Adeno-associated virus vector based gene therapy for hereditary angioedema

Abstract

To provide a method for treating hereditary angioedema (HAE).SOLUTION: The present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a codon-optimized SERPING1 sequence encoding a human C1-esterase inhibitor. The disclosure also provides a method of treating a subject having hereditary angioedema (HAE), comprising administering to the subject in need thereof a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and codon-optimized SERPING1 sequences encoding a human C1-esterase inhibitor.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 924,877, filed Oct. 23, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Hereditary angioedema (HAE) is a rare condition characterized by recurrent swelling in the face, throat, and most extremities. HAE is a potentially life - threatening disorder characterized by unpredictable recurrent attacks of vasodilation that manifest as subcutaneous and submucosal angioedema. HAE is associated in some cases with low levels of C1 - inhibitor in plasma (type I), and in other cases, the protein circulates in normal or elevated amounts but may be dysfunctional (type II). C1 inhibitor is the main regulator of plasma kallikrein activity. Symptoms of HAE attacks include swelling of the face, mouth, and / or airway, which can occur spontaneously or be induced by minor trauma. Edematous attacks affecting the airway can be life - threatening. In addition to acute inflammatory attacks, excessive plasma kallikrein activity is also associated with chronic conditions such as autoimmune diseases including lupus erythematosus.

[0003] For the treatment of C1 - INH deficiency or dysfunction, various strategies have been contemplated and developed, including, for example, inhibiting members of the contact system. For example, lanadelumab is a fully human monoclonal antibody inhibitor of plasma kallikrein and is approved for the treatment of HAE.

[0004] The treatment of diseases would benefit from the use of vectors that produce proteins in vivo, but is limited by various factors such as insufficient protein production after delivery to the subject.

[0005] To date, available treatments have not addressed issues including disease recurrence and the need for long-term continuous administration. Therefore, there is a need for a new and sustainable treatment approach for treating HAE. SUMMARY OF THE INVENTION

[0006] The present invention provides a recombinant adeno-associated virus (rAAV) vector that enables efficient and robust expression of human C1 esterase inhibitor (C1-INH or C1EI) in vivo.

[0007] In one aspect, the present invention provides a recombinant adeno-associated virus (rAAV) vector comprising, inter alia, an AAV8 capsid and a codon-optimized SERPING1 sequence encoding a C1 inhibitor (C1-INH).

[0008] In some embodiments, the codon-optimized SERPING1 sequence encoding C1-INH comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 2.

[0009] In some embodiments, the codon-optimized SERPING1 sequence encoding C1-INH comprises a sequence identical to SEQ ID NO: 2.

[0010] In some embodiments, the vector further comprises a liver-specific promoter.

[0011] In some embodiments, the liver-specific promoter is the transthyretin promoter (TTR).

[0012] In some embodiments, the vector further comprises a ubiquitous promoter.

[0013] In some embodiments, the vector further comprises one or more of the following: 5' and 3' terminal inverted repeats, an intron upstream of the sequence, and a cis-acting regulatory module (CRM).

[0014] In some embodiments, the vector further comprises a WPRE sequence.

[0015] In some embodiments, the WPRE sequence is modified.

[0016] In some embodiments, WPRE contains the mut6delATG modification.

[0017] In some embodiments, the intron is a murine minute virus (MVM) intron or an SV40 intron.

[0018] In some embodiments, the CRM is a liver-specific CRM.

[0019] In some embodiments, the CRM is CRM8.

[0020] In some embodiments, the vector comprises at least three CRMs.

[0021] In another aspect, the present invention provides a recombinant adeno-associated virus (rAAV) comprising, inter alia, an AAV8 capsid and an rAAV vector, the vector comprising a. a 5' terminal inverted repeat (ITR), b. a cis-acting regulatory module (CRM), c. a liver-specific promoter, d. murine minute virus (MVM), e. a SERPING1 sequence encoding C1 inhibitor (C1-INH), f. a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and g. a 3' ITR and comprising.

[0022] In some embodiments, the SERPING1 sequence is a wild-type sequence or a codon-optimized sequence.

[0023] In some embodiments, the codon-optimized SERPING1 sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 2.

[0024] In another aspect, the present invention provides, inter alia, a method of treating a subject having hereditary angioedema (HAE), the method comprising administering to a subject in need thereof an rAAV of any one of the preceding embodiments.

[0025] In another aspect, the present invention provides, inter alia, a method of treating a subject having hereditary angioedema (HAE), the method comprising administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a promoter operably linked to a nucleic acid sequence encoding C1 inhibitor (C1-INH), wherein administering the rAAV vector increases C1-INH enzyme activity in the subject.

[0026] In some embodiments, C1-INH is detected in the plasma of the subject.

[0027] In some embodiments, C1-INH is detected in the liver of the subject.

[0028] In some embodiments, C1-INH is maintained for at least 30, 60, 90, 120, 150, 180 days or more after a single administration.

[0029] In some embodiments, C1-INH activity is present in the subject after administration of the rAAV vector.

[0030] In some embodiments, the subject has a C4 level that has recovered to pre-attack levels.

[0031] In some embodiments, the AAV is administered intravenously.

[0032] In some embodiments, the AAV is administered intrathecally.

[0033] In some embodiments, the AAV is administered at a dose of at least about 5×10 9 vg.

[0034] In some embodiments, administration of the rAAV does not induce an immune response.

[0035] Various aspects of the invention are described in detail in the following sections. The use of the sections is not meant to limit the invention. Each section can be applied to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise indicated. As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

Brief Description of the Drawings

[0036]

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Mode for Carrying Out the Invention

[0037] Definition Adeno-associated virus (AAV): As used herein, the terms "adeno-associated virus" or "AAV" or recombinant AAV ("rAAV") include, but are not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 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 episomally without integrating into the host cell genome. AAV vectors are commonly used in gene therapy.

[0038] Administration: As used herein, the terms "administering" or "introducing" are used interchangeably in the context of delivering a therapeutic agent-encoding rAAV vector to a subject by a method or route that results in delivery of the rAAV vector. Methods for administering rAAV vectors are known in the art and include, for example, intravenous, subcutaneous, or percutaneous. Percutaneous administration of an rAAV vector can be performed using a "gene gun" or a biolistic particle delivery system. In some embodiments, the rAAV vector is administered via a non-viral lipid nanoparticle.

[0039] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal can be a transgenic animal, a genetically engineered animal, and / or a clone.

[0040] Approximately or about: As used herein, the terms "approximately" or "about", when applied to one or more target values, refer to a value that is similar to the specified reference value. In certain embodiments, the terms "approximately" or "about" refer to a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or lesser) of the recited reference value, unless otherwise indicated or not apparent from the context (except when the numerical value exceeds 100% of the possible values).

[0041] Functional equivalent or derivative: As used herein, the terms "functional equivalent" or "functional derivative" in the context of a functional derivative of an amino acid sequence mean a molecule that retains substantially similar biological activity (either function or structure) to the original sequence. A functional derivative or equivalent can be a natural derivative or can be prepared synthetically. Exemplary functional derivatives include amino acid sequences having one or more amino acid substitutions, deletions, or additions as long as the biological activity of the protein is preserved. The substituted amino acids preferably have chemical and physical properties similar to the amino acids being substituted. Desirable similar chemical and physical properties include similarities in charge, bulk, hydrophobicity, hydrophilicity, etc.

[0042] in vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, during cell culture, rather than within a multicellular organism.

[0043] in vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms such as humans and non-human animals. In a cell-based context, this term can be used to refer to events that occur within living cells (e.g., as contrasted with an in vitro system).

[0044] IRES: As used herein, the term "IRES" refers to any suitable internal ribosome entry site sequence.

[0045] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together via peptide bonds. This term is used to refer to amino acid chains of any length, although those skilled in the art will understand that this term is not limited to long chains and can also refer to the smallest chain containing two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides can be subject to processes and / or modifications.

[0046] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as distinct units. If a single polypeptide is a distinct functional unit and does not require a permanent or transient physical association with other polypeptides to form a distinct functional unit, the terms "polypeptide" and "protein" can be used interchangeably. If a distinct functional unit is composed of more than one polypeptide that physically associates with each other, the term "protein" refers to a plurality of polypeptides that are physically bound and function together as a distinct unit.

[0047] Regulatory element: As used herein, the term "regulatory element" refers to a transcriptional regulatory element capable of regulating and / or controlling the transcription of a gene, particularly a non-coding cis-acting transcriptional regulatory element. A regulatory element contains at least one transcription factor binding site, e.g., 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, a regulatory element increases or enhances promoter-driven gene expression as compared to gene transcription by the promoter alone without the regulatory element. Thus, a regulatory element particularly includes an enhancer sequence, but it should be understood that a regulatory element that enhances transcription is not typically limited to an enhancer sequence far upstream and can be present at any distance from the gene being regulated. As understood in the art, a sequence that regulates transcription can be located either upstream (e.g., within the promoter region) or downstream (e.g., within the 3'UTR) of the gene being regulated in vivo, and can be located close to or quite far from the gene. A regulatory element can include a naturally occurring sequence, a combination of such regulatory element(s) (part), or several copies of a regulatory element, e.g., any of non-naturally occurring sequences. Thus, regulatory elements include both naturally occurring sequences and regulatory elements optimized or engineered to achieve a desired expression level. It can be located either upstream (e.g., within the promoter region) or downstream (e.g., within the 3'UTR) of the gene being regulated in vivo, and can be located close to or quite far from the gene. A regulatory element can include a naturally occurring sequence, a combination of such regulatory element(s) (part), or several copies of a regulatory element, e.g., any of non-naturally occurring sequences. Thus, regulatory elements include both naturally occurring sequences and regulatory elements optimized or engineered to achieve a desired expression level.

[0048] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, bird, sheep, horse, or primate). Humans include pre- and postnatal forms. In many embodiments, the subject is a human. The subject can be a patient, and a patient refers to a human who visits a healthcare provider for the diagnosis or treatment of a disease. The term "subject" can be used interchangeably herein with "individual" or "patient". The subject can be one who has or is susceptible to having a disease or disorder, and may or may not exhibit symptoms of the disease or disorder.

[0049] Substantially: As used herein, the term "substantially" refers to a qualitative condition indicating that the characteristic or property of interest is within an overall or near overall range or degree. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, reach completion and / or proceed to completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to account for the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0050] Substantial homology: The phrase "substantial homology" is used herein to refer to a comparison between amino acids or nucleic acid sequences. As will be recognized by those of skill in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues that have suitably similar structural and / or functional characteristics. For example, as is well known to those of skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substituting one amino acid for another of the same type can often be considered a "homologous" substitution.

[0051] As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms 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. In addition to identifying homologous sequences, the programs described above typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be 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 the corresponding residues are homologous over a relevant stretch of residues. In some embodiments, the relevant stretch is the complete sequence.In some embodiments, the relevant stretch 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.

[0052] Substantial identity: The term "substantial identity" is used herein to refer to a comparison between amino acids or nucleic acid sequences. As will be appreciated by those of skill in the art, two sequences are generally considered to be "substantially identical" if they contain the same residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms 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 include 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 It is described in Biology, Vol. 132), Humana Press, 1999. In addition to identifying the same sequences, the above programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered 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 the corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is the complete sequence. In some embodiments, the relevant stretch 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.

[0053] Affected: An individual who is "affected" by a disease, disorder, and / or condition has been diagnosed with the disease, disorder, and / or condition or exhibits one or more of its symptoms.

[0054] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of one or more symptoms of a disease, disorder, and / or condition when administered to a subject who has or is at risk of having the disease, disorder, and / or condition. One of ordinary skill in the art will recognize that a therapeutically effective amount is typically administered via a dosing regimen that includes at least one unit dose.

[0055] To treat: As used herein, the terms "treating," "treatment," or "to treat" refer to any method used to partially or completely alleviate, improve, reduce, suppress, prevent, delay the occurrence of, decrease the severity of, and / or decrease the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit symptoms of the disease and / or a subject who exhibits only early symptoms of the disease for the purpose of reducing the risk of presenting pathological findings associated with the disease.

[0056] In this specification, the recitation of a numerical range by endpoints includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4, and 5). It should also be understood that all numbers and their fractions are considered to be modified by the term "about."

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

[0058] Various aspects of the invention are described in detail in the following sections. The use of the sections is not meant to limit the invention. Each section can be applied to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise indicated.

[0059] Detailed Description The present disclosure describes an efficient and robust recombinant adeno-associated virus (rAAV) vector that results in in vivo production of C1-INH for treating diseases associated with C1-INH deficiency, such as HAE.

[0060] Hereditary angioedema (HAE) HAE is characterized by a decrease in the level of C1-INH and an associated upregulation of bradykinin. HAE is an autosomal dominant genetic disorder that occurs at a rate of 1 in 10,000 to 1 in 50,000 individuals. The underlying cause of HAE (types I and II) is thought to be an autosomal dominant inheritance of mutations in the C1 esterase inhibitor gene (C1EI gene or SERPING1 gene) located on chromosome 11. There can be over 300 autosomal dominant mutations in the SERPING1 gene, but gene therapy is possible. 85% of HAE cases are type I, in which there is insufficient production of C1 esterase inhibitor (see, e.g., Gower et al., World Allergy Organ J., 4:S9-S21 (2011); Cungo et al., Trends Mol Med, 15:69-78 (2009); Gooptu et al., Annu Rev Biochem, 78:147-176 (2009); and Zuraw et al., J Allergy Clin Immunol Pract, 1:458-467 (2013)). The remaining cases are characterized by the expression of dysfunctional C1 esterase inhibitor.

[0061] The frequency, duration, and severity of attacks associated with HAE vary, with 30% of patients reporting more than one attack per month, 40% reporting 6-11 attacks per year, and the remaining 30% showing symptoms only rarely. Usually, symptoms progress transiently over 12-36 hours and resolve within 2-5 days, but some attacks may last for about a week. Although HAE episodes are self-limiting, the occurrence of unpredictable attacks is a significant burden on patients, often having a major impact on quality of life and, in some cases, being life-threatening.

[0062] To date, therapeutic agents have been applied for long-term prophylaxis, treatment of acute attacks, and short-term prophylaxis (i.e., before dental surgery), and these include agents such as danazol, C1 inhibitor replacement protein, bradykinin receptor antagonists, kallikrein inhibitors, fresh frozen plasma, and purified C1 inhibitor, which have a high harmful effect profile. These therapies can relieve symptoms and maximize quality of life, but disease recurrence and the need for long-term continuous administration still remain a major obstacle to treatment (see, for example, Aberer, Ann Med, 44:523-529 (2012); Charignon et al., Expert Opin Pharmacother, 13:2233-2247 (2012); Papadopoulou-Alataki, Curr Opin Allergy Clin Immunol, 10:20-25 (2010); Parikh et al., Curr Allergy Asthma Rep, 11:300-308 (2011); Tourangeau et al., Curr Allergy Asthma Rep, 11:345-351 (2011); Bowen et al., Ann Allergy Asthma Immunol, 100:S30-S40 (2008); Frank, Immunol Allergy Clin North Am, 26:653-668 (2006); Cicardi et al., J Allergy Clin Immunol, 99:194-196 (1997); Kreuz et al., Transfusion 49:1987-1995 (2009); Bork et al., Ann Allergy Asthma Immunol, 100:153-161 (2008); and Cicardi et al., J Allergy Clin Immunol, 87:768-773 (1991)).

[0063] The present invention provides, inter alia, methods and compositions for treating HAE using a recombinant adeno-associated virus (rAAV) vector comprising a codon-optimized SERPING1 sequence encoding hC1-INH. In particular, the present invention provides a method for treating HAE by administering an rAAV comprising a codon-optimized sequence SERPING1 encoding human C1-INH in an effective amount such that at least one symptom or feature of HAE is reduced in intensity, severity, or frequency. The gene therapy methods described herein have been particularly effective in expressing therapeutic levels of hC1-INH.

[0064] rAAV SERPING1 Vector Design In some embodiments, provided herein is a recombinant adeno-associated virus (rAAV) vector encoding C1-INH. The rAAV vector comprises a capsid and a SERPING1 sequence.

[0065] Schematic diagrams illustrating exemplary rAAV vectors of the present disclosure are shown in FIGS. 1A and 1B. As shown in FIG. 1A, in some embodiments, the rAAV vector of the present disclosure comprises a liver-specific promoter, 5' and 3' terminal inverted repeats (ITRs), a cis-acting regulatory module (CRM), and an intron.

[0066] The SERPING1 sequence of the vector can be wild-type or a codon-optimized variant. Thus, in some embodiments, the rAAV vector comprises a wild-type SERPING1 nucleotide sequence. In some embodiments, the rAAV vector comprises a codon-optimized SERPING1 sequence.

[0067] A preferred C1-INH of the present invention is any protein or portion of a protein that can replace at least some of the activity of naturally occurring C1-INH or relieve one or more phenotypes or symptoms associated with C1-INH deficiency.

[0068] In some embodiments, the C1-INH nucleotide sequence suitable for the present invention includes the SERPING1 sequence encoding the human C1-INH protein. The naturally occurring human C1-INH nucleotide sequence is shown in GenBank: AF435921.1. The corresponding human C1-INH amino acid sequence is shown in Table 1. [Table 1] Various types of promoters can be used for the rAAV vectors described herein. These 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), modified hTTR, (hTTR mod.), alpha-1 antitrypsin promoter, human factor IX pro / liver transcription factor-responsive oligomer, LSP, CMV / CBA promoter (1.1 kb), CAG promoter (1.7 kb), mTTR, modified mTTR, mTTR pro, mTTR enhancer, and 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 incorporated herein by reference.

[0069] In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is the chicken beta-actin promoter.

[0070] In some embodiments, the rAAV vector contains additional enhancers or regulatory elements (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, IRES, etc.) that promote mRNA transcription and / or translation. In some embodiments, the vector includes 5' and 3' terminal inverted repeats (ITRs). In some embodiments, the vector includes one or more enhancer elements. In some embodiments, the vector includes a polyA tail.

[0071] In some embodiments, the rAAV vector contains one or more small elements such as introns. Various introns are known in the art. Suitable introns 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 alpha globin intron 1. In some embodiments, the rAAV vector contains the MVM intron. In some embodiments, the rAAV vector contains the SV40 intron. In some embodiments, the intron can be exon 1-intron 1-partial exon 2 of the SERPING1 gene.

[0072] In some embodiments, the rAAV vector comprises a woodchuck hepatitis virus post - transcriptional regulatory element (WPRE) because the WPRE increases transgene expression of the viral vector in a number of tissues. Various optimizations or variant forms of the WPRE are known in the art, including, for example, WPRE wild - type, WPRE3, and WPREmut6delATG. The WPRE and related WPRE variants are described in U.S. Patent No. 10,179,918; U.S. Patent No. 7,419,829; U.S. Patent No. 9,731,033; U.S. Patent No. 8,748,169; U.S. Patent No. 7,816,131; U.S. Patent No. 8,865,881; U.S. Patent No. 6,287,814; U.S. Patent Application Publication No. 2016 / 0199412; U.S. Patent Application Publication No. 2017 / 0114363; U.S. Patent Application Publication No. 2017 / 0360961; U.S. Patent Application Publication No. 2019 / 0032078; U.S. Patent Application Publication No. 2018 / 0353621; International Publication No. WO2017201527; International Publication No. WO2018152451; International Publication No. WO2013153361; International Publication No. WO2014144756; European Patent No. EP1017785; and European Patent Application Publication No. 3440191. Each of the foregoing publications is hereby incorporated by reference in its entirety into this specification.

[0073] In some embodiments, the rAAV vector comprises one or more cis - regulatory elements (CREs). The CREs are modified versions of the backbone elements of the construct. Various optimizations or variant forms of the CREs are known in the art, including, for example, CRE4 and CRE6. The following publications describe various variants of the CREs and each is hereby incorporated by reference into this specification: International Publication No. WO2016146757, WO2014064277, WO2014063753, and WO2009130208.

[0074] In some embodiments, the rAAV vector comprises a cis - acting regulatory module (CRM). Various types of CRM are suitable for use in the vectors described herein, and examples include liver - specific CRM, neuron - specific CRM, and / or CRM8. In some embodiments, the vector comprises more than one CRM. For example, in some embodiments, the vector comprises 2, 3, 4, 5, or 6 CRMs. In some embodiments, the vector comprises 3 CRMs, for example, 3 CRM8s.

[0075] In some embodiments, the rAAV vector is sequence - optimized to enhance transcript stability, translate more efficiently, and reduce immunogenicity. In some embodiments, the rAAV vector is sequence - optimized to enhance transcript stability, translate more efficiently, and / or reduce immunogenicity. In some embodiments, SERPING1 is sequence - optimized.

[0076] In some embodiments, the rAAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or 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. In some embodiments, the rAAV vector has an optimized sequence. In some embodiments, the rAAV capsid is modified.

[0077] Exemplary element sequences are shown in Table 2 below. In some embodiments, the rAAV vector comprises an rAAV vector element comprising a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the vector element sequence 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.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

[0078] The sequence identities between the codon-optimized SERPING1 sequences (HA03, HA04, HA05, and HA06) and the wild-type SERPING1 sequence (HA01), and between one codon-optimized SERPING1 sequence and another codon-optimized sequence are shown in Table 3 below.

Table 3

[0079] Use of rAAV vectors encoding C1-INH for the treatment of diseases Described herein are methods of treating diseases associated with C1-INH deficiency. Thus, in some embodiments, the rAAV vectors described herein are suitable for treating subjects having a C1-INH deficiency, such as patients suffering from HAE. The method of treatment comprises administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) vector described herein.

[0080] The rAAV vectors described herein can be used to treat any disease associated with a deficiency or disorder of C1-INH.

[0081] 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 the CRISPR-Cas system.

[0082] In some embodiments, the pharmaceutical composition comprising the rAAV vector described herein is used in a subject in need thereof. The pharmaceutical composition containing the rAAV vector or particle of the present invention contains a pharmaceutically acceptable excipient, diluent or carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil-in-water emulsions, various types of wetting agents, sterile solutions, and the like. The pharmaceutical composition may be in a lyophilized form. Such carriers can be formulated by conventional methods and administered to the subject in a therapeutically effective amount.

[0083] The rAAV vector is administered via a route suitable for a subject in need thereof. In some embodiments, the rAAV vector is administered by intravenous, intraperitoneal, subcutaneous, or intradermal routes. In one embodiment, the rAAV vector is administered intravenously. In an embodiment, intradermal administration includes administration by use of a "gene gun" or a biolistic particle delivery system. In some embodiments, the rAAV vector 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 nanoparticles, such as lipid nanoparticles or inorganic nanoparticles.

[0084] In some embodiments, functional C1-INH is detectable in the plasma of the subject at about 1 to 6 weeks after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject at about 1 week. In some embodiments, functional C1-INH is detectable in the plasma of the subject at about 2 weeks. In some embodiments, functional C1-INH is detectable in the plasma of the subject at about 3 weeks. In some embodiments, functional C1-INH is detectable in the plasma of the subject at about 4 weeks. In some embodiments, functional C1-INH is detectable in the plasma of the subject at about 5 weeks. In some embodiments, functional C1-INH is detectable in the plasma of the subject at about 6 weeks. In some embodiments, functional C1-INH is detectable in the hepatocytes of the subject at about 1 to 6 weeks after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the hepatocytes of the subject more than 7 weeks after administration of the rAAV vector.

[0085] In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 1 month, 2 months, 3 months, 4 months, 5 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 C1-INH is detectable in the plasma of the subject for at least 3 months after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 6 months after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 12 months after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 2 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 3 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 4 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 5 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 6 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 7 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 8 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 9 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for at least 10 years after administration of the rAAV vector. In some embodiments, functional C1-INH is detectable in the plasma of the subject for the remainder of the subject's life after administration of the rAAV vector.

[0086] In some embodiments, administration of rAAV comprising SERPING1 produces the same amount of active C1-INH as is observed after administration of intravenously delivered purified C1-INH protein. In some embodiments, administration of rAAV comprising SERPING1 produces more active C1-INH as compared to administration of intravenously delivered purified C1-INH protein.

[0087] In some embodiments, administration of rAAV containing SERPING1 increases C1-INH in a subject. In some embodiments, the increase in C1-INH is detected in the plasma of the subject. In some embodiments, the increase in C1-INH is detected in the liver tissue of the subject. In some embodiments, the increase in C1-INH can be detected in one or more tissues / organs including the gallbladder, spleen, ovary, bladder, fat, placenta, lung, prostate, heart, lymph node, and endometrium. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or about 10% compared to the subject's C1-INH baseline level before administration of rAAV containing SERPING1. Thus, in some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 95%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 90%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 85%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 80%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 75%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 70%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 65%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 60%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 55%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 50%. In some embodiments, administration of rAAV containing SERPING1 increases the subject's C1-INH by about 45%.In some embodiments, administration of rAAV comprising SERPING1 results in an approximately 40% increase in C1-INH in the subject. In some embodiments, administration of rAAV comprising SERPING1 results in an approximately 35% increase in C1-INH in the subject. In some embodiments, administration of rAAV comprising SERPING1 results in an approximately 30% increase in C1-INH in the subject. In some embodiments, administration of rAAV comprising SERPING1 results in an approximately 25% increase in C1-INH in the subject. In some embodiments, administration of rAAV comprising SERPING1 results in an approximately 20% increase in C1-INH in the subject. In some embodiments, administration of rAAV comprising SERPING1 results in an approximately 15% increase in C1-INH in the subject. In some embodiments, administration of rAAV comprising SERPING1 results in an approximately 10% increase in C1-INH in the subject.

[0088] In some embodiments, the level of functional C1-INH detectable in circulation after administration of the AAV vector to the subject is approximately 2 to 20 times greater than the amount of functional C1-INH detectable in the subject prior to administration of rAAV comprising SERPING1.

[0089] In some embodiments, the detectable level of active C1-INH after administration of the AAV vector to the subject meets or exceeds the human therapeutic level. In some embodiments, the level of active C1-INH after administration of the rAAV vector is about 2 to 35 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 2 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 3 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 4 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 5 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 6 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 6 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 7 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 8 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 9 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 10 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 15 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 20 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 25 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 30 times the human therapeutic level. In some embodiments, the level of active C1-INH after administration is about 35 times the human therapeutic level.

[0090] In some embodiments, the rAAV.SERPING1 vector is delivered as a single administration per subject. In some embodiments, a minimum effective dose (MED) is delivered to the subject. As used herein, MED refers to the dose of the rAAV SERPING1 vector required to achieve C1-INH activity that results in an increase in the subject's C1-INH level.

[0091] 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 SERPING1 vector preparation. In one embodiment, the dose is about 1×10 11 vector genomes (vg) / kg body weight to about 2×10 13 vg / kg (including the endpoints).

[0092] In some embodiments, the dose is at least 5×10 9 vg / kg or more.

[0093] The rAAV SERPING1 vector composition can be formulated in dosage units containing an amount of replication-deficient virus within the range of about 1.0×10 9 vg to about 1.0×10 15 vg. As used herein, the term "dose" can refer to the total dose delivered to the subject during the course of treatment or the amount delivered in a single (multiple) administration.

[0094] In some embodiments, the dose is sufficient to increase the C1-INH level in the patient's plasma by 25% or more. In some embodiments, rAAV SERPING1 is administered in combination with one or more therapeutic agents for the treatment of HAE.

Example

[0095] Other features, objects, and advantages of the present invention will be apparent from the following examples. However, it should be understood that the examples are illustrative of embodiments of the present invention and are provided by way of illustration only, not limitation. Various modifications and variations within the scope of the present invention will be apparent to those skilled in the art from the examples.

[0096] Example 1. Vector Design An exemplary method and design for generating an rAAV expression construct (rAAV vector) containing the coding sequence of human C1-esterase inhibitor (C1-INH) and its variants are described in this example. In this study, hSERPING1 was used as the coding sequence of human C1-INH (hC1-INH), and recombinant AAV vector (rAAV8) was used as the vector. The basic design of the rAAV vector includes an expression cassette flanked at both ends by inverted terminal repeats (ITRs): 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 in vector-producing cells. Typically, the expression cassette contains a promoter, a coding sequence, a polyA tail, and / or a tag, as shown in FIG. 1A. An expression construct hSERPING1 encoding human hC1-INH was designed and prepared using standard molecular biology techniques. The coding sequence of hSERPING1 was inserted downstream of the promoter hTTR (human transthyretin promoter). Furthermore, a liver-specific cis-acting regulatory module (CRM) was inserted upstream of the promoter, and an intron sequence was inserted downstream of the promoter. As shown in the following examples, this combination of regulatory factors and promoter was tested for high transduction levels. This expression construct was then ligated into an AAV vector and tested by sequencing. The vector was packaged into virus particles and stored.

[0097] In another embodiment, a WPRE sequence was inserted downstream of the coding region. This element provides a tertiary structure that enhances mRNA stability. A schematic diagram of the expression construct described herein is shown in FIG. 1B. Any number of variant forms can be made according to the above scheme.

[0098] Codon-optimized construct Furthermore, the coding sequence of SERPING1 was 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 utilize more frequently used codons and potentially increase the expression level of the transgene product from the vector. CpG island sequences that can induce an immune response were reduced. Repetitive nucleotides were also removed. Any number of variant forms can be made according to the above scheme. For example, multiple promoters may be used. Furthermore, different combinations of regulatory regions, promoters, introns, and exons can also be contemplated.

Table 4-1

Table 4-2

[0099] Different expression constructs (HAE constructs) designed to treat human angioedema are listed in Table 4 and shown in FIGS. 2A-7B. The M construct is generally shown in FIG. 2A, and two M constructs (M01 and M01A) are shown in FIG. 2B. The constructs of M01 and M01A both contain the HA01 sequence, which is the human SERPING1 wild-type sequence (control construct). HA01 contains 24 CpGs and has a GC content of 53.4%. M01 contains intron: SERPING1 exon 1-intron 1-partial exon 2 (717 bp), while M01A contains the MVM (77 bp) intron.

[0100] The J constructs are generally shown in Figure 3A, and four J constructs: J01, J02, J03, and J04 are shown in Figure 3B. The J constructs are codon-optimized SERPING1 sequences. J01 contains the HA03 SERPING1 sequence, J02 contains the HA06 SERPING1 sequence, J03 contains the HA05 SERPING1 sequence, and J04 contains the HA04 SERPING1 sequence.

[0101] The S constructs are generally shown in Figure 4A, and nine S constructs: S01, S02, S03, S04, S05, S06, S07, S08, and S09 are shown in Figure 4B. The S constructs contain the HA06 SERPING1 sequence, an intron (either the MVM intron or exon 1-intron 1-partial exon 2), and a WPRE (either WPRE3 or WPREmut6delATG).

[0102] The N constructs are generally shown in Figure 5A, and nine N constructs: Naptune01, Naptune02, Naptune03, Naptune04, Naptune05, Naptune06, Naptune07, Naptun08, and Naptun09 are shown in Figure 5B. The N constructs contain the HA06 SERPING1 sequence and a novel promoter element such as hTTR, hTTR mod., mTTR pro, mTTR enhancer, CAG promoter, or CMV / CBA promoter.

[0103] The U constructs are generally shown in Figure 6A, and ten U constructs: U01, U02, U03, U04, U05, U06, U07, U08, U09, and U10 are shown in Figure 6B. The U constructs contain a codon-optimized sequence and WPREmut6delATG.

[0104] The P constructs are generally shown in Figure 7A, and six P constructs: P01, P02, P03, P04, P05, and P06 are shown in Figure 7B. The P constructs contain the HA06 SERPING1 sequence WPREmut6delATG, and the modified backbone elements CRE4 and / or CRE6.

[0105] Example 2. In vitro expression of glycosylated functional hC1-INH mediated by the AAV8.SERPING1 vector This example shows the efficacy of the AAV8.SERPING1 vector in the in vitro expression of hC1-INH.

[0106] An rAAV vector expressing hC1-INH (AAV8.SERPING1) or a control vector (as a negative control) was transfected into HepG2 cells (liver cells), and the supernatant was collected after 72 hours. The rAAV vector construct is shown in Figure 1A. A plasma-derived hC1-INH sample was used as a positive control. The hC1-INH expression in the cell supernatant was evaluated by immunoblot using standard Western blot analysis. As shown in Figure 8A, hC1-INH was detected in the supernatant obtained from cells treated with the rAAV vector. The results of this example show the expression of hC1-INH from the rAAV vector.

[0107] The hC1-INH expression level in HepG2 cells was determined by measuring the amount of hC1-INH present in the supernatant using ELISA. As shown in Figure 8B, the cells transfected with rAAV expressed a very large amount of hC1-INH compared to the control cells. The results from this example show that rAAV-transfected cells express functional hC1-INH.

[0108] Example 3. Dose-dependent expression of hC1-INH in vivo mediated by the AAV8.hSERPING1 vector This example demonstrates the efficacy of an rAAV (i.e., AAV8.hSERPING1) vector in the dose-dependent expression of hC1-INH in vivo.

[0109] To demonstrate in vivo expression, the AAV8.hSERPING1 vector encoding hC1-INH, designated as M01 in Figure 2B, was intravenously injected into mice (C57 / bl / 6). Three different vector doses (1×10 11 vg / kg, 4×10 11 vg / kg, and 4×10 12 vg / kg) were evaluated. A single dose was administered to each mouse. Plasma samples were collected on day 14 after injection.

[0110] The efficacy of rAAV was determined by monitoring the levels of hC1-INH in plasma. The levels of hC1-INH in plasma were expressed as a percentage of the levels of hC1-INH present in normal mice. The results are shown in Figure 9A. Mice administered the rAAV vector showed dose-dependent expression of hC1-INH. The group of mice administered the rAAV vector at a dose of 4×10 12 vg / kg expressed levels of hC1-INH corresponding to the clinical target level (i.e., 220 μg / mL).

[0111] Furthermore, the transduction efficiency and transcription efficiency of rAAV were determined by intravenously injecting the vector into mice. Three doses: 1×10 11 vg / kg, 4×10 11 vg / kg, and 4×10 12Mice (C57 / bl / 6) were administered rAAV at one of the doses of vg / kg, and the fourth group of mice was administered vehicle only. The rAAV vector construct is shown as M01 in Figure 2B. The animals were sacrificed on day 28 after injection, and the livers were harvested. The transduction efficiency and transcription efficiency of rAAV were compared with those of the group administered vehicle only. The results are shown in Figure 9B. It has been shown that there was significant transduction and transcription of hSERPING1 at all three doses. In fact, when hSERPING1 DNA was expressed as hSERPING1 DNA copies / cell, dose-dependent increased transduction was observed. As expected, transduction and transcription of hSERPING1 were not seen in mice administered vehicle only.

[0112] The results according to this example show that the rAAV.AAV8.hSERPING1 vector expresses C1-INH in a dose-dependent manner in vivo. This result also reveals that physiological levels of human C1-INH are present in wild-type mice.

[0113] Effect of codon optimization and screening of codon-optimized constructs Example 4. In vivo efficacy of rAAV8 vectors containing codon-optimized hSERPING1 sequences This example shows the in vivo efficacy of several codon-optimized rAAV8.C1-INH constructs in the expression of C1-INH in plasma.

[0114] To test the effect of codon optimization of the hSERPING1 sequence on the in vivo efficacy of the rAAV8 vector, four codon-optimized constructs were generated. The codon-optimized constructs were J01, J02, J03, and J04. An rAAV vector containing either M01A (wild-type C1-INH) or a codon-optimized C1-INH sequence was injected into mice (C57bl / 6). All five constructs are shown in Figure 10B. 4×10 11 vg / kg or 2×10 12Either vector at vg / kg was administered to mice, and plasma samples were collected before rAAV administration and on days 7, 14, and 28 after injection. For the dose response, only J04 was used for evaluation. Only male mice were used in this study. The level of hC1-INH in plasma was expressed as a percentage of the level of hC1-INH present in normal mice. The results are shown in Figure 10A and Table 5.

[0115] Mice administered the control vector (wild type) and mice administered the codon-optimized construct both expressed hC1-INH. With different constructs, codon optimization was shown to have a moderate effect on the ability to express hC1-INH.

Table 5

[0116] The codon-optimized construct J04 expressed hC1-INH in a dose-dependent manner throughout the study (i.e., on days 7, 14, and 28), as shown in Figure 10A.

[0117] The results of this example show that codon optimization moderately improves the effectiveness of the construct over the wild type (control vector). The codon-optimized construct also expresses hC1-INH in a dose-dependent manner. Based on this study, J02 was selected for subsequent in vivo studies.

[0118] Effect of WPRE Example 5. In Vivo Efficacy of rAAV8 Vectors Containing Codon-Optimized hSERPING1 Sequences and WPRE This example shows the in vivo efficacy of vectors containing the codon-optimized AAV8.C1-INH-co2 sequence combined with three different WPREs in the expression of C1-INH in plasma.

[0119] Different codon-optimized constructs: (1) J02 (without WPRE element), (2) S07 (containing WPRE3), and rAAV vectors containing S04 (containing WPREmut6delATG) were injected into mice. The rAAV vectors containing WPRE are shown in Figure 1B, and different codon-optimized constructs and related elements are listed in Table 4. 4×10 11 vg / kg of the vector was administered to the mice, and plasma samples were collected before rAAV administration and on days 7, 14, and 28 after injection. The levels of hC1-INH in the plasma were expressed as a percentage of the levels of hC1-INH present in normal mice. The results are shown in Figure 11.

[0120] Mice administered constructs containing WPRE expressed more than about 2.5-fold higher hC1-INH compared to codon-optimized constructs without WPRE.

[0121] The results of this example show that incorporating WPRE improves the effectiveness of the codon-optimized construct.

[0122] Effect of WPRE and intron Example 6. In vivo efficacy of rAAV8 vector containing HA06 (hSERPING1) sequence, WPRE, and intron This example shows the in vivo efficacy of an rAAV8 vector containing the HA06 (hSERPING1) sequence, WPRE, and intron with respect to the expression of hC1-INH in plasma.

[0123] To test the effects of WPRE and introns on the in vivo efficacy of rAAV8 vectors containing the HA06 array, different combinations of WPRE and introns were used. The introns used in this study were either the MVM intron or exon 1-intron 1-partial exon 2, and the WPREs used were either WPRE3 or WPREmut6delATG. Recombinant AAV vectors containing different combinations of WPRE, intron, and codon-optimized hSERPING1 array used in this study are shown in Figure 12B. 4×10 11 vg / kg or 2×10 12 vg / kg of either vector was administered to mice (C57bl / 6; male), and plasma samples were collected before rAAV administration and on days 7, 14, and 28 after injection. The levels of hC1-INH in plasma were expressed as a percentage of the levels of hC1-INH present in normal mice. The results are shown in Figure 12A.

[0124] Mice administered a construct containing the MVM intron, HA06 (SERPING1) array, and WPRE3 (e.g., S07) expressed more hC1-INH compared to the remaining animals administered other constructs at the same dose. Mice administered J04 at 2×10 12 vg / kg showed the highest hC1-INH expression.

[0125] The results according to this example show that incorporating the MVM intron and WPRE3 improves the efficacy of the HA06 (SERPING1) array.

[0126] Effect of WPRE and alternative promoter elements at low and high vector doses Example 7. In vivo efficacy of rAAV8 vectors containing the HA06 (hSERPING1) array, WPRE, and alternative promoter This example shows the in vivo efficacy of a construct containing the HA06 (hSERPING1) array, WPRE, and novel promoter in plasma.

[0127] To test the effect of WPRE and other novel promoters on the in vivo efficacy of rAAV8 vectors, different constructs containing different novel promoter elements were used. Seven different constructs (J02, J04, S04, N01, N02, N03, and C22) were tested. C22 included in this test is a control vector. All seven constructs are shown in Figure 13B. Three doses: 4×10 11 vg / kg, 1.2×10 13 vg / kg, and 2×10 12 vg / kg of one of the vectors were administered to mice (C57bl / 6; male), and plasma samples were collected before rAAV administration and on days 7, 14, and 28 after injection. The levels of hC1-INH in plasma were expressed as a percentage of the levels of hC1-INH present in normal mice. The results are shown in Figure 13A.

[0128] Mice administered a construct containing the HA06 (SERPING1) sequence and WPREmut6delATG (e.g., S04) expressed more hC1-INH compared to the remaining animals administered other constructs at the same dose. Mice administered J04 at 1.2×10 13 vg / kg showed the highest hC1-INH expression. The results according to this example indicate that incorporating WPREmut6delATG improves the efficacy of the HA06 (SERPING1) sequence.

[0129] In another example, two doses: 2×10 12 vg / kg and 2×10 13 vg / kg of S04 were administered to mice (C57bl / 6; male), and plasma was collected on days 14 and 28. Another group of mice was administered buffer as a control. The results are shown in Figure 13C.

[0130] The results according to this example indicate that S04 expresses hC1-INH in a dose-dependent manner at both time points.

[0131] Effect of codon optimization Example 8. In vivo efficacy of rAAV8 vector containing a construct comprising a codon-optimized hSERPING1 sequence This example demonstrates the in vivo efficacy of an rAAV8 vector containing a construct comprising a codon-optimized hSERPING1 sequence in plasma.

[0132] Ten different codon-optimized constructs (U01, U02, U03, U04, U05, U06, U07, U08, U09, and U10) were prepared. All ten constructs are shown in Figure 14B. The in vivo efficacy of all codon-optimized constructs was tested together with S04. Construct S04 is shown in Figure 4B. 2×10 12 vg / kg of the vector was administered to mice (C57bl / 6; male), and plasma samples were collected before rAAV administration and on day 28 after injection. The levels of hC1-INH in plasma were expressed as a percentage of the levels of hC1-INH present in normal mice. The results are shown in Figure 14A.

[0133] Mice administered U06 expressed hC1-INH equivalent to that of the group administered S04 at the same dose.

[0134] Effect of WPRE and alternative promoter elements at medium dose in long-term studies Example 9. In vivo efficacy of rAAV8 vector containing a construct comprising HA06 (hSERPING1) sequence, WPRE and an alternative promoter This example demonstrates the in vivo efficacy of an rAAV8 vector containing a construct comprising HA06 (hSERPING1) sequence, WPRE and a novel promoter element in plasma.

[0135] To test the impact of the WPRE and the novel promoter on the in vivo efficacy of the rAAV8 vector, different constructs with or without the WPRE and novel promoter elements were used. Seven different constructs (J02, S03, S04, S06, S07, N04, and N05) were tested. All seven constructs are shown in Figure 15B. A medium dose of 2×10 12 vg / kg was administered to mice (C57bl / 6; male), and plasma samples were collected before rAAV administration and on days 7, 14, 28, 49, 70, 91, 112, 133, 152, and 175 after injection. The levels of hC1-INH in plasma were expressed as a percentage of the levels of hC1-INH present in normal mice. The results are shown in Figure 15A.

[0136] Mice administered constructs containing the MVM intron (e.g., S07) expressed more hC1-INH compared to the group administered constructs without MVM (e.g., S06). In addition, the groups administered constructs containing WPRE3, a short form of WPRE, (e.g., S06 and S07) expressed more hC1-INH compared to the groups administered constructs containing WPREmut6delATG, a modified WPRE, (e.g., S03 and S04).

[0137] The results according to this example show that by incorporating the MVM intron and WPRE3, a short form of WPRE, the efficacy of the HA06 (SERPING1) sequence is improved. Construct S07, which incorporated both the MVM intron and WPRE3, showed the greatest efficacy and expressed hC1-INH over a period of 175 days. Equivalents and Scope

[0138] One of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein using only routine experimentation. The scope of the invention is not limited to the above "Modes for Carrying Out the Invention" but is set forth in the following claims. In certain embodiments, for example, the following are provided: (Item 1) A recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a codon-optimized SERPING1 sequence encoding a C1 inhibitor (C1-INH). (Item 2) The rAAV vector according to item 1, wherein the codon-optimized SERPING1 sequence encoding the C1-INH comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 2. (Item 3) The rAAV vector according to item 2, wherein the codon-optimized SERPING1 sequence encoding the C1-INH comprises the same sequence as SEQ ID NO: 2. (Item 4) The rAAV vector according to item 1, wherein the vector further comprises a liver-specific promoter. (Item 5) The rAAV vector according to item 4, wherein the liver-specific promoter is a transthyretin promoter (TTR). (Item 6) The rAAV vector according to any one of the preceding items, wherein the vector further comprises a ubiquitous promoter. (Item 7) The rAAV vector according to any one of the preceding items, wherein the vector further comprises one or more of the following: 5' and 3' terminal inverted repeats, an intron upstream of the sequence, and a cis-acting regulatory module (CRM). (Item 8) The rAAV vector according to any one of the preceding items, wherein the vector further comprises a WPRE sequence. (Item 9) The rAAV vector according to item 8, wherein the WPRE sequence is modified. (Item 10) The rAAV vector according to item 9, wherein the WPRE contains a mut6delATG modification. (Item 11) The rAAV vector according to item 7, wherein the intron is a murine minute virus (MVM) or SV40 intron. (Item 12) The rAAV vector according to item 7, wherein the CRM is a liver-specific CRM. (Item 13) The rAAV vector according to item 7, wherein the CRM is CRM8. (Item 14) The rAAV vector according to item 7, wherein the vector contains at least three CRMs. (Item 15) A recombinant adeno-associated virus (rAAV) comprising an AAV8 capsid and an rAAV vector, wherein the vector comprises: a. A 5' terminal inverted repeat (ITR), b. A cis-acting regulatory module (CRM), c. A liver-specific promoter, d. Murine minute virus (MVM), e. A SERPING1 sequence encoding C1 inhibitor (C1-INH), f. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and g. A 3' ITR The rAAV comprising the same. (Item 16) The rAAV according to item 15, wherein the SERPING1 sequence is a wild-type sequence or a codon-optimized sequence. (Item 17) The rAAV according to item 16, wherein the codon-optimized SERPING1 sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 2. (Item 18) A method for treating a subject having hereditary angioedema (HAE), the method comprising administering to a subject in need thereof the rAAV according to any one of the preceding items. (Item 19) A method of treating a subject having hereditary angioedema (HAE), the method comprising administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a promoter operably linked to a nucleic acid sequence encoding a C1 inhibitor (C1-INH), wherein administration increases the C1-INH enzyme activity in the subject. (Item 20) The method according to item 19, wherein the C1-INH is detected in the plasma of the subject. (Item 21) The method according to any one of items 18 to 20, wherein the C1-INH is detected in the liver of the subject. (Item 22) The method according to any one of items 18 to 21, wherein C1-INH is maintained for at least 30, 60, 90, 120, 150, 180 days or more after a single administration. (Item 23) The method according to any one of items 18 to 22, wherein C1-INH activity is present in the subject after administration of the rAAV vector. (Item 24) The method according to item 23, wherein the subject has a C4 level that has recovered to pre-attack levels. (Item 25) The method according to any one of items 18 to 24, wherein the AAV is administered intravenously. (Item 26) The method according to any one of items 18 to 24, wherein the AAV is administered intrathecally. (Item 27) The AAV is administered at a dose of at least about 5×10 9 vg, the method according to item 25 or 26. (Item 28) The method according to any one of items 18 to 27, wherein the administration of the rAAV does not induce an immune response.

Claims

[Claim 1] The invention described in this specification.

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