Adeno-associated virus vector-based gene therapy for hereditary angioedema

Abstract

The present disclosure provides, inter alia, a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a codon-optimized SERPING1 sequence encoding human C1-esterase inhibitor. The present disclosure also provides a method of treating a subject having hereditary angioedema (HAE), comprising administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a codon-optimized SERPING1 sequence encoding human C1-esterase inhibitor.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 924,877, filed October 23, 2019, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Hereditary angioedema (HAE) is a rare condition characterized by recurrent episodes of swelling in the face, throat, and most of the extremities. HAE is a potentially life-threatening disorder characterized by unpredictable, recurrent attacks of vasodilation that manifest as subcutaneous and submucosal angioedema. HAE is in some cases associated with low levels of C1-inhibitor in the plasma (Type I) and in other cases where the protein circulates in normal or high amounts but may be dysfunctional (Type II). C1-inhibitor is the main regulator of plasma kallikrein activity. Symptoms of an HAE attack include swelling of the face, mouth, and / or airways, which may occur spontaneously or be precipitated by minor trauma. Edematous attacks affecting the airways 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] Various strategies have been conceived and developed for the treatment of C1-INH deficiency or dysfunction, including, for example, inhibiting members of the contact system, such as lanadelumab, a fully human monoclonal antibody inhibitor of plasma kallikrein, which has been approved for the treatment of HAE.

[0004] For the treatment of diseases, it is desirable to use vectors that produce proteins in vivo, but this is limited by a variety of factors, including insufficient protein production after delivery to a subject.

[0005] To date, available treatments do not address challenges including disease recurrence and the need for continuous treatment over long periods of time. Thus, there is a need for novel and sustained therapeutic approaches to treat HAE. Summary of the Invention

[0006] The present invention provides recombinant adeno-associated virus (rAAV) vectors that enable 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 that includes, inter alia, an AAV8 capsid and a codon-optimized SERPING1 sequence encoding 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' inverted repeats, an intron upstream 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, the WPRE contains a mut6delATG modification.

[0017] In some embodiments, the intron is a minute virus of mouse (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 a rAAV vector, the vector comprising: a.5' inverted terminal repeat (ITR), b. cis-acting regulatory modules (CRMs); C. liver-specific promoter, D. Minute virus of mice (MVM), e. SERPING1 sequence encoding C1 inhibitor (C1-INH); f. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and g.3'ITR Includes.

[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), 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), 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), whereby C1-INH enzyme activity in the subject is increased.

[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 following a single administration.

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

[0030] In some embodiments, the subject has C4 levels restored to pre-stroke 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 at least about 5×10 9 It is administered in a dose of vg.

[0034] In some embodiments, administration of the rAAV does not elicit 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 may be applicable 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] [Figure 1] A is a schematic diagram of an expression cassette comprising a wild-type human C1-INH (hC1-INH) expression vector. B is a schematic diagram of an expression cassette designed for hC1-INH expression in the liver (hepatocytes). ITR: inverted terminal repeat; hTTR: human transthyretin promoter; CRM: cis-acting regulatory module; intron can be MVM intron (minute virus of mouse intron); polyA is an upstream enhancer. [Figure 2A] General schematic diagram of the M construct. The M construct is a control construct with the HA01 (SERPING1) sequence. HA01 is the human SERPING1 wild-type sequence, with 1503 bp, 24 CpGs, and 53.4% ​​GC content. [Figure 2B]Two M constructs are shown: M01 and M01A. M01 contains the HAO1 (SERPING1) sequence and exon 1-intron 1-partial exon 2 (717 bp) intron, while Mar01A contains the HAO1 (SERPING1) sequence and MVM (77 bp) intron. [Diagram 3] A is a general schematic diagram of the J construct. The J construct contains four codon-optimized SERPING1 sequences. B shows schematic diagrams of four J constructs: J01, J02, J03, and J04. J01 contains the codon-optimized SERPING1 sequence HA03. J02 contains the codon-optimized SERPING1 sequence HA06. J03 contains the codon-optimized SERPING1 sequence HA05. J04 contains the codon-optimized SERPING1 sequence HA04. [Figure 4A] General schematic of S constructs. All S constructs contain codon-optimized HA06 (SERPING1) sequences with different combinations of introns and WPREs. Introns contain either MVM introns or exon1-intron1-partial exon2. [Figure 4B] Schematic diagrams of the nine S constructs: S01, S02, S03, S04, S05, S06, S07, S08, and S09 are shown. [Figure 5A] General schematic of N constructs. All N constructs contain the codon-optimized HA06 (SERPING1) sequence and novel promoter elements. [Figure 5B] Schematic diagrams of nine N constructs: N01, N02, N03, N04, N05, N06, N07, N08, and N09 are shown. [Figure 6A] 1 is a general schematic diagram of the U constructs, which contain different codon-optimized SERPING1 sequences combined with WPREmut6delATG. [Figure 6B]Schematic diagrams of ten U constructs: U01, U02, U03, U04, U05, U06, U07, U08, U09, and U10 are shown. U01 contains the codon-optimized SERPING1 sequence HA11. U02 contains the codon-optimized SERPING1 sequence HA12. U03 contains the codon-optimized SERPING1 sequence HA13. U04 contains the codon-optimized SERPING1 sequence HA14. U05 contains the codon-optimized SERPING1 sequence HA15. U06 contains the codon-optimized SERPING1 sequence HA16. U07 contains the codon-optimized SERPING1 sequence HA17. U08 contains the codon-optimized SERPING1 sequence HA18. U09 contains the codon-optimized SERPING1 sequence HA19. U10 contains the codon-optimized SERPING1 sequence HA20. [Figure 7A] General schematic of P constructs. All P constructs contain the codon-optimized HA06 (SERPING1) sequence and WPREmut6delATG in combination with different novel promoter elements. The novel promoter elements include CRE4, CRE6, or CRE4 and CRE6. [Figure 7B] Schematic diagrams of six P constructs: P01, P02, P03, P04, P05, and P06 are shown. [Figure 8] (A) is an image of a Western blot showing expression of hC1-INH by the AAV8.SERPING1 vector in HepG2 cells. (B) is a graph of experimental data showing expression of functional C1-INH by AAV8.SERPING1 in HepG2 cells as measured by ELISA assay. [Figure 9](A) is a graph of experimental data showing dose-dependent expression of hCl-INH after a single intravenous administration of AAV8.SERPING1 vector to wild-type C57Bl / 6 albino mice. Levels of hCl-INH were measured in plasma 14 days after injection. (B) is a graph of experimental data showing dose-dependent expression of hSERPING1 DNA or RNA after a single intravenous administration of AAV8.SERPING1 vector to wild-type C57Bl / 6 albino mice. Levels of hCl-INH were measured in liver 28 days after injection. Expression levels of hSERPING1 DNA are expressed as copies / μg and copies / cell. [Figure 10] (A) is a graph of experimental data showing the in vivo efficacy of rAAV8 vectors containing four codon-optimized SERPING1 constructs (J01, J02, J03, and J04) in expressing C1-INH in plasma after a single intravenous administration of the different vectors to wild-type C57Bl / 6 albino mice. Expression levels of hC1-INH were measured 7, 14, and 28 days after injection. The M01A construct was used as a control. (B) shows a schematic diagram of the different constructs used in this study. [Figure 11] 1 is a graph of experimental data showing the in vivo efficacy of rAAV8 vectors containing the codon-optimized SERPING1 sequence and WPRE in expressing C1-INH in plasma after a single intravenous administration of the vector to wild-type C57Bl / 6 albino mice. Expression levels of hC1-INH were measured 7, 14, and 28 days after injection. A single vector dose of 4×10 vg / kg was administered. [Figure 12] (A) is a graph of experimental data showing the in vivo efficacy of rAAV8 vectors containing the HA06 (SERPING1) sequence, different WPREs and introns in expressing C1-INH in plasma after a single intravenous dose of the vector in C57Bl / 6 albino mice. Expression levels of hC1-INH were measured 7, 14 and 28 days after injection. (B) shows a schematic diagram of the different constructs used in this study. [Figure 13](A) is a graph of experimental data showing the in vivo efficacy of rAAV8 vectors containing the HA06 (SERPING1) sequence, WPRE and different promoter elements in expressing C1-INH in plasma after a single intravenous administration of the vector at a dose of 4×10 vg / kg in C57Bl / 6 albino mice. Expression levels of hC1-INH were measured 7, 14 and 28 days after injection. (B) shows a schematic diagram of the different constructs used in the study. (C) is a graph of experimental data showing the dose-dependent efficacy of S04, a construct containing the HA06 (SERPING1) sequence, WPRE mut6delATG and MVM intron, in expressing C1-INH in plasma after intravenous administration of the construct at low (2×10 vg / kg) and high (2×10 vg / kg) doses in C57Bl / 6 albino mice. The expression levels of hC1-INH were measured on days 14 and 28 after injection. In C, a schematic diagram of S04 is shown. [Figure 14] (A) is a graph of experimental data showing the in vivo efficacy of rAAV8 vectors containing the codon-optimized SERPING1 sequence in expressing C1-INH in plasma after a single intravenous dose of the vector at a dose of 2x1012 vg / kg in C57Bl / 6 albino mice. Expression levels of hC1-INH were measured 28 days after injection. Expression levels of the vector containing the codon-optimized SERPING1 sequence were compared to those of the S04 construct at the same dose. (B) shows a schematic diagram of the different constructs used in this study. [Figure 15] (A) is a graph of experimental data showing the in vivo efficacy of rAAV8 vectors containing the HA06 (SERPING1) sequence in combination with different WPRE and novel promoter elements in expressing C1-INH in plasma after a single intravenous administration of a medium dose of vector to C57Bl / 6 albino mice. Expression levels of hC1-INH were measured at 7, 14, 28, 49, 70, 91, 112, 133, 152, and 175 days after injection. (B) shows a schematic diagram of the different constructs used in this study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] definition Adeno-associated virus (AAV): As used herein, the term "adeno-associated virus" or "AAV" or recombinant AAV ("rAAV") includes, but is 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 extrachromosomally without integrating into the genome of the host cell. AAV vectors are often used in gene therapy.

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

[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 animals are mammals (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, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0040] Approximately or about: As used herein, the term "approximately" or "about" when applied to one or more target values ​​refers to a value that is similar to a specified reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls 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 less) of the stated reference value, unless otherwise indicated or clear from the context (except when the numerical value in question exceeds 100% of the possible values).

[0041] Functional equivalent or derivative: As used herein, the term "functional equivalent" or "functional derivative" in the context of a functional derivative of an amino acid sequence means a molecule that retains substantially similar biological activity (either function or structure) as the original sequence. The functional derivative or equivalent may be a natural derivative or is synthetically prepared. Exemplary functional derivatives include amino acid sequences with one or more amino acid substitutions, deletions, or additions, so long as the biological activity of the protein is preserved. The substituted amino acid desirably has similar chemical and physical properties as the amino acid that is substituted. Desirable similar chemical and physical properties include similarity in charge, bulkiness, hydrophobicity, hydrophilicity, and the like.

[0042] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0043] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

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

[0045] Polypeptide: The term "polypeptide" as used herein refers to a continuous chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of skill in the art will understand that the term is not limited to long chains, but can also refer to a minimal chain containing two amino acids linked together via a peptide bond. As known to those of skill in the art, polypeptides may be subject to processing and / or modification.

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

[0047] Regulatory element: As used herein, the term "regulatory element" refers to a transcriptional control element, particularly a non-coding cis-acting transcriptional control element, capable of regulating and / or controlling the transcription of a gene. A regulatory element comprises 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 compared to gene transcription by the promoter alone without the regulatory element. Thus, although regulatory elements include, in particular, enhancer sequences, it is understood that regulatory elements that enhance transcription are not limited to enhancer sequences, which are typically located far upstream, but may be located at any distance from the gene to be regulated. As is understood in the art, sequences that regulate transcription may be located either upstream (e.g., within the promoter region) or downstream (e.g., within the 3'UTR) of the gene to be regulated in vivo, and may be located in close proximity to the gene or at a considerable distance. Regulatory elements may include either naturally occurring sequences, combinations of (parts of) such regulatory elements, or several copies of regulatory elements, e.g., non-naturally occurring sequences. Thus, regulatory elements include naturally occurring sequences and regulatory elements that have been 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, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, which refers to a human visiting a health care provider for diagnosis or treatment of a disease. The term "subject" may be used interchangeably herein with "individual" or "patient." A subject may be a subject suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0049] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting a characteristic or property of interest in whole or in whole close extent or degree. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to and / or proceed to completion or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture 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 acid or nucleic acid sequences. As will be appreciated by those skilled 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, appropriately having similar structural and / or functional characteristics. For example, as will be familiar to those skilled 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. Substitution of one amino acid with another amino acid of the same type may 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, 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. In addition to identifying homologous sequences, the above-mentioned programs 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 the 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 phrase "substantial identity" is used herein to refer to comparison between amino acid or nucleic acid sequences. As those skilled in the art will recognize, 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 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., 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 identical sequences, the above-mentioned programs typically provide an indication of the degree of identity. 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 the corresponding residues are identical over the 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] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0054] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of a symptom(s) of the disease, disorder, and / or condition. One of skill in the art will recognize that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0055] Treating: As used herein, the terms "treat", "treatment", or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease and / or who show only early signs of the disease, with the aim of reducing the risk of developing pathology associated with the disease.

[0056] As used herein, the recitations of numerical ranges by endpoints include 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 is also understood that all numbers and fractions thereof are presumed 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 may be applicable 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 indicates 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 may be applicable 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 efficient and robust recombinant adeno-associated virus (rAAV) vectors that direct the in vivo production of C1-INH to treat diseases associated with C1-INH deficiency, such as HAE.

[0060] Hereditary Angioedema (HAE) HAE is characterized by reduced levels of C1-INH and concomitant upregulation of bradykinin. HAE is an autosomal dominant condition with an incidence of 1 in 10,000 to 1 in 50,000 individuals. The underlying cause of HAE (types I and II) is thought to be autosomal dominant inheritance of mutations in the C1 esterase inhibitor gene (C1EI gene or SERPING1 gene) located on chromosome 11. There may be more than 300 autosomal dominant mutations in the SERPING1 gene, but gene therapy is available. Eighty-five percent of HAE cases are type I, characterized by 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] Attacks associated with HAE vary in frequency, duration, and severity, with 30% of patients reporting attacks more than once per month, 40% reporting 6-11 attacks per year, and the remaining 30% only experiencing infrequent symptoms. Symptoms usually progress transiently over 12-36 hours and subside within 2-5 days, although some attacks may last up to a week. Although HAE episodes are self-limited, the unpredictable occurrence of attacks is a significant burden for patients, often significantly impacting their quality of life and even being life-threatening.

[0062] To date, therapeutic agents are indicated for long-term prophylaxis, treatment of acute attacks, and short-term prophylaxis (i.e., prior to dental surgery) and include agents such as danazol, C1 inhibitor replacement protein, bradykinin receptor antagonists, kallikrein inhibitors, fresh frozen plasma, and purified C1 inhibitor, all of which have a high adverse effect profile. Although these treatments can alleviate symptoms and maximize quality of life, disease recurrence and the need for long-term continuous medication remain major obstacles to treatment (e.g., 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 recombinant adeno-associated virus (rAAV) vectors comprising a codon-optimized SERPING1 sequence encoding hC1-INH. In particular, the present invention provides methods for treating HAE by administering rAAV comprising the codon-optimized SERPING1 sequence encoding human C1-INH in an effective amount such that at least one symptom or characteristic 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 disclosure are shown in Figures 1A and 1B. As shown in Figure 1A, in some embodiments, the rAAV vectors of the disclosure include a liver-specific promoter, 5' and 3' inverted repeats (ITRs), a cis-acting regulatory module (CRM), and an intron.

[0066] The SERPING1 sequence of the vector can be a wild-type or 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 suitable C1-INH of the present invention is any protein or portion of a protein that is capable of replacing at least a portion of the activity of naturally occurring C1-INH or relieving 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 comprises 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 in the rAAV vectors described herein. These include, for example, ubiquitous promoters, tissue-specific promoters, and regulated (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, human transthyretin promoter (TTR), modified hTTR, (hTTR mod.), alpha-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 contents of which are incorporated herein by reference.

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

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

[0071] In some embodiments, the rAAV vector comprises one or more small elements, such as an intron. Various introns are known in the art. Introns suitable for the rAAV vectors described herein include, for example, MVM intron, truncated F.IX intron, chimeric beta globin SD / immunoglobulin heavy chain SA intron, SV40 and / or alpha globin first intron. In some embodiments, the rAAV vector comprises an MVM intron. In some embodiments, the rAAV vector comprises an 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 multiple tissues. Various optimized or variant forms of the WPRE are known in the art, including, for example, WPRE wild type, WPRE3, and WPREmut6delATG, among others. 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 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 incorporated herein by reference in its entirety.

[0073] In some embodiments, the rAAV vector comprises one or more cis-regulatory elements (CRE). The CRE is a modified backbone element of the construct. Various optimized or variant forms of CRE are known in the art, including, for example, CRE4 and CRE6, among others. The following publications describe various variants of CRE, each of which is incorporated herein by reference: International Publication No. WO2016146757, International Publication No. WO2014064277, International Publication No. WO2014063753, and International Publication No. WO2009130208.

[0074] 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, including, for example, 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 three CRMs, for example, three CRM8s.

[0075] In some embodiments, the rAAV vector is sequence-optimized to enhance transcript stability, more efficient translation, and / or reduced immunogenicity. In some embodiments, the rAAV vector is sequence-optimized to enhance transcript stability, more efficient translation, and / or reduced 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 an AAV1. In some embodiments, the rAAV vector is an AAV2. In some embodiments, the rAAV vector is an AAV3. In some embodiments, the rAAV vector is an AAV4. In some embodiments, the rAAV vector is an AAV5. In some embodiments, the rAAV vector is an AAV6. In some embodiments, the rAAV vector is an AAV7. In some embodiments, the rAAV vector is an AAV8. In some embodiments, the rAAV vector is an AAV9. In some embodiments, the rAAV vector is an AAV10. In some embodiments, the rAAV vector is an AAV11. In some embodiments, the rAAV vector is sequence optimized. In some embodiments, the rAAV capsid is modified.

[0077] Exemplary element sequences are shown below in Table 2. 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 a vector element sequence shown in Table 2. In some embodiments, the rAAV vector comprises a vector element nucleotide sequence identical to a 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 identity between the codon-optimized SERPING1 sequences (HA03, HA04, HA05, and HA06) and the wild-type SERPING1 sequence (HA01), as well as the sequence identity between one codon-optimized SERPING1 sequence and the other codon-optimized sequences, are shown in Table 3 below. [Table 3]

[0079] Use of rAAV vectors encoding C1-INH for disease treatment Described herein is a method for treating diseases associated with C1-INH deficiency.Thus, in some embodiments, the rAAV vector described herein is suitable for treating subjects with C1-INH deficiency, such as patients suffering from HAE.The method for treating comprises administering to a subject in need thereof the 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 impairment 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 a subject. Such integration can be achieved by using various gene editing techniques, such as, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), ARCUS genome editing, and / or CRISPR-Cas systems.

[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 pharma- ceutically 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 lyophilized form. Such carriers can be formulated by conventional methods and administered to a subject in a therapeutically effective amount.

[0083] The rAAV vector is administered to a subject in need thereof via a suitable route. 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 using a "gene gun" or biolistic particle delivery system. In some embodiments, the rAAV vector is administered via a non-viral lipid nanoparticle. For example, a composition comprising a rAAV vector may include one or more diluents, buffers, liposomes, lipids, lipid complexes. In some embodiments, the rAAV vector is contained within a microsphere or nanoparticle, for example, a lipid nanoparticle or an inorganic nanoparticle.

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

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

[0086] In some embodiments, administration of a rAAV comprising SERPING1 produces active C1-INH to the same extent as that seen following administration of purified C1-INH protein delivered intravenously, in some embodiments, administration of a rAAV comprising SERPING1 produces more active C1-INH compared to administration of purified C1-INH protein delivered intravenously.

[0087] In some embodiments, administration of a rAAV comprising 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 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, ovaries, bladder, fat, placenta, lung, prostate, heart, lymph nodes, and endometrium. In some embodiments, administration of a rAAV comprising SERPING1 increases C1-INH in a subject 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 baseline C1-INH level prior to administration of a rAAV comprising SERPING1. Thus, in some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 95%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 90%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 85%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 80%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 75%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 70%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 65%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 60%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 55%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 50%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 45%.In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 40%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 35%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 30%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 25%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 20%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 15%. In some embodiments, administration of a rAAV comprising SERPING1 increases a subject's C1-INH by about 10%.

[0088] In some embodiments, the level of functional C1-INH detectable in the circulation after administration of the AAV vector to a subject is about 2-20 times greater than the amount of functional C1-INH detectable in the subject before administration of the rAAV containing 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 dose to each subject. In some embodiments, the subject is delivered a minimum effective dose (MED). As used herein, MED refers to the dose of rAAV SERPING1 vector required to achieve C1-INH activity that results in an increase in the subject's C1-INH level.

[0091] The vector titer is determined based on the DNA content of the vector preparation. In some embodiments, the DNA content of the rAAVSERPING1 vector preparation is determined using quantitative PCR or optimized quantitative PCR. In one embodiment, the dosage is about 1×10 11 Vector genome (vg) / kg body weight ~ approx. 2 × 10 13 vg / kg (endpoints included).

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

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

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

[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 intended to illustrate embodiments of the present invention and are provided by way of illustration rather than limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the examples.

[0096] Example 1. Vector design In this example, an exemplary method and design for generating rAAV expression constructs (rAAV vectors) containing the coding sequence of human C1-esterase inhibitor (C1-INH) and its variants are described. 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 rAAV vectors includes an expression cassette flanked by terminal inverted repeats (ITRs): 5'-ITR and 3'-ITR. These ITRs mediate the replication and packaging of the vector genome by AAV replication proteins Rep and associated factors in vector-producing cells. Typically, the expression cassette contains a promoter, coding sequence, polyA tail and / or tag, as shown in FIG. 1A. The expression construct hSERPING1 encoding human hC1-INH was designed and generated using standard molecular biology techniques. The coding sequence of hSERPING1 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 an intron sequence was inserted downstream of the promoter. This regulator-promoter combination was tested for high transduction levels, as shown in the following examples. This expression construct was then ligated into an AAV vector and tested by sequencing. The vector was packaged into viral 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 Figure 1B. Any number of variations can be made according to the above scheme.

[0098] Codon-optimized constructs Furthermore, the coding sequence of SERPING1 was codon-optimized based on multiple parameters, such as codon adaptation index (CAI), number of CpG sites, GC content, and repeated base 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 may induce an immune response were reduced. Repeated bases were also removed. Any number of variations can be made with 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 designed to treat human angioedema (HAE constructs) are listed in Table 4 and shown in Figures 2A-7B. The M construct is generally shown in Figure 2A, and two M constructs (M01 and M01A) are shown in Figure 2B. Both M01 and M01A constructs contain the human SERPING1 wild type sequence, the HAO1 sequence (control constructs). HAO1 contains 24 CpGs and a GC content of 53.4%. M01 contains the intron: SERPING1 exon 1-intron 1-partial exon 2 (717bp), while M01A contains the MVM (77bp) 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 (MVM intron or exon1-intron1-partial exon2), 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 novel promoter elements 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 modified backbone elements CRE4 and / or CRE6.

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

[0106] The rAAV vector expressing hC1-INH (AAV8.SERPING1) or the control vector (as a negative control) was transfected into HepG2 cells (liver cells), and the supernatant was collected 72 hours later. The rAAV vector construct is shown in FIG. 1A. A plasma-derived hC1-INH sample was used as a positive control. hC1-INH expression in cell supernatant was evaluated by immunoblotting using standard Western blot analysis. As shown in FIG. 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, rAAV transfected cells expressed significantly more hC1-INH than control cells.The results of this example show that rAAV transfected cells express functional hC1-INH.

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

[0109] To demonstrate in vivo expression, mice (C57 / bl / 6) were intravenously injected with the AAV8.hSERPING1 vector encoding hC1-INH, shown as M01 in Figure 2B. Three different vector doses (1 × 10 11 vg / kg, 4 × 10 11 vg / kg, and 4 × 10 12 The doses of 100 mg / kg of 1000 mg ...

[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 with rAAV vectors showed dose-dependent expression of hC1-INH. 4 x 10 12 Mice receiving rAAV vectors at a dose of 1000 vg / kg expressed levels of hC1-INH equivalent to the clinical target level (i.e., 220 μg / mL).

[0111] Additionally, the transduction and transcription efficiencies 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 dose of 1000 mg / kg, and a fourth group of mice received vehicle only. The rAAV vector construct is shown as M01 in Figure 2B. Animals were sacrificed 28 days after injection and livers were harvested. The transduction and transcription efficiencies of rAAV were compared to the group that received vehicle only. The results are shown in Figure 9B. It is shown that there was significant transduction and transcription of hSERPING1 at all three doses. Indeed, when hSERPING1 DNA was expressed as hSERPING1 DNA copies / cell, a dose-dependent increased transduction was observed. As expected, no transduction or transcription of hSERPING1 was observed in mice that received vehicle only.

[0112] Our results show that the rAAV.AAV8.hSERPING1 vector expresses C1-INH in a dose-dependent manner in vivo and also demonstrate the presence of physiological levels of human C1-INH 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 demonstrates the in vivo efficacy of several codon-optimized rAAV8.C1-INH constructs in expressing C1-INH in plasma.

[0114] To test the effect of codon optimization of the hSERPING1 sequence on the in vivo efficacy of rAAV8 vectors, four codon-optimized constructs were generated. The codon-optimized constructs were J01, J02, J03, and J04. Mice (C57bl / 6) were injected with rAAV vectors containing either M01A (wild type C1-INH) or the codon-optimized C1-INH sequence. All five constructs are shown in Figure 10B. 4x10 11 vg / kg or 2 × 10 12Mice were administered either vector at 1000 ng / kg and plasma samples were taken prior to rAAV administration and at 7, 14, and 28 days post-injection. Dose response was assessed using J04 only. Only male mice were used in this study. Levels of hC1-INH in plasma were expressed as a percentage of levels of hC1-INH present in normal mice. Results are shown in Figure 10A and Table 5.

[0115] Both mice receiving the control vector (wild type) and the codon-optimized constructs expressed hC1-INH, and the different constructs showed that codon optimization had 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., days 7, 14, and 28), as shown in FIG. 10A.

[0117] The results from this example show that codon optimization moderately improves the efficacy 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 biological testing.

[0118] Effects of WPRE Example 5. In vivo efficacy of rAAV8 vectors containing codon-optimized hSERPING1 sequences and WPRE This example demonstrates the in vivo efficacy of vectors containing the codon-optimized AAV8.C1-INH-co2 sequence in combination with three different WPREs in expressing C1-INH in plasma.

[0119] Mice were injected with rAAV vectors containing different codon-optimized constructs: (1) J02 (no WPRE element), (2) S07 (containing WPRE3), and S04 (containing WPREmut6delATG). The WPRE-containing rAAV vectors are shown in FIG. 1B, and the different codon-optimized constructs and associated elements are listed in Table 4. 11 Mice were administered 1000 ng / kg of vector and plasma samples were taken prior to administration of rAAV and at 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 FIG.

[0120] Mice administered the WPRE-containing construct expressed approximately 2.5-fold more hC1-INH compared to the codon-optimized construct without the WPRE.

[0121] The results from this example demonstrate that incorporation of a WPRE improves the efficacy of codon-optimized constructs.

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

[0123] To test the effect of WPRE and intron on the in vivo efficacy of rAAV8 vectors containing the HA06 sequence, different WPRE and intron combinations were used. The introns used in this study were either MVM intron or exon1-intron1-partial exon2, and the WPREs used were either WPRE3 or WPREmut6delATG. The recombinant AAV vectors containing different combinations of WPRE, intron, and codon-optimized hSERPING1 sequence used in this study are shown in Figure 12B. 11 vg / kg or 2 × 10 12 Mice (C57bl / 6; male) were administered either vector at 1000 vg / kg, and plasma samples were taken before administration of rAAV and at 7, 14, and 28 days 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 receiving constructs containing the MVM intron, HA06 (SERPING1) sequence, and WPRE3 (e.g., S07) expressed more hC1-INH compared to the remaining animals receiving other constructs at the same dose. 12 Mice treated with vg / kg showed the highest hC1-INH expression.

[0125] The results of this example show that the incorporation of the MVM intron and WPRE3 improves the efficacy of the HA06 (SERPING1) sequence.

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

[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 study, is a control vector. All seven constructs are shown in Figure 13B. Three doses: 4x10 11 vg / kg, 1.2×10 13 vg / kg, and 2 × 10 12 Mice (C57bl / 6; male) were administered one vector at 1000 ng / kg, and plasma samples were taken before administration of rAAV and at 7, 14, and 28 days 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 constructs 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. 13 Mice administered 1000 mg / kg showed the highest hC1-INH expression. The results of this example show that the incorporation of WPREmut6delATG improves the efficacy of the HA06 (SERPING1) sequence.

[0129] In another embodiment, two doses: 2×10 12 vg / kg and 2 × 10 13 Mice (C57bl / 6; male) were administered S04 at 100 mg / kg, 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 from this example show 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 vectors containing constructs containing codon-optimized hSERPING1 sequences This example demonstrates the in vivo efficacy of rAAV8 vectors containing constructs containing codon-optimized hSERPING1 sequences 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 FIG. 14B. The in vivo efficacy of all codon-optimized constructs was tested along with S04. Construct S04 is shown in FIG. 4B. 2×10 12 Mice (C57bl / 6; male) were administered 1000 vg / kg of vector, and plasma samples were taken before administration of rAAV and 28 days 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 treated with U06 expressed hC1-INH levels comparable to those of mice treated with the same dose of S04.

[0134] Effect of WPRE and alternative promoter elements at intermediate doses in long-term studies Example 9. In vivo efficacy of rAAV8 vectors containing constructs containing the HA06 (hSERPING1) sequence, WPRE, and alternative promoters This example demonstrates the in vivo efficacy in plasma of rAAV8 vectors containing constructs containing the HA06 (hSERPING1) sequence, WPRE and novel promoter elements.

[0135] To test the effect of the WPRE and the new promoter on the in vivo efficacy of rAAV8 vectors, different constructs were used, with or without the WPRE and the new promoter elements. Seven different constructs (J02, S03, S04, S06, S07, N04, and N05) were tested. All seven constructs are shown in Figure 15B. 2x10 12 A medium dose of 1000 mg / kg was administered to mice (C57bl / 6; male) and plasma samples were taken prior to administration of rAAV and at 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 than those administered constructs without the MVM (e.g., S06). In addition, mice administered constructs containing the short WPRE, WPRE3 (e.g., S06 and S07) expressed more hC1-INH than those administered constructs containing the modified WPRE, WPREmut6delATG (e.g., S03 and S04).

[0137] The results of this example show that the incorporation of the MVM intron and the short form of the WPRE, WPRE3, improves the efficacy of the HA06 (SERPING1) sequence. Construct S07, which incorporates both the MVM intron and WPRE3, showed the greatest efficacy, expressing hC1-INH for 175 days. Equivalence and Scope

[0138] Those skilled 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 limited to the above Detailed Description, 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 C1 inhibitor (C1-INH). (Item 2) 2. The rAAV vector of item 1, wherein 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. (Item 3) 3. The rAAV vector of item 2, wherein the codon-optimized SERPING1 sequence encoding C1-INH comprises a sequence identical to SEQ ID NO:2. (Item 4) 2. The rAAV vector of item 1, wherein the vector further comprises a liver-specific promoter. (Item 5) 5. The rAAV vector of item 4, wherein the liver-specific promoter is a transthyretin promoter (TTR). (Item 6) The rAAV vector of any one of the preceding items, wherein the vector further comprises a ubiquitous promoter. (Item 7) The rAAV vector of any one of the preceding items, wherein the vector further comprises one or more of the following: 5' and 3' inverted repeats, an intron upstream of the sequence, and a cis-acting regulatory module (CRM). (Item 8) The rAAV vector of any one of the preceding items, wherein the vector further comprises a WPRE sequence. (Item 9) 9. The rAAV vector of item 8, wherein the WPRE sequence is modified. (Item 10) 10. The rAAV vector of item 9, wherein the WPRE contains a mut6delATG modification. (Item 11) 8. The rAAV vector of item 7, wherein the intron is a minute virus of mouse (MVM) or SV40 intron. (Item 12) The rAAV vector of item 7, wherein the CRM is a liver-specific CRM. (Item 13) The rAAV vector of item 7, wherein the CRM is CRM8. (Item 14) 8. The rAAV vector of item 7, wherein the vector comprises at least three CRMs. (Item 15) A recombinant adeno-associated virus (rAAV) comprising an AAV8 capsid and a rAAV vector, the vector comprising: a.5' inverted terminal repeat (ITR), b. cis-acting regulatory modules (CRMs); C. liver-specific promoter, D. Minute virus of mice (MVM), e. SERPING1 sequence encoding C1 inhibitor (C1-INH); f. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and g.3'ITR The rAAV comprising: (Item 16) The rAAV of item 15, wherein the SERPING1 sequence is a wild-type sequence or a codon-optimized sequence. (Item 17) The rAAV of item 16, wherein the codon-optimized SERPING1 sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:2. (Item 18) A method for treating a subject having hereditary angioedema (HAE), comprising administering to a subject in need thereof an rAAV described in any one of the preceding items. (Item 19) 1. A method of treating a subject having hereditary angioedema (HAE), 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 the administration increases C1-INH enzyme activity in the subject. (Item 20) 20. The method of claim 19, wherein the C1-INH is detected in the plasma of the subject. (Item 21) 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) 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 dose. (Item 23) 23. The method of any one of items 18 to 22, wherein C1-INH activity is present in the subject after administration of the rAAV vector. (Item 24) 24. The method of claim 23, wherein the subject has C4 levels restored to pre-stroke levels. (Item 25) 25. The method of any one of items 18 to 24, wherein the AAV is administered intravenously. (Item 26) 25. The method of any one of items 18 to 24, wherein the AAV is administered intrathecally. (Item 27) The AAV is at least about 5×10 9 27. The method of claim 25 or 26, wherein the administration is at a dose of vg. (Item 28) 28. The method of any one of items 18 to 27, wherein said administration of said rAAV does not elicit an immune response.

Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid, 5' and 3' inverted terminal repeat (ITR) sequences, an intron upstream of the sequences, a liver-specific promoter which is a transthyretin (TTR) promoter, a codon-optimized SERPING1 sequence encoding C1 inhibitor (C1-INH) having any one of the sequences of SEQ ID NOs: 5, 7, or 13, a cis-acting regulatory module (CRM), and a WPRE sequence.

2. The rAAV vector of claim 1, wherein the WPRE sequence is WPRE3 of SEQ ID NO:

23.

3. The rAAV vector of claim 1, wherein the WPRE contains a mut6delATG modification, and the WPRE containing the mut6delATG modification comprises a sequence identical to SEQ ID NO:

24.

4. 2. The rAAV vector of claim 1, wherein the intron is a minute virus of mouse (MVM) intron or an SV40 intron.

5. The rAAV vector of claim 1 , wherein the CRM is a liver-specific CRM.

6. The rAAV vector of claim 1 , wherein the CRM is CRM8.

7. The rAAV vector of claim 1 , wherein the vector comprises at least three CRMs.

8. 2. The rAAV vector of claim 1, wherein the SERPING1 sequence is a wild-type sequence or a codon-optimized sequence.

9. 2. The rAAV vector of claim 1, wherein the codon-optimized SERPING1 sequence has the sequence of any one of SEQ ID NOs: 5, 7, or 13.

10. A composition comprising the rAAV vector of any one of claims 1 to 9 for treating a subject with hereditary angioedema (HAE).

11. 1. A composition for treating a subject having hereditary angioedema (HAE), comprising a recombinant adeno-associated virus (rAAV) vector, the recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a promoter operably linked to a nucleic acid sequence having any one of SEQ ID NOs: 4-17 encoding C1 inhibitor (C1-INH), wherein administration of the composition increases C1-INH activity in the subject.

12. The composition of claim 11, wherein the C1-INH is detected in the plasma of the subject.

13. The composition of any one of claims 10 to 12, wherein the C1-INH is detected in the liver of the subject.

14. The composition of any one of claims 10 to 13, wherein C1-INH is maintained for at least 30, 60, 90, 120, 150, or 180 days after a single dose.

15. The composition of any one of claims 10 to 14, wherein C1-INH activity is present in the subject following administration of the rAAV vector.

16. 16. The composition of claim 15, wherein the subject has C4 levels restored to pre-stroke levels.

17. The composition according to any one of claims 10 to 16, characterized in that the composition is administered intravenously.

18. The composition according to any one of claims 10 to 16, characterized in that the composition is administered intrathecally.

19. The AAV is at least about 5×10 ９ 19. The composition according to claim 17 or 18, characterized in that it is administered in a dose of 1.5 vg.

20. The composition according to any one of claims 10 to 19, characterized in that said administration of said composition does not elicit an immune response.

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