Adeno-Associated Virus Vector for the Treatment of Hunter Disease

Abstract

Among other things, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid or an AAV9 capsid and a codon-optimized sequence encoding the human iduronate-2-sulfatase (I2S) enzyme. The present disclosure also provides a method of treating a subject with Hunter syndrome (MPS II), comprising administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid or an AAV9 capsid and a promoter operably linked to a nucleic acid sequence encoding iduronate-2-sulfatase (I2S), thereby increasing I2S enzyme activity in the subject.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Patent Application No. 62 / 945,920, filed on December 10, 2019, the content of which is incorporated herein by reference.

Background Art

[0002] Hunter syndrome (also known as mucopolysaccharidosis type II (MPS II)) is a lysosomal storage disease caused by the deficiency or absence of the enzyme iduronate - 2 - sulfatase (I2S). Iduronate - 2 - sulfatase is involved in the breakdown and recycling of certain mucopolysaccharides, also known as glycosaminoglycans or GAGs. Thus, in Hunter syndrome, GAGs accumulate in cells throughout the body, thereby interfering with the normal function of various cells and organs within the body, resulting in a number of severe symptoms. In many cases of Hunter syndrome, the massive accumulation of GAGs in the neurons and meninges of affected individuals leads to various forms of central nervous system (CNS) symptoms, cognitive impairment, and developmental delays.

[0003] In the management of Hunter syndrome, various treatment options, including enzyme replacement therapy (ERT), are used. An approved treatment regimen with ERT includes the intravenous administration of recombinant I2S enzyme. However, the I2S enzyme administered intravenously has various limitations, including poor distribution to the cells and tissues of the CNS, as well as poor distribution to the cells of deep somatic tissues such as the heart, lungs, and bones. The treatment of Hunter syndrome remains a challenging problem.

[0004] For the treatment of diseases, it is desirable to use vectors that produce therapeutic proteins in vivo, but it is limited by various factors, including poor in vivo production of the desired therapeutic proteins.

Summary of the Invention

[0005] The present invention provides an efficient and robust recombinant adeno-associated virus (rAAV) vector encoding I2S (throughout this application, I2S or IDS). The present invention is based in part on the surprising discovery that an optimized rAAV vector containing the I2S sequence robustly expresses I2S in vivo.

[0006] In some embodiments, the present invention provides a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a sequence encoding a human iduronate-2-sulfatase (I2S) enzyme.

[0007] In some embodiments, the present invention provides a recombinant adeno-associated virus (rAAV) vector comprising an AAV9 capsid and a sequence encoding a human iduronate-2 sulfatase (I2S) enzyme.

[0008] In some embodiments, the rAAV encodes a codon-optimized human I2S enzyme. In some embodiments, the codon-optimized human I2S has a nucleotide sequence selected from SEQ ID NO: 11 or 12.

[0009] In some embodiments, the sequence encoding the human I2S enzyme comprises a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% identity to SEQ ID NO: 6. In some embodiments, the human I2S enzyme is encoded by a nucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% identity to SEQ ID NO: 6. In some embodiments, the human I2S enzyme is encoded by the nucleotide sequence of SEQ ID NO: 6.

[0010] In some embodiments, the amino acid sequence of the human I2S enzyme comprises a sequence identical to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the human I2S enzyme is identical to SEQ ID NO: 1.

[0011] In some embodiments, the amino acid sequence of the human I2S enzyme comprises a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% identity to SEQ ID NO: 2.

[0012] In some embodiments, the sequence encoding the human I2S enzyme comprises a sequence identical to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the human I2S enzyme is a sequence identical to SEQ ID NO: 2.

[0013] In some embodiments, the codon-optimized sequence encoding the human I2S enzyme comprises a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% identity to SEQ ID NO: 11 or 12. In some embodiments, the codon-optimized sequence encoding the human I2S enzyme comprises a sequence identical to SEQ ID NO: 11 or 12.

[0014] In some embodiments, the vector further comprises a liver-specific promoter.

[0015] In some embodiments, the liver-specific promoter is the transthyretin promoter (TTR).

[0016] In some embodiments, the vector further comprises a 5' inverted terminal repeat (ITR) and a 3' ITR, an intron upstream of the I2S sequence, and a cis-acting regulatory module (CRM).

[0017] In some embodiments, the vector further comprises a ubiquitous promoter.

[0018] In some embodiments, the vector further comprises a 5' inverted terminal repeat and a 3' inverted terminal repeat, an intron upstream of the I2S sequence, and a cis-acting regulatory module (CRM).

[0019] In some embodiments, the rAAV vector comprises sulfatase modifying factor 1 (SUMF1).

[0020] In some embodiments, there is an internal ribosome entry site (IRES) upstream of the SUMF1.

[0021] In some embodiments, the vector further comprises a WPRE sequence. In some embodiments, the WPRE sequence is a variant WPRE sequence or an optimized WPRE sequence. In some embodiments, the WPRE sequence is encoded by a nucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% identity to SEQ ID NO: 7. In some embodiments, the WPRE sequence is encoded by a nucleotide sequence having SEQ ID NO: 7.

[0022] In some embodiments, the intron is an intron of murine minute virus (MVM) or SV40. In some embodiments, the intron is a β-globin / IgG chimeric intron.

[0023] In some embodiments, the CRM is a liver-specific CRM.

[0024] In some embodiments, the CRM is a neuron-specific CRM. In some embodiments, the CRM is a muscle-specific CRM.

[0025] In some embodiments, the CRM is CRM8.

[0026] In some embodiments, the vector comprises at least three CRMs.

[0027] In some embodiments, the present invention provides a recombinant adeno-associated virus (rAAV) comprising an AAV8 capsid and an rAAV vector, the vector comprising: a) a 5' inverted terminal repeat (ITR); b) a cis-acting regulatory module (CRM); c) a liver-specific promoter; d) murine minute virus (MVM); e) a sequence encoding a human iduronate-2-sulfatase (I2S) enzyme; f) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and g) a 3' ITR.

[0028] In some embodiments, the sequence encoding the human I2S enzyme is a wild-type sequence or a codon-optimized sequence.

[0029] In some embodiments, the nucleotide sequence encoding the human I2S enzyme is a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding human I2S is identical to SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the human I2S enzyme is a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 11 or 12. In some embodiments, the nucleotide sequence encoding the human I2S enzyme is identical to the sequence of SEQ ID NO: 11 or 12.

[0030] In some embodiments, it comprises a sequence encoding sulfatase modifying factor 1 (SUMF1) and an internal ribosome entry site (IRES).

[0031] In some embodiments, the present invention provides a recombinant adeno-associated virus (rAAV) comprising an AAV9 capsid and an rAAV vector, the vector comprising: a) a 5' inverted terminal repeat (ITR); b) a cis-acting regulatory module (CRM); c) a ubiquitous promoter; d) murine minute virus (MVM); e) a sequence encoding a human iduronate-2-sulfatase (I2S) enzyme; f) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and g) a 3' ITR.

[0032] In some embodiments, the sequence encoding the human I2S enzyme is a wild-type sequence or a codon-optimized sequence.

[0033] In some embodiments, the sequence includes a sequence encoding sulfatase modifying factor 1 (SUMF1) and an internal ribosome entry site (IRES).

[0034] In some embodiments, the rAAV vector does not contain ApoB.

[0035] A method for treating a subject with Hunter syndrome (MPS II), the method comprising administering to a subject in need thereof an rAAV according to any one of the preceding claims.

[0036] A method for treating a subject with Hunter syndrome (MPS II), the method comprising administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid or an AAV9 capsid and a promoter operably linked to a nucleic acid sequence encoding iduronate-2-sulfatase (I2S), wherein administration thereof improves I2S enzyme activity in the subject.

[0037] In some embodiments, the improvement in I2S activity is detected in the serum of the subject.

[0038] In some embodiments, the improvement in I2S activity is detected in the liver of the subject.

[0039] In some embodiments, I2S activity is detected in the central nervous system (CNS).

[0040] In some embodiments, the improvement in I2S activity is detected in the brain of the subject.

[0041] In some embodiments, the improvement in I2S activity is detected in the hippocampus, thalamus, corpus callosum, cortex, cerebellum or striatum of the brain.

[0042] In some embodiments, an improvement in I2S activity is detected in the kidney of a subject, among other organs. In some embodiments, an improvement in I2S activity is detected in the heart of a subject. In some embodiments, an improvement in I2S activity is detected in the lung of a subject. In some embodiments, an improvement in I2S activity is detected in the bone marrow of a subject. In some embodiments, an improvement in I2S activity is detected in the kidney of a subject.

[0043] In some embodiments, the improvement in I2S activity is maintained for at least 30, 60, 90, 120, 150, 180 days or more days after a single administration.

[0044] In some embodiments, the I2S activity level is measured by a heparin sulfate assay.

[0045] In some embodiments, the I2S activity level is measured by a dermatan sulfate assay.

[0046] In some embodiments, administration of the AAV reduces the level of glycosaminoglycan (GAG) in the subject.

[0047] In some embodiments, administration of the AAV reduces the level of GAG in the serum of the subject.

[0048] In some embodiments, administration of the AAV reduces the level of GAG in the liver of the subject.

[0049] In some embodiments, administering the AAV reduces the level of GAG in the kidney of the subject, among other organs. In some embodiments, administering the AAV reduces the level of GAG in the heart of the subject. In some embodiments, administering the AAV reduces the level of GAG in the lung of the subject. In some embodiments, administering the AAV reduces the level of GAG in the bone marrow of the subject. In some embodiments, administering the AAV reduces the level of GAG in the kidney of the subject.

[0050] In some embodiments, administering the AAV reduces the level of GAG in the CNS of the subject.

[0051] In some embodiments, administering the AAV reduces the level of GAG in the brain of the subject.

[0052] In some embodiments, administering the AAV reduces the level of GAG in the hippocampus, thalamus, corpus callosum, cortex, cerebellum, or striatum of the brain.

[0053] In some embodiments, the AAV is administered intravenously.

[0054] In some embodiments, the AAV is administered intrathecally.

[0055] In some embodiments, the AAV is administered at a dose of about 5×10 9 vg.

[0056] In some embodiments, administering the rAAV does not induce an immune response.

[0057] Various aspects of the present invention are described in detail in the following sections. The use of the sections is not intended to limit the present invention. Each section can be applied to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise specified. As used herein, unless the context clearly indicates otherwise, the singular forms with "a", "an", and "the" include both singular and plural referents.

Brief Description of the Drawings

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[0059] Definitions Adeno-associated virus (AAV): As used herein, the terms "adeno-associated virus", "AAV" or "recombinant AAV ("rAAV")" include, but are not limited to, AAV type 1, AAV type 2, AAV type 3 (including 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV (see, e.g., Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers), Gao et al., J. Virology 78:6381-6388 (2004), Mori et al., Virology 330:375-383 (2004)). Typically, AAV can infect both dividing and non-dividing cells and can exist in an episomal state without integrating into the host cell genome. AAV vectors are commonly used in gene therapy.

[0060] Administration: As used herein, the terms "administer" or "introduce" are used synonymously in the context of delivering an rAAV vector encoding I2S to a subject by a method or route that efficiently delivers the rAAV vector. Various methods for the administration of rAAV vectors (including, for example, intravenous, subcutaneous, or transdermal administration) are known in the art. Transdermal administration of an rAAV vector can be performed by using a "gene gun" or a biolistic particle delivery system. In some embodiments, a composition of any of an rAAV vector and / or a transgene expression cassette and / or an optimized IDS transgene sequence and / or its gene expression cassette is administered via non-viral chemical particles such as lipid nanoparticles, non-viral biomolecules such as exosomes, and / or extracellular vesicles.

[0061] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0062] Known immunoglobulin polypeptides include kappa and lambda light chains, and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu heavy chains, or equivalents in other species. The "light chain" of a full-length immunoglobulin (about 25 kDa or about 214 amino acids) includes a variable region of about 110 amino acids at the NH2 terminus and a kappa or lambda constant region at the COOH terminus. The "heavy chain" of a full-length immunoglobulin (about 50 kDa or about 446 amino acids) similarly includes a variable region (about 116 amino acids) and one of the heavy chain constant regions described above, such as a gamma constant region (about 330 amino acids).

[0063] Approximately or about: As used herein, the terms "approximately" or "about" as applied to one or more values of interest refer to values that are comparable to the recited reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the recited reference value in either direction (positive or negative), unless otherwise indicated or otherwise apparent from the context (provided such numbers do not exceed 100% of the possible values).

[0064] Biologically active: As used herein, the phrase "biologically active" refers to the characteristic of any agent that has activity in a biological system and particularly in a living organism. For example, an agent that has a biological effect on a living organism when administered to that organism is considered to be biologically active. In certain embodiments where a peptide is biologically active, the portion of the peptide that has at least one of the biological activities of the peptide is typically referred to as the "biologically active" portion.

[0065] Functional equivalent or functional derivative: As used herein, the terms "functional equivalent" or "functional derivative" in the context of a functional derivative of an amino acid sequence refer to a molecule that retains a biological activity (either a function or a structure) that is substantially the same as the biological activity of the original sequence. A functional derivative or functional equivalent may be a natural derivative or may be prepared synthetically. Exemplary functional derivatives include amino acid sequences in which one or more amino acids have been substituted, deleted or added, provided that the biological activity of the protein is conserved. The substituting amino acid preferably has physicochemical properties similar to those of the amino acid being substituted. Desirable similar physicochemical properties include similar charge, bulk, hydrophobicity, hydrophilicity, etc.

[0066] In vitro: As used herein, the term "in vitro" refers to events that occur not within a multicellular organism but in an artificial environment, such as a test tube, reaction vessel, cell culture medium, etc.

[0067] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism such as a human and non-human animals. In the context of cell-based systems, this term may be used to refer to events that occur within living cells (e.g., as contrasted with an in vitro system).

[0068] IRES: As used herein, the term "IRES" refers to any suitable internal ribosome entry site sequence.

[0069] Isolation: As used herein, the term "isolated" means (1) separated from at least a portion of the components that were associated therewith when initially produced (whether in nature and / or in an experimental setting), and / or (2) a substance and / or entity that has been generated, prepared, and / or manufactured by human hand. An isolated substance and / or entity may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100% or 100% of the other components that were initially associated with the substance and / or entity. In some embodiments, the purity of the isolated agent is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100% or 100%. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the term "isolated cell" refers to a cell that is not contained within a multicellular organism.

[0070] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked by peptide bonds. This term is used for the purpose of referring to amino acid chains of any length, and one of ordinary skill in the art will understand that this term is not limited to long chains and can also refer to the shortest chain containing two amino acids linked by a peptide bond. As is known to those of ordinary skill in the art, polypeptides may be processed and / or modified.

[0071] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as discrete units. The terms "polypeptide" and "protein" may be used interchangeably where one polypeptide is a discrete functional unit and does not need to permanently or transiently physically associate with other polypeptides for the purpose of forming a discrete functional unit. Where a discrete functional unit is composed of two or more polypeptides that physically associate with each other, the term "protein" refers to a plurality of polypeptides that physically associate and function together as a discrete unit.

[0072] Regulatory element: As used herein, the term "regulatory element" refers to a transcriptional control element, particularly a non-coding cis-acting transcriptional control element that can regulate and / or control the transcription of a gene. A regulatory element includes at least one transcription factor binding site, for example, at least one binding site for a tissue-specific transcription factor. In the embodiments described herein, the regulatory element has at least one binding site for a liver-specific transcription factor. Typically, in the absence of a regulatory element, a regulatory element increases or enhances gene expression driven by a promoter as compared to transcribing a gene from the promoter alone. That is, a regulatory element particularly includes an enhancer sequence, but it should be understood that a regulatory element that enhances transcription is not limited to a typical enhancer sequence that is far upstream, and a regulatory element can be present regardless of the distance from the gene it regulates. As is understood in the art, a sequence that regulates transcription can be either upstream (e.g., promoter region) or downstream (e.g., 3'UTR) of the gene being regulated in vivo, and can be located either in close proximity to or far from the gene. A regulatory element can include any of a natural sequence, such a regulatory element or some copies of a regulatory element, e.g., a combination including a non-natural sequence (portion). Thus, a regulatory element includes natural regulatory elements and optimized or engineered regulatory elements to achieve a desired expression level.

[0073] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include both prenatal and postnatal forms. In many embodiments, the subject is a human. The subject can be a patient, where a patient refers to a human who is receiving medical care from a healthcare provider for the diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". The subject can be afflicted with, or be susceptible to, a disease or disorder, and may or may not exhibit symptoms of the disease or disorder.

[0074] Substantially: As used herein, the term "substantially" refers to a qualitative state where the relevant feature or characteristic is at a complete or nearly complete degree or extent. In biological and chemical phenomena, those skilled in the art in the biological field will understand that it is rare, if at all, for a biological or chemical phenomenon to be complete and / or proceed to a complete state, or for a result without doubt to be achieved or avoided. Thus, the term "substantially" is used herein for the purpose of indicating the potential lack of completeness that pertains to many biological and chemical phenomena.

[0075] Substantial identity: The term "substantial identity" is used herein for the purpose of referring to the result of comparison between amino acid sequences or nucleic acid sequences. As will be apparent to those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain homologous residues at corresponding positions. The homologous residues may be identical residues. Alternatively, the homologous residues may be non-identical residues having similar structural and / or functional characteristics as appropriate. For example, as is well known to those skilled in the art, certain amino acids are typically classified as "hydrophobic" amino acids or "hydrophilic" amino acids, and / or amino acids having "polar" side chains, or amino acids having "non-polar" side chains. Substituting one amino acid with another amino acid of the same type is often considered to be a "homologous" substitution.

[0076] As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990, Altschul, et al., Methods in Enzymology, Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402, 1997, Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998, and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. The above programs typically provide a measure of the degree of homology in addition to identifying homologous sequences. In some embodiments, two sequences are considered substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over the relevant residue regions. In some embodiments, the relevant region is the complete sequence.In some embodiments, the relevant region is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0077] Substantial identity: The term "substantial identity" is used herein for the purpose of referring to the result of comparison between amino acid sequences or nucleic acid sequences. As will be apparent to those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary of such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990, Altschul, et al., Methods in Enzymology, Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997, Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998 and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. The above programs typically provide a measure of the degree of identity in addition to identifying identical sequences. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant residue regions. In some embodiments, the relevant region is the complete sequence.In some embodiments, the associated region is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0078] Suffering from: An individual "suffering from" a disease, disorder and / or condition has been diagnosed with that disease, disorder and / or condition or exhibits one or more symptoms of that disease, disorder and / or condition.

[0079] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent, when administered to a subject suffering from or susceptible to a disease, disorder and / or condition, is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of the symptoms of that disease, disorder and / or condition. It will be apparent to those skilled in the art that a therapeutically effective amount is typically administered by a dosing regimen that includes at least one unit dose.

[0080] Treatment: As used herein, the terms "treating", "treatment" or "treat" refer to any method used for the purpose of partially or completely alleviating, ameliorating, reducing, inhibiting, preventing, delaying the onset of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder and / or condition. Treatment may be performed on a subject in whom no signs of the disease are present and / or in whom only early signs of the disease are present, for the purpose of reducing the risk of developing the pathological conditions associated with that disease.

[0081] When a numerical range is indicated by endpoint values in this specification, all numbers and fractions included within that range are included (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4, and 5). It should also be understood that all such numbers and fractions are presumed to be modified by the term "about".

[0082] Various aspects of the present invention are described in detail in the following sections. The use of sections does not limit the present invention. Each section is applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated. As used in this specification, unless the context clearly indicates otherwise, the singular forms with "a", "an", and "the" include both singular and plural referents.

[0083] Various aspects of the present invention are described in detail in the following sections. The use of sections does not limit the present invention. Each section is applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated.

Mode for Carrying Out the Invention

[0084] The present disclosure describes an efficient and robust recombinant adeno-associated virus (rAAV) vector for in vivo production of I2S for the treatment of diseases associated with I2S deficiency (such as Hunter syndrome).

[0085] Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal storage disorder caused by a deficiency of the enzyme iduronate-2-sulfatase (I2S). I2S cleaves the terminal 2-O-sulfate moiety from the glycosaminoglycans (GAGs) dermatan sulfate and heparan sulfate. In patients with Hunter syndrome, the absence or deficiency of the I2S enzyme causes GAGs to progressively accumulate in the lysosomes of various types of cells, resulting in cell swelling, organ enlargement, tissue destruction, and organ system dysfunction.

[0086] Generally, the physical manifestations in people with Hunter syndrome include both physical and neurological symptoms. For example, in some cases of Hunter syndrome, lesions in the central nervous system (CNS) cause developmental delays and neurological problems. Symptoms such as neurodegeneration and mental retardation appear in childhood, and Hunter syndrome patients with neurological effects often die at a young age due to brain organ damage. Similarly, the accumulation of GAGs can have an adverse effect on the body's organ systems. Initially, it manifests as hypertrophy of the heart, lungs, and airway walls, as well as abnormal enlargement of the liver, spleen, and kidneys, and these significant changes can ultimately lead to extensive and catastrophic organ failure. As a result, Hunter syndrome is always a severe and progressive fatal disease.

[0087] Enzyme replacement therapy (ERT) is a therapy approved for the treatment of Hunter syndrome (MPS II) and involves administering exogenous replacement I2S enzyme to Hunter syndrome patients. However, ERT has various drawbacks, among which, for example, the limited distribution of I2S to various target organs can be cited.

[0088] The vectors described herein robustly express I2S in various organs including, for example, the liver, kidney, spleen, heart, lungs, and central nervous system. Thus, in some embodiments, the vectors described herein express I2S in the liver. In some embodiments, the vectors described herein express I2S in the kidney. In some embodiments, the vectors described herein express I2S in the spleen. In some embodiments, the vectors described herein express I2S in the heart. In some embodiments, the vectors described herein express I2S in the lungs. In some embodiments, the vectors described herein express I2S in the central nervous system. In some embodiments, the vectors described herein express I2S in the plasma.

[0089] Design of the rAAV I2S Vector In some embodiments, provided by the present invention is a recombinant adeno-associated virus (rAAV) vector encoding iduronate-2-sulfatase (I2S) protein. A schematic diagram showing an exemplary rAAV vector of the present disclosure is shown in FIG. 1B. As shown in FIG. 1B, in some embodiments, the rAAV vector of the present disclosure includes a liver-specific promoter, 5' inverted terminal repeat (ITR) and 3' ITR, cis-acting regulatory module (CRM), intron and WPRE sequence.

[0090] In some embodiments, the vector also includes the sulfatase modifying factor 1 (SUMF) gene. In some embodiments, the vector includes an internal ribosome entry site (IRES).

[0091] The iduronate-2-sulfatase (I2S) of the vector can be a wild-type or codon-optimized variant. Thus, in some embodiments, the rAAV vector includes a wild-type I2S nucleotide sequence. In some embodiments, it includes a codon-optimized I2S sequence.

[0092] Suitable I2S for the present invention is any protein or protein moiety that can replace at least a partial activity of the native iduronate-2-sulfatase (I2S) protein or can rescue one or more of the phenotypes or symptoms associated with I2S deficiency. As used herein, the terms "I2S enzyme" and "I2S protein", as well as grammatically equivalent terms, are used synonymously.

[0093] Typically, human I2S protein is produced in a precursor form. The precursor form of human I2S contains a signal peptide (amino acid residues 1 - 25 of its full-length precursor), a propeptide (amino acid residues 26 - 33 of its full-length precursor), and a chain that can be further processed into a 42 kDa chain (residues 34 - 455 of its full-length precursor) and a 14 kDa chain (residues 446 - 550 of its full-length precursor) (residues 34 - 550 of its full-length precursor). Typically, its precursor form, also referred to as the full-length precursor or full-length I2S protein, contains 550 amino acids. The amino acid sequences of a typical wild-type, i.e., natural, human I2S protein in its mature form (SEQ ID NO: 1) with the signal peptide removed and the full-length precursor (SEQ ID NO: 2) are shown in Table 1. The signal peptide is underlined.

[0094]

Table 1

[0095] In the rAAV vectors described herein, various types of promoters can be used. Those promoters include, for example, ubiquitous promoters, tissue-specific promoters, and regulatable (e.g., inducible or repressible) promoters. In some embodiments, the promoter is a liver-specific promoter. Examples of liver-specific promoters are known in the art and include, for example, the human transthyretin promoter (TTR), the α1-antitrypsin promoter, the human precursor form of factor IX / liver transcription factor-responsive oligomer, LSP, and the basic albumin promoter. Liver-specific promoters are described, for example, in Zhijian Wu et al., Molecular Therapy vol 16, no 2, February 2008, the content of which is hereby incorporated by reference in its entirety.

[0096] In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is a chicken β-actin promoter.

[0097] In some embodiments, the rAAV vector includes additional enhancer or regulatory elements (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, IRES, etc.) to promote transcription and / or translation of its mRNA. In some embodiments, the vector includes 5' inverted terminal repeats (ITRs) and 3' ITRs. In some embodiments, the vector includes one or more enhancer elements. In some embodiments, the vector includes a poly(A) tail.

[0098] In some embodiments, the rAAV vector includes one or more small elements such as introns. In the art, various introns are known. Examples of introns suitable for the rAAV vectors described herein include, for example, MVM intron, truncated F.IX intron, chimeric β-globin SD / immunoglobulin heavy chain SA intron, SV40 and / or the first intron of α-globin. In some embodiments, the rAAV vector includes the MVM intron. In some embodiments, the rAAV vector includes the SV40 intron.

[0099] In some embodiments, the rAAV vector includes the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In the art, various optimized or variant forms of WPRE are known, including, inter alia, WPRE3, WPREmut6delATG. Other variant WPRE forms include, for example, WPRE2, WPRE_wt (GenBank accession number J04514), WPRE_wt (GenBank accession number J02442), and WPREmut6.

[0100] In some embodiments, the rAAV vector comprises a cis-acting regulatory module (CRM). Various types of CRMs are suitable for use in the vectors described herein, and examples of such CRMs include liver-specific CRMs and neuron-specific CRMs. In some embodiments, the vectors described herein include a hepatocyte-specific CRM, such as CRM8. In some embodiments, the vector includes two or more CRMs. For example, in some embodiments, the vector includes two, three, four, five, or six CRMs. In some embodiments, the vector includes three CRMs, for example, three CRM8s.

[0101] In some embodiments, the rAAV vector is optimized in sequence to improve the stability of the transcript and reduce immunogenicity for improved translation efficiency. In some embodiments, the I2S is optimized in sequence.

[0102] In some embodiments, the rAAV vector is an AAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV10 vector, or an AAV11 vector. In some embodiments, the rAAV vector is AAV1. In some embodiments, the rAAV vector is AAV2. In some embodiments, the rAAV vector is AAV3. In some embodiments, the rAAV vector is AAV4. In some embodiments, the rAAV vector is AAV5. In some embodiments, the rAAV vector is AAV6. In some embodiments, the rAAV vector is AAV7. In some embodiments, the rAAV vector is AAV8. In some embodiments, the rAAV vector is AAV9. In some embodiments, the rAAV vector is AAV10. In some embodiments, the rAAV vector is AAV11.

[0103] An exemplary array of elements is shown in Table 2 below. In some embodiments, the rAAV vector comprises an rAAV vector element having a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the vector element array shown in Table 2. In some embodiments, the rAAV vector comprises a vector element nucleotide sequence identical to the vector element nucleotide sequence shown in Table 2. In the table, Xn(60-100) means a DNA titer measurement tag containing 60 to 100 nucleotides.

[0104] [Table 2-1]

[0105] [Table 2-2]

[0106] [Table 2-3]

[0107] [Table 2-4]

[0108] In some embodiments, the rAAV I2S vector comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the nucleotide sequence shown in Table 3 below. In some embodiments, the rAAV I2S vector comprises a sequence identical to the nucleotide sequence shown in Table 3 below.

[0109] [Table 3-1]

[0110]

Table 3-2

[0111]

Table 3-3

[0112]

Table 3-4

[0113]

Table 3-5

[0114]

Table 3-6

[0115]

Table 3-7

[0116]

Table 3-8

[0117]

Table 3-9

[0118]

Table 3-10

[0119]

Table 3-11

[0120]

Table 3-12

[0121]

Table 3-13

[0122]

Table 3-14

[0123]

Table 3-15

[0124]

Table 3-16

[0125]

Table 3-17

[0126]

Table 3-18

[0127]

Table 3-19

[0128]

Table 3-20

[0129]

Table 3-21

[0130]

Table 3-22

[0131]

Table 3-23

[0132]

Table 3-24

[0133]

Table 3-25

[0134]

Table 3-26

[0135]

Table 3-27

[0136]

Table 3-28

[0137]

Table 3-29

[0138]

Table 3-30

[0139]

Table 3-31

[0140] Use of an rAAV vector encoding I2S for the treatment of a disease Described herein are methods for treating diseases associated with a deficiency of I2S enzyme. Thus, in some embodiments, the rAAV vectors described herein are suitable for treating subjects with a deficiency of I2S, such as Hunter syndrome (MPSII). The treatment method includes administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) vector as described herein.

[0141] Any disease or disorder associated with an I2S deficiency can be treated using the rAAV vectors described herein.

[0142] In some embodiments, the rAAV vector remains episomal after administration to a subject in need thereof. In some embodiments, the rAAV vector does not remain episomal after administration to a subject in need thereof. For example, in some embodiments, the rAAV vector is integrated into the genome of the subject. Such integration can be achieved, for example, by using various gene editing techniques such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), ARCUS genome editing, and / or CRISPR-Cas systems.

[0143] In some embodiments, a subject in need of treatment is treated using a pharmaceutical composition comprising the rAAV vectors described herein. The pharmaceutical composition comprising the rAAV vectors or particles of the present invention comprises a pharmaceutically acceptable excipient, diluent, or carrier. Examples of suitable pharmaceutical carriers are well known in the art and include, as carriers, phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to the subject in a therapeutically effective amount.

[0144] The rAAV vector of the present invention is administered to a subject in need of treatment via an appropriate route. In embodiments, the rAAV vector is administered by intravenous administration, intraperitoneal administration, subcutaneous administration, or intradermal administration. In embodiments, the rAAV vector is administered intravenously. In embodiments, the intradermal administration includes administering by using a "gene gun" or a biolistic particle delivery system. In some embodiments, the rAAV vector of the present invention is administered via non-viral lipid nanoparticles. For example, the composition containing the rAAV vector may include one or more diluents, buffers, liposomes, lipids, lipid complexes. In some embodiments, the rAAV vector is contained within microspheres or within nanoparticles such as lipid nanoparticles. In some embodiments, the rAAV vector and / or the transgene expression cassette and / or the optimized IDS transgene sequence and / or any composition of the gene expression cassette thereof is administered via non-viral chemical particles such as lipid nanoparticles, non-viral biomolecules such as exosomes, and / or extracellular vesicles.

[0145] In some embodiments, functional I2S is detectable in the plasma or serum of the subject about 2 to 6 weeks after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at about 2 weeks. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at about 3 weeks. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at about 4 weeks. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at about 5 weeks. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at about 6 weeks. In some embodiments, functional I2S is detectable in the hepatocytes of the subject about 2 to 6 weeks after administration of the rAAV vector.

[0146] In some embodiments, functional I2S is detectable in the plasma of the subject at least 3 months, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years or 10 years after administration of the rAAV vector. Thus, in some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 3 months after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 6 months after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 12 months after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 2 years after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 3 years after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 4 years after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 5 years after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 6 years after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 7 years after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 8 years after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 9 years after administration of the rAAV vector. In some embodiments, functional I2S is detectable in the plasma or serum of the subject at least 10 years after administration of the rAAV vector.In some embodiments, functional I2S is detectable in the plasma or serum of the subject for the remainder of the subject's life after administration of the rAAV vector.

[0147] In some embodiments, administration of the rAAV comprising the I2S produces active I2S to the same extent as seen after intravenous administration of purified I2S protein. In some embodiments, administration of the rAAV comprising the I2S produces a greater amount of active I2S than when purified I2S protein is administered intravenously.

[0148] In some embodiments, administration of rAAV containing I2S reduces glycosaminoglycan (GAG) in the subject. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15% or about 10% compared to the baseline GAG level of the subject before administration of rAAV containing I2S. Thus, in some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 95%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 90%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 85%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 80%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 75%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 70%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 65%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 60%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 55%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 50%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 45%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 40%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 35%. In some embodiments, administration of rAAV containing I2S reduces GAG in the subject by about 30%.In some embodiments, administration of rAAV comprising I2S results in a reduction of GAG in the subject by about 25%. In some embodiments, administration of rAAV comprising I2S results in a reduction of GAG in the subject by about 20%. In some embodiments, administration of rAAV comprising I2S results in a reduction of GAG in the subject by about 15%. In some embodiments, administration of rAAV comprising I2S results in a reduction of GAG in the subject by about 10%.

[0149] In some embodiments, administration of rAAV comprising I2S results in a reduction of GAG in the subject for at least about 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years or more than 5 years.

[0150] In some embodiments, after administration of the AAV vector to the subject, the level of functional I2S detectable in the circulating blood is about 2 to 10 times higher than the amount of functional I2S detectable in the subject prior to administration of rAAV comprising I2S.

[0151] In some embodiments, after administration of the AAV vector to the subject, the level of active I2S that is detectable meets or exceeds human therapeutic levels. In some embodiments, the level of active I2S after administration of the rAAV vector is about 2 to 35 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 2 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 3 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 4 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 5 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 6 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 6 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 7 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 8 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 9 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 10 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 15 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 20 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 25 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 30 times the human therapeutic level. In some embodiments, the level of active I2S after administration is about 35 times the human therapeutic level.

[0152] That is, administration of the rAAV vector containing I2S results in robust and sustained expression compared to administering purified I2S once to the required subject.

[0153] In some embodiments, the rAAV I2S vector is delivered as a single dose per subject. In some embodiments, the subject is delivered a minimum effective dose (MED). As used herein, MED refers to the dose of the rAAV I2S vector required to produce I2S activity that reduces GAG levels in the subject.

[0154] Vector titer is determined based on the DNA content of the vector preparation. In some embodiments, quantitative PCR or optimized quantitative PCR is used to determine the DNA content of the rAAV I2S vector preparation. In one embodiment, the dosage is about 1×10 11 vector genomes (vg) per kg of body weight to about 1×10 13 vg / kg (including the end points).

[0155] In one embodiment, the dosage is selected in the range of 1×10 9 vg / kg to 3×10 15 vg / kg (e.g., 1×10 9 vg / kg, 3×10 9 vg / kg, 1×10 10 vg / kg, 3×10 10 vg / kg, 1×10 11 vg / kg, 3×10 11 vg / kg, 1×10 12 vg / kg, 3×10 12 vg / kg, 1×10 13 vg / kg, 3×10 13 vg / kg, 1×10 14 vg / kg, 3×10 14 vg / kg, 1×10 15 vg / kg, 3×10 15 vg / kg). In some embodiments, the dosage is 5×10 13 vg / kg. In another embodiment, the dosage is 5×10 12 vg / kg. In a specific embodiment, the dosage of rAAV administered to the subject is at least 5×10 11 vg / kg, 1×10 12 vg / kg, 1.5×10 12vg / kg, 2.0×10 12 vg / kg, 2.5×10 12 vg / kg, 3.0×10 12 vg / kg, 3.5×10 12 vg / kg, 4.0×10 12 vg / kg, 4.5×10 12 vg / kg, 5.0×10 12 vg / kg, 5.5×10 12 vg / kg, 6.0×10 12 vg / kg, 6.5×10 12 vg / kg, 7.0×10 12 vg / kg or 7.5×10 12 vg / kg.

[0156] In some embodiments, the rAAV I2S vector composition can be formulated in a dosage unit to contain an amount of replication-deficient virus in the range of about 1.0×10 9 vg to about 1.0×10 15 vg. As used herein, the term "dosage" can refer to the total dosage delivered to a subject during a course of treatment, or the amount delivered in a single administration (of multiple administrations).

[0157] In some embodiments, the dosage is an amount sufficient to reduce plasma GAG levels by 25% or more in a patient. In some embodiments, rAAV I2S is administered in combination with one or more therapies for treating Hunter syndrome.

[0158] Production of rAAV viral vectors Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. Patent No. 7,790,449, U.S. Patent No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and US7588772B2. In one system, producer cell lines are transiently transfected with a construct encoding a transgene flanked by ITRs and a construct (s) encoding rep and cap. In a second system, packaging cell lines that stably supply rep and cap are transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or helper herpesvirus, and the rAAV needs to be separated from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to recover AAV, i.e., the system also supplies adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, as well as the herpesvirus polymerase in trans. In these new systems, the helper function can be supplied by transiently transfecting cells with a construct encoding the necessary helper function, or the cells can be engineered to stably contain the gene encoding the helper function, the expression of which can be controlled at the transcriptional or post-transcriptional level.

[0159] In some embodiments, infection with a baculovirus-based vector introduces an expression cassette flanked by ITRs and the rep / cap genes into a desired cell or cell line.

[0160] In some embodiments, infection of a baculovirus-based vector introduces an expression cassette flanked by ITRs and the rep / cap genes into insect cells. For a review of these production systems, generally see, for example, Zhang et al, 2009, “Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production,” Human Gene Therapy 20:922-929 (the contents of which are incorporated herein by reference in their entirety). Also, methods of making and using the above and other AAV production systems are described in U.S. Pat. Nos. 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065 (the contents of each are incorporated herein by reference in their entirety). Generally see, for example, Grieger & Samulski, 2005, “Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications,” Adv. Biochem. Engin / Biotechnol. 99:119-145, Buning et al, 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med 10:717-733, and the references cited below (each incorporated herein by reference in their entirety).

[0161] The methods used to construct any of the embodiments of the present invention are known to those with knowledge in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for generating rAAV virions are well known, and the selection of an appropriate method is not limiting of the present invention. See, for example, K. Fisher et al, (1993) J. Virol, 70:520-532 and U.S. Patent No. 5,478,745.

[0162] Many plasmids that can be used in accordance with the present invention, as well as other cloning vectors and expression vectors, are well known to those skilled in the art and are readily available to them. Furthermore, those skilled in the art can readily construct any number of other plasmids suitable for use in the present invention. The characteristics, construction, and methods of use of such plasmids and other vectors in the present invention will be readily apparent to those skilled in the art from the present disclosure.

[0163] In one embodiment, the production plasmid is as described herein or as described in WO2012 / 158757 (incorporated herein by reference). In the art, various plasmids are known for use in the production of rAAV vectors and are useful in the present invention. The production plasmid is cultured in a host cell such that the cell expresses the AAV cap protein and / or rep protein. In the host cell, each rAAV genome is rescued and packaged into its capsid protein or envelope protein to form infectious virus particles.

[0164] In certain embodiments, the rAAV expression cassette, vector (such as an rAAV vector), virus (such as rAAV), and production plasmid include AAV inverted terminal repeat sequences, codon-optimized nucleic acid sequences encoding IDS and / or SUMF-1, and expression control sequences that induce expression of the encoded protein in a host cell. In another embodiment, the rAAV expression cassette, virus, vector (such as an rAAV vector), and production plasmid further include one or more of an intron, Kozak sequence, polyA, post-transcriptional regulatory element, and the like. In one embodiment, the post-transcriptional regulatory element is the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

[0165] In the art, various methods are known for the production and purification of AAV vectors. See, for example, Mizukami, Hiroaki, et al. A Protocol for AAV vector production and purification, U.S. Patent Application Publication Nos. 20070015238 and 20120322861. For example, a plasmid containing the gene may be combined with one or more helper plasmids containing, for example, a rep gene (such as a gene encoding Rep78, Rep68, Rep52, and Rep40) and a cap gene (a gene encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), transfected into a recombinant cell to package the rAAV, and later purified.

[0166] In some embodiments, the packaging is performed in helper cells or producer cells such as mammalian cells or insect cells. Exemplary mammalian cells include, but are not limited to, HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (see, for example, ATCC® CRL-1573™, ATCC® CRL-1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-10™, or ATCC® CCL-61™). Exemplary insect cells include, but are not limited to, Sf9 cells (see, for example, ATCC® CRL-1711™). The helper cells may contain a rep gene and / or a cap gene encoding a Rep protein and / or a Cap protein used in the methods described herein. In some embodiments, the packaging is performed in vitro.

[0167] In some embodiments, the plasmid containing the gene is combined with one or more helper plasmids containing, for example, a rep gene of a first serotype and a cap gene of the same or a different serotype, and transfected into helper cells so that the rAAV is packaged.

[0168] In some embodiments, one or more of the helper plasmids include a first helper plasmid containing a rep gene and a cap gene, and a second helper plasmid containing one or more of the helper genes, namely, the Ela gene, the Elb gene, the E4 gene, the E2a gene, and the VA gene. For clarity, the helper genes are genes encoding the helper proteins Ela, Elb, E4, E2a, and VA. In some embodiments, the cap gene is modified such that one or more of the VP1 protein, the VP2 protein, and the VP3 protein are not expressed. In some embodiments, the cap gene is modified such that VP2 is not expressed. Methods for making such modifications are known in the art (Lux et al. (2005), J Virology, 79:11776-87).

[0169] Helper plasmids, and methods of making helper plasmids, are generally known in the art and are generally commercially available (e.g., pDF6, pRep, pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory (Bielefeld, Germany), other products and services available from Vector Biolabs (Philadelphia, PA), Cellbiolabs (San Diego, CA), Agilent Technologies (Santa Clara, Ca), and Addgene (Cambridge, MA), pxx6, Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760, Kem, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081., Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850, Kronenberg et al. (2005); see also).

Example

[0170] Other features, objects, and advantages of the present invention will become apparent from the following examples. However, it should be understood that the examples, while showing embodiments of the present invention, are presented by way of illustration only and are not limiting. Various changes and modifications within the scope of the present invention will be apparent to those skilled in the art from the examples.

[0171] Example 1. Vector Design In this example, an exemplary method for creating an rAAV expression construct (rAAV vector) containing the coding sequence of human iduronate-2-sulfatase (IDS or I2S) and its variant forms, and its exemplary design are shown. In this study, a recombinant AAV vector (rAAV8) was used. The basic design of the rAAV vector includes an expression cassette flanked by inverted terminal repeats (ITRs), namely the 5'-ITR and 3'-ITR. These ITRs mediate the replication and packaging of the vector genome by the AAV replication protein Rep and related factors within the vector producer cells. Typically, as shown in Figure 1A, the expression cassette includes a promoter, a coding sequence, a polyA tail, and / or a tag. The expression construct encoding human IDS (hIDS) was designed and prepared using standard molecular biology techniques. The coding sequence of hIDS was inserted downstream of the promoter, hTTR (human transthyretin promoter). In addition, a liver-specific cis-acting regulatory module (CRM) was inserted upstream of the promoter, and the mouse minute virus (MVM) intron sequence was inserted downstream of the promoter. As shown in the following examples, this combination of regulatory module and promoter was tested for high transduction levels. Furthermore, the WPRE sequence was inserted downstream of the coding region. Without wishing to be bound by theory, this element creates a tertiary structure that improves the stability of the mRNA. Other functional mechanisms have been described for WPRE, including, for example, enhancement of transcription termination and facilitation of nuclear export of mRNA. Figure 1B shows a schematic diagram of the above expression construct. Subsequently, the expression construct was cloned into the AAV plasmid backbone and confirmed by sequencing. The vector was packaged into virus particles and stored.

[0172] One or several variants of the above-described scheme can be carried out. An alternative construct can be obtained by replacing the WPRE sequence with the SUMF1 (sulfatase modifying factor 1) coding sequence located in front of the internal ribosome entry site (IRES), as shown in Figure 2. According to the published data (Fraldi A et al. 2007. Biochem J. 403:305-312), the presence of SUMF1 improves I2S activity when tested in cell-based in vitro assays. Additional in vivo data published in the same study showed that sulfamidase (SGSH) activity was further significantly improved in the presence of SUMF1. If SUMF1 is required to activate the formylglycine catalytic residues of the substrates of SUMF1 (including IDS) in the endoplasmic reticulum, co-expressing IDS and SUMF1 from the same vector can be designed to increase the amount of SUMF1 and the amount of IDS processed at the cellular level, so that the activated IDS has an improved possibility of being processed before transport to the lysosome or exiting the cell and moving to other cells for update.

[0173] Codon optimization In addition, the coding sequences of IDS or SUMF1 were codon-optimized based on multiple parameters such as the codon adaptation index (CAI), the number of CpG sites, the GC content, and repetitive nucleotide sequences. A high CAI was preferred to use more frequently used codons and potentially increase the expression level of the transgene product from the vector. CpG sites that could induce an immune response were reduced. Repetitive nucleotides were also removed. For this purpose, a web-based multi-purpose optimization platform for synthetic gene design (referred to as COOL (Codon Optimization Online)) and an internal codon usage frequency table were used. In addition, potential splicing sites were manually removed. The characteristics of the optimized hIDS coding sequence and SUMF1 coding sequence are summarized in Table 4, and a schematic diagram of a representative construct of hIDS-WPRE is shown in Figure 1B, and a schematic diagram of a representative construct of hIDS-IRES-SUMF1 is shown in Figure 2. Variations of the above scheme can be made in any number of ways. For example, two or more promoters may be used, and / or an IRES sequence may be introduced upstream of the coding region. In addition, different combinations of regulatory regions, promoters, and introns are also contemplated.

[0174]

Table 4

[0175] Example 2. In vivo expression of hIDS-WPRE driven by rAAV This example illustrates the efficacy of in vivo expression of IDS driven by rAAV in an optimized construct. Mice were injected with a control vector (rAAV-XL032) (Group A), or the rAAV-XL024 (hIDS wt-WPRE) construct (Group B) or rAAV-XL026 (hIDS-AUSopt-WPRE) construct (Group C) of the test sample as shown in Figures 1A and 1B. Six-week-old mice were injected with the vector in a volume of 200 μl at 5×10 9In the vg group, administration was performed via the caudal vein, and serum samples were collected 2 days, 7 days, 21 days, 8 weeks, and 12 weeks after injection. The mice were sacrificed at 12 weeks of age, and their tissue samples were excised. A group of wild-type mice with age-matched were used as a positive control, and a group of untreated IDS-KO mice with age-matched were used as a negative control. The experimental design is summarized in Table 5 below.

[0176]

Table 5

[0177] The expression level mediated by the vector was determined by ELISA using the B85 antibody. The results are shown in Figure 3. In the mice injected with the rAAV vectors of the optimized constructs rAAV-XL024 (Group B) and rAAV-XL026 (Group C), the hIDS concentration in the serum was higher than that in the mice injected with the control vector rAAV-XL032 (a vector encoding codon-optimized hIDS by a different algorithm that does not reduce the CpG site and a truncated form of the wild-type WPRE sequence) (Group A). In all groups injected with the vectors (Groups A - C), the hIDS concentration increased until day 21, and the hIDS level was maintained for up to 12 weeks (84 days) after a single injection. Since human IDS was not expressed in all WT groups and IDS-KO groups, hIDS was not detected.

[0178] As shown in Figure 4A, when the level of hIDS activity was tested, similar results were obtained. In all groups injected with the vectors (Groups A - C), the activity level increased until day 21. Similar to the expression results, the hIDS activity in the mouse groups injected with the rAAV vectors of the optimized constructs (Groups B and C) was higher than that in the mouse group of the control vector rAAV-XL032 (Group A). The hIDS activity level in the WT mouse group was relatively stable over the test period. The rAAV vector used in this example was rAAV8.

[0179] As shown in Figure 4B, hI2S was measured in tissues using ELISA. In rAAV-XL024 (Group B), hI2S was detectable in the liver and kidney but not in the brain. In this group, the heart and spleen were not analyzed. In rAAV-XL026 (Group C), hI2S was detectable in the liver, kidney, heart, spleen, and brain. In rAAV-XL032 (Group A), hI2S was detectable in the liver, kidney, and brain. In this group, the heart and spleen were not analyzed.

[0180] As shown in Figure 4C, hI2S was measured in tissues using activity. In rAAV-XL024 (Group B), hI2S activity was detectable in the liver, kidney, and brain. In this group, the heart and spleen were not analyzed. In rAAV-XL026 (Group C), hI2S was detectable in the liver, kidney, heart, spleen, and brain. In rAAV-XL032 (Group A), hI2S was detectable in the liver, kidney, and brain. In this group, the heart and spleen were not analyzed. As shown in Figure 4D, the enzyme activity levels were compared with the WT group. Higher activity levels than WT were seen in all vector administration groups in serum and somatic tissues. In the brain, the levels of rAAV-XL026 and rAAV-XL032 were lower than WT.

[0181] Example 3. GAG Clearance by Gene Therapy Using the hIDS-WPRE Construct The enzyme iduronate-2-sulfatase (IDS) removes sulfate groups from the glycosaminoglycans (GAGs) dermatan sulfate and heparan sulfate, and its deficiency or inactivity causes mucopolysaccharidosis type II (MPSII), i.e., Hunter syndrome, a lysosomal storage disorder. Therefore, GAG clearance was measured to evaluate the efficacy of hIDS expressed by the optimized rAAV construct. At week 12, tissues of the brain, liver, and kidney were excised from the mouse groups shown in Table 5, and the GAG levels in each tissue were measured.

[0182] As shown in FIG. 5A, in the brain, in the case of rAAV-XL026 (Group C) and rAAV-XL032 (Group A), the GAG level was slightly decreased compared to untreated IDS-KO mice. As shown in FIGS. 5B and 5C, when an rAAV vector encoding hIDS was injected into mice, the GAG level was significantly decreased in the liver and kidney. The GAG level was comparable to the GAG level found in untreated WT mice.

[0183] Example 4. Reduction of heparan sulfate (HS) and dermatan sulfate (DS) by gene therapy using the hIDS-WPRE construct This example illustrates that in mice, by expressing hIDS, the levels of heparan sulfate and dermatan sulfate are decreased. The enzyme iduronate-2-sulfatase (IDS) removes sulfate groups from the glycosaminoglycans (GAGs) dermatan sulfate and heparan sulfate, and the lack or inactivity thereof causes GAGs to accumulate, resulting in the development of mucopolysaccharidosis type II (MPSII), i.e., Hunter syndrome, a lysosomal storage disorder.

[0184] To evaluate the efficacy of hI2S expressed by the optimized rAAV construct, GAG clearance was measured.

[0185] Using a liquid chromatography-mass spectrometry (LC / MS) assay capable of detecting heparan sulfate (HS) and dermatan sulfate (DS), glycosaminoglycans (GAGs) were measured in the tissues of the liver, kidney, heart, spleen, and brain excised at 12 weeks from the mouse groups shown in Table 5. The results are shown in FIGS. 6A - B.

[0186] In rAAV-XL024 (Group B), HS GAG and DS GAG were decreased in the liver and kidney compared to untreated KO (Group E), and also decreased in the brain, although not to the same extent. The heart and spleen were not analyzed in this group. In rAAV-XL026 (Group C), HS GAG and DS GAG were decreased in the liver, kidney, heart, and spleen compared to untreated KO (Group E), and also decreased in the brain, although not to the same extent. In rAAV-XL032 (Group A), HS GAG and DS GAG were decreased in the liver and kidney compared to untreated KO (Group E), and also decreased in the brain, although not to the same extent.

[0187] As shown in Figure 6B, the levels of HS GAG and DS GAG were normalized with untreated IKO at a reduction rate of 0% and WT at a reduction rate of 100%.

[0188] Overall, in all vector administration groups, a decrease in the proportion of HS GAG and DS GAG in somatic tissues and a decrease in the proportion of HS GAG and DS GAG in the brain were observed. These results indicated that the expression of I2S decreased the GAG level in mouse tissues.

[0189] Example 5. Reduction of Lysosomal Accumulation Compartments by Gene Therapy Using the hIDS-WPRE Construct in Mice In this example, it is exemplified that in mice, during the treatment of, for example, mucopolysaccharidosis (MPSII), the lysosomal accumulation compartment is reduced when detected by LAMP1 staining by enzyme replacement therapy using the hIDS-WPRE construct.

[0190] Briefly stated, using LAMP1 staining, lysosomal accumulation compartments in mice were measured. Various tissues such as the liver, and the hippocampus, thalamus, corpus callosum, cortex, cerebellum, and striatum of the brain were stained. In this example, the control mouse group was administered the I2S enzyme 5 times during the experimental period on days 0, 7, 14, 21, and 28 by intrathecal (IT) injection (group F in Table 5). The decrease in LAMP1 staining indicates a decrease in the substrate and an improvement in the pathological condition of the KO mice.

[0191] Those results are shown in FIGS. 7A and 7B. As seen in FIG. 7A, a significant decrease in LAMP1 was observed in the liver tissue of the mice injected with the vector. The effect in the liver was comparable to that in WT mice and the group of mice treated with ERT.

[0192] The brain is shown in FIG. 7B. Compared with untreated KO (group E), the decrease in the positive rate of LAMP1 staining was statistically significant in the hippocampus with rAAV-XL026, in the corpus callosum with rAAV-XL024 and rAAV-XL026, and in the thalamus with rAAV-XL024 and rAAV-XL026.

[0193] Overall, those results indicated that in the liver and brain tissues of mice, administration of IDS reduced the lysosomal accumulation compartment as measured by LAMP1 staining.

[0194] Example 6. In vivo expression of hIDS-IRES-SUMF1 driven by rAAV In this example, in mice, the in vivo expression and activity of hIDS when the hIDS-IRES-SUMF1 vector was administered were compared with those of the hIDS-WPRE vector.

[0195] Since SUMF1 is required to activate the FGly catalytic residue of IDS, a comparison was made using a vector expressing hIDS and SUMF1 against a vector expressing hIDS-WPRE.

[0196] Mice were injected with rAAV vectors expressing the hIDS AUSopt-WPRE construct (rAAV-XL026, group C), the hIDS-IRES-SUMF1 construct (group G was rAAV-XL027, group H was XL029), or the SUMF1 construct as a negative control (rAAV-XL030, group I). Schematic diagrams of these constructs are shown in FIGS. 1B and 2.

[0197] Six-week-old mice were administered the vector at 5×10 9 vg in a volume of 200 μl via the tail vein, and serum samples were collected 2 days, 7 days, and 21 days after injection. Groups of age-matched wild-type mice were used as positive and negative controls, respectively. An exemplary in vivo test is summarized in Table 6.

[0198] [Table 6]

[0199] The expression level of IDS in serum was quantified by ELISA. The results are shown in FIG. 8. Unexpectedly, in mice injected with rAAV-XL027 and rAAV-XL029, which express both hIDS and SUMF1, the hI2S concentration in serum at day 21 was lower than that in mice injected with the rAAV-XL026 vector (group C), which expresses only hIDS.

[0200] As shown in Fig. 9, when the activity level of hI2S was tested, similar results were obtained. In all groups (groups G - H) injected with vectors expressing both hIDS and SUMF1, the activity level was lower than that in the group injected with the vector expressing hIDS-WPRE (group C). Without wishing to be bound by a particular theory, it is thought that the activity increases because the WPRE element in the rAAV-XL026 vector brings about a tertiary structure that improves mRNA stability, which then increases protein expression. Other functional mechanisms have been described for WPRE, including, for example, enhancement of transcriptional termination and facilitation of mRNA nuclear export. On the other hand, SUMF1 gives rise to a more active form of hIDS, but the amount of hIDS expressed by the WPRE element is significantly higher. The rAAV vector used in this example is rAAV8.

[0201] From these results, it was shown that the level and activity of the IDS enzyme were higher with expression from the hIDS-WPRE vector than from the hIDS-IRES-SUMF1 vector.

[0202] Example 7. Long-term in vivo expression of hIDS-WPRE driven by rAAV in mouse serum and tissues In this example, the in vivo expression and activity of hIDS were compared in mice administered one of three doses of hIDS-WPRE. Expression levels and activity were evaluated in serum and tissues over approximately 12 - 13 months.

[0203] Mice were injected with an rAAV vector expressing the hIDS AUSopt-WPRE construct (rAAV-XL026) at doses of 5×10 9 vg (group D), 2.5×10 10 vg (group E), 1.25×10 11 vg (group F), and as a negative control, with a null vector construct rAAV-MY011 at a dose of 1.25×10 11 vg (group G). Schematic diagrams of these constructs are shown in Figs. 1B and 2.

[0204] Mice at 5 - 7 weeks of age were injected with the vector via the tail vein in a volume of 200 μl. Serum samples were collected 7 days, 14 days, 28 days, 56 days, 84 days, 112 days, 140 days, 168 days, 196 days, 224 days, 252 days, 280 days, 308 days, 336 days, and 364 days after the injection. A group of wild - type mice with age - matched was used as a positive control, and a group of age - matched untreated IDS - KO mice was used as a negative control. An exemplary in vivo test is shown in Table 7.

[0205]

Table 7

[0206] Levels and activities of hI2S in serum: The expression level of hI2S in serum was determined by ELISA. The results are shown in Figure 10. The concentration of hI2S increased as the dose of the rAAV - XL026 vector (groups D, E, F) increased, and hI2S remained detectable until the last time point, 364 days after the injection. The rAAV vector used in this example is rAAV8.

[0207] As shown in Figure 11, when the level of hI2S activity was tested, similar results were observed. The concentration of hI2S activity increased as the dose of the rAAV - XL026 vector (groups D, E, F) increased, and hI2S activity remained detectable until the last time point, 364 days after the injection.

[0208] Levels and activities of hI2S in tissues: In tissues, as shown in Figure 12A, hI2S was measured using ELISA. In rAAV - XL026 (groups D, E, F), hI2S was detectable in the liver, spleen, kidney, heart, lung, and brain and increased with increasing dose.

[0209] In the tissue, the activity of hI2S was measured as shown in Figure 12B. In rAAV-XL026 (groups D, E, F), the activity of hI2S was detectable in the liver, spleen, kidney, heart, lung, and brain, and increased with increasing dose.

[0210] Using a liquid chromatography-mass spectrometry (LC / MS) assay capable of detecting heparan sulfate (HS) and dermatan sulfate (DS), glycosaminoglycans (GAGs) were measured in the liver, kidney, heart, spleen, lung, and brain tissues excised on day 364 from the mouse groups shown in Table 7. The results are shown in Figure 12C.

[0211] In those somatic tissues, the levels of HS GAG and DS GAG were significantly decreased with rAAV-XL026 at all doses (groups D, E, F). As shown in Figure 12D, the levels of HS GAG and DS GAG were normalized with untreated IKO at a reduction rate of 0% and WT at a reduction rate of 100%. With rAAV-XL026 at all three doses (groups D, E, F), the reduction rates of HS GAG and DS GAG in somatic tissues were 100%. In the brain, the reduction rate of HS GAG was 17% in group D of rAAV-XL026, 38% in group E of rAAV-XL026, and 50% in group F of rAAV-XL026.

[0212] Various tissues such as the liver, and the hippocampus, thalamus, white matter, cortex, cerebellum, and striatum of the brain were stained with LAMP1. The brain is shown in Figure 12E. Compared with untreated KO (group H), the decrease in the LAMP1 staining positive rate was statistically significant in the hippocampus in groups D, E, and F of rAAV-XL026, the striatum in groups E and F of rAAV-XL026, the white matter in groups E and F of rAAV-XL026, the thalamus in groups E and F of rAAV-XL026, the cortex in groups E and F of rAAV-XL026, and the cerebellum in groups E and F of rAAV-XL026.

[0213] Bone volume: The body structure was examined using micro-computed tomography (micro-CT). As shown in FIGS. 13A and 13B, the bone volume was measured over time in mice at 7 months, 9 months, 11 months, and 13 months of age using micro-CT.

[0214] In FIGS. 13A and 13B, line A corresponds to group D of rAAV-XL026, line B corresponds to group E of rAAV-XL026, line C corresponds to group F of rAAV-XL026, line D corresponds to group G of rAAV-MY011 (negative control), line E corresponds to untreated IDS-KO group H (negative control), and line F corresponds to untreated wild-type group I (positive control).

[0215] In the humerus (FIG. 13A), the bone volume measurements in groups D, E, and F of rAAV-XL026 exceeded those of the wild-type group I, which is the positive control, and the volumes in these groups were smaller than those of group G of rAAV-MY011, which is the negative control, and the untreated IDS-KO group H.

[0216] In the zygomatic arch of the skull at 13 months (FIG. 13B), the bone volume measurements in groups D, E, and F of rAAV-XL026 exceeded those of the wild-type group I, which is the positive control, and the volumes in these groups were smaller than those of group G of rAAV-MY011, which is the negative control, and the untreated IDS-KO group H.

[0217] These results showed that the bone volume of the humerus and zygomatic arch was smaller when I2S was expressed, and this was maintained over 13 months.

[0218] Collectively, these results showed that the levels of I2S expression and activity in serum and tissues, and consequently, the effects on bone volume, were maintained over approximately 12 - 13 months.

[0219] Example 8. Results of monitoring in vivo expression of hIDS-WPRE driven by rAAV in mice for 3 months after administration at a low dose In this example, the in vivo expression and activity of hI2S in mice administered with hI2S at a dose of 5×10 6 ~5×10 9 vg were exemplified over 3 months.

[0220] Mice were injected with an rAAV vector expressing the hIDS AUSopt-WPRE construct (rAAV-XL026) at a dose of 5×10 6 vg (Group C), 5×10 7 vg (Group D), 5×10 8 vg (Group E), 5×10 9 vg (Group F), and as a negative control, a null vector construct rAAV-MY011 was injected at a dose of 5×10 9 vg (Group B).

[0221] A 200 μl vector was administered to 5- to 7-week-old mice via the tail vein, and serum samples were collected 14 days, 28 days, 56 days, and 84 days after injection. A group of age-matched wild-type mice was used as a positive control, and a group of age-matched untreated IDS-KO mice was used as a negative control. An exemplary in vivo test is summarized in Table 8.

[0222]

Table 8

[0223] Levels and activities of IDS in serum: The expression level of hIDS in the serum of mice administered with a low-dose hIDS-WPRE vector was determined by ELISA and shown in Fig. 14A. The concentration of hI2S decreased as the dose of the rAAV-XL026 vector decreased in Groups D, E, and F, and hI2S was undetectable in Group C. The rAAV vector used in this example was rAAV8.

[0224] The activity level of I2S in serum was measured as shown in Figure 14B. The concentration of hI2S decreased as the dose of rAAV-XL026 vector decreased in groups D, E, and F, and hI2S was undetectable in group C.

[0225] Overall, in group D administered with a dose of 5×10 8 vg, and group E administered with a dose of 5×10 9 vg, the expression and activity of I2S were higher than those in untreated mice, and the levels and activities of I2S were maintained over 84 days or 3 months.

[0226] Levels and activities of I2S in tissues: In the liver, spleen, kidney, heart, lung, bone marrow, quadriceps femoris, and brain, measured by ELISA, the groups of untreated KO (group A) and untreated WT (group J), as well as the negative control rAAV-MY011 (group B), did not contain detectable human I2S (hI2S) protein (Figure 14C). At 5×10 7 vg - 5×10 9 vg, groups D, E, and F of rAAV-XL026 contained higher concentrations of hI2S in tissues as the dose increased.

[0227] As shown in Figure 14D, I2S activity was measured in the liver, spleen, kidney, heart, lung, bone marrow, quadriceps femoris, and brain. In rAAV-MY011 (group B), I2S activity was at the same level as that in untreated KO (group A) in all tissues. At 5×10 7 vg - 5×10 9 vg, groups D, E, and F of rAAV-XL026 contained higher concentrations of I2S activity in tissues as the dose increased. As shown in Figure 14E, the I2S activity levels in each tissue of the administered KO mice could be comparable to the untreated WT levels.

[0228] In mice administered with a low dose of the rAAV vector containing hI2S-WPRE, the levels and activities of I2S in tissues increased in a dose-dependent manner.

[0229] Tissue GAG levels: Glycosaminoglycan (GAG) was measured in the liver, spleen, kidney, heart, lung, bone marrow, quadriceps femoris, skin, and brain tissues excised on day 84 from the mouse groups shown in Table 8 using a liquid chromatography-mass spectrometry (LC / MS) assay capable of detecting heparan sulfate (HS) and dermatan sulfate (DS). As shown in Tables 9 and 10, the levels of HS GAG and DS GAG were normalized with untreated IKO at a reduction rate of 0% and WT at a reduction rate of 100%.

[0230] 5×10 8 In the rAAV-XL026 group E at 5×10 8 vg, the levels of HS and GAG decreased by more than 90% in all somatic tissues measured. In the brain, in the rAAV-XL026 group E at 5×10 9 vg, the level of HS in the brain decreased by 8.8% and the level of GAG decreased by 41%. In the rAAV-XL026 group F at 5×10 9 vg, the levels of HS and GAG decreased by more than 95% in all somatic tissues measured. In the brain, in the rAAV-XL026 group F at 5×10

[0231] The levels of HS and GAG decreased in somatic tissues and the brain when rAAV containing IDS-WPRE was administered at 5×10 7 、5×10 8 or 5×10 9 vg.

[0232]

Table 9

[0233]

Table 10

[0234] Lysosome accumulation: Various tissues such as the liver, spleen, kidney, heart, and the hippocampus, thalamus, white matter, cortex, cerebellum, and striatum of the brain were stained with LAMP1. Somatic tissues are shown in Figure 14F. In somatic tissues including the liver, spleen, and heart, the positive rate of LAMP1 IHC staining was significantly lower in Group D with a dose of 5×10 7 vg, Group E with a dose of 5×10 8 vg, and Group F with a dose of 5×10 9 vg compared to the naive KO control. In the kidney, the positive rate of LAMP1 staining was significantly lower in Group E with a dose of 5×10 8 vg and Group F with a dose of 5×10 9 vg compared to the naive KO control.

[0235] LAMP1 IHC staining in the brain regions is shown in Figure 14G. In the brain, the positive rate of LAMP1 staining was measured in the hippocampus, striatum, thalamus, white matter, cortex, and cerebellum. In none of these regions was there a significant difference in the positive rate of LAMP1 staining between the rAAV-XL026 dose groups C, D, E, and F and the untreated KO control group A.

[0236] These results showed that administration of a low dose of IDS-WPRE reduced lysosome accumulation in somatic tissues but not significantly in the brain.

[0237] Collectively, these results showed that in mice, low-dose administration of rAAV containing hIDS-WPRE maintained the expression and activity of I2S in serum and tissues for approximately 3 months, and lysosome accumulation was reduced in somatic tissues as determined by LAMP.

[0238] Example 9. Serum expression level of the hI2S transgene product in non-human primates (NHP) This example illustrates an exemplary PK / distribution study in non-human primates administered with the hI2S transgene product.

[0239] Male non-human primates (aged approximately 1.8 - 2 years) were administered the hI2S product by intravenous injection. Animals in the low-dose cohort were administered a low dose of hI2S at 1.25×10 12 vg / kg (n = 3), and animals in the high-dose cohort were administered a higher dose of hI2S at 6.25×10 12 vg / kg (n = 6). Control animals were administered only the formulation buffer.

[0240] Serum samples were collected at various time points (starting from before administration, after collection before administration of the hI2S transgene, and then every 20 days until day 240). At 3 months, 3 animals were sacrificed from each of the low-dose and high-dose cohorts.

[0241] Figure 15A shows the serum concentration of the hI2S transgene product obtained from the low-dose cohort in NHP serum at necropsy from 1 day to 3 months after administration. It was observed that the concentration of the hI2S transgene product was maintained at 1.25×12 vg / kg (low dose).

[0242] In contrast, Figure 15B shows that the level of enzyme activity of the hI2S transgene product in serum was maintained for up to 3 months. At the 3-month time point, a level of I2S enzyme activity approximately 5 times higher was observed compared to the activity level of endogenous I2S in NHP measured in control animals administered only the formulation buffer.

[0243] Figure 15C shows the serum concentration of the hI2S transgene product in the high-dose cohort in 3 NHP at necropsy from 1 day to approximately 90 days, i.e., 3 months, after administration, and in the remaining high-dose animals from 1 day to approximately 240 days, i.e., 8 months, after administration.

[0244] Variations in the hI2S transgene product profile were shown among the individual animals, and the variations were observed at the obtained initial peak serum concentration levels. In the animals followed up to necropsy at the 3-month time point, a sharp decrease in the serum concentration of the hI2S transgene product was observed. Since the corresponding serum hI2S enzyme activity decreased to a plateau level of about 5200 - 7500 nmol / hr / mL after improvement and after the 21st day (Figure 15D), it was shown that in the 3 animals sacrificed at the 3-month time point, although the concentration of the I2S transgene product decreased, the hI2S enzyme activity in the serum was maintained.

[0245] In the animals in which the concentration of the hI2S transgene product in the serum and the I2S activity in the serum were followed up to about 240 days (8 months), variations were seen in the concentration profile of the hI2S transgene product. In 2 animals, by about the 8th month, the concentration of the I2S transgene product decreased to 100 ng / mL or less. This was also associated with the observation that by about the 8th month, the hI2S enzyme activity decreased to almost the endogenous level (in the case of the formulation buffer). In 1 of the animals in this study, the concentration and enzyme activity of the hI2S transgene product were maintained up to about 8 months.

[0246] Collectively, these data showed that in non-human primates, the expression of the hI2S transgene product persisted even in the absence of immunosuppressants.

[0247] Example 10. Comparison of the serum concentration of the hI2S transgene product and the I2S enzyme activity in non-human primates at low and high doses, with the anti-transgene product antibody (alias, anti-hI2S ADA (anti-drug antibody)) and the anti-AAV8 ADA titration values In this example, the comparison results are exemplified of plotting the data of anti-hI2S ADA and anti-AAV8 ADA obtained from individual non-human primates against the concentration and enzyme activity of the hI2S transgene product.

[0248] Data for anti-hI2S ADA and anti-AAV8 ADA obtained from individual animals were plotted against the concentration and enzyme activity of the hI2S transgene product (Figure 16). In these graphs, only ADA data up to 3 months were shown. In the absence of anti-hI2S ADA, an initial decrease in hI2S levels was observed within 1 month in some animals (2001, 3002, 4003, 4001, and 4002). The decrease in hI2S levels was not dose-dependent. The initial decrease (within 1 month) in the concentration of the hI2S transgene product in NHP serum varied among animals (10% - 80%).

[0249] In the cohort administered high doses of hI2S, the decrease in the hI2S transgene product in serum after the equilibrium state was correlated with the presence of anti-hI2S ADA. The "equilibrium state" or "reconstructed state" refers to the "steady state" or plateau level of the I2S enzyme or I2S enzyme activity. In some animals (3002 and 4001), the presence of anti-AAV8 ADA allowed avoidance of readministration. In none of the animals in the low-dose cohort was the presence of anti-hI2S ADA detected in serum.

[0250] These results showed that in non-human primates administered high doses of rAAV-XL026 containing hIDS-WPRE, the decrease in serum I2S levels was correlated with the presence of anti-hI2S ADA.

[0251] Example 11. Concentration Profile of hI2S Transgene Product in the Liver of Individual Non-Human Primates In this example, the concentration profile of the hI2S transgene product in the liver of non-human primates is illustrated.

[0252] At 1 and 2 months, liver biopsies were performed on both low-dose (Figure 17A) and high-dose (Figure 17B) animals, and at 3 months, liver samples were collected at the time of euthanasia. For 3 high-dose cohort animals assuming necropsy at 12 months, biopsies were performed at 3 and 6 months.

[0253] Each biopsy specimen was taken from the left and right lobes. The average concentration of the hI2S transgene product in the left and right lobes of the liver from the low-dose cohort was in the range of 38.2 ng / mL to 42.5 ng / mL for the 1-month and 3-month averages.

[0254] The concentration of the hI2S transgene product in the liver from the high-dose cohort was in the range of 175.1 ng / mL to 367.6 ng / mL at 1 month. In one of these animals (Animal 3001), there was a large difference between the left lobe (101.8 ng / mL) and the right lobe (528.3 ng / mL).

[0255] At 2 months, in two animals (Animal 3001 and Animal 3002), the concentration of the hI2S transgene product in the liver decreased significantly from 1 month to 2 months. In other animals including Animal 3003, it also decreased from 2 months to 6 months, but the degree of decrease was smaller than the above.

[0256] Overall, these results showed the individual concentration profiles of the I2S transgene product in the tissues of non-human primates from 1 month to 3 months after administration of rAAV-XL026.

[0257] Example 12. Relative Concentration Profile of hI2S Transgene Product in Tissues of Non-Human Primates In this example, the relative concentration profile of the hI2S transgene product in the tissues of non-human primates is illustrated.

[0258] At 3 months, tissue dissection was performed on the animals in the low-dose cohort and three animals in the high-dose cohort.

[0259] The concentration of the hI2S transgene product was measured by ELISA in tissue homogenates from various organs including the kidney, spleen, lung, heart, and bone marrow. In the serum of the high-dose cohort at 3 months, in the presence of anti-hI2S ADA, significantly lower levels were seen compared to the low-dose cohort (above 640 ng / mL) (less than 250 ng / mL), but the average hI2S concentrations in the kidney (Figure 18A) and spleen (Figure 18B) were higher in the high-dose cohort than in the low-dose case.

[0260] In the presence of anti-hI2S ADA, in three animals of the high-dose cohort, the concentration of the hI2S transgene product was low in the lung (Figure 18C) and heart (Figure 18D), but in the bone marrow (Figure 18E), the tissue concentration of hI2S was comparable between the high-dose and low-dose cohort animals. For example, in the formulation buffer group, since the tissue was from healthy NHP, endogenous I2S was detected in the lung.

[0261] From these results, relative I2S concentration profiles were seen in various non-human primate tissues after treatment with rAAV-XL026.

[0262] Example 13. Relative Tissue Exposure of hI2S to IDS Knockout Mice in Non-Human Primates In this example, relative I2S concentrations and enzyme activities in various target tissues are illustrated between non-human primates and IDS KO mice.

[0263] In IDS KO mice, 2.5x10 11 vg / kg and 2.5x10 10 The hI2S enzyme activity in tissues and the corresponding HS GAG reduction rate (%) at vg / kg are shown in Table 11.

[0264] In mice, at approximately 2.5x10 10At a dose of vg / kg, more than a 95% decrease in HS GAG was observed in most tissues except the kidney, and in the kidney, a decrease of approximately 91% was observed. In the heart and bone marrow, from data obtained from non-human primates in the low-dose cohort, the tissue concentration and enzyme activity of hI2S were 2.5x10 10 vg / kg, which was shown to be higher than that in IDS KO mice.

[0265] In lung tissue, in IDS KO mice, it was only 30% of the WT I2S enzyme activity, but at this level, the reduction rate of HS GAG was 97%. In non-human primates administered low-dose hI2S, the enzyme activity in the lung was even higher as a percentage of the WT hI2S enzyme activity, at 40%. By administering rAAV-XL026 to NHP by IV injection at 1.25×10 12 vg / kg, the lung was sufficiently exposed to the hI2S transgene product, and as a result, the reduction rate of HS GAG in the disease model can be 95% or more.

[0266]

Table 11

[0267] Equivalents and ranges One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as defined in the appended claims.

Claims

1. a. an AAV8 capsid; and b. a codon-optimized sequence encoding human iduronate-2-sulfatase (I2S) enzyme; and c. a 5' inverted terminal repeat (ITR); and d. a cis-acting regulatory module (CRM); and e. a liver-specific promoter; and f. a murine minute virus (MVM) intron; and g. a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and h. a 3' ITR A recombinant adeno-associated virus (rAAV) vector comprising the same.

2. The rAAV vector according to claim 1, wherein the codon-optimized sequence encoding the human I2S enzyme comprises a sequence having at least 90% identity with SEQ ID NO:

6.

3. The rAAV vector according to claim 1, wherein the codon-optimized sequence encoding the human I2S enzyme comprises a sequence identical to SEQ ID NO: 11 or 12.

4. The rAAV vector according to claim 1, wherein the liver-specific promoter is a transthyretin promoter (TTR).

5. The rAAV vector according to any one of claims 1 to 4, wherein the sequence of the MVM intron is upstream of the codon-optimized sequence encoding the I2S enzyme.

6. The rAAV vector according to any one of claims 1 to 5, further comprising sulfatase modifying factor 1 (SUMF1).

7. The rAAV vector according to claim 6, wherein there is an internal ribosome entry site (IRES) before the SUMF1.

8. The rAAV vector according to any one of claims 1 to 7, wherein the CRM is a liver-specific CRM.

9. The rAAV vector according to any one of claims 1 to 7, wherein the CRM is a neuron-specific CRM.

10. The rAAV vector according to any one of claims 1 to 7, wherein the CRM is a cis-acting regulatory module 8 (CRM8).

11. The rAAV vector according to any one of claims 1 to 10, comprising at least three CRMs.

12. The rAAV vector according to any one of claims 1 to 11, wherein the rAAV vector does not contain ApoB.

13. A medicament for treating a subject with Hunter syndrome (MPS II), comprising the rAAV vector according to any one of claims 1 to 12.

14. A medicament for treating a subject with Hunter syndrome (MPS II), comprising the rAAV vector according to any one of claims 1 to 12, wherein administration of the rAAV vector improves I2S enzyme activity in the subject.

15. The medicament according to claim 14, wherein the improvement of the I2S enzyme activity is detected in the serum of the subject.

16. The medicament according to claim 14 or 15, wherein the improvement of the I2S enzyme activity is detected in the liver of the subject.

17. The medicament according to any one of claims 14 to 16, wherein the improvement of the I2S enzyme activity is detected in the central nervous system (CNS).

18. The medicament according to any one of claims 14 to 17, wherein the improvement of the I2S enzyme activity is detected in the brain of the subject.

19. The medicament according to claim 18, wherein the improvement of the I2S enzyme activity is detected in the hippocampus, thalamus, corpus callosum, cortex, cerebellum or striatum of the brain of the subject.

20. The agent according to any one of claims 14 to 19, wherein the improvement in I2S enzyme activity is detected in the kidney of the subject.

21. The agent according to any one of claims 14 to 20, wherein the improvement in I2S enzyme activity is maintained for at least 30 days after a single administration.

22. The agent according to any one of claims 14 to 21, wherein the level of I2S enzyme activity is measured by a heparin sulfate assay.

23. The agent according to any one of claims 14 to 22, wherein the level of I2S enzyme activity is measured by a dermatan sulfate assay.

24. The agent according to any one of claims 14 to 23, wherein administration of the agent reduces the level of glycosaminoglycan (GAG) in the subject.

25. The agent according to claim 24, wherein administration of the agent reduces the level of GAG in the serum of the subject.

26. The agent according to claim 24 or 25, wherein administration of the agent reduces the level of GAG in the liver of the subject.

27. The agent according to any one of claims 24 to 26, wherein administration of the agent reduces the level of GAG in the kidney of the subject.

28. The agent according to any one of claims 24 to 27, wherein administration of the agent reduces the level of GAG in the CNS of the subject.

29. The agent according to any one of claims 24 to 28, wherein administration of the agent reduces the level of GAG in the brain of the subject.

30. The agent according to claim 29, wherein administration of the agent reduces the level of GAG in the hippocampus, thalamus, corpus callosum, cortex, cerebellum or striatum of the brain of the subject.

31. The agent according to any one of claims 14 to 30, which is administered intravenously.

32. The agent according to any one of claims 14 to 30, which is administered intrathecally.

33. The agent according to claim 31 or 32, wherein the agent is administered at a dose of 5 × 10 ９ vg.

34. The agent according to any one of claims 14 to 33, wherein administration of the agent does not induce an immune response.

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