 (2006); 230-235), and Hulting et al., (FEMS Microbiol Lett., 298 (2009), 44-50).

[0161] Glycosidases are enzymes that hydrolyze glycosidic bonds in complex carbohydrates. There are generally two major categories, namely exoglycosidases and endoglycosidases. Glycosidases cleave and thus release glycans / oligosaccharides from glycoproteins (e.g. antibodies).

[0162] Exoglycosidases useful in the present invention include, for example, but not limited to, N-acetylglucosaminidase, fucosidase, galactosidase, glucosidase, mannosidase, neuraminidase and xylosidase. Endoglycosidases useful in the present invention include, for example, but not limited to, EndoS, Endo D, Endoglycosidase-H, Endo F1, Endo F2 and Endo F3.

[0163] Thus, in certain embodiments, the glycosidase comprises an endoglycosidase.In certain embodiments, the glycosidase comprises an exoglycosidase.

[0164] Examples of endoglycosidases include, for example but not limited to, Endo S. In certain embodiments, the endoglycosidase comprises the sequence shown in any one of SEQ ID NOs: 44-47 or a modified variant thereof.

[0165] In certain embodiments, the EndoS polypeptide comprises an EndoS polypeptide, a fragment of an EndoS polypeptide, a variant of an EndoS polypeptide, or a variant of a fragment of an EndoS polypeptide, provided that the polypeptide, fragment, variant, or variant of a fragment has immunoglobulin (Ig) endoglycosidase activity.

[0166] In certain embodiments, the EndoS polypeptide is Streptococcus pyogenes EndoS. A variant of the EndoS polypeptide may be an EndoS polypeptide from another organism, such as another bacterium. In certain embodiments, the bacterium is a streptococcus, such as Streptococcus equi, Streptococcus zooepidemicus, or Streptococcus pyogenes. Alternatively, the variant may be from Corynebacterium pseudotuberculosis, such as a CP40 protein; for example, a CP40 protein; Enterococcus faecalis, such as an EndoE protein; or Elizabethingia meningoseptica (formerly Flavobacterium meningosepticum), such as an EndoF2 protein.

[0167] The EndoS polypeptide may comprise or consist of: (a) any one of the amino acid sequences of SEQ ID NOs: 44-47; (b) a fragment of (a) having Ig endoglycosidase activity; or (c) a variant of (a) having at least 50% identity to any one of the amino acid sequences of SEQ ID NOs: 44-47 and having Ig endoglycosidase activity; or (d) a variant of (b) having at least 50% identity to the corresponding portion of any one of the amino acid sequences of SEQ ID NOs: 44-47 and having Ig endoglycosidase activity.

[0168] In certain embodiments, the variant polypeptide has at least about 60% or more identity (e.g., 60-70%, 70-80%, or 80-90% identity) to any of the amino acid sequences in SEQ ID NOs: 44-47 or fragments thereof having Ig endoglycosidase activity. In certain embodiments, the variant polypeptide has 90-100% identity to any of the amino acid sequences in SEQ ID NOs: 44-47 or fragments thereof having Ig endoglycosidase activity.

[0169] The protease may comprise or consist of: (a) any one of the amino acid sequences in SEQ ID NO: 3-43 or 48; (b) a fragment of (a) having protease activity; or (c) a variant of (a) having at least 50% identity to any one of the amino acid sequences in SEQ ID NO: 3-43 or 48 and having protease activity; or (d) a variant of (b) having at least 50% identity to the corresponding portion of any one of the amino acid sequences in SEQ ID NO: 3-43 or 48 and having protease activity. In certain embodiments, the variant polypeptide has at least about 60% or more identity (e.g., 60-70%, 70-80% or 80-90% identity) to any one of the amino acid sequences in SEQ ID NO: 3-43 or 48 or a fragment thereof having protease activity. In certain embodiments, the variant polypeptide has 90-100% identity to any one of the amino acid sequences in SEQ ID NO: 3-43 or 48 or a fragment thereof having protease activity.

[0170] In certain embodiments, the protease or glycosidase lacks its native signal sequence and has an additional N-terminal methionine, such as SEQ ID NO: 48, which is the mature form of IdeS of Streptococcus pyogenes (without the signal sequence, but with an added N-terminal methionine). Any protease or glycosidase used in the methods of the invention may include an added N-terminal methionine in place of the native signal sequence.

[0171] In certain embodiments, the protease or glycosidase has a non-native N-terminal signal or leader peptide sequence in place of its native signal sequence. Any protease or glycosidase used in the methods of the invention may include a non-native N-terminal signal sequence in place of the native signal sequence. In certain embodiments, the non-native N-terminal signal sequence is selected from SEQ ID NO: 49-51 or a functional conservative variant thereof.

[0172] In certain embodiments, the immunoglobulin G degrading enzyme polypeptide is capable of degrading adenovirus neutralizing antibodies present in a patient.

[0173] In certain embodiments, the IgG degrading enzyme polypeptide is capable of degrading AAV neutralizing antibodies present in a patient.

[0174] In certain embodiments, the immunoglobulin G degrading enzyme polypeptide is an immunoglobulin G degrading enzyme (IdeS) polypeptide of Streptococcus pyogenes.

[0175] In certain embodiments, the immunoglobulin G degrading enzyme polypeptide is an immunoglobulin G degrading enzyme (IdeS) polypeptide of Streptococcus pyogenes, which is administered to degrade adenovirus neutralizing antibodies present in a patient.

[0176] In certain embodiments, the IgG degrading enzyme polypeptide is an IgG degrading enzyme (IdeS) polypeptide of Streptococcus pyogenes, which is administered to degrade AAV neutralizing antibodies present in the patient.

[0177] Thus, in certain embodiments, the present invention relates to the use of an immunoglobulin G degrading enzyme (IdeS) polypeptide of Streptococcus pyogenes in treating a disease that is treatable by administering a gene therapy vector to a patient in need thereof.

[0178] Thus, in certain embodiments, the present invention relates to the use of an immunoglobulin G degrading enzyme (IdeS) polypeptide of Streptococcus pyogenes in treating a disease to be treated by administering a gene therapy vector to a patient in need thereof, wherein the IdeS is administered to the patient to degrade neutralizing anti-AAV antibodies present in the patient.

[0179] In certain embodiments, the patient is a vector seropositive patient, and the present invention relates to immunoglobulin G degrading enzyme (IdeS) polypeptides of Streptococcus pyogenes for use in treating AAV seropositive patients.

[0180] In certain embodiments, the patient is a vector seronegative patient, and the present invention relates to immunoglobulin G degrading enzyme (IdeS) polypeptides of Streptococcus pyogenes for use in treating AAV seronegative patients.

[0181] In certain embodiments, the IdeS polypeptide according to the present invention is a variant or fragment of Streptococcus pyogenes IdeS, or a Streptococcus pyogenes IdeS that retains cysteine ​​protease activity. The variant may be an IdeS polypeptide from another organism (e.g., another bacterium). The bacterium is preferably a streptococcus. The streptococcus is preferably a group A streptococcus, a group C streptococcus, or a group G streptococcus. In particular, the variant may be an IdeS polypeptide from a group C streptococcus (e.g., Streptococcus equi or Streptococcus zooepidemicus). Alternatively, the variant may be a recombinant variant from Pseudomonas putida or from other bacterial strains. In certain embodiments, the IdeS polypeptide according to the present invention comprises any one of SEQ ID NOs: 3-43 or 48.

[0182] In certain embodiments, the cysteine ​​protease activity of the polypeptide has the ability to cleave all four human IgG subclasses at glycine residue 237 in the lower hinge region of the IgG heavy chain, while sparing IgA, IgM, IgD, and IgE isotypes.

[0183] In certain embodiments, the IdeS polypeptide comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 3-18, 23, or 48, or a functional conservative variant thereof.

[0184] In certain embodiments, the polypeptide according to the present invention may differ from any of SEQ ID NOs: 3-18, 23 or 48 by 1, 2, 3, 4 or 5 amino acids.

[0185] In certain embodiments, the invention includes polypeptides that are function conservative variants of the polypeptide of any one of SEQ ID NOs: 3-18, 23, or 48.

[0186] In certain embodiments, a polypeptide of the invention comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to any one of SEQ ID NOs: 3-18, 23, or 48, and is still capable of cleaving all four human IgG subclasses at glycine residue 237 in the lower hinge region of the IgG heavy chain while not cleaving IgA, IgM, IgD, and IgE isotypes.

[0187] In certain embodiments, a polypeptide of the invention consists of an amino acid sequence as shown in any one of SEQ ID NOs: 3-18, 23 or 48, or a variant thereof, said variant comprising at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to any one of SEQ ID NOs: 3-18, 23 or 48, and still capable of cleaving all four human IgG subclasses at glycine residue 237 in the lower hinge region of the IgG heavy chain without cleaving IgA, IgM, IgD and IgE isotypes.

[0188] In certain embodiments, the polypeptide of the present invention is a fragment of the polypeptide of any one of SEQ ID NOs: 3-18, 23 or 48, which is still capable of cleaving all four human IgG subclasses at glycine residue 237 in the lower hinge region of the IgG heavy chain without cleaving IgA, IgM, IgD and IgE isotypes.

[0189] In certain embodiments, the polypeptides of the invention comprise residues Lys-55 and / or Cys-65 and / or His-233 and / or Asp-255 and / or Asp-257 corresponding to the amino acid residues of any of SEQ ID NOs: 3-18, 23, or 48. In certain embodiments, the polypeptides of the invention comprise each of the residues corresponding to Lys-55, Cys-65, His-233, Asp-255, and Asp-257 of SEQ ID NO: 4. In certain embodiments, the polypeptides of the invention comprise at least one, at least two, at least three, at least four, or all five residues corresponding to Lys-55, Cys-65, His-233, Asp-255, and Asp-257 of SEQ ID NO: 4.

[0190] In order to verify whether the polypeptide or fragment of the present invention is still able to cleave the four human subclasses of IgG, each polypeptide or fragment can be tested. For example, after incubating the tested polypeptide or fragment with human IgG, the digestion fragments will be studied by Western blotting. No detection of intact IgG and detection of human IgG digestion fragments will indicate that the tested polypeptide or fragment has the ability to cleave all four human IgG subclasses.

[0191] As used herein, the term "functional conservative variant" refers to those in which a given amino acid residue in a protein or enzyme has been changed (inserted, deleted or substituted) without changing the overall conformation and function of the peptide. The variant includes proteins with amino acid changes (e.g., deletions, insertions and / or substitutions). "Deletion" refers to the absence of one or more amino acids in a protein. "Insertion" refers to the addition of one or more amino acids in a protein. "Substitution" refers to the replacement of one or more amino acids in a protein by another amino acid residue. Typically, a given amino acid is replaced by an amino acid with similar properties (e.g., polarity, hydrogen bonding potential, acidity, alkalinity, hydrophobicity, aromaticity, etc.). The given amino acid can be a natural amino acid or a non-natural amino acid. In addition to those indicated as conservative, the amino acids in a protein may be different, so that the percentage of protein or amino acid sequence similarity between any two proteins with similar functions may be different, for example, according to an alignment scheme (e.g., determined by a clustering method, wherein the similarity is based on a MEGALIGN algorithm), it may be 70%-99%. "Function conservative variants" also include polypeptides having at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, also preferably at least 90%, even more preferably at least 95%, and having the same or substantially similar properties or functions as the native or parent protein to which it is compared. Two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80%, preferably greater than 85%, preferably greater than 90% of the amino acids are identical, or greater than about 90%, preferably greater than 95% are similar (functionally identical) over the entire length of the shorter sequence. Preferably, similar or homologous sequences are identified by alignment using, for example, an alignment program of GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) or any sequence comparison algorithm (e.g., BLAST, FASTA, etc.).

[0192] In certain embodiments, a polypeptide of the invention comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 48, wherein one or more residues have been conservatively substituted with functionally similar residues, and which exhibits functional aspects of the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 48 as described herein.

[0193] In certain embodiments, the polypeptide of the invention comprises any one of SEQ ID NOs: 3-18, 23 or 48, wherein one or more residues have been conservatively substituted with functionally similar residues, and exhibits the functional aspects of the polypeptide of SEQ ID NOs: 3-18, 23 or 48 as described herein.

[0194] Examples of conservative substitutions include substitution of one nonpolar (hydrophobic) residue (e.g., isoleucine, valine, leucine, or methionine) for another; substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; substitution of one basic residue (such as lysine, arginine, or histidine) for another, or substitution of one acidic residue (such as aspartic acid or glutamic acid) for another.

[0195] The term "conservative substitution" also includes the use of chemically derived residues to replace non-derivatized residues. "Chemical derivatives" refers to subject peptides having one or more residues chemically derived by reaction of functional side groups. Examples of such derivative molecules include, for example, those in which free amino groups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbonyl groups, tert-butyloxycarbonyl groups, chloroacetyl groups, or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters, or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Chemical derivatives also include peptides containing one or more natural amino acid derivatives of twenty standard amino acids. For example: 4-hydroxyproline may replace proline; 5-hydroxylysine may replace lysine; 3-methylhistidine may replace histidine; homoserine may replace serine; and ornithine may replace lysine. The term "conservative substitution" also includes the use of non-natural amino acids intended to control and stabilize the secondary structure of peptides or proteins. These non-natural amino acids are chemically modified amino acids, such as proline, β-amino acids, N-methyl amino acids, cyclopropyl amino acids, α, α-substituted amino acids as described below. These non-natural amino acids may also include fluorinated, chlorinated, brominated or iodinated modified amino acids.

[0196] In certain embodiments, the polypeptide of the invention differs from any of SEQ ID NO: 4 or SEQ ID NO: 3-18, 23 or 48 in at least 1, 2, 5, 10, 20, 30, 50 or more mutations (which may be substitutions, deletions or insertions of amino acids). For example, 1 to 50, 2 to 30, 3 to 20 or 5 to 10 amino acid substitutions, deletions or insertions may be made. The modified polypeptide generally retains activity as an IgG-specific cysteine ​​protease. In specific embodiments, the amino acid substitutions are conservative substitutions.

[0197] In certain embodiments, the polypeptides of the present invention may further include a signal sequence.

[0198] In certain embodiments, the signal sequence comprises or consists of the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 or a functional conservative variant thereof.

[0199] In certain embodiments, the polypeptides of the present invention may include a tag. A tag is a sequence containing a tag that can be used to purify a peptide. It is attached by various techniques (such as affinity chromatography) and is used to locate the peptide or polypeptide in a cell or tissue sample using immunolabeling techniques, detect the peptide or polypeptide by immunoblotting, etc. Examples of tags commonly used in the art are GST (glutathione-S-transferase) tags, FLAG TM -Tag, Strep-Tag TM , V5 tag, myc tag, His tag, etc.

[0200] In certain embodiments, the polypeptides of the present invention can be labeled with fluorescent dyes. Fluorescent peptides labeled with dyes are important tools in cell research. The peptides can be labeled at the N-terminal side or the C-terminal side.

[0201] N-terminal peptide labeling using amine-reactive fluorescent dyes: Amine-reactive fluorescent probes are widely used to modify peptides at the N-terminus or lysine residues. Many fluorescent amino-reactive dyes have been developed to label various peptides, and the resulting conjugates are widely used in biological applications. Three major classes of amine-reactive fluorescent reagents are currently used to label peptides: succinimidyl esters (SE), isothiocyanates, and sulfonyl chlorides.

[0202] C-terminal labeling using amine-containing fluorescent dyes: Amine-containing dyes are used to modify peptides using water-soluble carbodiimides (such as EDC) to convert the carboxyl groups of the peptides into amide groups. Either NHS or NHSS can be used to improve the conjugation efficiency of EDC-mediated protein-carboxylic acid coupling.

[0203] In certain embodiments, the polypeptides used in the therapeutic methods according to the present invention can be modified to improve their therapeutic efficacy. Such modifications of therapeutic compounds can be used to reduce toxicity, increase circulation time, change biodistribution, or reduce immunogenicity. For example, the toxicity of potentially important therapeutic compounds can be significantly reduced by combining with a variety of drug carriers that change biodistribution.

[0204] A strategy to improve drug delivery is to utilize water-soluble polymers. Various water-soluble polymers have been shown to alter biodistribution, improve cellular uptake, change permeability through physiological barriers; and alter clearance from the body. To achieve targeted or sustained release, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendant groups on the polymer chain.

[0205] In view of its high biocompatibility and ease of modification, polyethylene glycol (PEG) has been widely used as a drug carrier. Attachment to various drugs, proteins and liposomes has been shown to improve residence time and reduce toxicity. PEG can be coupled to active agents through hydroxyl groups at the end of the chain and other chemical methods; however, PEG itself is limited to a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids are explored as new biomaterials that will retain the biocompatibility of PEG, but have the additional advantage of numerous attachment points per molecule (providing a larger drug loading), and can be synthetically designed to be suitable for a variety of applications.

[0206] Those skilled in the art are aware of the PEGylation technology for effective modification of drugs. For example, VectraMed (Plainsboro, NJ) has used a drug delivery polymer composed of an alternating polymer of PEG and a trifunctional monomer (e.g., lysine). The PEG chain (typically 2000 daltons or less) is connected to the a- and e-amino groups of lysine by a stable carbamate bond. The copolymer retains the desired properties of PEG while providing reactive side groups (the carboxylic acid group of lysine) at a strictly controlled predetermined interval along the polymer chain. The reactive side groups can be used for derivatization, cross-linking or conjugation with other molecules. By changing the molecular weight of the polymer, the molecular weight of the PEG segment, and the cleavable bond between the drug and the polymer, these polymers can be used to produce stable long-circulation prodrugs. The molecular weight of the PEG fragment affects the interval of the drug / linker complex and the drug amount of the conjugate per molecular weight (the smaller the PEG fragment, the greater the drug loading). Generally, increasing the total molecular weight of the block copolymer conjugate will increase the circulation half-life of the conjugate. However, the conjugate must be easily degraded or the molecular weight must be below the threshold (e.g., less than 45kDa) that limits glomerular filtration.

[0207] In addition, because the polymer backbone is important for maintaining the circulation half-life and biodistribution, a joint can be used to maintain the therapeutic agent in a prodrug form until released from the main chain polymer by a specific trigger (typically the enzymatic activity in the target tissue). For example, the drug delivery of this type of tissue activation is particularly useful in the case where it is necessary to be delivered to a specific site of biodistribution and the therapeutic agent is released at or near the pathological site. The linking group library for the activation of drug delivery is well known to those skilled in the art, and can be based on the universality of enzyme kinetics, active enzymes and the cutting specificity of selected disease-specific enzymes (for example, referring to technologies of established by VectraMed, Plainsboro, NJ). This joint can be used for modifying the derivative peptides described herein for therapeutic delivery.

[0208] According to the present invention, polypeptides can be produced by conventional automated peptide synthesis methods or by recombinant expression.The general principles of designing and preparing proteins are well known to those skilled in the art.

[0209] The polypeptides of the present invention can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The peptides of the present invention can also be synthesized by solid phase techniques, which use an exemplary peptide synthesizer, such as the 433A model of Applied Biosystems Inc. Reverse phase HPLC analysis can be used to determine the proteins produced by automatic peptide synthesis or recombinant methods. The chemical authenticity of each peptide can be established by any method well known to those skilled in the art.

[0210] As an alternative to automated peptide synthesis, recombinant DNA techniques may be employed in which a nucleotide sequence encoding a selection protein is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression as described below. For the production of longer polypeptides, recombinant methods are particularly preferred.

[0211] A variety of expression vector / host systems can be used to contain and express peptide or protein coding sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cell systems infected with viral expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids; see, e.g., Babe et al., 2000); or animal cell systems. Those skilled in the art are aware of various techniques for optimizing mammalian protein expression, see, e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that can be used for recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (e.g., COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293 cells. Exemplary protocols for recombinantly expressing peptide substrates or fusion polypeptides in bacteria, yeast, and other invertebrates are known to those skilled in the art and are briefly described below. U.S. Patent No. 6,569,645; U.S. Patent No. 6,043,344; U.S. Patent No. 6,074,849; and U.S. Patent No. 6,579,520 provide specific examples of recombinantly produced peptides, and these teachings of these patents are expressly incorporated herein by reference. Mammalian host systems for expressing recombinant proteins are also well known to those skilled in the art. Host cell strains can be selected to have specific abilities to process expressed proteins or to produce certain post-translational modifications that can be used to provide protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein may also be important for correct insertion, folding and / or function. Different host cells (such as CHO, HeLa, MDCK, 293, WI38, etc.) have specific cellular machinery and characteristic mechanisms to carry out such post-translational activities, and these host cells can be selected to ensure the correct modification and processing of the introduced foreign protein.

[0212] In the recombinant production of the derived polypeptide of the present invention, it will be necessary to adopt a vector comprising a polynucleotide molecule to encode the derived peptide. The method of preparing such a vector and producing a host cell transformed with such a vector is well known to those skilled in the art. The polynucleotide molecule used for this attempt can be combined with a vector, which generally includes a selectable marker and a replication origin to breed in the host. These elements of the expression construct are well known to those skilled in the art. Generally, an expression vector includes a DNA encoding a given protein, which is operably connected to a suitable transcription or translation regulatory sequence, such as those derived from mammals, microorganisms, viruses or insect genes. Examples of regulatory sequences include transcription promoters, operators or enhancers, mRNA ribosome binding sites and suitable sequences for controlling transcription and translation.

[0213] The terms "expression vector," "expression construct," or "expression cassette" are used interchangeably throughout this specification and are intended to include any type of genetic construct comprising a nucleic acid encoding a gene product wherein part or all of the nucleic acid coding sequence can be transcribed.

[0214] The selection of an appropriate expression vector for expressing the peptides or polypeptides of the invention will of course depend on the particular host cell used, and is within the skill of the ordinary artisan. Methods for constructing mammalian expression vectors are described, for example, in Okayama et al., 1983; Cosman et al., 1986; Cosman et al., 1984; EP-A-0367566; and WO91 / 18982. Additional considerations for producing expression vectors are described in detail, for example, in Makrides et al., 1999; Kost et al., 1999. Wurm et al., 1999, which is incorporated herein as a teaching factor to consider large-scale transient expression in mammalian cells for the production of recombinant proteins.

[0215] Expression needs to provide appropriate signals in the vector, such as enhancers / promoters from viruses and mammalian sources, which can be used to drive the expression of target nucleic acids in host cells. Typically, the expressed nucleic acid is under the transcriptional control of the promoter." Promoter " includes a DNA sequence required for specific transcription of genes that is recognized by the synthetic mechanism of the cell or the synthetic mechanism introduced. When the regulatory sequence is functionally relevant to the DNA (i.e., 4N1K, variants, etc.) encoding the target peptide, the nucleotide sequence is operably connected. Therefore, if the transcription of the promoter nucleotide sequence guide sequence, the promoter nucleotide sequence is operably connected to a given DNA sequence.

[0216] Similarly, the phrase "under transcriptional control" means that the promoter is in the correct position and orientation relative to the nucleic acid to control the initiation and expression of the RNA polymerase of the gene. Any promoter that will drive the expression of the nucleic acid can be used. The specific promoter used to control the expression of the target nucleic acid sequence is not important, as long as it can direct the expression of the nucleic acid in the target cell. Therefore, in the case of targeting human cells, it is preferred that the nucleic acid coding region is adjacent to a promoter that can be expressed in human cells and is under the control of the promoter. In general, such promoters can include human or viral promoters. Common promoters include, for example, human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat sequence, β-actin, rat insulin promoter, phosphoglycerol kinase promoter, human α-1 antitrypsin promoter, transthyretin promoter, TBG promoter and other liver-specific promoters, desmin promoter and similar muscle-specific promoters, EF1-α promoter, CAG promoter and other constitutive promoters, hybrid promoters with multiple tissue specificities, promoters specific to neurons (such as synaptophysin) and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are well-known and easily available promoters to those skilled in the art, and can be used to obtain high-level expression of the coding sequence. It is also considered to use other viral or mammalian cell or bacterial phage promoters well known in the art to achieve expression of the target coding sequence, provided that the expression level is sufficient to produce a recoverable yield of the target protein. By adopting a promoter with well-known properties, the level and pattern of target protein expression after transfection or transformation can be optimized. Inducible promoters can also be used.

[0217] Another regulatory element for protein expression is an enhancer. These are genetic elements that increase transcription of a promoter located at a distant position of the same DNA molecule. When the expression construct employs a cDNA insert, it will typically be necessary to include a polyadenylation signal sequence to achieve appropriate polyadenylation of the gene transcript. Any polyadenylation signal sequence recognized by cells of a selected transgenic animal species is suitable for use in the present invention, such as human or bovine growth hormone and SV40 polyadenylation signal.

[0218] As described above, the use of immunoglobulin G degrading enzyme polypeptides (such as Ides) may be useful in treating diseases that are treated by gene therapy using AAV vectors.

[0219] Therapeutic Compositions

[0220] In certain embodiments, the present invention relates to a therapeutic composition comprising an immunoglobulin G degrading enzyme polypeptide or a nucleic acid encoding an immunoglobulin G degrading enzyme polypeptide or an expression vector comprising a nucleic acid encoding an immunoglobulin G degrading enzyme polypeptide according to the present invention, for treating a disease treatable by gene therapy using a vector in a patient in need thereof.

[0221] Any of the therapeutic agents of the invention can be combined with a pharmaceutically acceptable excipient and, optionally, a sustained release matrix (eg, a biodegradable polymer) to form a therapeutic composition.

[0222] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when properly administered to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0223] The form of the pharmaceutical composition, route of administration, dosage and regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, and the like.

[0224] The pharmaceutical compositions of the present invention may be formulated for topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration, and the like.

[0225] The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable medium for a preparation that can be injected. These may be particularly isotonic sterile saline solutions (sodium dihydrogen phosphate or sodium dihydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts), or dry, especially freeze-dried compositions, which, as the case may be, allow the formation of injection solutions after the addition of sterile water or normal saline. The dosage for use may be adjusted according to various parameters, particularly according to the mode of administration used, the associated pathology or the desired treatment duration.

[0226] Additionally, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; time-release capsules; and any other form currently available for use.

[0227] The pharmaceutical compositions of the present invention may contain other therapeutically active agents.

[0228] The protease or glycosidase can be administered to a subject at any suitable dose. For example, a suitable dose can be about 0.05 mg / kg to about 5 mg / kg of the subject's body weight, or about 0.1 mg / kg to about 4 mg / kg of the subject's body weight.

[0229] In certain embodiments, IdeZ is administered at a dose of about 0.01 mg / kg to about 10 mg / kg of subject body weight. For example, a suitable dose may be about 0.05 mg / kg to about 5 mg / kg of subject body weight, or about 0.1 mg / kg to about 4 mg / kg of subject body weight.

[0230] In certain embodiments, IdeS is administered at a dose of about 0.01 mg / kg to about 10 mg / kg of subject body weight. For example, a suitable dose may be about 0.05 mg / kg to about 5 mg / kg of subject body weight, or about 0.1 mg / kg to about 4 mg / kg of subject body weight.

[0231] In certain embodiments, EndoS is administered at a dose of about 0.01 mg / kg to about 10 mg / kg of subject body weight. For example, a suitable dose may be about 0.05 mg / kg to about 5 mg / kg of subject body weight, or about 0.1 mg / kg to about 4 mg / kg of subject body weight.

[0232] Unless otherwise indicated herein, the method according to the present invention may be performed in any suitable order. In certain embodiments, the method may include first (a) administering to the subject a protease and / or glycosidase that effectively degrades or digests neutralizing antibodies; then (b) administering a recombinant viral vector to the subject. In certain embodiments, (b) administering a recombinant viral vector to the subject is performed between about 1 minute to about 90 days after (a) administering a protease and / or glycosidase that effectively degrades or digests neutralizing antibodies to the subject. In certain embodiments, (a) administering to the subject a protease and / or glycosidase that effectively degrades or digests neutralizing antibodies, and (b) administering a recombinant viral vector to the subject is performed at about the same time.

[0233] In certain embodiments, the method may include first (a) administering to a subject a recombinant viral vector carrying a heterologous polynucleotide, and then (b) administering to the subject an amount of a protease and / or glycosidase that is effective in degrading or digesting antibodies that bind to the recombinant viral vector and / or the heterologous polynucleotide or a protein or peptide encoded by the heterologous polynucleotide. In certain embodiments, (b) administering to the subject an amount of a protease and / or glycosidase (which is effective in degrading or digesting antibodies that bind to the recombinant viral vector and / or the heterologous polynucleotide or a protein or peptide encoded by the heterologous polynucleotide) is performed between about 1 minute and about 90 days after (a) administering to the subject a recombinant viral vector carrying a heterologous polynucleotide. In certain embodiments, (a) administering to the subject a recombinant viral vector carrying a heterologous polynucleotide, and (b) administering to the subject an amount of a protease and / or glycosidase (which is effective in degrading or digesting antibodies that bind to the recombinant viral vector and / or the heterologous polynucleotide or a protein or peptide encoded by the heterologous polynucleotide) are performed at about the same time.

[0234] Antibodies, such as neutralizing antibodies, may pre-exist and may be present in the subject at levels that inhibit or reduce transduction of cells by the recombinant viral vector even before administration of the viral vector. Alternatively, antibodies may be produced in the subject after exposure to the virus on which the recombinant viral vector is based. Still further, antibodies, such as neutralizing antibodies, or antibodies that bind to a heterologous polynucleotide or a protein or peptide encoded by a heterologous polynucleotide encapsidated by the viral vector, may be produced in the subject after administration of the recombinant viral vector.

[0235] Therefore, the methods herein are applicable to subjects with pre-existing antibodies and subjects without pre-existing antibodies. As described herein, such subjects include subjects who have been exposed to wild-type viruses and have produced pre-existing antibodies to viral vectors based on the wild-type viruses, and subjects who have received viral vector gene therapy and have produced antibodies and can subsequently be treated with one or more other doses of the same viral vector gene therapy (referred to as re-administration), or subjects treated with different gene therapies (e.g., different heterologous polynucleotides) using the same viral vector to deliver gene therapy treatment.

[0236] The subject's antibodies can be tested before administering the viral vector and / or before administering the protease glycosidase. Non-limiting examples of antibodies to be tested include neutralizing antibodies, antibodies that bind proteases or glycosidases as described herein, antibodies that bind heterologous polynucleotides, and antibodies that bind proteins or peptides encoded by heterologous polynucleotides. Therefore, prior to administering the recombinant viral vector and / or prior to administering the protease glycosidase, the subject's neutralizing antibodies, antibodies that bind proteases or glycosidases as described herein, antibodies that bind heterologous polynucleotides, or antibodies that bind proteins or peptides encoded by heterologous polynucleotides can be screened.

[0237] Subjects with pre-existing antibodies (e.g., IgG) that bind to proteases or glycosidases as described herein can optionally be excluded from initial treatment by the methods according to the invention. However, not all subjects that have or produce antibodies that bind to proteases or glycosidases need to be excluded from the treatment methods of the invention. For example, subjects with detectable anti-IdeS IgG with a titer of less than 15 mg / liter can still be treated using the methods according to the invention.

[0238] After administration of the recombinant viral vector, the subject may also be screened for neutralizing antibodies, antibodies that bind to a heterologous polynucleotide, or antibodies that bind to a protein or peptide encoded by a heterologous polynucleotide. Such subjects may optionally be monitored over a period of time after administration of the recombinant viral vector to determine whether such antibodies are generated or prevented in subjects in which no pre-existing antibodies are detected, or in the case of subjects with pre-existing antibodies, whether proteases and / or glycosidases reduce or eliminate such pre-existing antibodies.

[0239] In certain embodiments, a protease and / or glycosidase is administered to a subject after testing positive for the presence of a neutralizing antibody, an antibody that binds a protease or glycosidase as described herein, an antibody that binds a heterologous polynucleotide, or an antibody that binds a protein or peptide encoded by a heterologous polynucleotide. In certain embodiments, a protease and / or glycosidase is administered to a subject before testing positive for the presence of a neutralizing antibody, an antibody that binds a protease or glycosidase as described herein, an antibody that binds a heterologous polynucleotide, or an antibody that binds a protein or peptide encoded by a heterologous polynucleotide.

[0240] In certain embodiments, the subject is not tested for antibodies before or after administration of the protease and / or glycosidase. Thus, after administration of the protease and / or glycosidase, testing for neutralizing antibodies, antibodies that bind to a protease or glycosidase as described herein, antibodies that bind to a heterologous polynucleotide, or antibodies that bind to a protein or peptide encoded by a heterologous polynucleotide, or in the therapeutic methods of the invention, administration of a recombinant viral vector is optional.

[0241] The protease or glycosidase may be administered to the subject any number of times. For example, the protease and / or glycosidase may be administered to the subject 2 to 5 times, 2 to 10 times, 2 to 15 times.

[0242] Protease or glycosidase can be administered to a subject on a regular basis (e.g., consecutive days or alternating days) or irregular basis for any duration. In certain embodiments, after administration of the recombinant viral vector, protease and / or glycosidase is administered for about 1 to 12 weeks, or about 1 to 10 weeks, or about 1 to 8 weeks, or about 1 to 6 weeks, or about 1 to 4 weeks, or about 1 to 2 weeks, or about 2 weeks.

[0243] In certain embodiments, the recombinant viral vector is used before or after a protease or glycosidase is applied to a subject. In certain embodiments, the recombinant viral vector is applied to a subject for example 1-12, 12-24 or 24-48 hours or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50 or more than 50 days after a protease or glycosidase is applied to a subject. In certain embodiments, the recombinant viral vector is applied to a subject for example 1-12, 12-24 or 24-48 hours or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50 or more than 50 days after a protease or glycosidase is applied to a subject.

[0244] The recombinant viral vector, protease and / or glycosidase can be administered alone or in combination. In certain embodiments, the recombinant viral vector and protease and / or glycosidase are administered separately to the subject. In certain embodiments, the recombinant viral vector and protease and / or glycosidase are administered in combination to the subject.

[0245] In certain embodiments, a mixture of proteases and glycosidases is administered to a subject one or more times. In certain embodiments, two or more proteases or glycosidases are administered to a subject one or more times.

[0246] In certain embodiments, at least one immunosuppressant is administered to the subject before, substantially simultaneously with, or after the recombinant viral vector, protease, or glycosidase is administered to the subject. In certain embodiments, the immunosuppressant is an anti-inflammatory agent, such as a steroid. In certain embodiments, the immunosuppressant is prednisone, cyclosporine (e.g., cyclosporine A), mycophenolate mofetil, rituximab, rapamycin, or a derivative thereof.

[0247] Other strategies to reduce humoral immunity include methods of removing, depleting, capturing and / or inactivating antibodies, commonly referred to as apheresis, more particularly plasmapheresis of blood products. Apheresis or plasmapheresis is a process in which the plasma of a human subject is circulated ex vivo (extracorporeally) by a device that modifies the plasma by adding, removing and / or replacing components before the plasma is returned to the patient. Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from blood products (e.g., plasma). The procedure depletes, captures, inactivates, reduces or removes immunoglobulins (antibodies) that bind to recombinant viral vectors, bind to heterologous polynucleotides, bind to proteins or peptides encoded by heterologous polynucleotides, bind to proteases and / or bind to glycosidases, thereby reducing the titer of antibodies in the subject of treatment, which can contribute to the neutralization of, for example, viral vectors. An example is a device composed of an AAV capsid affinity matrix column. Passing a blood product (e.g., plasma) through such an AAV capsid affinity matrix will only result in the binding of AAV antibodies and all isotypes (including IgG, IgM, etc.). Immunoadsorption (U.S. Patent Application Publication US2018 / 0169273 A1) can also be used to deplete immunoglobulins, more particularly anti-AAV antibodies. Affinity ligands (International Patent Application Publication WO / 2018 / 158397) can also be used to deplete immunoglobulins, more particularly anti-AAV antibodies. Any of the above strategies can be used before, substantially simultaneously with, or after administering a recombinant viral vector, protease, or glycosidase to a subject.

[0248] Other strategies to reduce (overcome) or avoid humoral immunity to AAV in systemic gene transfer include using AAV empty capsid particles and / or capsid proteins as bait to adsorb anti-AAV antibodies. Other strategies are described in Mingozzi et al., 2013, Blood, 122: 23-36.

[0249] According to the present invention, the protease and / or glycosidase may be encapsulated or complexed with a liposome, a nanoparticle, a lipid nanoparticle, a polymer, a microparticle, a microcapsule, a micelle or an extracellular vesicle.

[0250] According to the present invention, the viral particles may be encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles or extracellular vesicles.

[0251] In certain embodiments, liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles, or extracellular vesicles may be used in the present invention for non-viral delivery of gene therapy nucleic acids.

[0252] "Lipid nanoparticles" or "LNPs" refer to lipid-based vesicles that are used to deliver recombinant viral vectors and / or proteases and / or glycosidases, and have nanoscale sizes, i.e., about 10 nm to about 1000 nm, or about 50 to about 500 nm, or about 75 to about 127 nm. Without being bound by theory, LNPs are believed to provide proteases, glycosidases or recombinant viral vectors that partially or completely shield the immune system. Shielding allows proteases, glycosidases or recombinant viral vectors to be delivered to tissues or cells while avoiding inducing a substantial immune response to proteases, glycosidases or recombinant viral vectors in vivo. Shielding can also allow repeated administration without inducing a substantial immune response to proteases, glycosidases or recombinant viral vectors in vivo (e.g., in a subject (such as a person)). Shielding can also improve or increase delivery efficiency, duration of therapeutic effect and / or therapeutic efficacy in vivo.

[0253] The pI (isoelectric point) of AAV is in the range of about 6 to about 6.5. Therefore, the surface of AAV carries a slight negative charge. Therefore, it may be beneficial for LNPs to contain cationic lipids (e.g., amino lipids). Exemplary amino lipids have been described in U.S. Pat. Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122 and 7,745,651 and in U.S. Patent Publication Nos. 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411 and 2010 / 0117125.

[0254] The terms "cationic lipid" and "amino lipid" are used interchangeably herein to include lipids and salts thereof having one, two, three or more fatty acids or fatty alkyl chains and pH titratable amino groups (e.g., alkylamino or dialkylamino groups). Cationic lipids are typically protonated (i.e., positively charged) at a pH lower than the pKa of the cationic lipid, and are substantially neutral at a pH higher than the pKa. Cationic lipids can also be titratable cationic lipids. In certain embodiments, the cationic lipid comprises: a protonable tertiary amine (e.g., pH titratable) group; a C18 alkyl chain, wherein each alkyl chain independently has 0-3 (e.g., 0, 1, 2, or 3) double bonds; and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0255] The cationic lipids may include, but are not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linoleyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA, also known as DLin-C2K-DMA, X TC2 and C2K), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleyl-3-trimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-M-C3-DMA, also known as MC3), their salts and mixtures thereof. Other cationic lipids also include, but are not limited to, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-DLen-C2K-DMA and (DLin-MP-DMA) (also known as 1-B11).

[0256] Further cationic lipids may include, but are not limited to, 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpiperazinyl-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinolenoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinolenoyloxy- 3-morpholinepropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propane (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2, 3-Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (D MRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propane trifluoroacetate (DOSPA), dioctadecylaminoglycidyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxopentaoxy]-3-dimethyl -1-(cis,cis-9',1-2'-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermidine (DS) and disubstituted spermine (D2S) or mixtures thereof.

[0257] Many commercial preparations of cationic lipids are available, e.g. (including DOTMA and DOPE available from GIBCO / BRL) and (including DOSPA and DOPE available from GIBCO / BRL).

[0258] In certain embodiments, the cationic lipid may be present in an amount from about 10 wt % of the LNP to about 85 wt % of the lipid nanoparticle, or from about 50 wt % of the LNP to about 75 wt % of the LNP.

[0259] Sterol can give LNP mobility.As used herein, " sterol " refers to any naturally occurring sterol of plant (phytosterol) or animal (animal sterol) source and non-naturally occurring synthetic sterol, all of which are characterized by the presence of hydroxyl on the 3-position of sterol A ring.Sterol can be any sterol conventionally used in liposome, lipid vesicle or lipid particle preparation field, most commonly cholesterol.Phytosterol can include campesterol, sitosterol and stigmasterol.Sterol also includes sterol-modified lipids, such as those described in U.S. Patent Application Publication 2011 / 0177156.In certain embodiments, sterol can be present in an amount of about 5 % by weight of LNP to about 50 % by weight of lipid nanoparticle or about 10 % by weight of LNP to about 25 % by weight of LNP.

[0260] LNP can comprise neutral lipid.Neutral lipid can comprise any lipid species existing in uncharged or neutral zwitterionic form at physiological pH.Such lipid includes but is not limited to diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside.The selection of neutral lipid is usually guided by especially considering particle diameter and necessary stability.In certain embodiments, neutral lipid component can be lipid (for example, diacylphosphatidylcholine and diacylphosphatidylethanolamine) with two acyl groups.

[0261] The lipid of acyl chain with various chain lengths and saturation is available, or can be separated or synthesized by well-known technology. In certain embodiments, lipids containing saturated fatty acids with carbon chain length in the range of C14-C22 can be used. In certain embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain length in the range of C14-C22 are used. In addition, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include but are not limited to 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-ethanolamine (DOPE), 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC) or any related phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin or phospholipids with other head groups (such as serine and inositol).

[0262] In certain embodiments, the neutral lipid may be present in an amount from about 0.1 wt % of the lipid nanoparticle to about 75 wt % of the LNP, or from about 5 wt % of the LNP to about 15 wt % of the LNP.

[0263] The LNP-encapsulated protease, glycosidase or recombinant viral vector can be incorporated into a pharmaceutical composition, such as a pharmaceutically acceptable carrier or excipient. Such a pharmaceutical composition is particularly useful for administering and delivering the LNP-encapsulated protease, glycosidase or recombinant viral vector to a subject in vivo or ex vivo.

[0264] Formulations of LNPs may be combined with additional components which may include, for example but not limited to, polyethylene glycol (PEG) and sterols.

[0265] The term "PEG" refers to polyethylene glycol, a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weight; for example, PEG 2000 has an average molecular weight of about 2,000 Daltons, while PEG5000 has an average molecular weight of about 5,000 Daltons. PEGs are commercially available from Sigma Chemical Co. and other companies, and include, for example, the following functional PEGs: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol succinate (MePEG-S), monomethoxy polyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-tribenzoate (MePEG-TRES), and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0266] In certain embodiments, PEG can be a polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons, and is optionally substituted by an alkyl, alkoxy, acyl or aryl group. In certain embodiments, PEG can be substituted by a methyl group at the terminal hydroxyl position. In certain embodiments, PEG can have an average molecular weight of about 750 to about 5,000 daltons, or about 1,000 to about 5,000 daltons, or about 1,500 to about 3,000 daltons, or about 2,000 daltons or about 750 daltons. PEG can be optionally substituted by an alkyl, alkoxy, acyl or aryl group. In certain embodiments, the terminal hydroxyl group can be substituted by a methoxy or methyl group.

[0267] The lipids modified by PEG- are included in the PEG-dialkoxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397. Useful PEG-modified lipids (or lipid-polyoxyethylene conjugates) can have a variety of "anchoring" lipid moieties to fix the PEG moiety to the surface of the lipid vesicle. The examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) described in U.S. Patent Nos. 5,820,873, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropane-3-amine. In certain embodiments, the lipids modified by PEG can be diacylglycerols and dialkylglycerols modified by PEG. In certain embodiments, the amount of PEG can be about 0.5% by weight of LNP to about 20% by weight of LNP, or about 5% by weight of LNP to about 15% by weight of LNP.

[0268] In addition, LNP can be PEG-modified and sterol-modified LNP. The LNP combined with other components can be the same or separate LNP. In other words, the same LNP can be PEG-modified and sterol-modified, or the first LNP can be PEG-modified and the second LNP can be sterol-modified. Optionally, the first and second modified LNPs can be combined.

[0269] In certain embodiments, before encapsulation, the size of the LNP can be about 10nm to 500nm, or about 50nm to about 200nm, or 75nm to about 125nm. In certain embodiments, the size of the LNP-encapsulated protease, glycosidase or recombinant viral vector can be about 10nm to 500nm.

[0270] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, such as small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, reverse splicing RNA or antisense RNA).

[0271] Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single, double, or triple, linear or circular, and can be of any length. When discussing nucleic acids, the sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0272] "Heterologous" nucleic acid sequence refers to a polynucleotide inserted into a plasmid or vector for the purpose of vector-mediated transfer / delivery of polynucleotides into cells. Heterologous nucleic acid sequences are different from viral nucleic acids, that is, they are non-natural relative to viral nucleic acids. Once transferred / delivered into cells, the heterologous nucleic acid sequence contained in the vector can be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the heterologous polynucleotides transferred / delivered in the cells contained in the vector do not need to be expressed. Although the term "heterologous" is not always used herein to refer to nucleic acid sequences and polynucleotides, even if there is no modifier "heterologous", it is intended to include heterologous nucleic acid sequences and polynucleotides, whether or not there are omissions, when referring to nucleic acid sequences or polynucleotides.

[0273] As used herein, "transgenic" conveniently refers to a nucleic acid that is intended to be or has been introduced into a cell or organism. A transgenic includes any nucleic acid, such as a heterologous polynucleotide sequence or a heterologous nucleic acid encoding a protein or peptide. The terms transgenic and heterologous nucleic acid / polynucleotide sequence are used interchangeably herein.

[0274] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of: GAA (acid alpha-glucosidase) for treating Pompe disease; ATP7B (copper transporting ATPase 2) for treating Wilson's disease; alpha galactosidase for treating Fabry disease; ASS1 (arginine succinate synthase) for treating citrullinemia type 1; beta-glucocerebrosidase for treating Gaucher disease type 1; beta-hexosaminidase A for treating Tay-Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for treating hereditary angioedema (HAE, also known as C1 inhibitor deficiency type I and type II); and glucose-6-phosphatase for treating glycogen storage disease type I (GSDI).

[0275] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiogenin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF) , transforming growth factor alpha (TGFα), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGFβ, activins, inhibins, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophins, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0276] In certain embodiments, the heterologous polynucleotide encodes acid alpha-glucosidase (GAA). Administration of a recombinant viral vector comprising a heterologous polynucleotide encoding GAA to a subject suffering from Pompe disease or another glycogen storage disease can result in expression of the GAA protein. Expression of the GAA protein in the patient can be used to prevent, inhibit or reduce the accumulation of glycogen, prevent the accumulation of glycogen or degrade glycogen, which in turn can reduce or reduce one or more adverse reactions of Pompe disease or another glycogen storage disease.

[0277] In certain embodiments, the heterologous polynucleotide encodes a protein selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-36, etc.), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β and γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules.

[0278] In certain embodiments, the heterologous polynucleotide encodes CFTR (cystic fibrosis transmembrane regulator), blood coagulation (coagulation) factors (factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), an increase in functional coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globulin, α-globulin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, β-25 glucocorticoid glycosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial cell-derived growth factor, transforming growth factor alpha and beta, cytokines, interferon alpha, interferon beta, interferon gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid aminotransferase (Gyrate Atrophy), retinoic acid relaxin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin A, GM2-AP, NPC1, VPC2, saposin, one or more zinc finger nucleases for genome editing, and one or more donor sequences for use as repair templates for genome editing.

[0279] In certain embodiments, the heterologous polynucleotide encodes erythropoietin (EPO) for treating anemia; interferon-α, interferon-β and interferon-γ for treating various immune diseases, viral infections and cancers; interleukins (ILs) for treating various inflammatory diseases or immune deficiencies, including any one of IL-1 to IL-36, and corresponding receptors; chemokines for treating immune diseases, including chemokine (CXC motif) ligand 5 (CXCL5); granulocyte colony stimulating factor (G-CSF) for treating immune diseases (such as Crohn's disease); Granulocyte macrophage colony stimulating factor (GM-CSF) for the treatment of inflammatory diseases; macrophage colony stimulating factor (M-CSF) for the treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for the treatment of epithelial tissue damage; chemokines (such as monocyte chemoattractant protein-1 (MCP-1)) for the treatment of recurrent miscarriage, HIV-related complications and insulin resistance; tumor necrosis factor (TNF) and receptors for the treatment of various immune diseases; alpha-1-antitrypsin for the treatment of emphysema or chronic obstructive pulmonary disease (COPD); for the treatment of mucopolysaccharidosis I (MPS I); ornithine transcarbamylase (OTC) for the treatment of OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia lyase (PAL) for the treatment of phenylketonuria (PKU); lipoprotein lipase for the treatment of lipoprotein lipase deficiency; apolipoprotein for the treatment of apolipoprotein (Apo) AI deficiency; low-density lipoprotein receptor (LDL-R) for the treatment of familial hypercholesterolemia (FH); albumin for the treatment of hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate synthetase; arginine aminosuccinate lyase; arginase; fumarate acetoacetate hydrolase; porphobilinogen deaminase; cystathionine beta synthase for the treatment of homocystinuria; branched-chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl-CoA carboxylase; methylmalonyl-CoA mutase; glutaryl-CoA dehydrogenase; insulin; pyruvate carboxylase; liver phosphorylase; phosphorylase kinase; glycine decarboxylase; H protein; T protein; cystic fibrosis transmembrane regulator (CFTR); ATP-binding cassette, subfamily A (ABC1), member 4 (ABCA4) for the treatment of Stargardt disease; or dystrophin.

[0280] The terms "polypeptide", "protein" and "peptide" are used interchangeably herein. "Polypeptides", "proteins" and "peptides" encoded by "polynucleotide sequences" include full-length native sequences and naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants, as long as the subsequences, modified forms or variants retain a certain degree of function of the native full-length protein. In the present invention, these polypeptides, proteins and peptides encoded by polynucleotide sequences can be, but are not necessarily, identical to endogenous proteins that are lacking, under-expressed or lacking in the treated mammal.

[0281] In certain embodiments, the heterologous polynucleotide encodes an inhibitory nucleic acid selected from the group consisting of siRNA, antisense molecules, miRNA, RNAi, ribozymes, and shRNA.

[0282] In certain embodiments, the inhibitory nucleic acid binds to a gene, a transcript of a gene, or a gene associated with a multinucleotide repeat disease, the gene being selected from the group consisting of a huntingtin (HTT) gene, a gene associated with dentate nucleus red nucleus pallidum Lewy body atrophy (atrophin1, ATN1), androgen receptor on chromosome X in spinal cord muscular atrophy, human Ataxin-1, -2, -3 and -7, Ca v2.1 P / Q voltage-dependent calcium channel (CACNA1A), TATA-binding protein, Ataxin 8 reverse chain (ATXN8OS), serine / threonine protein phosphatase 2A55 in spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, 17) kDa regulatory subunit Bβ isoform, FMR1 (fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMR1 (fragile X mental retardation 2) in fragile XE mental retardation, or AF4 / FMR2 family member 2; troponin kinase (MT-PK) in myotonic dystrophy; Frataxin in Friedreich's ataxia; mutants of the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transcriptional gene transactivator in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene in HIV infection; CC chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute renal failure in renal transplantation or renal injury with delayed graft function; protein kinase N3 (PKN3) in late recurrent or metastatic solid malignancies; LMP2, LMP2 also known as proteasome subunit beta 9 type (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta 8 type (PSMB 8), metastatic melanoma; MECL1 also known as proteasome subunit beta 10 type (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis inhibitory B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2 adrenergic receptor, glaucoma; RTP801 / Redd1, also known as DNA damage-inducible transcript 4 protein in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6A N17K mutant protein in congenital hyperthyroidism;Influenza A virus genome / gene sequence in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence in SARS infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola virus virus genome / gene sequence in Ebola virus infection; hepatitis B and hepatitis C virus genome / gene sequence in hepatitis B and hepatitis C infection; herpes simplex virus (HSV) genome / gene sequence in HSV infection; coxsackie virus B3 genome / gene sequence in coxsackie virus B3 infection; pathogenic allele silencing (allele-specific silencing) of genes, such as trunk protein A (TOR1A) in primary dystonia, specific pan I class and HLA alleles in transplantation; and mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP). ;

[0283] The recombinant viral vector may be administered in any suitable dose. Typically, the dose is at least 1×10 8 or higher, such as 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or 1×10 14 or higher vector genomes / kg of subject weight (vg / kg) to achieve therapeutic effect. The dose range of AAV in mice is 1×10 10 -1×10 11 vg / kg and 1×10 12 -1×10 13 More particularly, a dose of about 1 x 10 11 vg / kg-approx. 5x10 14 vg / kg (inclusive), or about 5x10 11 vg / kg-approx. 1x10 14 vg / kg (inclusive), or about 5x10 11 vg / kg-approx. 5x10 13 vg / kg (inclusive), or about 5x10 11 vg / kg-approx. 1x10 13 vg / kg (inclusive), or about 5x10 11 vg / kg-approx. 5x10 12 vg / kg (inclusive), or about 5x10 11 vg / kg-approx. 1x10 12 vg / kg (inclusive). The dose may be, for example, about 5×10 14 vg / kg, or less than about 5×1014 vg / kg, for example about 2x10 11 - Approx. 2x10 14 vg / kg dose (including endpoints), particularly for example about 2x10 12 vg / kg, about 6x10 12 vg / kg, or about 2x10 13 vg / kg.

[0284] In certain embodiments, administering a protease and / or glycosidase to a subject reduces the dose of the recombinant viral vector comprising a heterologous polynucleotide required to effectively treat the subject. In certain embodiments, administering a protease and / or glycosidase to a subject allows for the administration of an increased dose of a recombinant viral vector comprising a heterologous polynucleotide.

[0285] The dosage may vary and depends on the type, onset, progression, severity, frequency, duration or likelihood of the disease for which the subject is being treated, the desired clinical endpoint, prior or concurrent treatment, general health, age, sex, race or immune competence, and other factors that will be understood by those skilled in the art. The dosage, amount, frequency or duration may be increased or decreased proportionally, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy, and the condition of the subject. The skilled person will understand the factors that may affect the dosage and time required to provide an amount sufficient to provide a therapeutic or preventive benefit.

[0286] The dosage to achieve a therapeutic effect, e.g., the dosage in the vector genome per kilogram of body weight (vg / kg), will vary based on several factors, including, but not limited to: the route of administration, the heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease being treated, any host immune response to the recombinant viral vector, the host immune response to the heterologous polynucleotide or the expression product (protein or peptide), and the stability of the expressed protein or peptide. A person skilled in the art can determine the dosage range of the recombinant viral vector genome for treating a patient with a specific disease or condition based on the above factors and other factors.

[0287] An "effective amount" or "sufficient amount" refers to an amount that provides, alone or in combination with one or more other compositions, treatments, regimens or therapeutic regimen agents, a detectable response of any duration (long or short term), any measurable or detectable degree, or an expected or desired result or benefit to a subject for any duration (e.g., minutes, hours, days, months, years, or cure) in a single dose or multiple doses, alone or in combination. An "effective amount" or "sufficient amount" dose for treatment (e.g., to ameliorate or provide a therapeutic benefit or improvement) is generally effective to provide a measurable range of responses to one, multiple or all adverse symptoms, consequences or complications of a disease, e.g., one or more adverse symptoms, conditions, diseases, pathologies or complications caused by or associated with the disease, although reducing, diminishing, inhibiting, preventing, limiting or controlling the progression or worsening of the disease is a satisfactory result.

[0288] An effective amount or sufficient amount may, but need not, be provided in a single administration, may require multiple administrations, and may, but need not, be administered alone or in combination with another composition (e.g., an agent), treatment, regimen, or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the needs of the subject, the type, state, and severity of the disease to be treated, or the side effects of the treatment, if any. In addition, if provided in a single dose or multiple doses without a second composition (e.g., another drug or agent), treatment, regimen, or therapeutic regimen, an effective amount or sufficient amount may not necessarily be effective or insufficient, because another dose, an amount or duration higher than or exceeding such a dose, or another composition (e.g., a drug or agent), treatment, regimen, or therapeutic regimen may be included to be considered effective or sufficient in a given subject. It is believed that an effective amount also includes an amount that results in a reduction in the use of another treatment, therapeutic regimen, or regimen, such as the administration of recombinant GAA to treat lysosomal storage diseases (e.g., Pompe disease) or the administration of recombinant coagulation factor proteins (e.g., FVIII or FIX) to treat coagulation disorders (e.g., hemophilia A (HemA) or hemophilia B (HemB)).

[0289] For Pompe disease, an effective amount will be an amount of GAA that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal, reduces lysosomal changes in a subject's body tissue, or, for example, improves the subject's muscle tone and / or muscle strength and / or respiratory function. For example, an effective amount can be determined by determining the kinetics of myoblast uptake of GAA from plasma. Myoblast GAA uptake rates (K uptake) of about 141-147 nM appear to be effective (see, e.g., Maga et al., J. Biol. Chem. 2012). In animal models, it has been observed that GAA activity levels in plasma greater than about 1,000 nmol / hr / mL, e.g., about 1,000-about 2,000 nmol / hr / mL are therapeutically effective.

[0290] For HemA and HemB, in general, it is believed that in order to achieve a therapeutic effect, a concentration of coagulation factor greater than 1% of that found in normal individuals is required to change the severe disease phenotype to a moderate disease phenotype. The severe phenotype is characterized by joint damage and life-threatening bleeding. In order to convert the moderate disease phenotype to a mild disease phenotype, a concentration of coagulation factor greater than 5% of normal is believed to be required.

[0291] The level of FVIII and FIX in a normal human is about 150-200 ng / mL plasma, but may be lower (e.g., about 100-150 ng / mL range) or higher (e.g., about 200-300 ng / mL range) and still be considered normal due to functional coagulation as determined, for example, by the activated partial thromboplastin time (aPTT) one-stage coagulation assay. Thus, a therapeutic effect may be achieved such that the total amount of FVIII or FIX in a subject / human is greater than 1% of the FVIII or FIX present in a normal subject / human, e.g., 1% of 100-300 ng / mL.

[0292] The composition can be administered to a subject as a combined composition, or administered separately, such as delivering or administering a recombinant viral vector comprising a heterologous polynucleotide simultaneously or consecutively or sequentially (before or after). The invention provides combinations in which the methods or uses of the invention are combined with any compound, agent, drug, treatment policy, treatment regimen, method, therapy, or composition described herein or known to those skilled in the art. The compound, agent, drug, treatment policy, treatment regimen, method, therapy, or composition can be administered or performed before, substantially simultaneously, or after administering a recombinant viral vector comprising a heterologous polynucleotide to a subject.

[0293] Therefore, the present invention particularly includes methods and uses that result in a reduction in the need or use of another compound, reagent, drug, treatment policy, treatment regimen, method or therapy. For example, for coagulation disorders, if a lower frequency or reduced dose is applied in a given subject or a recombinant coagulation factor protein is eliminated to supplement the endogenous coagulation factor that is defective or lacking (abnormal or mutated) in the subject, the treatment method of the present invention has a therapeutic benefit. In another example, for lysosomal storage diseases (such as Pompe disease), even if a recombinant viral vector comprising GAA has been previously applied at a lower frequency or reduced dose, or continues to be applied to the subject, the treatment method of the present invention still has a therapeutic benefit. Therefore, the need or use of reducing another treatment or therapy is included in the present invention.

[0294] An effective amount or sufficient amount need not be effective in every treated subject in a given group or population, nor need it be effective in the majority of treated subjects. An effective amount or sufficient amount is effective or sufficient in a specific subject rather than a group or general population. As is typical of such methods, some subjects show a greater response, or a lesser response, or no response, to a given treatment method or use.

[0295] The term "improvement" refers to a detectable or measurable improvement in a subject's disease or its symptoms or potential cellular responses. Detectable or measurable improvements include subjectively or objectively reducing, reducing, inhibiting, preventing, limiting or controlling the occurrence, frequency, severity, progression or duration of a disease, or complications caused by or associated with the disease, or improving the symptoms, root causes or consequences of a disease, or the reversal of a disease. For example, for Pompeii, an effective amount will be an amount that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal, improves muscle tone and / or muscle strength and / or respiratory function. For example, for HemA or HemB, an effective amount will be an amount that reduces the frequency or severity of acute bleeding episodes in a subject, or, for example, reduces the amount of clotting time measured by a coagulation assay.

[0296] Therefore, the pharmaceutical compositions of the present invention include compositions containing effective amounts of active ingredients to achieve the intended therapeutic purpose. It is well within the capabilities of skilled medical practitioners to determine a therapeutically effective dose using techniques known in the art and guidance using the teachings provided herein.

[0297] The therapeutic dose will depend on factors such as the age and general condition of the subject, the severity of the abnormal phenotype, and the strength of the control sequences regulating the expression level. Therefore, the therapeutically effective amount in humans will fall within a wide range that can be determined by a physician based on the response of an individual patient to vector-based therapy. This dose can be alone or in combination with an immunosuppressant or drug.

[0298] Compositions (such as pharmaceutical compositions) can be delivered to a subject to allow transgene expression and optionally production of the encoded protein. In certain embodiments, the pharmaceutical composition comprises genetic material sufficient to enable a subject to produce a therapeutically effective amount of a coagulation factor to improve hemostasis in the subject. In certain embodiments, the pharmaceutical composition comprises a heterologous polynucleotide sufficient to enable a subject to produce a therapeutically effective amount of GAA.

[0299] In certain embodiments, the therapeutic effect in the subject lasts for a period of time, such as 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30 or 30-50 days or longer, such as 50-75, 75-100, 100-150, 150-200 days or longer. Therefore, in certain embodiments, the recombinant viral vector provides a therapeutic effect.

[0300] In certain embodiments, the recombinant viral vector provides a therapeutic effect in the absence of an immunosuppressant. In certain embodiments, at least one immunosuppressant is administered to a subject prior to, substantially simultaneously with, or after administration of the recombinant viral vector to the subject.

[0301] In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a sterol. In certain embodiments, the immunosuppressant is prednisone, cyclosporine (e.g., cyclosporine A), mycophenolate mofetil, rituximab, rapamycin, or a derivative thereof. Other specific agents include stable compounds. Other immunosuppressants that can be used in accordance with the method of the present invention include, for example, but not limited to, B cell targeting antibodies, such as rituximab; proteasome inhibitors, such as bortezomib; mammalian target rapamycin (mTOR) inhibitors, such as rapamycin; tyrosine kinase inhibitors, such as ibrutinib; B cell activating factor (BAFF) inhibitors; and inhibitors of proliferation-inducing ligands (APRIL).

[0302] The composition can be administered in any sterile biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, dextrose and water. The composition can be administered to the patient alone or in combination with other agents that affect dosage, frequency of administration and / or therapeutic efficacy.

[0303] Methods and uses of the present invention include delivery and administration systemically, regionally or locally, or by any route, such as by injection or infusion. Delivery of pharmaceutical compositions in vivo can usually be accomplished by injection using conventional syringes, although other delivery methods, such as convection-enhanced delivery (see, for example, U.S. Patent No. 5,720,720) are contemplated. For example, compositions can be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intrapleurally, intraarterially, orally, intrahepatically (via portal vein) or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal administration. Clinicians who specialize in the treatment of coagulation disorders can determine the best approach to use adenovirus-associated vectors based on many criteria, including but not limited to: the patient's condition and therapeutic purposes (e.g., increased GAA, enhanced coagulation, etc.).

[0304] Therapeutic methods according to the present invention include combination therapies, which include the additional use of any compound, agent, drug, treatment or other treatment policy or regimen with a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combined compositions and treatments include second active substances, such as biologics (proteins), agents (e.g., immunosuppressants) and drugs. Such biologics (proteins), agents, drugs, treatments and therapies may be administered or performed prior to, substantially simultaneously with, or after any other therapeutic methods according to the present invention, for example, a method for treating a lysosomal storage disease (e.g., Pompeii) in a subject, or a method for treating a coagulation disease (e.g., HemA or HemB) in a subject.

[0305] Compounds, agents, drugs, treatments or other treatment strategies or regimens may be administered as a combined composition, or separately, such as by delivering or administering nucleic acids, vectors, recombinant vectors (e.g., recombinant viral vectors) or recombinant viral particles simultaneously or continuously or sequentially (before or after). Thus, the invention provides combinations in which the methods or uses according to the invention are combined with any compound, agent, drug, treatment strategy, treatment regimen, method, therapy or composition described herein or known to those skilled in the art. According to the invention, compounds, agents, drugs, treatment strategies, treatment regimens, methods, therapies or compositions may be administered or performed prior to, substantially simultaneously with or after administering nucleic acids, vectors, recombinant vectors (e.g., recombinant viral vectors) or recombinant viral particles to a subject.

[0306] In certain embodiments, administration of a protease and / or glycosidase to a subject can result in preventing the formation of neutralizing antibodies, antibodies that bind to heterologous polynucleotides, and / or antibodies that bind to proteins or peptides encoded by heterologous polynucleotides. As described herein, administration of a protease and / or glycosidase to the subject can be performed prior to administration of a viral vector to the subject, substantially simultaneously with administration of a viral vector, or after administration of a viral vector.

[0307] In certain embodiments, the administration of proteases and / or glycosidases with pre-existing antibodies to a subject results in a reduction in neutralizing antibodies, antibodies that bind to heterologous polynucleotides, and / or antibodies that bind to proteins or peptides encoded by heterologous polynucleotides. After administration of the proteases and / or glycosidases, the recombinant viral vectors can then be administered to these subjects according to the methods herein. After administration of the recombinant viral vectors, such subjects can be optionally assessed for the presence of remaining pre-existing antibodies. Alternatively, the recombinant viral vectors can be administered to such subjects after the proteases and / or glycosidases have reduced or eliminated a predetermined amount of any such pre-existing antibodies in the subject.

[0308] In certain embodiments, administration of a protease and / or glycosidase to a subject can result in at least 20%-50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the neutralizing antibodies, antibodies that bind to heterologous polynucleotides, and / or antibodies that bind to proteins or peptides encoded by heterologous polynucleotides being degraded or digested, as reflected by measuring such antibodies in a biological sample obtained from a subject to which a recombinant viral vector is administered. In certain embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the neutralizing antibodies and / or antibodies that bind to heterologous polynucleotides and / or antibodies that bind to proteins or peptides encoded by heterologous polynucleotides are degraded or digested according to the methods of the invention.

[0309] Non-limiting examples of biological samples from a subject that can be analyzed include whole blood, serum, plasma, and the like, and combinations thereof. The biological sample may be cell-free, or may include cells (eg, red blood cells, platelets, and / or lymphocytes).

[0310] In certain embodiments, the neutralizing antibodies present in the biological sample of the subject can be degraded or digested to less than about 1:25, wherein 1 part of the biological sample diluted in 25 parts of buffer results in 50% neutralization of the recombinant viral vector. In certain embodiments, the neutralizing antibodies present in the biological sample of the subject can be degraded or digested to less than about 1:20, less than about 1:15, less than about 1:10, less than about 1:5, less than about 1:4, less than about 1:3, less than about 1:2 or less than about 1:1, wherein 1 part of the biological sample diluted in 20, 15, 10, 5, 4, 3, 2 or 1 parts of buffer, respectively, results in 50% neutralization of the recombinant viral vector.

[0311] Exemplary analysis and measurement of AAV neutralizing antibodies in biological samples are disclosed herein and are also described in U.S. Patent Application Publication No. 2016 / 0123990. Binding of an antibody to an Fc receptor can be measured by determining the equilibrium binding constant. A decrease in Fc receptor binding of an antibody is determined by an increase in the equilibrium binding constant for the IgG:FcR interaction.

[0312] The method according to the present invention is applicable to loss of function and increase and functional genetic defects. As used herein, the term "loss of function" related to genetic defects refers to any mutation in a gene, wherein the protein (i.e., mutant protein) encoded by the gene shows some or all of the loss of function usually associated with wild-type proteins. As used herein, the term "increase of function" related to genetic defects refers to any mutation in a gene, wherein the protein (i.e., mutant protein) encoded by the gene obtains a function that is usually unrelated to the protein (i.e., wild-type protein) to cause or contribute to a disease or illness. Function increase mutation can be the deletion, addition or replacement of one or more nucleotides in a gene, which can cause changes in the function of the encoded protein. In certain embodiments, the function increase mutation changes the function of the mutant protein or causes interaction with other proteins. In certain embodiments, for example, by the interaction of the mutant protein changed with the normal wild-type protein, the function increase mutation causes a reduction or removal of the normal wild-type protein.

[0313] Diseases and disorders that can be treated by the methods according to the invention include, for example, but are not limited to, pulmonary diseases (e.g., cystic fibrosis), bleeding diseases (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemias, blood diseases (e.g., anemias), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartylglucosuria, Batten disease, late infantile neuronal lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2-ganglioneuropathies type I (Tay et al., 2003), and glycogen storage disease type II (Pompe disease). Sachs disease), GM2-ganglioneuroses type II (Sandhoff disease), seborrheic lesions type I (sialidosis type I and II), type II (I cell disease), type III (pseudo-Hurler disease) and type IV, mucopolysaccharidoses (Hurler disease and variants, Hunter, Sanfilippo types A, B, C, D, Morquio types A and B, Maroteaux-Lamy, and Sly diseases), Niemann-Pick disease types A / B, C1, and C2, and Schindler disease types I and II), hereditary angioedema (HAE), copper or iron storage diseases (e.g., Wilson's disease or Menkes' disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), solid organ (e.g., brain, liver, kidney, heart) diseases, or infectious viral (e.g., hepatitis B and C, HIV, etc.), bacterial, or fungal diseases.

[0314] Type II glycogen storage disease, also known as Pompe disease, can be treated by the method according to the invention. Pompe disease is an autosomal recessive genetic disease caused by mutations in the coding gene of the lysosomal enzyme acid α-glucosidase (GAA) that catalyzes glycogen degradation. The resulting enzyme deficiency leads to pathological accumulation of glycogen and lysosomal changes in all tissues of the body, resulting in cardiac, respiratory and skeletal muscle dysfunction (van der Ploeg & Reuser, 2008).

[0315] Coagulation disorders that can be treated by the methods according to the invention include, for example, but are not limited to, hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any of the coagulation factors: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency or gamma-carboxylase deficiency.

[0316] Other diseases and conditions that can be treated by the methods according to the invention include, for example, but are not limited to, anemia; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharides, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors), or platelet disorders (such as Bernard Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency).

[0317] Other diseases and conditions that can be treated by the methods according to the invention include, for example, but are not limited to, proliferative diseases (cancer, tumors, dysplasia, etc.), Crigler-Najjar and metabolic diseases, such as metabolic diseases of the liver, Friedreich's ataxia, infectious diseases, viral diseases (e.g., caused by hepatitis B or C viruses, HIV, herpes, retroviruses, etc.), genetic diseases (cystic fibrosis, nutritional sugar diseases, myopathies, such as Duchenne myopathy or muscular dystrophy, myotubular myopathy, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubular myopathy, motor neuron diseases, such as spinal muscular atrophy (SMA), spinal glomerular muscular atrophy or Charcot-Marie-Tooth disease, arthritis, severe combined immunodeficiency (such as RSV-S), myopathy, ... CID, ADA-SCID or X-SCID), Wiskott-Aldrich syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatous disease, etc.), coagulation factor deficiency, cardiovascular disease (restenosis, ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.), ophthalmological diseases such as retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy and Stargardt disease; lysosomal storage diseases such as Sanfilippo syndrome; hyperbilirubinemia (e.g. CN type I or II or Gilbert's syndrome), glycogen storage diseases (e.g. GSDI, GSDII (Pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII or fatal congenital cardiac glycogen storage disease.

[0318] In certain embodiments, the subject suffers from a disease that affects or originates from the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. Non-limiting examples of CNS or neurodegenerative diseases include Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, polyglutamine repeat disease or Parkinson's disease. In certain embodiments, the disease is mental illness, addiction (e.g., tobacco, alcohol or medicine), epilepsy, Canavan disease or adrenoleukodystrophy. In certain embodiments, CNS or neurodegenerative diseases are polyglutamine repeat diseases, such as and without limitation spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7 or SCA17).

[0319] The present invention can be used for human and veterinary medical applications. Therefore, suitable subjects include mammals such as humans, and non-human mammals. The term "subject" refers to an animal, typically a mammal, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry (such as chickens and ducks), horses, cattle, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, teenagers and adult subjects. Subjects also include animal disease models, for example, mice and other animal models of protein / enzyme deficiency (such as Pompe disease (GAA deficiency), glycogen storage disease (GSD)) and other diseases known to those skilled in the art.

[0320] The present invention provides compositions, such as kits, which include packaging materials and one or more components therein. The kit typically includes a label or package insert that includes a description of the components or instructions for use of the components therein in vitro, in vivo or ex vivo. The kit may include a collection of such components, such as nucleic acids, recombinant vectors, viral (e.g., AAV, lentiviral) vectors or viral particles, and proteases and / or glycosidases that degrade or digest antibodies.

[0321] Kit refers to the physical structure that houses one or more components of the kit. The packaging material can sterilely hold the components and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0322] The label or insert may include identification information of one or more components, dosage of the active ingredient, clinical pharmacology, including mechanism of action, pharmacokinetics and pharmacodynamics. The label or insert may include information identifying the manufacturer, batch number, place of manufacture and date, expiration date. The label or insert may include information identifying manufacturer information, batch number, manufacturer location and date. The label or insert may include information on diseases for which the kit components may be used. The label or insert may include instructions for clinicians or subjects to use one or more kit components in a method, use, or treatment regimen or treatment policy. The instructions may include dosage, frequency or duration, and instructions for implementing any method, use, treatment regimen or prevention or treatment policy described herein.

[0323] The label or insert may include information on any benefits that the composition may provide, such as a prophylactic or therapeutic benefit. The label or insert may include information on potential adverse side effects, complications, or reactions, such as to provide a warning to the subject or clinician regarding situations in which use of a particular composition would not be appropriate. Adverse side effects or complications may also occur when the subject has, will, or is taking one or more other medications that may be incompatible with the composition, or when the subject has, will, or is undergoing another treatment regimen or treatment policy that would be incompatible with the composition, and therefore, information about such incompatibilities may be included in the instructions.

[0324] Labels or inserts include "printed matter", such as paper or cardboard, either alone or attached to a component, kit or packaging material (such as a box), or attached to an ampoule, tube or vial containing a kit component.

[0325] The present invention also relates to the following items:

[0326] Item 1. A method for treating a subject in need of treatment for a disease caused by loss of protein function or activity, comprising: (a) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide encoding a protein or peptide that provides or supplements the protein function or activity; and (b) administering to the subject an amount of a protease or glycosidase that effectively degrades or digests and / or inhibits or reduces the effector function of antibodies bound to the recombinant viral vector and / or the protein or peptide encoded by the heterologous polynucleotide.

[0327] Item 2. A method for treating a subject in need of treatment for a disease caused by increased protein functional activity or expression, comprising: (a) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide, which is transcribed into a nucleic acid that inhibits, reduces or decreases the increase in protein function, activity or expression; and (b) administering to the subject a protease or glycosidase that effectively degrades or digests and / or inhibits or reduces the effector function of an antibody bound to the recombinant viral vector.

[0328] Item 3. A method for treating a subject in need of treatment for a disease caused by loss of protein function or activity, comprising: (a) administering to the subject a protease or glycosidase that effectively degrades or digests and / or inhibits or reduces the effector function of a viral vector-bound antibody; and (b) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide encoding a protein or peptide that provides or supplements the protein function or activity.

[0329] Item 4. A method for treating a subject in need of treatment for a disease caused by increased protein functional activity or expression, comprising: (a) administering to the subject a protease or glycosidase that effectively degrades or digests and / or inhibits or reduces the effector function of a viral vector-bound antibody; and (b) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, decreases or reduces the increase in protein function, activity or expression.

[0330] Item 5. The method of Item 1 or Item 2, wherein step (b) is performed within about 90 days after step (a).

[0331] Item 6. The method according to any one of Items 1 to 5, wherein the protease comprises a cysteine ​​protease or a thiol protease.

[0332] Item 7. The method according to any one of Items 1 to 5, wherein the protease comprises a protease from Streptococcus pyogenes, Streptococcus equi, or Mycoplasma canis.

[0333] Item 8. The method according to any one of Items 1 to 5, wherein the protease comprises IdeS shown in any one of SEQ ID NOs: 3-18, 23 or 48 or a modified variant thereof.

[0334] Item 9. The method according to any one of Items 1-5, wherein the glycosidase comprises an endoglycosidase.

[0335] Item 10. The method according to Item 9, wherein the endoglycosidase comprises a sequence shown in any one of SEQ ID NOs: 44-47.

[0336] Item 11. The method according to any one of Items 1-10, wherein the protease or glycosidase degrades or digests and / or inhibits or reduces the effector function of the human antibody.

[0337] Item 12. The method according to any one of Items 1-11, wherein the viral vector comprises a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

[0338] Item 13. The method of Item 12, wherein the lentiviral vector comprises an envelope protein to which the antibody binds.

[0339] Item 14. The method of Item 12, wherein the AAV vector comprises an antibody-bound capsid protein.

[0340] Item 15. The method according to Item 12, wherein the AAV vector comprises antibody-bound VP1, VP2 and / or VP3 capsid proteins.

[0341] Item 16. A method according to any one of items 12-15, wherein the AAV vector comprises VP1, VP2 and / or VP3 capsid proteins having 60% or higher sequence identity with VP1, VP2 and / or VP3 capsid proteins selected from the following group: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, Rh10, Rh74, SEQ ID NO: 1 and SEQ ID NO: 2 VP1, VP2 and / or VP3 capsid proteins.

[0342] Item 17. A method according to any one of items 12-15, wherein the AAV vector comprises VP1, VP2 and / or VP3 capsid proteins having 100% sequence identity with VP1, VP2 and / or VP3 capsid proteins selected from the following group: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, Rh10, Rh74, SEQ ID NO: 1 and SEQ ID NO: 2 VP1, VP2 and / or VP3 capsid proteins.

[0343] Item 18. The method of any one of Items 1, 3, and 5-17, wherein the subject has antibodies that bind to the viral vector.

[0344] Item 19. The method according to any one of Items 1, 3, and 5-17, wherein the subject does not have antibodies that bind to the viral vector.

[0345] Item 20. The method according to any one of items 1, 3, and 5-19, wherein the subject has antibodies that bind to the protein or peptide encoded by the heterologous polynucleotide.

[0346] Item 21. The method according to any one of items 1-20, wherein the antibody comprises IgG, IgM, IgA, IgD and / or IgE.

[0347] Item 22. The method according to any one of items 1-21, further comprising determining the presence, quantifying the amount or effector function of viral vector-binding antibodies present in the subject before performing step (a), after performing step (a) but before performing step (b), and / or after performing steps (a) and (b).

[0348] Item 23. The method according to any one of items 1-21 further includes analyzing the presence, amount or effector function of viral vector-binding antibodies present in a biological sample from the subject before performing step (a), after performing step (a) but before performing step (b), and / or after performing steps (a) and (b).

[0349] Item 24. The method according to Item 23, wherein the biological sample from the subject is a blood product.

[0350] Item 25. The method according to any one of items 1-24, wherein the method results in a reduction of the viral vector binding antibodies by 20-50%, 50-75%, 75-90%, 90-95% or 95% or more.

[0351] Item 26. A method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:100,000, wherein 1 part of the biological sample or blood product diluted in 100,000 parts of buffer results in 50% viral vector neutralization.

[0352] Item 27. The method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:50,000, wherein 1 part of the biological sample or blood product diluted in 50,000 parts of buffer results in 50% viral vector neutralization.

[0353] Item 28. A method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:10,000, wherein 1 part of the biological sample or blood product diluted in 10,000 parts of buffer results in 50% viral vector neutralization.

[0354] Item 29. A method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:1,000, wherein 1 part of the biological sample or blood product diluted in 1,000 parts of buffer results in 50% viral vector neutralization.

[0355] Item 30. A method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:100, wherein 1 part of the biological sample or blood product diluted in 100 parts of buffer results in 50% viral vector neutralization.

[0356] Item 31. A method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:10, wherein 1 part of the biological sample or blood product diluted in 10 parts of buffer results in 50% viral vector neutralization.

[0357] Item 32. A method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:5, wherein 1 part of the biological sample or blood product diluted in 5 parts of buffer results in 50% viral vector neutralization.

[0358] Item 33. A method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:4, wherein 1 part of the biological sample or blood product diluted in 4 parts of buffer results in 50% viral vector neutralization.

[0359] Item 34. A method according to Item 23 or Item 24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:3, wherein 1 part of the biological sample or blood product diluted in 3 parts of buffer results in 50% viral vector neutralization.

[0360] Item 35. A method according to any one of items 1-24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:2, wherein 1 part of the biological sample or blood product diluted in 2 parts of buffer results in 50% viral vector neutralization.

[0361] Item 36. A method according to any one of items 1-24, wherein the viral vector binding antibodies present in the biological sample or blood product from the subject are less than about 1:1, wherein 1 part of the biological sample or blood product diluted in 1 part of buffer results in 50% viral vector neutralization.

[0362] Item 37. The method according to any one of items 1, 3 and 5-36 further includes determining the presence or quantifying the amount of antibodies that bind to the polypeptide or peptide encoded by the heterologous polynucleotide after performing step (a) but before performing step (b) and / or after performing steps (a) and (b).

[0363] Item 38. The method according to any one of items 2 and 4-36 further includes determining the presence or quantifying the amount of antibodies bound to the nucleic acid after performing step (a) but before performing step (b) and / or after performing steps (a) and (b).

[0364] Item 39. The method according to any one of items 1-38, wherein step (b) is performed within about 60 days before or after step (a).

[0365] Item 40. The method according to any one of items 1-38, wherein step (b) is performed within about 45 days before or after step (a).

[0366] Item 41. The method according to any one of items 1-38, wherein step (b) is performed within about 30 days before or after step (a).

[0367] Item 42. The method according to any one of items 1-38, wherein step (b) is performed within about 21 days before or after step (a).

[0368] Item 43. The method according to any one of items 1-38, wherein step (b) is performed within about 14 days before or after step (a).

[0369] Item 44. The method according to any one of items 1-38, wherein step (b) is performed within about 7 days before or after step (a).

[0370] Item 45. The method according to any one of items 1-38, wherein step (b) is performed within about 72 hours before or after step (a).

[0371] Item 46. The method according to any one of items 1-38, wherein step (b) is performed within about 48 hours before or after step (a).

[0372] Item 47. The method according to any one of items 1-38, wherein step (b) is performed within about 24 hours before or after step (a).

[0373] Item 48. The method according to any one of items 1-38, wherein step (b) is performed within about 12 hours before or after step (a).

[0374] Item 49. The method according to any one of items 1-38, wherein step (b) is performed within about 6 hours before or after step (a).

[0375] Item 50. A method according to any one of items 1-49, wherein the subject suffers from a lung disease (e.g., cystic fibrosis), a bleeding disease (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disease (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, a lysosomal storage disease (e.g., aspartylglucosuria, Batten disease, late infantile neuronal lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease type I, II, and III, glycogen storage disease II (Pompe disease), GM2-ganglioneuropathies type I (Tay et al., Sachs disease), GM2-ganglioneuroses type II (Sandhoff disease), seborrheic lesions type I (sialidosis type I and II), type II (I cell disease), type III (pseudo-Hurler disease) and type IV, mucopolysaccharidoses (Hurler disease and variants, Hunter, Sanfilippo types A, B, C, D, Morquio types A and B, Maroteaux-Lamy, and Sly diseases), Niemann-Pick disease types A / B, C1, and C2, and Schindler disease types I and II), hereditary angioedema (HAE), copper or iron storage diseases (e.g., Wilson's disease or Menkes' disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), solid organ (e.g., brain, liver, kidney, heart) diseases, or infectious viral (e.g., hepatitis B and C, HIV, etc.), bacterial, or fungal diseases.

[0376] Item 51. The method according to any one of Items 1-49, wherein the subject suffers from a coagulation disorder.

[0377] Item 52. A method according to any one of items 1-49, wherein the subject suffers from hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any coagulation factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or γ-carboxylase deficiency.

[0378] Item 53. A method according to any one of items 1-49, wherein the disease is anemia; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors) or platelet diseases (such as Bernard Soulier syndrome, Glanzmann thrombasthenia or storage pool deficiency).

[0379] Item 54. A method according to any one of items 1 or 3-53, wherein the heterologous polynucleotide encodes a protein selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiogenin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (ESF), fibroblast growth factor (GF ... Growth factor (EGF), transforming growth factor alpha (TGFα), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), TGFβ, activins, inhibins, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophins, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0380] Item 55. A method according to any one of items 1 or 3-53, wherein the heterologous polynucleotide encodes a protein selected from the following group: thrombopoietin (TPO), interleukins (IL1 to IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β and γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules.

[0381] Item 56. A method according to any one of items 1 or 3-53, wherein the heterologous polynucleotide encodes CFTR (cystic fibrosis transmembrane regulator), blood coagulation (coagulation) factors (factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), an increase in functional coagulation factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globulin, α-globulin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase enzymes, beta-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial cell-derived growth factor, transforming growth factor alpha and beta, cytokines, interferon alpha, interferon beta, interferon gamma, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony-stimulating factor, lymphotoxins, suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid aminotransferase (Gyrate Atrophy), retinoic acid relaxin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin A, GM2-AP, NPC1, VPC2, saposin, one or more zinc finger nucleases for genome editing, and one or more donor sequences for use as repair templates for genome editing.

[0382] Item 57. A method according to any one of items 2, 4-49, wherein the heterologous polynucleotide encodes an inhibitory nucleic acid.

[0383] Item 58. The method according to Item 57, wherein the inhibitory nucleic acid is selected from the group consisting of siRNA, antisense molecules, miRNA, RNAi, ribozymes and shRNA.

[0384] Item 59. The method according to Item 57, wherein the inhibitory nucleic acid binds to a gene, a transcript of a gene, or a transcript of a gene associated with a polynucleotide repeat disease, wherein the gene is selected from the group consisting of a huntingtin (HTT) gene, a gene associated with atrophy of the dentate nucleus, rubral nucleus, and globus pallidus with Lewy bodies (atrophin 1, ATN1), androgen receptor on chromosome X in spinal cord muscular atrophy, human Ataxin-1, -2, -3, and -7, Ca v2.1 P / Q voltage-dependent calcium channel (CACNA1A), TATA-binding protein, Ataxin 8 reverse chain (ATXN8OS), serine / threonine protein phosphatase 2A55 in spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, 17) kDa regulatory subunit Bβ isoform, FMR1 (fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMR1 (fragile X mental retardation 2) in fragile XE mental retardation, or AF4 / FMR2 family member 2; troponin kinase (MT-PK) in myotonic dystrophy; Frataxin in Friedreich's ataxia; mutants of the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, human immunodeficiency virus transcriptional gene transactivator in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene in HIV infection; CC chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute renal failure in renal transplantation or renal injury with delayed graft function; protein kinase N3 (PKN3) in late recurrent or metastatic solid malignancies; LMP2, LMP2 also known as proteasome subunit beta 9 type (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta 8 type (PSMB 8), metastatic melanoma; MECL1 also known as proteasome subunit beta 10 type (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis inhibitory B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2 adrenergic receptor, glaucoma; RTP801 / Redd1, also known as DNA damage-inducible transcript 4 protein in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6A N17K mutant protein in congenital hyperthyroidism;Influenza A virus genome / gene sequence in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence in SARS infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola virus virus genome / gene sequence in Ebola virus infection; hepatitis B and hepatitis C virus genome / gene sequence in hepatitis B and hepatitis C infection; herpes simplex virus (HSV) genome / gene sequence in HSV infection; coxsackie virus B3 genome / gene sequence in coxsackie virus B3 infection; pathogenic allele silencing (allele-specific silencing) of genes, such as trunk protein A (TOR1A) in primary dystonia, specific panI class and HLA alleles in transplantation; and mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP). ;

[0385] Item 60. A method according to any one of items 1, 3 or 5-59, wherein the protein encoded by the heterologous polynucleotide comprises a gene editing nuclease.

[0386] Item 61. A method according to Item 60, wherein the gene editing nuclease comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN).

[0387] Item 62. A method according to Item 61, wherein the gene editing nuclease comprises a functional type II CRISPR-Cas9.

[0388] Item 63. The method according to any one of items 1-62, wherein step (a) and / or step (b) is performed two or more times.

[0389] Item 64. The method according to any one of Items 1-63, wherein the subject is human.

[0390] Item 65. A kit comprising:

[0391] (a) a recombinant viral vector comprising a heterologous polynucleotide encoding a protein or peptide;

[0392] (b) proteases or glycosidases that degrade or digest the antibody; and

[0393] (c) a label with instructions for performing the method according to any one of items 1-64, wherein (a) and (b) are in separate or the same container.

[0394] Item 66. A kit comprising:

[0395] (a) a recombinant viral vector comprising a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, reduces or decreases protein expression;

[0396] (b) proteases or glycosidases that degrade or digest the antibody; and

[0397] (c) a label with instructions for performing the method according to any one of items 1-64, wherein (a) and (b) are in separate or the same container.

[0398] Item 67. Immunoglobulin G degrading enzyme polypeptide for use in treating a disease treatable by a gene therapy vector in a patient in need thereof.

[0399] Item 68. An immunoglobulin G degrading enzyme polypeptide for use according to Item 67, wherein the vector is an adeno-associated virus (AAV) vector, a lentiviral vector or an adenoviral vector.

[0400] Item 69. An immunoglobulin G degrading enzyme polypeptide for use according to Item 68, wherein the vector is an AAV vector comprising a capsid protein selected from the group consisting of AAV1, AAV2, AAV2 variant, AAV3, AAV3 variant, AAV3B, AAV3B variant, AAV4, AAV5, AAV6, AAV6 variant, AAV7, AAV8, AAV9, AAV10, AAVcy10, AAVrh10, AAVrh74, AAVdj, AAV-Anc80, SEQ ID NO: 1, SEQ ID NO: 2, or AAV2i8.

[0401] Item 70. An immunoglobulin G degrading enzyme polypeptide for use according to Items 67-69, wherein the immunoglobulin G degrading enzyme polypeptide comprises any one of SEQ ID NOs: 3-18, 23 or 48.

[0402] Item 71. An immunoglobulin G degrading enzyme polypeptide for use according to Item 70, wherein the immunoglobulin G degrading enzyme polypeptide further comprises any one of the non-native signal sequences of SEQ ID NOs: 49-51.

[0403] Item 72. An immunoglobulin G degrading enzyme polypeptide for use according to Items 67-71, wherein the disease treated by the gene therapy vector is selected from the group consisting of proliferative diseases (cancer, tumors, dysplasia, etc.), Crigler-Najjar and metabolic diseases, such as metabolic diseases of the liver, Frederick's ataxia, infectious diseases, addiction (e.g., tobacco, alcohol or drugs), epilepsy, Canavan disease, adrenoleukodystrophy, viral diseases (e.g., caused by hepatitis B or C virus, HIV, herpes, retroviruses, etc.), genetic diseases (cystic fibrosis, malnutrition diabetes, myopathy, such as Duchenne myopathy or muscular dystrophy, myotubular myopathy, hemophilia A, hemophilia B, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubular myopathy, motor neuron disease, such as spinal muscular atrophy (SMA), spinal glomerular muscular atrophy or Charcot -Marie-Tooth disease, arthritis, severe combined immunodeficiency (such as RS-SCID, ADA-SCID, or X-SCID), Wiskott-Aldrich syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatous disease, etc.), coagulation factor deficiency, cardiovascular disease (restenosis, ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.), ophthalmological diseases such as retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy, and Stargardt disease; lysosomal storage diseases such as Sanfilippo syndrome; hyperbilirubinemia (such as CN type I or II or Gilbert syndrome), Fabry disease, glycogen storage diseases (such as GSDI, GSDII (Pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, and fatal congenital cardiac glycogen storage disease.

[0404] Item 73. An immunoglobulin G degrading enzyme polypeptide for use according to Items 67-72, wherein the vector comprises a therapeutic polynucleotide suitable for treating the disease described in Item 72.

[0405] Item 74. A nucleic acid sequence encoding an immunoglobulin G degrading enzyme polypeptide for use in treating a disease treatable by a gene therapy vector in a patient in need thereof.

[0406] Item 75. An expression vector comprising a nucleic acid sequence encoding an immunoglobulin G degrading enzyme polypeptide for use in treating a disease treatable by a gene therapy vector in a patient in need thereof.

[0407] Item 76. A therapeutic composition for treating a disease treatable by a gene therapy vector in a patient in need thereof, the therapeutic composition comprising an immunoglobulin G degrading enzyme polypeptide comprising any one of SEQ ID NOs: 3-18, 23 or 48, or a nucleic acid sequence encoding an immunoglobulin G degrading enzyme polypeptide comprising any one of SEQ ID NOs: 3-18, 23 or 48, or an expression vector comprising a nucleic acid sequence encoding an immunoglobulin G degrading enzyme polypeptide comprising any one of SEQ ID NOs: 3-18, 23 or 48.

[0408] Item 77. A method for treating a disease treated by a gene therapy vector, comprising administering a therapeutically effective amount of an immunoglobulin G degrading enzyme polypeptide to a subject in need thereof.

[0409] A number of embodiments of the present invention have been described. However, those skilled in the art may make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, the following examples are intended to illustrate in any way, but not to limit the scope of the invention claimed. DETAILED DESCRIPTION

[0410] Example 1

[0411] Materials and methods

[0412] AVV carrier

[0413] AAV vectors were prepared as previously described (Ayuso et al. (2010) Gene Ther. 17, 503-10). Genome-containing vectors were produced in roller bottles using a three-step transfection protocol and cesium chloride gradient purification (Ayuso et al. (2010)). AAV vector titration was performed by real-time PCR (qPCR) using Absolute ROX mix (Taqman, Thermo Fisher Scientific, Waltham, MA) in an ABI PRISM 7900HT sequence detector, except that empty capsids were first titrated by SDS-PAGE, then silver stained and quantified by densitometry for AAV capsids used as standards. For in vitro neutralization assays, an AAV8 vector encoding luciferase (AAV8-Luc) was used as a reporter gene as previously described (Miao et al. (2006) Blood. 108, 19-27). The AAV vectors used in the in vivo experiments expressed human FIX or Gaussia luciferase under the control of a liver-specific promoter (Manno et al. (2006); Mingozzi et al. (2013) Gene Ther. 20, 417-24).

[0414] Design and production of IdeS endopeptidase

[0415] The IdeS coding sequence (CDS) was cloned into a pEX-N-His-tagged cloning vector (OriGene Technologies, Inc., Rockville MD) and converted into BL21 competent Escherichia coli (NEB, Ipswich, MA) following the manufacturer's instructions. Protein expression was induced at 37°C for 4 hr by adding 0.5 mM IPTG to the bacterial culture. Anti-FabRICATOR antibody A3-AF1-010 (Genovis) and nickel affinity column (GE Healthcare) were used for protein detection and purification, respectively. Endotoxin removal was performed using a poly (ε-lysine) resin centrifugal column (Thermo Scientific, Waltham, MA), and residual endotoxin concentration was assessed by LAL test using Endosafe Cartridge Technology (Charles River Laboratories) according to the manufacturer's instructions. IdeS endopeptidase was diluted in an injectable solution buffer consisting of 50 mM sodium phosphate, 150 mM NaCl, 0.25% human albumin, pH 6.6.

[0416] Cell culture

[0417] 2V6.11 cells (ATCC CRL-2784) were maintained at 37°C, 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM GlutaMAX (Invitrogen Life Technology, Carlsbad, CA) and used in the AAV neutralization assay.

[0418] In vitro digestion of IgG by IdeS

[0419] Serum from AAV8 seropositive humans (NAb titer: 1:31.6) and monkeys (NAb titer: 1:100) or IVIg (NAb titer: 1:316) was diluted to 4-4.5 mg / mL IgG in PBS. Fifty microliters of diluted IgG (200 μg) were incubated with IdeS (100 IU, 50 IU / μL) or PBS at 37°C for 22 hours. Anti-AAV8 IgG was measured by ELISA and neutralization assays. In the case of monkey IgG, an antibody specific for the γ chain of monkey IgG conjugated to horseradish peroxidase (HRP) was used (Fitzgerald, Acton, MA USA).

[0420] Anti-human IgG ELISA

[0421] ELISA plates were coated with goat anti-human κ antibody (2.5 μg / mL in PBS) and blocked with PBS-1% bovine serum albumin (BSA). IgG samples were incubated at a 1 / 10 dilution using a commercially available IVIg preparation as a standard. Then, intact IgG was displayed using a secondary HRP-conjugated mouse anti-human IgG-Fc antibody and o-phenylenediamine dihydrochloride (OPD) substrate.

[0422] AAV antibody assay

[0423] Anti-AAV IgG capture assay was performed as previously described (Mingozzi et al. (2013) Sci Transl Med. 5, 194ra92). Briefly, recombinant AAV empty capsids were diluted to 2×10 per mL in coating buffer (35 mM bicarbonate, 13 mM carbonate, pH>9.2). 10 The final concentration of each carrier particle was 50 microliters. 50 microliters were added to each well in a 96-well Nunc Maxisorp immunoplate (Thermofisher, Waltham, USA). A standard curve made of purified human IgG (Tégéline, LFB, France) was added directly to the plate. The plate was coated overnight at 4°C. The next day, the plate was washed 3 times with washing buffer (PBS, 0.05% Tween-20) and then blocked with blocking buffer (PBS, 6% skim milk) for 2 hours at room temperature. The plate was washed 3 times with washing buffer (PBS, 0.05% Tween-20) again and then incubated at 37°C for 1 hour with serum diluted in blocking buffer. After washing 3 times, an antibody specific for the human IgG heavy chain conjugated to HRP (Southern Biotech, Birmingham, USA) was incubated at 37°C for 1 hour. After incubation, the plate was washed 3 times with washing buffer and displayed with OPD substrate (Sigma, Saint Louis, USA). The reaction was terminated with 3M H2SO4 solution and the plate was analyzed using an ELISA reader (ENSPIRE TM , Perkin Elmer, Waltham, USA) at 492nm for optical density (OD) measurements. Anti-AAV IgG concentrations were determined for a specific standard curve using 4-parameter regression, and the results are expressed as μg / mL of IgG. In NHP studies, monkey IgG purified for a standard curve and antibodies specific to the γ chain of monkey IgG conjugated with horseradish peroxidase (HRP) (Fitzgerald, Acton, MA USA) were used for visualization.

[0424] AAV neutralization assay

[0425] NAb assays were performed as previously described (Meliani et al. (2015) Hum Gene Ther Methods. 26, 45-53). Briefly, on day 1, 96-well plates were plated at 2×10 4 2V6.11 cells / well were inoculated for 24 hours. Recombinant AAV8-CMV-luciferase was diluted in serum-free DMEM (Life Technologies, Carlsbad, USA) and incubated with half-log serial dilutions of serum samples and then incubated at 37°C for 1 hour. Subsequently, the serum-vector mixture was added to the cells. Each dilution was tested in duplicate or triplicate. After 24 hours, the cells were lysed and the luminometer (ENSPIRE TM Luciferase activity was measured on a 400 μM luciferase assay (Perkin Elmer, Waltham, USA). Conversion efficiency was measured in relative light units (RLU) per second. Neutralization titers were reported as the highest serum dilution that inhibited AAV transduction by ≥50% compared to a serum-free control (100% transduction).

[0426] Mouse studies

[0427] Mouse studies were performed in accordance with French and European regulations on animal care and experimentation (2010 / 63 / EU and CE12-037) and were approved by the local institutional ethics committee (protocol number 2017-014-B).

[0428] Male C57BL / 6 mice or male and female hemophilia B (HB) mice deficient in factor IX (FIX) at 6-8 weeks of age were used. In all experiments, animals were passively immunized by intravenous injection of 9 mg IVIg (PRIVIGEN, CSL Behring, US-PA) in a final volume of 100 μL. 30 minutes later, mice received 250 UI, 500 UI, or 1250 UI of IdeS (Promega, Madison, US-WI) by retro-orbital injection. The next day, 30 hours after the injection of IdeS, all mice were injected with a dose of 1x10 12 vg / kg(2x10 10vg / mouse) of AAV8 vectors. Blood samples were collected from the retro-orbital plexus in heparin-coated capillaries (Sarstedt, Nümbrecht, Germany) on D-7, D+7, D+14, and D+28, or samples were collected from the mandibular approach on D-1+15mn and D0. At euthanasia, whole livers were collected and snap-frozen for subsequent vector genome copy number assessment.

[0429] NHP Research

[0430] All procedures using NHPs were approved by the Institutional Animal Care and Use Committee (protocol number 2018120409565637, APAFIS18092) and performed at the Nantes Veterinary School (Oniris, Nantes, France). Two macaques (Macaca facscicularis, 2–3 years old) were selected based on their anti-AAV8 neutralizing antibody titers (1:10 to 1:31.6). On day -2, one animal (NHP2) received a slow intravenous infusion of IdeS (500 μg / kg). 24 h later (D-1), a second injection of IdeS was repeated following the same protocol. The next day, 24 h after the second IdeS injection, 2 × 10 12 vg / kg dose to 2 monkeys (NHP1, control animals; NHP2 treated animals) infused with AAV8-hFIX vector. Before (D-2), during treatment and before AAV administration (D0), blood samples were collected on D-7, D-2, D-2 + 10h, D-1, D-1 + 10h, D0 to evaluate the efficacy of IdeS treatment on IgG antibody degradation. Other blood samples were collected to track the humoral response to anti-AAV8 and transgenic expression on D4, D7, D13, D21, D28, D35, D50 days. At euthanasia (D50), 4 wedge biopsy samples taken from 4 lobes of the liver were collected and snap-frozen for subsequent vector genome copy number studies.

[0431] For the re-administration study, 2 AAV8 seropositive monkeys were infused with 5x10 12 vg / k of AAV8-hFIX vector (D0). Before the vector injection, one monkey received a single injection of IdeS (500 μg / kg) on ​​D-1. Subsequently, two monkeys received two injections of IdeS (500 μg / kg) on ​​days 80 and 81, and 5x10 12 vg / kg of AAV8-hFIX vector was injected. Blood samples were collected at different time points during the experiment. At euthanasia (D105), liver biopsies were collected from 4 liver lobes for VGCN assessment.

[0432] Anti-Monkey IgG ELISA

[0433] Monkey IgG levels were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, we used goat anti-human IgG F(ab')2 (Thermo Scientific) and goat anti-monkey IgG-HRP (Fitzgerald) as capture and detection antibodies, respectively. OPD peroxidase substrate system (SIGMAFAST TM -OPD, Sigma) for display.

[0434] Human FIX ELISA

[0435] Human FIX (hFIX) protein levels in mouse plasma were measured according to the manufacturer's instructions. Capture HRP and secondary HRP antibodies for ELISA assays were purchased from Affinity Biological (Ancaster, Canada). In NHP samples, anti-hFIX antibody (MAI-43012, Thermo Fisher Scientific, Waltham, MA) and anti-hFIX-HRP antibody (CL20040APHP, Tebu-bio, France) were used for coating and detection, respectively. SIGMAFAST was added according to the manufacturer's instructions. TM OPD substrate (Sigma-Aldrich) and measuring OD at an absorbance of 492 nm to determine hFIX antigen levels.

[0436] Vector genome copy number (VGCN)

[0437] Using qPCR assay, the liver vector genome copy number was determined with primers and probes specific to the liver promoter and mouse Titin or eGlobin genes. Promoter-specific primers and probes were synthesized by Thermo Scientific (Waltham, MA, USA). Mouse Titin and eGlobin were used as normalization genes in mouse and NHP studies, respectively, and synthesized by ThermoScientific (Waltham, MA, USA). DNA was extracted from tissues after whole organs (mouse) or biopsies (NHP) were homogenized using FastPrep machines (MP Biomedicals) and GentraPuregen Blood Kit (Qiagen, ref 158467). Each sample was tested in triplicate, and the genomic copy number of each diploid genome was determined according to the standard curve of the linearized plasmid.

[0438] Gaussia luciferase assay

[0439] 50 μL of plasma samples diluted in PBS (1:50) were loaded onto a white 96-well microplate (OptiPlate-96 Perkin Elmer, Waltham, USA). TM , Perkin Elmer, Waltham, USA) on the Gaussia luciferase activity was measured. The measurement was performed within 1 second after the automatic injection of 50 μL substrate solution (Coelenterazine, Sigma Aldrich). The signal background was subtracted from each value. Gaussia luciferase activity is expressed as RLU / sec, and the values ​​are the mean + / -SD of the replicate samples from the replicate determination.

[0440] Tail clip bleeding assay

[0441] Anesthetized animals were delivered a mixture of ketamine and xylazine by intraperitoneal injection (IP) and placed in the prone position. The 3 mm distal portion of the tail was removed with a scalpel. The tail was immediately immersed in a 50 mL Falcon tube containing isotonic saline preheated to 37°C. The position of the tail was vertical with the tip approximately 2 cm below the body horizon. Even if bleeding stopped, each animal was monitored for 20 minutes to detect if there was any recurrence of bleeding. The bleeding time was determined using a stopped clock. If bleeding on / off cycles occurred, the sum of the bleeding times within 20 minutes was used. At the end of the experiment, the animals were killed by cervical dislocation.

[0442] Example 2

[0443] Results for AAV8

[0444] We first investigated whether IdeS hydrolyzes human IgG in vivo after passive immunization of wild-type mice, i.e., in the presence of endogenous mouse IgG. Wild-type mice were injected with human IgG (IVIg) and 30 minutes later with IdeS. Intact human IgG (IVIg) was then tested in serum collected 1, 6, and 24 hours later. Figure 1 ). In untreated mice, the level of human IgG gradually decreased over time, from 9.9±1.2 mg / mL (mean±SD) at 0 hours to 3.7±0.6 mg / mL at 24 hours, consistent with the half-life of human IgG in mice (Roopenian et al. (2003) J Immunol. 170, 3528-33). In contrast, human IgG in mice treated with IdeS showed rapid elimination from 11.1±1.0 mg / mL at 0 hours to 0.7±0.03 mg / mL at 24 hours. Thus, treatment with IdeS resulted in a 5-fold decrease in human IgG concentrations at 6 hours and 24 hours after injection.

[0445] To investigate the ability of IdeS to reduce anti-AAV8 IgG levels in vivo, we used a previously described passive immunization model (Scallan et al. (2006)). Wild-type C57BL / 6 mice were injected with 9 mg IVIg. Thirty minutes later, mice were injected with IdeS (250 UI) or PBS as an untreated control. Thirty hours after IdeS injection, mice were treated with 1x10 12 The mice were injected with AAV8 vector encoding human FIX transgene at a dose of vg / kg. Figure 2 The mice were divided into 4 groups (n=6 mice / group) as shown in the following table of treatment groups:

[0446] Group IVIg IdeS AAV8-hFIX 1 + + + 2 + - + 3 - + + 4 - - +

[0447] Animals were followed for four weeks after vector delivery and plasma was collected on days -7, 7, 14, and 28 to measure FIX transgene expression levels ( Figure 2 Anti-AAV8 IgG and NAb in serum were measured from blood collected after reconstitution with human IgG (D-1+15min) and after injection of IdeS or PBS (D0). As previously published (Scallan et al. (2006)), IVIg contains high levels of functionally relevant anti-AAV8 IgG, as measured by ELISA ( Figure 3A , for Group 1 and Group 2, 2.56±0.21μg / mL and 2.83±0.22μg / mL, mean±SD) and neutralization assay ( Figure 3B , 1:22.6 [range: 1:10 to 31.6] titers and 1:19 [range: 1:10 to 31.6] titers) were detected. Injection of IdeS resulted in a dramatic decrease in circulating levels of anti-AAV8 IgG. After 24 hours, the decreases in anti-AAV8 IgG (0.39±0.10 μg / mL and 1.67±0.42 μg / mL for Group 1 and Group 2, respectively) and AAV8 NAb were 4.3-fold and 10-fold, respectively. In PBS-treated mice (Group 2), the decrease in anti-AAV8 IgG overtime levels indicates natural elimination of human IgG in mice.

[0448] Wild-type mice reconstituted with IVIg or PBS and treated with IdeS or PBS were then injected with AAV8 vectors encoding hFIX, and hFIX plasma levels were measured 7, 14, and 28 days later. As expected, unreconstituted mice lacking anti-AAV8 IgG showed constant production and accumulation of hFIX in the blood over a 4-week period ( Figure 4A This was accompanied by a high number of gene copies in the mouse liver ( Figure 4B In contrast, mice reconstituted with human IgG containing anti-AAV8 neutralizing IgG ( Figure 3A) failed to produce hFIX. Interestingly, treatment of human IgG reconstituted mice with IdeS was able to restore hFIX production after gene therapy. As assessed by measuring the area under the curve, the level of hFIX produced in IdeS-treated mice represented 50.31% of the level produced in control non-reconstituted mice ( Figure 4A ). Therefore, in the liver of mice treated with IdeS reconstituted with human IgG, the gene copy number was 2.016±0.620 vg / diploid cell ( Figure 4B ), whereas in mice reconstituted with human IgG that did not receive IdeS, the gene copy number was 0.005±0.002 vg / diploid cell (P=0.002).

[0449] IdeS hydrolyzes IgG from different species, including nonhuman primates. Therefore, we investigated whether incubation of IdeS with AAV8-seropositive human and nonhuman primate (NHP, monkey) serum samples hydrolyzes neutralizing anti-AAV8 IgG. Figure 5A As shown, IdeS indeed hydrolyzed anti-AAV8 IgG in human and monkey serum, with anti-AAV8 IgG levels in monkey serum reduced 15-fold within 22 hours of incubation (0.42 ± 0.01 μg / mL and 6.29 ± 0.10 μg / mL when treated with IdeS or PBS, respectively). Anti-AAV8 NAb titers were also reduced in human serum incubated with IdeS ( Figure 5B No decrease in anti-AAV8 NAb titers was observed after incubation of monkey sera with IdeS, but the neutralizing capacity of these antibodies decreased from 76% to 64% after incubation with IdeS ( Figure 5B The lack of decreased anti-AAV NAb titers may be due to the retention of functional F(ab')2 fragments of high-affinity monkey anti-AAV8 IgG in the incubation mixture.

[0450] Example 3

[0451] Results for AAV2 and AAV9

[0452] Anti-AAV2 and anti-AAV9 IgG and NAb ( Figure 2 ). For example, ELISA (for Group 1 and Group 2, respectively: Figure 3C , anti-AAV2 IgG 9.05±1.20μg / mL and 8.92±0.63μg / mL, mean±SD; Figure 3E, anti-AAV9 IgG 5.51±0.492μg / mL and 5.145±0.691μg / mL) and neutralization assays (for Group 1 and Group 2, respectively: Figure 3D , 1:886 and 1:772 [range: 1:316 to 1000] anti-AAV2 NAb titers; Figure 3F IVIg contained high levels of functionally relevant anti-AAV2 and anti-AAV9 IgG, as measured by anti-AAV9 NAb titers (range: 1:10 to 31.6, 1:15.4, and 1:24.4 [range: 1:10 to 31.6]). Injection of IdeS resulted in a dramatic decrease in circulating levels of anti-AAV2 and AAV9 IgG. After 24 hours, anti-AAV2 IgG decreased 6-fold (1.51±0.31μg / mL and 4.58±0.45μg / mL for Groups 1 and 2, respectively), and anti-AAV2 NAb decreased 28-fold. After 24 hours, anti-AAV9 IgG decreased 36-fold (0.153±0.074μg / mL and 2.92±0.50μg / mL for Groups 1 and 2, respectively), and anti-AAV9 NAb decreased 20-fold. The levels of anti-AAV2 and AAV9 IgG in PBS-treated mice (Group 2) decreased over time, representing the natural elimination of human IgG in mice.

[0453] Example 4

[0454] Gaussia luciferase results

[0455] Wild-type mice passively immunized with pooled human IgG received IdeS (1250 UI / mouse) or PBS, followed by injection of AAV8 encoding GLuc. GLuc expression was measured in mouse plasma after 7, 14, and 28 days. Unreconstituted mice lacking anti-AAV8 IgG exhibited constant production and accumulation of GLuc in the blood over 4 weeks ( Figure 4C This was accompanied by a high number of gene copies in the mouse liver ( Figure 4D In contrast, mice reconstituted with human IgG containing anti-AAV8 neutralizing IgG failed to produce GLuc ( Figure 4C Interestingly, in the case of GLuc, treatment of passively immunized mice with IdeS restored almost complete gene therapy efficiency ( Figure 4C ).

[0456] Therefore, in the liver of mice treated with human IgG-reconstituted IdeS, the gene copy number was 0.759 ± 0.148 vg / diploid cell ( Figure 4D ), whereas in mice reconstituted with human IgG that did not receive IdeS, the gene copy number was 0.0006±0.0004 vg / diploid cell (P<0.0001).

[0457] Example 5

[0458] HB mouse studies

[0459] Factor IX-deficient hemophilia B (HB) mice reconstituted with IVIg or PBS were treated with IdeS (1250 UI / mouse) or PBS and injected with AAV8 vectors encoding hFIX, and hFIX plasma levels were measured 7 and 14 days later. Unreconstituted HB mice lacking anti-AAV8 IgG showed production and accumulation of hFIX in the blood within 2 weeks ( Fig. 6A In contrast, mice reconstituted with human IgG containing anti-AAV8 neutralizing IgG failed to produce hFIX ( Fig. 6A Interestingly, passively immunized mice treated with IdeS restored full gene therapy efficiency and achieved circulating hFIX levels equivalent to those observed in control non-reconstituted mice ( Fig. 6A ). The endogenously produced hFIX levels achieved after combined IdeS / AAV8 treatment protected mice from acute bleeding as measured by tail clip. In fact, the blood loss was not statistically significant compared to unimmunized mice and wild-type C57BL / 6 mice treated with AAV8 (dashed line). In contrast, passively immunized mice that did not receive IdeS lost significantly more blood than mice in other groups.

[0460] Example 6

[0461] NHP Research

[0462] We investigated the efficacy of IdeS treatment in non-human primates. A cynomolgus monkey (NHP2) was treated with two IV injections of IdeS (500 μg / kg) as Figure 7 Intact IgG was measured in serum by ELISA using purified monkey IgG as a standard and is expressed in mg / mL ( Fig. 8A ). The data showed depletion of total NHP2 IgG, with some intact IgG remaining even after the second injection of IdeS (D-1+10h, D0). Recovery of IgG began on D4 after treatment, with a total recovery of 21 days. Anti-AAV8 IgG was measured before and after IdeS treatment. Anti-AAV8 IgG levels in treated monkeys (NHP2) decreased 2.7-fold (from 2.86 to 1.05 μg / mL) after the second injection of IdeS as measured by ELISA ( Figure 8B Similarly, anti-AAV8 NAb titers measured in the neutralization assay decreased 3-fold (titer from 17.2 to 5.4) ( Figure 8C ).

[0463] NHP2 and control monkeys (NHP1) not treated with IdeS received AAV8 encoding hFIX (2x10 12 vg / kg), and the levels of hFIX in plasma were measured over time over 50 days. Compared with the control monkeys (NHP1), the monkeys treated with IdeS (NHP2) had significantly higher hFIX expression levels over time until death ( Fig.9A ). This result is consistent with the assessment of liver VGCN. In fact, treated monkeys (NHP2) showed more efficient liver transduction than untreated control monkeys (NHP1) ( Fig. 9B , 10.1±1.6 vs.1.6±0.2). We also tracked the levels of neutralizing anti-AAV8 IgG in the blood of NHP1 and NHP2 monkeys after AAV8-mediated gene therapy. In IdeS-treated monkeys (NHP2), the anti-AAV8 IgG humoral response was reduced by 11-fold compared with that in untreated monkeys (NHP1). Fig. 10A , 75.4±0.3 vs. 816.7±122). A similar reduction in humoral response was observed with anti-AAV8 NAb ( Fig. 10B :NHP2,1:316 vs.NHP1,1:3160).

[0464] We then developed a regimen to re-administer IdeS and AAV8 ( Fig.11 To this end, two monkeys with similar initial levels of anti-AAV8 IgG were treated with AAV8 encoding hFIX, with (NHP4) or without (NHP3) prior IdeS treatment. As expected, after the first AAV8 injection, endogenous levels of hFIX were higher in the IdeS / AAV8-treated monkey (NHP4) than in the AAV8-treated control monkey that did not receive IdeS (NHP3). Fig. 12A ). Both monkeys then received two Ides injections and were re-administered with AAV8 encoding hFIX. NHP3 failed to produce endogenous hFIX, which was associated with elevated levels of neutralizing anti-AAV8 IgG, poor liver transduction efficiency, and production of anti-hFIX antibodies (data not shown). In contrast, NHP4 showed increased hFIX production after a second treatment with IdeS / AAV8, consistent with low levels of anti-AAV8 IgG and NAbs. Interestingly, hFIX levels were more stable after vector re-administration than after the first vector injection. These results are consistent with the assessment of hepatic VGCN ( Fig. 12B We tracked the levels of neutralizing anti-AAV8 IgG in the monkeys’ blood over 105 days. In monkeys treated with IdeS (NHP4), anti-AAV8 IgG humoral responses were barely detectable at the end of the experiment ( Fig.13A, 2.25±0.071μg / mL for NHP4 and 1552.5±337.9μg / mL for NHP3). A similar reduction in humoral response was observed with anti-AAV8 NAb ( Fig. 13B , NHP4 is 1:17.2, and NHP3 is 1:10000).

[0465] in conclusion

[0466] The data show that Ides treatment can effectively reduce neutralizing antibodies that inhibit gene therapy using AAV8 vectors encoding factor IX in the context of hemophilia B (see Examples 1, 4, and 5). This data supports the use of IdeS treatment for gene therapy with other AAV serotypes (see Example 2).

[0467] Example 7

[0468] method

[0469] In vitro cleavage of immunoglobulin G (IgG) by endopeptidase: Human serum or non-human primate (NHP) plasma samples with or without neutralizing antibodies (NAbs) against Spk2 capsid were incubated with increasing doses of immunoglobulin degrading enzyme (IdeS; Promega) from Streptococcus pyogenes at 37°C for 1 hour. According to Promega's instructions, one unit is defined as 1 μg of recombinant monoclonal IgG cleaved ≥95% within 30 minutes at 37°C. The reaction volume was adjusted with PBS. Cleavage of total IgG was assessed by SDS-PAGE and Coomassie Brilliant Blue staining.

[0470] SDS-PAGE analysis of cleaved IgG: Prepare cleaved samples using Non-reducing SDS-PAGE was performed using LDS sample buffer (4X) (ThermoFisher Scientific) and heated to 70°C for 10 min. Then, MOPS SDS Running buffer was used to The samples were analyzed on Novex 4-12% Bis-Tris gels. The gels were stained with Coomassie blue.

[0471] Anti-AAV capsid neutralizing antibody (NAb) titers: Neutralizing antibodies against AAV-Spk1 or AAV-Spk2 capsids were quantified using a cell-based in vitro assay and AAV-Spk1 or AAV-Spk2 reporter vectors encapsulating a Renilla luciferase transgene, respectively. Briefly, early passage (less than passage #26) 293E4 cells were thawed and plated at 2x10 4Cells / 200 μL / well were plated in a flat-bottom white 96-well plate. Ecdysone A (Invitrogen; cat. # H101-01) was added to each well at a final concentration of 1 μg / mL to induce the expression of the helper virus protein human adenovirus E4. The cells were then cultured overnight in a 37°C / 5% CO2 incubator. The next day, the samples were heat inactivated at 56°C for 30 minutes, and then 4 dilutions (from 1:1 to 1:5) were prepared using FBS as a diluent. Factor assay control plasma (FACT; King George Bio-Medical, Inc.) was prepared in 3.16-fold (half-log) serial dilutions to evaluate assay performance. The AAV luciferase vector was diluted to 7.5x10 7 vg / mL and then added to the FACT controls and samples. The vehicle and controls / samples were incubated at 37°C for 60 minutes. A volume of 7.5 μL per well of the "neutralized" control / sample was transferred to each well of the plate seeded with cells, and the cells were returned to the incubator and cultured overnight. The next day, the cells were washed once in PBS, lysed in Renilla assay lysis buffer, and luciferase activity was measured using the Renilla luciferase assay system (Promega) and read on a SpectraMax L microplate reader.

[0472] Artificial immunization mouse model using IVIg: To generate artificial NAb titers in male C57BL / 6 mice, intravenous immunoglobulin (IVIg; Gamunex) containing 10% immunoglobulin G (IgG) purified from human blood was injected intraperitoneally one day before intravenous (IV) administration of vector. To determine whether in vitro cleavage of IgG by IdeS or IdeZ (a similar endopeptidase from Streptococcus equi that has increased activity against mouse IgG2a and IgG3 compared to IdeS) could rescue transduction efficiency in vivo, IVIg was treated with 125 units of IdeZ overnight at 37°C and then administered IP. Cleavage of total IgG was assessed by SDS-PAGE and Coomassie Brilliant Blue staining. One day after IVIg administration, 2x10 12 vg / kg of vector (AAV-Spk1-GAA). Mouse plasma samples were collected weekly and analyzed for transgene (GAA enzyme) activity. To determine whether in vivo cleavage of IgG by IdeS could rescue transduction efficiency in vivo, 300 mg / kg IVIg was infused via IP administration. 24 hours later, 0.4 or 4 mg / kg IdeS was infused via IV administration. Then, 24 hours after IdeS infusion, 2x10 12 Vector (AAV-Spk1-GAA) was administered at vg / kg.

[0473] GAA activity assay: GAA activity was assessed by measuring the cleavage of the substrate 4-methyl-umbelliferyl-α-D-glucoside at pH 4 (Galjaard et al., Clin Chim Acta 1973; 49 (3): 361-75). Briefly, the reaction was initiated by adding 20 μL of substrate to 10 μL of plasma sample diluted 1:250 in MilliQ water. The reaction mixture was incubated at 37°C for 1 hour and then subsequently terminated by carbonate buffer at pH 10.5. Thereafter, the standard curve was plated with 4-methylumbelliferyl (a blue fluorescent dye released from 4-methyl-umbelliferyl-α-D-glucoside that produces fluorescence at 440 nm when excited at 370 nm).

[0474] Anti-AAV capsid IgG antibodies: The formation of total anti-AAV capsid IgG was measured using a capture assay. The ELISA plate wells were coated with 50 μL of a solution containing 1 μg / mL AAV-Spk1 capsid particles. Total human IgG (Southern Biotech, 0150-01) was diluted to generate a 10-point standard curve from 10,000 ng / mL to 0.5 ng / mL and added to the plate. After reverse calculation, the limit of quantification of the assay was 460 ng / mL. Three levels of quality control samples were prepared and included on each plate to evaluate the performance of the assay. The capsid particles, standards, and quality controls (QC) were incubated overnight at 4°C. After washing, the wells were blocked with a 2% BSA, 0.05% Tween-20 solution in PBS at room temperature for 2 hours. Then, serial dilutions of the samples in blocking buffer were loaded onto the plate and incubated at room temperature for 2 hours. Horseradish peroxidase (HRP)-conjugated sheep anti-human IgG antibody (GE Healthcare, cat. #NA933V) diluted 1:5000 in blocking buffer was used as detection antibody and incubated on the plate for 1 hour at room temperature. After washing, peroxidase activity was shown after incubation with 3,3',5,5'-tetramethylbenzidine substrate (TMB) for 10 minutes at room temperature. The reaction was terminated with 1M sulfuric acid, and the optical density (OD) at 450nm of the plate was then read by an absorbance plate reader. IgG concentration was determined for a standard curve prepared from a serial dilution of purified total human IgG.

[0475] Example 8

[0476] IdeS cleaves IgG from human, NHP and hamster samples in vitro

[0477] Immunoglobulin G (IgG) degrading enzyme (IdeS) from Streptococcus pyogenes is a cysteine ​​protease that cleaves all four human IgG subclasses with high specificity. IdeS hydrolyzes human IgG at Gly236 in the lower hinge region of the IgG heavy chain.

[0478] To analyze the ability of IdeS to cleave IgG in serum, increasing doses of IdeS (0-100 units; Promega) were added to human serum and NHP (rhesus monkey) plasma samples with or without anti-Spk2 NAb titers. In human samples with naive (<1:1) or relatively mid-range (1:5-1:10) or relatively high (1:20-1:40) NAb titers, the lowest dose of IdeS cleaved all IgG (~150 kD) to release the Fc fragment (~25 kD) ( Fig.14A In NHP samples, IgG was cleaved similarly in all NAb titer groups (<1:1, 1:50-1:100, >1:100, Fig. 14B ).

[0479] It was expected that hamsters would provide a better model than mice for examining IdeS processing in response to AAV re-administration. The ability of IdeS to cleave hamster IgG was tested by incubating increasing amounts of IdeS (0-50 units; Promega) with pooled hamster plasma (pooled human plasma as a positive control). Samples were analyzed by non-reducing SDS-PAGE and Coomassie blue staining. IdeS efficiently cleaved IgG in both pooled hamster plasma and pooled human plasma in vitro ( Fig. 14C ).

[0480] These results demonstrate that IdeS is a highly efficient and specific protease for human IgG, rhesus monkey IgG, and hamster IgG.

[0481] Example 9

[0482] Reduction of NAb titers in vitro by IgG cleavage by IdeS

[0483] To analyze whether cleavage of IgG by IdeS is sufficient to reduce neutralization, the transduction efficiency of AAV vectors was determined in vitro. In this assay, the presence of neutralizing antibodies against AAV capsids can be assessed in serum or plasma samples from various species by preincubating AAV vectors encoding Renilla luciferase with plasma or serum, transducing human cells in culture with these mixtures, and subsequently assessing the level of luciferase activity.

[0484] Human patient serum samples that were naive (<1:1) or had high titers of anti-Spk2 NAb (1:10-1:20) were pretreated with or without excess IdeS (50 units) and then assessed for Nab titers. Interestingly, in one study, patient samples with previously reported NAb titers of 1:10-1:20 pretreated with IdeS showed at least a two-fold decrease to Nab titers of 1:5-1:10 (Table 1). These results suggest that AAV vector transduction increases when IgG antibodies are cleaved by IdeS.

[0485] Table 1 - Anti-Spk2 NAb titer analysis of human sera incubated with excess IdeS.

[0486] Study 1

[0487]

[0488] Study 2

[0489] sample# Spark ID IdeS Carrier Previous titer Titer range S1 FBS no Spk2 NA <1:1 S2 FBS yes Spk2 NA <1:1 S3 BRH1450399 no Spk2 1:2.5 1:2.5 S4 BRH1450399 yes Spk2 1:2.5 1:1 S5 BRH1450436 no Spk2 ～1:5 1:5 S6 BRH1450436 yes Spk2 ～1:5 1:1 S7 BRH1450427 no Spk2 1:10 1:10 S8 BRH1450427 yes Spk2 1:10 1:10

[0490] Analysis of anti-Spk2 NAb titers after treatment with IdeS endopeptidase. Human patient samples (designated by Spark ID) were pre-treated with and without IdeS. Subsequently, NAb titers were assessed by in vitro vector transduction assay.

[0491] Example 10

[0492] IdeZ degradation of IVIg in vitro increases the transduction efficiency of the vector in vivo

[0493] The effector functions of IgG antibodies, such as cytotoxicity and complement fixation, are mediated by the Fc portion. Neutralization relies on the specificity of the variable regions of the heavy and light chains for the antigen. Although the F(ab')2 fragments still contain intact antigen binding regions, data indicate that F(ab')2 fragments released by IdeS or IdeZ (similar endopeptidases in Streptococcus equi, which have improved activity against mouse IgG2a and IgG3) result in reduced stability without the Fc portion, and therefore F(ab')2 fragments are cleared from the circulation faster than intact IgG. This assay tests whether administration of neutralizing antibodies pre-cut by IdeS or IdeZ should result in a reduction in neutralizing activity against AAV in an in vivo setting.

[0494] Mice were immunized with IVIg, which was a mixture of human IgG including anti-AAV capsid neutralizing antibodies pretreated with or without 0.1 mg / kg IdeZ. Then, mice were administered 2x10 12vg / kg AAV-Spk1-GAA. Mice treated with 1.0 mg or 5.0 mg IVIg resulted in decreased GAA activity levels in plasma (10,951±1,554 nmol / hr / mL and 1,041±553 nmol / hr / mL) compared with control mice (33,551±13,635 nmol / hr / mL), indicating that neutralization of the vector by IVIg was dose-dependent ( Fig.15 ).

[0495] Pretreatment of 40 mg / kg IVIg with IdeZ rescued transduction efficiency, producing GAA activity levels comparable to controls (37,707 ± 11,449 nmol / hr / mL). IdeZ pretreatment of 200 mg / kg IVIg partially alleviated vector neutralization (13,440 nmol / hr / mL ± 15,543), with one animal fully recovering activity (41,025 nmol / hr / mL). Of note, IdeZ itself did not interfere with AAV vector transduction efficiency. IVIg dosing retentates were analyzed by SDS-PAGE with Coomassie Brilliant Blue staining to confirm IgG cleavage. These results suggest that neutralizing antibodies against AAV capsids cleaved in vitro by IdeS / IdeZ can rescue AAV vector transduction and transgene expression in vivo.

[0496] Embodiment 11

[0497] In vivo degradation of IVIg by IdeS increases the transduction efficiency of the vector in vivo

[0498] To analyze whether cleaved IgG in vivo would affect vector transduction and transgene expression / activity in plasma, mice were first infused with intact IVIg to generate artificial titers of human anti-capsid neutralizing IgG. 24 hours later, mice were infused with two concentrations (0.4 mg / kg or 4 mg / kg) of IdeS, and then 24 hours after IdeS infusion, all mice were administered 2x10 12 vg / kg AAV-Spk1-GAA. Anti-Spk1 NAb titers and IgG levels were analyzed before and after IdeS infusion (immediately before vector administration).

[0499] IdeS infusion induced NAb ( Fig.16 ) and a dose-dependent decrease in IgG levels ( Fig.17 ). The highest dose of IdeS (4 mg / kg) was able to reduce the NAb titer from at least 1:40 to <1:1.

[0500] When GAA activity was measured one week after vector infusion, control mice administered only the vector exhibited a GAA activity level of 49,387 ± 7,345 nmol / hr / mL ( Fig.18 ). Mice injected with 300 mg / kg IVIg exhibited GAA transgene activity levels in plasma consistent with nearly complete inhibition of transduction (1,702±336 nmol / hr / mL). IdeS showed a dose-dependent rescue of transgene activity levels; 0.4 mg / kg IdeS resulted in a rescue of 70% of GAA activity (34,408±10,562 nmol / hr / mL), while 4 mg / kg rescued 99% of GAA activity (48,948±5,322 nmol / hr / mL). These results demonstrate that in vivo IdeS treatment reduces neutralizing antibody titers and allows dosing and transduction of AAV vectors in refractory animals.

[0501] Example 12

[0502] In vivo degradation of IVIg by IdeS increases the transduction efficiency of the vector in vivo

[0503] The ability of IdeS to cleave higher titers of anti-capsid IgG in vivo and rescue AAV transduction in the presence of a higher degree of AAV vector neutralization was evaluated. Mice (male C57BL / 6) were infused with different doses of whole human IVIg (300 mg / kg (low), 800 mg / kg (medium), or 1600 mg / kg (high)) to generate artificial titers of human anti-capsid neutralizing IgG. After 24 hours, mice were infused with IdeS at three concentrations (0.4 mg / kg (low), 1 mg / kg (medium), or 2 mg / kg (high). 24 hours after IdeS infusion, mice were infused with 2x10 12 AAV-Spk1-GAA was administered to mice at 100 vg / kg. Anti-Spk1 NAb titers were determined before and after IdeS infusion (immediately before vector administration) using the anti-AAV capsid NAb titer assay described in Example 1. AAV transduction was assessed two weeks after vector administration by measuring transgene product (GAA) activity in plasma using a GAA activity assay as described in Example 1.

[0504] Pretreatment with IdeS resulted in a dose-dependent reduction in AAV NAb titers for all doses of IVIg (Table 2). Table 2 lists the neutralizing anti-Spk1 antibody (NAb) titers for each animal in each group before and after IdeS infusion. AAV NAb titers were designated as low (<1:1, 1:1–1:2.5), low to mid-range (1:2.5–1:5), mid-to-high range (1:5–1:10), and high (>1:10–1:20). The highest dose of IdeS (2 mg / kg) was able to reduce NAb titers of >1:160 (generated with 1600 mg / kg IVIg) to 1:1-1:2.5.

[0505] Table 2 - Anti-NAb titers in mouse plasma before and after IdeS infusion.

[0506]

[0507]

[0508]

[0509] The results of GAA activity measured two weeks after vector infusion are shown in Fig.19 . Plasma from negative control mice (administered only the vector) showed GAA activity levels of 26,689±12,420 nmol / hr / mL. Mice injected with 300 mg / kg (or higher) IVIg and not receiving IdeS had plasma GAA activity levels of only 436±41 nmol / hr / mL, consistent with inhibition of AAV vector transduction by NAb. Consistent with the results in the previous examples, IdeS pretreatment resulted in a dose-dependent rescue of AAV vector transduction as measured by GAA activity levels in plasma: 0.4 mg / kg IdeS resulted in GAA activity levels of 7,702±4,710 nmol / hr / mL, 1 mg / kg IdeS resulted in GAA activity levels of 15,444±4,226 nmol / hr / mL, and 2 mg / kg IdeS resulted in GAA activity levels of 14,375±2,572 nmol / hr / mL.

[0510] The groups receiving higher doses of IVIg (800 or 1600 mg / kg IVIg) showed a similar trend of dose-dependent increases in GAA activity levels with increasing IdeS. For 800 mg / kg IVIg, 0.4 mg / kg IdeS resulted in GAA activity levels of 4,188±2,549 nmol / hr / mL, 1 mg / kg IdeS resulted in GAA activity levels of 17,813±11,283 nmol / hr / mL, and 2 mg / kg IdeS resulted in GAA activity levels of 26,846±7,354 nmol / hr / mL. For 1600 mg / kg IVIg, 0.4 mg / kg IdeS resulted in GAA activity levels of 580 ± 217 nmol / hr / mL, 1 mg / kg IdeS resulted in GAA activity levels of 12,511 ± 1,602 nmol / hr / mL, and 2 mg / kg kg IdeS resulted in GAA activity levels of 11,573 ± 1,313 nmol / hr / mL. At the highest IVIg dose (1600 mg / kg), the lowest IdeS dose (0.4 mg / kg) failed to rescue vector transduction. However, at the 1600 mg / kg IVIg dose, supraphysiological levels of total IgG were present in the circulation, which may reduce the effectiveness of IdeS at the 0.4 mg / kg dose due to an increase in the number of its substrates.

[0511] These results demonstrate that in vivo IdeS treatment reduces neutralizing antibody titers and allows administration and transduction of viral vectors in animals that are refractory to viral vector gene therapy approaches.

[0512] Example 13

[0513] This co-therapy has a wide range of applications. For example, by overcoming NAb into AAV capsids or other gene delivery methods through IdeS, it is possible to treat patients with pre-existing neutralizing antibody titers, and to re-administer to patients who have been previously administered gene therapy products (where effective levels have not been reached or have lost effective levels due to time or other confounding issues). In addition, the method herein enables liver gene transfer to pediatric populations, which have been considered refractory to gene therapy due to the potential loss of transgenic expression during the expansion and development of hepatocytes. These results show that IdeS administration will lead to a reduction or elimination of neutralizing antibodies to AAV capsids, and make it possible to treat patients who were previously considered unsuitable for gene therapy or who produced AAV antibodies after AAV gene therapy.

[0514] Embodiment 14

[0515] Hamster Model of AAV Readministration

[0516] Hamsters are infused with rAAV vector particles carrying a transgene of interest, IdeS is administered after the production of anti-AAV NAbs (e.g., 4 weeks), and additional rAAV particles carrying another transgene are infused. This model allows analysis of transduction efficiency at different stages, including before and after IdeS treatment and before and after re-administration with rAAV.

[0517] To evaluate the ability of Ides to degrade or reduce the effects of neutralizing antibodies against AAV capsids, studies were performed in Syrian golden hamsters (a species whose IgG is efficiently cleaved by the endopeptidase IdeS). Hamsters were first infused with 2x10 12 vg / kgSpk1-FVIII, Spk1-FIX or other Spk1 encapsidated vectors. In addition to measuring Nab formation of Spk1 capsids by anti-Spk1 IgG ELISA or cell-based neutralizing antibody assays, the expression of transgenic products (i.e., FVIII, FIX, etc.) in animal plasma is also monitored. After NAb titers are generated, animals are infused with single or multiple increasing doses of IdeS: 0, 0.5, 1 and 2 mg / kg (and higher doses) by intravenous, subcutaneous, intraperitoneal or other routes within 3-5 weeks of vector infusion. After administration of IdeS, animals are tracked by measuring anti-Spk1 capsid IgG and / or Nabs against Spk1. When animals show a sufficient reduction in NAb levels, they are infused with 2x10 12 vg / kg Spk1-GAA. After transduction, GAA expression in plasma was measured by GAA activity assay and / or GAA antigen level measurement to determine the transduction level achieved.

[0518] These studies show whether IdeS can reduce AAV capsid-specific NAbs in vivo to levels low enough to warrant re-administration. Measurement of anti-FVIII IgG (if produced in vivo) provides information about the effectiveness of IdeS in reducing NAbs targeting the transgene product and the number of IdeS re-administration rounds allowed before loss of effectiveness.

[0519] Embodiment 15

[0520] Cynomolgus monkey model of AAV re-administration

[0521] To evaluate the ability of IdeS to degrade or reduce the effects of Nabs against AAV capsids in a large animal model, studies were conducted in cynomolgus monkeys (macaques). Monkeys were first screened for pre-existing NAbs against Spk1 capsids. NAb-positive animals may be caused by exposure to naturally occurring AAV in the wild or in collective feeding grounds. Animals were grouped according to negative or positive Nab titers, and if positive, how high the pre-existing Nab titers were.

[0522] In the re-dosing group of the study, animals were given 2x10 12 vg / kg Spk1-FIX or other Spk1 encapsidated vectors. In addition to measuring the NAb formation of Spk1 capsid by anti-Spk1 IgG ELISA or cell-based NAb assays, the expression of transgenic products (ie, FIX or others) in animal plasma is also monitored. After the generation of NAb titers, within 3-5 weeks of vector infusion, animals are infused with IdeS doses of 0 or 0.5, 1 and 2 mg / kg and higher by intravenous, subcutaneous, intraperitoneal or other routes of administration. After administration of IdeS, animals are tracked by measuring anti-Spk1 capsid IgG and / or Nabs against Spk1. When animals show a sufficient reduction in NAb levels, they are infused with 2x10 12 vg / kg Spk1-GAA. After transduction, GAA expression in plasma was measured by GAA activity assay and / or GAA antigen level measurement to determine the transduction level achieved.

[0523] Another arm of the study evaluated the ability of IdeS to overcome pre-existing Nab titers. Animals showing different NAb titer levels were grouped according to titer and infused with single or multiple ascending doses of IdeS: 0, 0.5, 1 and 2 mg / kg (and higher doses) by intravenous, subcutaneous, intraperitoneal or other routes of administration. After administration of IdeS, animals were tracked by measuring anti-Spk1 capsid IgG and / or Nabs against Spk1, and when animals showed a sufficient reduction in NAb levels, they were infused with 2x10 12 vg / kg Spk1-GAA. After transduction, GAA expression in plasma was measured by GAA activity assay and / or GAA antigen level measurement to determine the transduction level achieved.

[0524] These studies show whether IdeS can reduce AAV capsid-specific NAbs in vivo to low levels sufficient for re-administration in cynomolgus monkeys, a species that is an excellent model for human AAV administration. These studies also show that maximal pre-existing NAb titers can be overcome by IdeS administration. Measurement of anti-FVIII IgG (if generated in vivo) provides information about the effectiveness of IdeS in reducing NAbs targeting transgene products and the number of IdeS re-administration rounds allowed before loss of effectiveness.

[0525] Example 16

[0526] Mouse studies using IdeS and AAV-Spk1-hFVIII

[0527] Two different IdeS formulations (Batch 1 and Batch 2) were tested in mice with artificial titers of human anti-capsid neutralizing IgG. C57BL / 6 mice were injected with 300 mg / kg IVIg on day -2, followed by 1 mg / kg IdeS on day -1 (pre-dosing with AAV), and finally 5x10 of an AAV-Spk1 vector encoding human Factor VIII (AAV-Spk1-hFVIII) on day 0 (post-dosing). 10 Negative control animals did not receive IVIg or IdeS treatment, and the "no IdeS" group received only IVIg and AAV-Spk1-hFVIII vector. Using an anti-AAV capsid neutralization assay similar to that described in Example 7, samples were titrated at 8 points (1:1 to 1:160), and antibody titers were measured in plasma before and after IdeS administration, and Luminescence was read on a Discover Microplate Reader (Promega). Titers were determined as the highest dilution or range in which luminescence was inhibited by >50%. NAb titers before and after IdeS treatment showed that both batches of IdeS effectively reduced NAb titers in mice ( Fig. 20 ).

[0528] Human FVIII antigen levels were measured by ELISA before vector infusion and one and two weeks after vector infusion ( Fig.21 ). In vivo treatment of both batches of IdeS reduced neutralizing antibody titers and allowed co-administration with AAV vectors and transgene expression.

[0529] Embodiment 17

[0530] Mouse studies with anti-AAV-Spk1 IgG

[0531] IVIg was provided to C57BL / 6 mice to induce artificial titers of human anti-capsid neutralizing IgG. Three concentrations of IVIg (300 mg / kg (low), 800 mg / kg (middle) and 1600 mg / kg (high)) were used, and in each IVIg group, animals were treated with increasing doses of IdeS (0, 0.4, 1.0, 2.0 mg / kg). Anti-Spk1 capsid IgG levels were assessed by ELISA. In brief, 96-well plates were coated with Spk1 empty capsids, then blocked with BSA, washed, and incubated with plasma, diluted 1:100, and kept for 2 hours. After incubation, the plates were washed and incubated with a second antibody conjugated with HRP for 1 hour. Subsequently, the plates were washed again and developed using TMB substrate. The optical density (OD) of the plates was read at 450 nm on an absorbance plate reader. Luminescence was compared with the standard curve of human IgG to determine antibody concentration. All three concentrations of IdeS (0.4, 1.0, 2.0 mg / kg) abolished or significantly reduced serum levels of anti-Spk1 capsid IgG against all three concentrations (low, medium, and high) of IVIg ( Fig. 22 ).

[0532] Embodiment 18

[0533] Spk1 (SEQ ID NO: 1):

[0534] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0535] Spk2(SEQ ID NO:2):

[0536] MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

[0537] The IdeS sequence, which includes an N-terminal methionine and a signal sequence (SEQ ID NO: 3, NCBI reference sequence number WP_010922160.1):

[0538] MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL GVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLLSTGQDSWNQTN

[0539] Mature IdeS sequence lacking the N-terminal methionine and signal sequence (GenBank Accession No. ADF13949.1). (SEQ ID NO: 4):

[0540] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGY RLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFLTLSTGQDSWNQTN

[0541] SEQ ID NOs: 5-18 are sequences of exemplary IdeS polypeptides from Table C of WO 2016 / 128558.

[0542] SEQ ID NO:5:

[0543] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFRGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHIVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0544] SEQ ID NO:6:

[0545] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFKGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHIVIDMFINGYRLSLTNHGPTPVKRGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0546] SEQ ID NO:7:

[0547] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFRGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKKGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0548] SEQ ID NO:8:

[0549] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLREHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELDEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0550] SEQ ID NO:9:

[0551] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLKEHPEKQKINFNGEQMFDVKEAIRTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELEEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0552] SEQ ID NO:10:

[0553] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIERYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEKNIGAQVLGLFTLSTGQKSWNQTN

[0554] SEQ ID NO:11:

[0555] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLKEHPEKQKINFRGEQMFDVKEAIRTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKSELENGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0556] SEQ ID NO:12:

[0557] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLKEHPEKQKINFRGEQMFDVKEAIRTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKKGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELEEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0558] SEQ ID NO:13:

[0559] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIERYLEEHPEKQKINFRGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKKGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQKSWNQTN

[0560] SEQ ID NO:14:

[0561] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSW

[0562] SEQ ID NO:15:

[0563] SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0564] SEQ ID NO:16:

[0565] SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFKGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0566] SEQ ID NO:17:

[0567] SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIERYLEEHPEKQKINFKGEQMFDVKKAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQKSWNQTN

[0568] SEQ ID NO:18:

[0569] DDYQRNATEAYAKEVPHQITSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIERYLEEHPEKQKINFKGEQMFDVKKAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQKSWNQTNGGGHHHHHH

[0570] The IgdE of Streptococcus agalactiae is specific for human IgG1 (WO2017134274) (SEQ ID NO:19):

[0571] NQNNIQETNLVEKNSEDKFIQELNRYKTEIPNFKGFNVWILGDKGYYKNLINLEEIKNIQATLKKERNEEYVFVKLNGKIAHDTTVFLMNKKHKLLKNIEEFKTITQKRLTERGKFPYDTVHSTFEIKDENFIMERLKSSGLSMGKPVDYMGVNGIPIYTKTLSIDNKFAFENNSKDSSYSSNINISEDKIKENDQKILDLIVKSGANNQNLTDEEKVIAFTKYIGEITNYDNEAYRARNVDTEYYRASDLFSVTERKLAMCVGYSVTAARAFNIMGIPSYVVSGKSPQGISHAAVRAYYNRSWHIIDITASTYWKNGNYKTTYSDFIKEYCIDGYDVYDPAKTNNRFKVKYMESNEAFENWIHNNGSKSMLFINESAALKDKKPKDDFVPVTEKEKNELIDKYKKLLSQIPENTQNPGEKNIRDYLKNEYEEILKKDNLFEHEHAEFKESLNLNESFYLQLKKEEKKPSDNLKKEEKPRENSVKERETPAENNDFVSVTEKNNLIDKYKELLSKIPENTQNPGEKNIRNYLEKEYEELLQKDKLFKHEYTEFTKSLNLNETFYSQLKEGEMKLSENPEKGETNTN

[0572] IgdE of Streptococcus suis pseudoporcinus degrades human IgG1 and porcine IgG (WO2017134274) (SEQ ID NO:20):

[0573] RENENVRQLQSENKQMKAVNLQEFSEKLKGEIAENQQFHIFKLGLNNYYIGGVRINELSDLAKNHDFIMIDNRATHNKYGVPHIIIMNKDDVIVHNQEDYNKEMAELTFAGDKPIQSDSYLPQK KRIHALFEIIGLDSNRRQLLNAAGLKTPENSVIELDTFKIYSHGLAVDNKYYDEYSHFNNNTNVNITKQRFTENDNLIHNLITTSTAKDQPTDRDKVKTFVMYVANHTIYDWNAANNAVSNISDV NYYLGSDLFSITERKKAMCVGFSTTAARAFNMLGIPAYVVEGKNAQGVDHATARVYYNGKWHTIDGTGFINGNRTRSTLYTESHFRSVGEDSYQLVGLNEDIPFDRNYMKIDKVYEEWAPKQKT ADLLLVNKDKSLVGLDRVAYVEPVYVDKNRQDALTQIYKKLKETMESSSKKNPSSGGFSSLLGSASSDIAKLEGSSQLTQEEYDKIHRSMTSILTFFAQLDKDAAEAFEKGNDYKNYLATTKHAQ

[0574] The complete sequence of IdeZ is available as NCBI reference sequence number WP 014622780.1 (SEQ ID NO: 21). The sequence includes an N-terminal methionine followed by a 33 amino acid secretion signal sequence. The N-terminal methionine and signal sequence (34 amino acids total at the N-terminus) are typically removed to form the mature IdeZ protein:

[0575] MKTIAYPNKPHSLSAGLLTAIAIFSLASSNITYADDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSA RQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFFTLSSGKDIWQKLS

[0576] IdeZ sequence without the 34 amino acids from the N-terminus of the complete sequence (SEQ ID NO:22):

[0577] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFING YYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFFTLSSGKDIWQKLS

[0578] IdeS / Z hybrid sequence with the N-terminal portion based on IdeZ, without the N-terminal methionine and signal sequence (34 amino acids total at the N-terminus) (SEQ ID NO:23):

[0579] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVFHAPYVANQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGDNMFDVKKAIDTKNHQLDSKLFNYFKEKAFPGLSARRIGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKNKNLNDISTIIKQELTKGKALGLSHTYANVSINHVINLWGADFNAEGNLEAIYVTDSDSNASIGMKKYFVGVNAHGHVAISAKKIEGENIGAQVLGLFTLSTGQDSWQKLS

[0580] SEQ ID NOs: 24 - 43 correspond to peptides having modifications relative to IdeZ of SEQ ID NO: 22.

[0581] SEQ ID NO: 24:

[0582] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHAPYLANQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKEAIRTKDSQTNSQLFEYFRDKAFPYLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVKRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKEELTKGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINKHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLN

[0583] SEQ ID NO: 25:

[0584] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKTFNGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNEELFDLKAAIDTKDSQTNSQLFNYFKEKAFPNLSTRQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDFKEKGLKDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFDAEGNLKAIYVTDSDANASIGMKKYFVGINAHGKVAISAKKIEGENIGAQVLGLFTLSSGKDIWQQLS

[0585] SEQ ID NO:26:

[0586] DSFSANQEIRYSEVTPYHVTSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0587] SEQ ID NO:27:

[0588] SVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0589] SEQ ID NO:28:

[0590] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0591] SEQ ID NO:29:

[0592] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0593] SEQ ID NO:30:

[0594] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0595] SEQ ID NO:31:

[0596] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIINNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0597] SEQ ID NO:32:

[0598] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0599] SEQ ID NO:33:

[0600] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIIRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0601] SEQ ID NO:34:

[0602] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLANQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKEAIRTKDSQTNSQLFEYFRDKAFPYLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVKRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKEELTKGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINKHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLN

[0603] SEQ ID NO:35:

[0604] SVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0605] SEQ ID NO:36:

[0606] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGADNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLSSEQ ID NO:37:

[0607] SVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGADNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0608] SEQ ID NO:38:

[0609] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0610] SEQ ID NO:39:

[0611] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFTQGEDVFHAPYVANQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGENMFDVKKAIDTKNHQLDSKLFNYFKEKAFPYLSAKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKNKNLNDISTIIKQELTKGKALGLSHTYANVRINHVINLWGADFNAEGNLEAIYVTDSDSNASIGMKKYFVGVNAHGHVAISAKKIEGENIGAQVLGLFTLSTGQDSWQKLS

[0612] SEQ ID NO:40:

[0613] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFTQGEDVFHAPYVANQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFRGENMFDVKEAIRTKNHQLDSKLFEYFKEKAFPYLSAKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKKGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKNKNLNDISTIIKSELTNGKALGLSHTYANVRINHVINLWGADFNAEGNLEAIYVTDSDSNASIGMKKYFVGVNKHGHVAISAKKIEGENIGAQVLGLFTLSTGQDSWQKLN

[0614] SEQ ID NO:41:

[0615] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKQELTEGKALGLSHTYANVRINHVINLWGADFDAEGNLKAIYVTDSDSNASIGMKKYFVGVNAAGKVAISAKKIEGENIGAQVLGLFTLSTGQDSWNQTS

[0616] SEQ ID NO:42:

[0617] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFRGEQMFDVKEAIRTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKKGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKEKNLKEISDLIKEELTKGKALGLSHTYANVRINHVINLWGADFDAEGNLKAIYVTDSDSNASIGMKKYFVGVNKAGKVAISAKKIEGENIGAQVLGLFTLSTGQDSWNQTN

[0618] SEQ ID NO:43:

[0619] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYVANQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFRGEQMFDVKKAIDTKNHQLDSKLFNYFKEKAFPGLSARRIGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGNQSKLLTSRHDFKNKNLNDISTIIKQELTKGKALGLSHTYANVSINHVINLWGADFNAEGNLEAIYVTDSDSNASIGMKKYFVGVNAHGHVAISAKKIEGENIGAQVLGLFTLSTGQDSWQKLS

[0620] The protein sequence of endoglycosidase EndoS49 from Streptococcus pyogenes is shown in U.S. Patent 9,493,752 (SEQ ID NO:44):

[0621] MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKTDQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMAEVPKEVDILFVFHDHTASDSPFWSELKDSYVHKLHQQGTALVQTIGVNELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFTNKRTPEEDARALNVFKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFKGIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQFMIGFSFFEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKAGIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKTLMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGDKIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGMTGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKLLMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEHDILTDFVFGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFRKDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLNIGSGAIMMENLAKGAKVIGTSGDFEQAKKIFDGEKSDRFFTWGQTNWIAFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLEKMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDGGASSYYPQYTELQILGQRLSNDVANTLKD

[0622] The protein sequence of mature endoglycosidase S (EndoS) from Streptococcus pyogenes is shown in U.S. Patent Nos. 8,889,128 and 9,707,279 (SEQ ID NO:45):

[0623] EEKTVQVQKGLPSIDSLHYLSENSKKEFKEELSKAGQESQKVKEILAKAQQADKQAQELAKMKIPEKIPMKPLHGPLYGGYFRTWHDKTSDPTEKDKVNSMGELPKEVDLAFIFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIPWRFLAGGDNSGIAEDTSKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVDVEHDSIPKVDKKEDTAGVERSIQVFEEIGKLIGPKGVDKSRLFIMDSTYMADKNPLIERGAPYINLLLVQVYGSQGEKGGWEPVSNRPEKTMEERWQGYSKYIRPEQYMIGFSFYEENAQEGNLWYDINSRKDEDKANGINTDITGTRAERYARWQPKTGGVKGGIFSYAIDRDGVAHQPKKYAKQKEFKDATDNIFHSDYSVSKALKTVMLKDKSYDLIDEKDFPDKALREAVMAQVGTRKGDLERFNGTLRLDNPAIQSLEGLNKFKKLAQLDLIGLSRI TKLDRSVLPANMKPGKDTLETVLETYKKDNKEEPATIPPVSLKVSGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDTMLSTISNHVGSNEQTVKFDKQKPTGHYPDTYGKTSLRLPVANEKVDLQSQLLFGTVTNQGTLINSEADYKAYQNHKIAGRSFVDSNYHYNNFKVSYENYTVKTDSTLGTTTDKTLATDKEETYKVDFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVIGGSADPVNARKVFDGQLGSETDNISLGWDSKQSIIFKLKEDGLIKHWRFFNDSARNPETTNKPIQEASLQIFNIKDYNLLENPNKFDDEKYWITVDTYSAQGERATAFSNTLNNITSKYWRVVFDTKGDRYSSPVVPELQILGYPLPNADTIMKTVTTAKELSQQKDKFSQKMLDELKIKEMALETSLNSKIFDVTAINANAGVLKDCIEKRQLLKK

[0624] Includes the complete sequence of the secretion signal of the endoglycosidase EndoS from Streptococcus pyogenes (GenBank Accession No. AAK00850.1). (SEQ ID NO: 46):

[0625] MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTTVKAEEKTVQVQKGLPSIDSLHYLSENSKKEFKEELSKAGQESQKVKEILAKAQQADKQAQELAKMKIPEKIPMKPLHGPLYGGYFRTWHDKTSDPTEKDKVNSMGELPKEVDLAFIFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIPWRFLAGGDNSGIAEDTSKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVDVEHDSIPKVDKKEDTAGVERSIQVFEEIGKLIGPKGVDKSRLFIMDSTYMADKNPLIERGAPYINLLLVQVYGSQGEKGGWEPVSNRPEKTMEERWQGYSKYIRPEQYMIGFSFYEENAQEGNLWYDINSRKDEDKANGINTDITGTRAERYARWQPKTGGVKGGIFSYAIDRDGVAHQPKKYAKQKEFKDATDNIFHSDYSVSKALKTVMLKDKSYDLIDEKDFPDKALREAVMAQVGTRKGDLERFNGTLRLDNPAIQSLEG LNKFKKLAQLDLIGLSRITKLDRSVLPANMKPGKDTLETVLETTYKKDNKEEPATIPPVSLKVSGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDTMLSTISNHVGSNEQTVKFDKQKPTGHYPDTYGKTSLRLPVANEKVDLQSQLLFGTVTNQGTLINSEADYKAYQNHKIAGRSFVDSNYHYNNFKVSYENYTVKTDSTLGTTTDKTLATDKEETYKVDFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVIGGSADPVNARKVFDGQLGSETDNISLGWDSKQSIIFKLKEDGLIKHWRFFNDSARNPETTNKPIQEASLQIFNIKDYNLLENPNKFDDEKYWITVDTYSAQGERATAFSNTLNNITSKYWRVVFDTKGDRYSSPVVPELQILGYPLPNADTIMKTVTTAKELSQQKDKFSQKMLDELKIKEMALETSLNSKIFDVTAINANAGVLKDCIEKRQLLKK

[0626] The protein sequence of EndoS isolated from S. pyogenes AP1, including the signal sequence, is described in U.S. Patents 8,889,128 and 9,707,279 (SEQ ID NO:47):

[0627] MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTTVKAEEKTVQVQKGLPSIDSLHYLSENSKKEFKEELSKAGQESQKVKEILAKAQQADKQAQELAKMKIPEKIPMKPLHGPLYGGYFRTWHDKTSDPTEKDKVNSMGELPKEVDLAFIFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIPWRFLAGGDNSGIAEDTSKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVDVEHDSIPKVDKKEDTAGVERSIQVFEEIGKLIGPKGVDKSRLFIMDSTYMADKNPLIERGAPYINLLLVQVYGSQGEKGGWEPVSNRPEKTMEERWQGYSKYIRPEQYMIGFSFYEENAQEGNLWYDINSRKDEDKANGINTDITGTRAERYARWQPKTGGVKGGIFSYAIDRDGVAHQPKKYAKQKEFKDATDNIFHSDYSVSKALKTVMLKDKSYDLIDEKDFPDKALREAVMAQVGTRKGDLERFNGTLRLDNPAIQSLEG LNKFKKLAQLDLIGLSRITKLDRSVLPANMKPGKDTLETVLETTYKKDNKEEPATIPPVSLKVSGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDTMLSTISNHVGSNEQTVKFDKQKPTGHYPDTYGKTSLRLPVANEKVD...

Claims

1. Use of an immunoglobulin G degrading enzyme (IdeS) polypeptide of Streptococcus pyogenes in the preparation of a medicament for treating a humoral immune response caused by administering a recombinant gene therapy adeno-associated virus (AAV) vector, wherein the humoral immune response includes neutralizing antibodies that bind to the recombinant AAV vector.

2. Use of an immunoglobulin G degrading enzyme (IdeS) polypeptide of Streptococcus pyogenes in the preparation of a medicament for treating a disease treated by a recombinant gene therapy adeno-associated virus (AAV) vector in combination with a recombinant gene therapy AAV vector in a subject in need thereof, wherein the patient has neutralizing anti-AAV antibodies that inhibit cell transduction by the recombinant gene therapy AAV vector.

3. The use according to any one of claims 1-2, wherein the recombinant AAV vector comprises a capsid protein selected from the group consisting of AAV1, AAV2, AAV2 variant, AAV3, AAV3 variant, AAV3B, AAV3B variant, AAV4, AAV5, AAV6, AAV6 variant, AAV7, AAV8, AAV9, AAV10, AAVcy10, AAVrh10, AAVrh74, AAVdj, AAV-Anc80, SEQ ID NO: 1, SEQ ID NO: 2 or AAV2i8.

4. The use according to any one of claims 1-2, wherein the IdeS polypeptide is capable of cleaving all four human IgG subclasses at glycine residue 237 in the lower hinge region of the IgG heavy chain, while not cleaving IgA, IgM, IgD and IgE isotypes. 5 . The use according to any one of claims 1 to 2 , wherein the IdeS polypeptide comprises the sequence of SEQ ID NO: 4 or SEQ ID NO:

48. The use according to claim 5, wherein the IdeS polypeptide further comprises any non-native signal sequence in SEQ ID NOs: 49-51.

7. The use according to claim 2, wherein the disease treated by the recombinant gene therapy AAV vector is selected from the group consisting of proliferative diseases (cancer, tumors, dysplasia, etc.), Crigler-Najjar and metabolic diseases, such as metabolic diseases of the liver, Friedrich's ataxia, infectious diseases, addiction (e.g., tobacco, alcohol or drugs), epilepsy, Canavan disease, adrenoleukodystrophy, viral diseases (e.g., caused by hepatitis B or C virus, HIV, herpes, retroviruses, etc.), genetic diseases (cystic fibrosis, malnutrition diabetes, myopathy, such as Duchenne myopathy or muscular dystrophy, myotubular myopathy, hemophilia A, hemophilia B, hemophilia A with inhibitors, hemophilia B with inhibitors, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubular myopathy, motor neuron diseases, such as spinal muscular atrophy (SMA), spinal glomerular muscular atrophy or Charcot-Marie-Too th disease, arthritis, severe combined immunodeficiency (such as RS-SCID, ADA-SCID or X-SCID), Wiskott-Aldrich syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatous disease, etc.), coagulation factor deficiency, cardiovascular disease (restenosis, local ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.), ophthalmological diseases such as retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy and Stargardt disease; hereditary angioedema (HAE); lysosomal storage diseases such as Sanfilippo syndrome; hyperbilirubinemia (such as CN type I or II or Gilbert syndrome), Fabry disease, glycogen storage diseases (such as GSDI, GSDII (Pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and fatal congenital cardiac glycogen storage disease.

8. The use according to any one of claims 1-2, wherein the recombinant gene therapy AAV vector comprises a therapeutic polynucleotide suitable for treating the disease according to claim 7.

9. The use according to claim 2, wherein the patient is a subject who has been previously administered the recombinant gene therapy AAV vector and has produced antibodies that bind to and / or neutralize the recombinant gene therapy AAV vector, and is to be re-administered or re-administered with the recombinant gene therapy AAV vector.

10. The use according to claim 2, wherein the patient is a human.

11. The use according to any one of claims 1-2, wherein the sample is analyzed for the presence or amount of recombinant gene therapy AAV vector binding antibodies present in the biological sample from the subject before administration of the IdeS polypeptide, after administration of the IdeS polypeptide but before administration of the recombinant gene therapy AAV vector, and / or after administration of the IdeS polypeptide and the recombinant gene therapy AAV vector.

12. The use according to claim 11, wherein the biological sample is a blood product.

13. Use of a nucleic acid encoding an immunoglobulin G degrading enzyme (IdeS) polypeptide of Streptococcus pyogenes in the preparation of a medicament for use in combination with a recombinant gene therapy adeno-associated virus (AAV) vector for treating a disease treated by the recombinant gene therapy AAV vector in a subject in need thereof, wherein the patient has neutralizing anti-AAV antibodies that inhibit cell transduction by the recombinant gene therapy AAV vector. The use according to claim 13 , wherein the IdeS polypeptide comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 48.

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