Compositions and methods for treating hereditary angioedema - Patents.com

Abstract

Nucleic acids encoding C1 inhibitor are described. Expression cassettes, vectors, cells, and cell lines containing the nucleic acids, as well as methods of using the nucleic acids to treat complement-mediated disorders, such as hereditary angioedema, are also described.

Description

[Technical field]

[0001] The present invention relates to the field of gene therapy. In particular, the present invention relates to an optimized cassette for the expression of human C1 inhibitor and methods of using same to treat complement-mediated disorders, particularly hereditary angioedema.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 142,121, filed January 27, 2021, U.S. Provisional Patent Application No. 63 / 201,466, filed April 30, 2021, and U.S. Provisional Patent Application No. 63 / 261,603, filed September 24, 2021. The entire contents of the foregoing applications are incorporated herein by reference, including all text, tables, sequence listings, and figures.

[0003] Reference to an Electronically Submitted Sequence Listing This application contains a sequence listing submitted electronically via EFS-Web as an ASCII formatted sequence listing with file name "SequenceListing5WO1", a creation date of January 27, 2022, and having a size of 727 kb. The sequence listing submitted via EFS-Web is a part of this specification and is incorporated by reference in its entirety. [Background technology]

[0004] Hereditary angioedema (HAE), also known as C1 esterase inhibitor deficiency, C1 inhibitor deficiency, HANE, Quincke's edema, and secondary angioneurotic edema, is a rare but potentially life-threatening autosomal dominant genetic disorder that occurs in approximately 1 in 50,000 people. HAE is characterized by recurrent episodes of swelling that most frequently affect the skin or mucosal tissues of the upper respiratory and gastrointestinal tracts (Banerji, Ann Allergy Asthma Immunol, 111: 329-336 (2013); Aygoren-Pursun et al., Orphanet J Rare Dis., 9: 99 (2014)).

[0005] HAE occurs when plasma leaks through blood vessels into tissues as a result of overproduction of bradykinin. The disease mechanism is thought to involve cleavage of prekallikrein (PKK) by activated factor XII (F12) and release of active plasma kallikrein, which activates even more F12. Plasma kallikrein then cleaves kininogen, releasing bradykinin that binds to the B2 bradykinin receptor on endothelial cells, increasing endothelial permeability. Normally, C1 esterase inhibitor (encoded by the SERPING1 gene) controls bradykinin production by inhibiting activation of plasma kallikrein and F12 (Busse et al., N Engl J Med., 382(12): 1136-1148 (2020)).

[0006] There are two types of HAE: HAE-C1-INH and HAE-nl-C1-INH. HAE-C1-INH results in a mutation in the SERPING1 gene (which codes for C1 inhibitor), resulting in low levels of active C1 inhibitor in the blood. There are two subtypes of HAE-C1-INH: type 1 HAE accounts for 85% of cases and is caused by the inability to secrete mutant C1 inhibitor protein into the blood, and type II HAE accounts for the remaining 15% of cases and is caused by a mutant C1 inhibitor that is secreted but dysfunctional. HAE-nl-C1-INH was first described in 2000 and is still not fully understood. Patients with HAE-nlC1-INH may also have a mutation in clotting factor XII, but most patients have no known mutations and have a normal C1 inhibitor gene. (Website: www.angioedemacenter.com / patient-resources / angioedema-types / ).

[0007] Currently, therapeutic agents are indicated for long-term prevention, treatment for acute attacks, and short-term prevention (e.g., before dental surgery) and include agents such as danazol, plasma-derived or recombinant C1 inhibitor replacement protein, bradykinin receptor antagonists (e.g., icatibant), kallikrein inhibitors (e.g., ecallantide), fresh frozen plasma, and purified C1 inhibitor, which have a high adverse effect profile. Although these therapies can alleviate symptoms and maximize quality of life, disease recurrence and the need for long-term continuous dosing remain major barriers to treatment (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)).

[0008] There is a need for effective and long-lasting therapeutic approaches to treat the angioedema associated with C1 inhibitor and F12 deficiency. Summary of the Invention

[0009] Disclosed herein are optimized cassettes for liver-directed expression of secretable versions of human C1 inhibitor.These optimizations of cassettes result in increased C1 inhibitor secretion from the liver, allowing hepatic gene transfer to achieve sufficient circulating levels of C1 inhibitor in subjects to cross-correct C1 inhibitor deficiency systemically.These cassettes are useful as gene therapy treatments for subjects with hereditary angioedema (HAE) and other diseases and disorders that can be treated with C1 inhibitor.

[0010] In one general aspect, the invention relates to a polynucleotide comprising a nucleic acid encoding a C1 inhibitor, the nucleic acid being selected from the group consisting of: (1) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO:105; (2) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO:106; (3) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO:107; (4) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO:108; (5) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO:109; (6) a polynucleotide having at least 84% sequence identity to the sequence of SEQ ID NO:110; (7) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO:111; (8) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO:112; (9) a polynucleotide having at least 82% sequence identity to the sequence of SEQ ID NO:113; (10) a polynucleotide having at least 82% sequence identity to the sequence of SEQ ID NO:114. (11) a polynucleotide having at least 82% sequence identity to the sequence of SEQ ID NO: 115; (12) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 116; (13) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 117; (14) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO: 118; (15) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 119. (16) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 120; (17) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 121; (18) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO: 122; (19) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO: 123; (20) a polynucleotide having at least 92% sequence identity to the sequence of SEQ ID NO: 124;(21) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 125; (22) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 126; (23) a polynucleotide having at least 92% sequence identity to the sequence of SEQ ID NO: 127; (24) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 128; (25) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 129; (26) a polynucleotide having at least 91% sequence identity to the sequence of SEQ ID NO: 130; (27) a polynucleotide having at least 92% sequence identity to the sequence of SEQ ID NO: 131; (28) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO: 132; (29) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO: 133; (30) a polynucleotide having at least 87% sequence identity to the sequence of SEQ ID NO: 134 (31) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 135; (32) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO: 136; (33) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO: 137; (34) a polynucleotide having at least 87% sequence identity to the sequence of SEQ ID NO: 138; (35) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 139; (36) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 140; and (37) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 141, and optionally the C1 inhibitor comprises the amino acid sequence of SEQ ID NO: 181 (mature C1 inhibitor; without signal peptide) or SEQ ID NO: 192 (nascent C1 inhibitor; with signal peptide);

[0011] In certain embodiments, the nucleic acid comprises fewer than 24 CpG dinucleotides, and optionally, comprises 0 CpG dinucleotides.

[0012] In certain embodiments, the nucleic acid encoding C1 inhibitor has a sequence at least 85%, at least 90%, at least 95% or 100% identical to any of SEQ ID NOs:105-142, 145-147, 156, 171, 172, 230 and 231.

[0013] In certain embodiments, the invention relates to nucleic acids encoding mutant C1 inhibitors, including truncated C1 inhibitors, fusions of two or more C1 inhibitors, or fusions of a C1 inhibitor with an Fc region or domain.

[0014] In certain embodiments, the nucleic acid has the sequence of any of SEQ ID NOs: 143-144, 158, and 165-170, and optionally the mutant C1 inhibitor comprises the amino acid sequence of any of SEQ ID NOs: 193-201, or a variant thereof.

[0015] In certain embodiments, the polynucleotide comprises a signal peptide sequence located at the 5' end of the nucleic acid encoding the C1 inhibitor.

[0016] In certain embodiments, the signal peptide sequence is a heterologous signal peptide sequence.

[0017] In certain embodiments, the signal peptide sequence is the endogenous or native C1 inhibitor signal peptide sequence.

[0018] In certain embodiments, the signal peptide is selected from the group consisting of C1 inhibitor signal peptide, human chymotrypsinogen B2 signal peptide, ALB signal peptide, ORM1 signal peptide, TF signal peptide, AMBP signal peptide, LAMP1 signal peptide, BTN2A2 signal peptide, CD300 signal peptide, NOTCH2 signal peptide, STRC signal peptide, AHSG signal peptide, SYN1 signal peptide, SYN2 signal peptide, SYN3 signal peptide, and SYN4 signal peptide, or variants thereof.

[0019] In certain embodiments, the signal peptide has a coding sequence of one of SEQ ID NOs:84-103, and optionally, the signal peptide comprises the amino acid sequence of any of SEQ ID NOs:203-218 or a variant thereof.

[0020] In one embodiment, the present invention relates to a signal peptide comprising any of the sequences of SEQ ID NOs: 215-218.

[0021] In one embodiment, the present invention relates to a nucleic acid encoding a signal peptide comprising any of the sequences of SEQ ID NOs: 100 to 103.

[0022] In certain embodiments, a signal peptide of the invention conforms to any one, two, three, or all four of the following definitions: (1) an amino-terminal N-region of 2-5 amino acids having a net positive charge, (2) a hydrophobic H-region of 6-15 amino acids, (3) a carboxyl-terminal C-region of 5-10 amino acids (wherein the amino acid at position -3 from the C-terminus of the signal peptide is uncharged) and an amino acid at position -1 from the C-terminus of the signal peptide that includes a short side chain, and (4) a leucine residue at position -10 from the C-terminus of the signal peptide.

[0023] In one embodiment, the invention relates to an expression cassette comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor operably linked to an expression control element.

[0024] In certain embodiments, the expression control element is a liver-specific expression control element.

[0025] In certain embodiments, the expression cassette further comprises one or more tissue-specific elements, the tissue-specific element being a promoter, which optionally is a hAAT promoter sequence.

[0026] In one embodiment, the expression cassette further comprises one or more efficacy elements, the efficacy elements being an enhancer or polyA sequence, the enhancer being ApoE, 2xApoE, 4xApoE, hAAT enhancer, WPRE, WPRE3, and optionally an intron that is a human hemoglobin beta (HBB) derived intron, and the polyA sequence is optionally a bovine growth hormone (bGH) polyadenylation (polyA) sequence.

[0027] In certain embodiments, the expression control elements of the expression cassette are located 5' to the polynucleotide, and optionally, the expression control elements include an ApoE / hAAT enhancer / promoter sequence.

[0028] In one embodiment, the expression cassette further comprises one or more response elements, wherein the response element is a miRNA binding site, a regulated Ire1-dependent decay (RIDD) sequence that drives degradation of the mRNA, an acute phase response element (APRE), or another 5' UTR or 3' UTR sequence, wherein the miRNA binding site is optionally miR-142-3p, wherein the RIDD sequence is selected from the group consisting of 1xRIDD, 3xRIDD, and RIDD1xBlos, wherein the APRE is selected from the group consisting of SERPING1 5' UTR, APRE 5' UTR, and SAA2 5' UTR, and wherein the other 5' UTR or 3' UTR sequence is optionally a SAA2 3' UTR sequence.

[0029] In certain embodiments, the tissue-specific element, efficacy element, and / or response element has reduced CpGs compared to the wild-type tissue-specific element, efficacy element, and / or response element.

[0030] In certain embodiments, the present invention relates to an adeno-associated virus (AAV) vector comprising the polynucleotide or expression cassette.

[0031] In one embodiment, an AAV vector comprises (a) one or more AAV capsids and (b) one or more AAV inverted terminal repeats (ITRs), where the AAV ITRs flank the 5' and / or 3' ends of the polynucleotide or expression cassette.

[0032] In one embodiment, at least one or more of the ITRs of the AAV vector are modified to reduce CpGs.

[0033] In certain embodiments, the AAV vector has a capsid serotype that includes modified or mutated AAV VP1, VP2, and / or VP3 capsids that have 90% or more, 95% or more, or 100% sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:188), AAV3B, AAV-2i8, SEQ ID NO:226, SEQ ID NO:189, SEQ ID NO:190, and / or LK03 (SEQ ID NO:191).

[0034] In one embodiment, the AAV vector comprises one or more ITRs of any of the AAV serotypes, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B, or a combination thereof.

[0035] In one embodiment, the present invention provides (1) a 5' AAV ITR, optionally an AAV2 5' ITR, optionally a 5' ITR comprising the polynucleotide sequence of SEQ ID NO: 70 or 72; (2) one or more tissue-specific elements, wherein the tissue-specific element is a promoter, and optionally the promoter comprises the polynucleotide sequence of SEQ ID NO: 79 or 80; (3) one or more efficacy elements, wherein the one or more efficacy elements is an enhancer or polyA sequence, and optionally, the one or more efficacy elements has a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 225, 74-76, 81-83, and 173-178; (4) one or more response elements, wherein the one or more response elements are an miRNA binding site, a regulated Ire1-dependent decay (RIDD) sequence, an acute phase response element (APRE), or a 5' UTR or 3' UTR sequence, optionally wherein the one or more response elements have a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 77-78, and 179, 180, and 182-187; (5) a nucleic acid encoding a signal peptide, which optionally encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 203 to 218, and which optionally has a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 84 to 103; (6) A nucleic acid encoding at least one of C1 inhibitor, mutant C1 inhibitor, and fusions or combinations thereof, a. optionally a C1 inhibitor having the amino acid sequence of SEQ ID NO:181 or 192, and optionally a polynucleotide sequence selected from the group consisting of SEQ ID NOs:104-142, 145-147, 156, and 171-172; b. optionally a mutant C1 inhibitor having an amino acid sequence selected from the group consisting of SEQ ID NOs: 193-201, and optionally a polynucleotide selected from the group consisting of SEQ ID NOs: 143-144, 158, and 165-170; and (7) A 3' AAV ITR, optionally an AAV2 3' ITR, optionally comprising the polynucleotide sequence of SEQ ID NO: 71 or SEQ ID NO: 73. The present invention relates to an AAV vector comprising the

[0036] In one embodiment, the AAV vector comprises a polynucleotide sequence of one of SEQ ID NOs:1-69 and 227-229.

[0037] In certain embodiments, the invention relates to a non-viral vector comprising the polynucleotide or expression cassette.

[0038] In one embodiment, the present invention relates to a pharmaceutical composition comprising multiple AAV or non-viral vectors in a biologically compatible carrier or excipient.

[0039] In certain embodiments, the pharmaceutical composition further comprises an empty AAV capsid.

[0040] In certain embodiments, the pharmaceutical composition further comprises a surfactant.

[0041] In one embodiment, the present invention relates to a method of treating a subject in need of C1 inhibitor, comprising administering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, AAV vector, non-viral vector, or pharmaceutical composition, wherein the C1 inhibitor is expressed in the subject.

[0042] In certain embodiments, the subject is a human.

[0043] In certain embodiments, the subject has hereditary angioedema (HAE).

[0044] In certain embodiments, the polynucleotide, expression cassette, AAV vector, non-viral vector, or pharmaceutical composition is administered to a subject intravenously, intraarterially, intracavitary, intramucosally, or via a catheter.

[0045] In one embodiment, the AAV vector is about 1 x 10 per kilogram of subject body weight. 8 ~Approx. 1×10 14 The vector is administered to the subject in the range of 1000 mg / kg of vector genome (vg / kg).

[0046] In certain embodiments, the method reduces, reduces or inhibits the need for C1 inhibitor or one or more symptoms of HAE; or prevents or reduces the progression or worsening of the need for C1 inhibitor or one or more symptoms of HAE; or stabilizes the need for C1 inhibitor or one or more symptoms of HAE; or ameliorates the need for C1 inhibitor or one or more symptoms of HAE.

[0047] In certain embodiments, the present invention relates to a cell comprising a polynucleotide or expression cassette of the present invention.

[0048] In one embodiment, the present invention relates to a cell that produces an AAV vector of the present invention.

[0049] In one embodiment, the invention relates to a method of producing an AAV vector of the invention, comprising: (a) introducing an AAV vector genome comprising a polynucleotide or expression cassette of the invention into a packaging helper cell; and (b) culturing the helper cell under conditions for producing the AAV vector. [Brief description of the drawings]

[0050] The foregoing summary section, as well as the following detailed description of the invention section, will be better understood when read in conjunction with the accompanying drawings. It is understood that the invention is not limited to the precise embodiments shown in the drawings. [Figure 1]FIG. 1 shows a schematic diagram of an exemplary expression vector described herein. [Diagram 2] FIG. 2 represents graphs showing mean ± standard deviation C1-INH antigen levels in mouse plasma for low dose (1.0×10 vg / kg, groups 7-11) (FIG. 2A) and high dose (4.0×10 vg / kg, groups 1-5) (FIG. 2B) vector cohorts of Example 2 as measured by ELISA; grey shaded areas represent mean ± standard deviation of all PNP measurements (low dose: 170.30 ± 20.36 μg / mL; high dose: 181.0 ± 35.62 μg / mL); values ​​considering upper and lower limits of quantification were excluded from the calculation of mean ± standard deviation; SERPING1 = SEQ ID NO:1, Syn1 = SEQ ID NO:13, Syn4 = SEQ ID NO:16, AHSG = SEQ ID NO:12, sp7 = SEQ ID NO:2. [Diagram 3] FIG. 3 is a graph showing steady-state C1-INH antigen levels in mouse plasma for low-dose (1.0×10 vg / kg, groups 7-11) (FIG. 3A) and high-dose (4.0×10 vg / kg, groups 1-5) (FIG. 3B) vector cohorts of Example 2, presented as mean ± standard deviation; steady state was defined as no statistically significant difference in mean antigen levels across the time points evaluated (linear regression analysis not shown); statistical comparisons to the native SERPING1 signal peptide were performed by Kruskal-Wallis test with post-hoc Dunn's multiple comparison test (ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001); SERPING1 = SEQ ID NO:1, Syn1 = SEQ ID NO:13, Syn4 = SEQ ID NO:16, AHSG = SEQ ID NO:12, sp7 = SEQ ID NO:2. [Figure 4]FIG. 4 shows C1-INH activity in mouse plasma from the high dose cohort of Example 2, specifically, individual C1-INH activity values ​​are shown for the high dose cohort, including data from weeks 22, 25, and 27 (circles, triangles, and squares, respectively) with values ​​shown as the mean±standard deviation; statistical comparisons were performed against the native SERPING1 signal peptide by one-way ANOVA with post-hoc Dunnett's multiple comparison test (****, p<0.0001); SERPING1 = SEQ ID NO:1, Syn1 = SEQ ID NO:13, Syn4 = SEQ ID NO:16, AHSG = SEQ ID NO:12, sp7 = SEQ ID NO:2. [Diagram 5] FIG. 5 shows the correlation between C1-INH antigen and C1-INH activity in mouse plasma from the high dose cohort of Example 2; specifically, individual values ​​for C1-INH activity were plotted with C1-INH activity and antigen using data from weeks 22, 25, and 27 measured by chromogenic assay and ELISA, respectively, against each animal's respective C1-INH antigen levels; values ​​were log transformed and a linear fit was generated using regression analysis (R2 = 0.771), and a two-tailed Pearson correlation showed significance (Pearson r = 0.8778; p < 0.0001); SERPING1 = SEQ ID NO: 1, Syn1 = SEQ ID NO: 13, Syn4 = SEQ ID NO: 16, AHSG = SEQ ID NO: 12, sp7 = SEQ ID NO: 2. [Figure 6]FIG. 6 is a graph showing vector genome concentrations from liver tissue at the endpoint of Example 2, where vector genome copy numbers were normalized to mouse Foxp1 in low-dose (FIG. 6A) and high-dose (FIG. 6B) cohorts, values ​​are reported as BGHpA copies per Foxp1 copy and are expressed as mean ± standard deviation; statistical comparisons were performed against the native SERPING1 signal peptide by Kruskal-Wallis test with post-hoc Dunn's multiple comparison test (**, p<0.01; ***, p<0.001); endpoints were defined as 18 weeks for low-dose cohorts (groups 7-11) and 28 weeks for high-dose cohorts (groups 1-5); SERPING1 = SEQ ID NO:1, Syn1 = SEQ ID NO:13, Syn4 = SEQ ID NO:16, AHSG = SEQ ID NO:12, sp7 = SEQ ID NO:2. [Figure 7] FIG. 7 shows a graph depicting the correlation between C1-INH antigen and genome concentration at the endpoint of Example 2, with endpoint C1-INH antigen values ​​versus vector genome concentration (normalized to Foxp1) at the endpoint in each mouse; endpoints were defined as week 18 for the low dose cohort (groups 7-11) and week 28 for the high dose cohort (groups 1-5); values ​​were log transformed and a linear fit was generated using regression analysis (R2 = 0.866), and a two-tailed Pearson correlation showed significance (Pearson r = 0.9306; p<0.0001); SERPING1 = SEQ ID NO:1, Syn1 = SEQ ID NO:13, Syn4 = SEQ ID NO:16, AHSG = SEQ ID NO:12, sp7 = SEQ ID NO:2. [Figure 8]FIG. 8 is a graph showing steady-state C1-INH antigen levels versus vector genome concentration at the end of Example 2, with steady-state C1-INH antigen levels plotted relative to endpoint liver vector genome (VG) concentration (normalized to Foxp1) in the low-dose (FIG. 8A) and high-dose (FIG. 8B) cohorts; steady-state C1-INH antigen was defined as no statistically significant difference in levels across the time points tested (statistical analysis not shown, 3-18 weeks and 3-28 weeks for low-dose and high-dose cohorts, respectively); values ​​are expressed as mean ± standard deviation; statistical comparisons were performed against the native SERPING1 signal peptide by Kruskal-Wallis test with post-hoc Dunn's multiple comparison test (*, p<0.05); endpoints were defined as 18 weeks for low-dose cohorts (groups 7-11) and 28 weeks for high-dose cohorts (groups 1-5); SERPING1 = SEQ ID NO:1, Syn1 = Sequence number 13, Syn4 = sequence number 16, AHSG = sequence number 12, sp7 = sequence number 2. [Figure 9] FIG. 9 shows graphs showing C1-INH antigen levels in mouse plasma for Study A (FIG. 9A) and Study B (FIG. 9B) of Example 3, measured by ELISA; groups were followed for 7 weeks (Study A) or 6 weeks (Study B) after vector administration; grey shaded areas indicate the mean ± standard deviation of all PNP measurements (Study A, 179.2 ± 20.88 μg / mL; Study B, 165.9 ± 20.70 μg / mL); SERPING1 = SEQ ID NO:1, I3 = SEQ ID NO:18, I4 = SEQ ID NO:19, I7 = SEQ ID NO:20, I20 = SEQ ID NO:21, I21 = SEQ ID NO:22, I24 = SEQ ID NO:23; I2 = SEQ ID NO:17, H9 = SEQ ID NO:24, H13 = SEQ ID NO:25, BC2 = SEQ ID NO:26. [Figure 10]FIG. 10 is a graph showing steady-state C1-INH antigen levels in mouse plasma for Study A (FIG. 10A) and Study B (FIG. 10B) as measured by ELISA; groups were followed for 7 weeks (Study A) or 6 weeks (Study B) after vector administration; values ​​are expressed as mean ± standard deviation; statistical comparisons against AAV-encapsidated SEQ ID NO:1 were performed by one-way ANOVA with post-hoc Dunnett's multiple comparison test (*, p<0.05; **, p<0.001, ***, p<0.001); SERPING1 = SEQ ID NO:1, I3 = SEQ ID NO:18, I4 = SEQ ID NO:19, I7 = SEQ ID NO:20, I20 = SEQ ID NO:21, I21 = SEQ ID NO:22, I24 = SEQ ID NO:23, I2 = SEQ ID NO:17, H9 = SEQ ID NO:24, H13 = SEQ ID NO:25, BC2 = SEQ ID NO:26. [Figure 11]FIG. 11 is a graph showing plasma bradykinin antigen levels in mice at week 7 as assessed by ELISA; bradykinin was assessed in mice administered AAV-encapsidated SEQ ID NO:20 (group 4) and SEQ ID NO:22 (group 6) codon-optimized variants, which are associated with higher levels of C1-INH antigen expression; FIG. 11A shows bradykinin values ​​in the vehicle, SEQ ID NO:20 variant, and SEQ ID NO:22 variant groups of Study A; grey shaded area indicates the mean ± standard deviation of all pooled normal plasma (PNP) measurements (35010.16 ± 8093.98 pg / mL); values ​​are expressed as the mean ± standard deviation; post-hoc Statistical comparisons to SEQ ID NO:1 were performed by one-way ANOVA with Dunnett's multiple comparison test (**, p<0.01); Figure 11B shows individual C1-INH antigen values ​​in mice treated with AAV-encapsidated SEQ ID NO:20 and SEQ ID NO:22 at week 7 plotted relative to their respective plasma bradykinin antigen levels (total samples n=9), and two-tailed Pearson correlation showed significance for the SEQ ID NO:22 group (Pearson r = -0.9417, p<0.0168, R2 = 0.8867); One animal in the vehicle group was euthanized prior to the end of the study after exhibiting a necrotic tail and head tilt. One animal administered AAV-encapsidated SEQ ID NO:20 was removed from the analysis after using the ROUT method for outlier detection; I7 = SEQ ID NO:20, I21 = SEQ ID NO:22. [Figure 12]FIG. 12 shows graphs depicting vector genome concentrations in liver tissue at endpoint for Study A (FIG. 12A) and Study B (FIG. 12B) of Example 3, with vector genome copy numbers normalized to mouse Foxp1, and values ​​expressed as mean±standard deviation; Study end is defined as week 7 and week 6 for Study A and Study B, respectively; Statistical comparisons compared to the native SERPING1 transgene were performed by Kruskal-Wallis test with post-hoc Dunn's multiple comparison test (ns, p>0.05); SERPING1 = SEQ ID NO:1, I3 = SEQ ID NO:18, I4 = SEQ ID NO:19, I7 = SEQ ID NO:20, I20 = SEQ ID NO:21, I21 = SEQ ID NO:22, I24 = SEQ ID NO:23, I2 = SEQ ID NO:17, H9 = SEQ ID NO:24, H13 = SEQ ID NO:25, BC2 = SEQ ID NO:26. [Figure 13] FIG. 13 shows the correlation between vector genome concentration at the endpoint and C1-INH antigen in individual mice; correlation was performed on the codon-optimized variant from study A with the highest C1-INH expression; values ​​were log-transformed and a linear fit was generated using regression analysis (R2 = 0.7243); two-tailed Pearson correlation showed significance (Pearson r = 0.8510, p<0.0001); SERPING1 = SEQ ID NO:1, I7 = SEQ ID NO:20, I21 = SEQ ID NO:22, I24 = SEQ ID NO:23. [Figure 14]FIG. 14 is a graph showing steady-state C1-INH antigen levels compared to vector genome concentration (normalized to Foxp1) at the endpoint (week 7) of Study A in Example 3; steady-state C1-INH antigen was defined as no statistically significant difference in levels across the time points tested (statistical analysis not shown, weeks 3-7); values ​​are expressed as mean ± standard deviation; statistical comparisons compared to the native SERPING1 signal peptide were performed by Kruskal-Wallis test with post-hoc Dunn's multiple comparison test (*, p<0.05); SERPING1 = SEQ ID NO:1, I3 = SEQ ID NO:18, I4 = SEQ ID NO:19, I7 = SEQ ID NO:20, I20 = SEQ ID NO:21, I21 = SEQ ID NO:22, I24 = SEQ ID NO:23. [Figure 15] FIG. 15 shows graphs depicting C1-INH antigen levels in mouse plasma for the study of Example 4; specifically, C1-INH antigen levels are plotted as a function of time (FIG. 15A) and week 8 (FIG. 15B) for all groups measured by ELISA; pooled normal human plasma (PNP) was included as a control to monitor inter-assay variability; the grey shaded area represents the mean ± standard deviation of all measurements of PNP (175.4 ± 41.3 μg / mL); values ​​are expressed as mean ± standard deviation; values ​​below the limit of detection were excluded from the calculation of the mean ± standard deviation; one male and three females (all low dose) showed no detectable C1-INH antigen over the course of the study; I7 = SEQ ID NO:20. [Figure 16] FIG. 16 is a graph showing the correlation between C1-INH antigen and C1-INH activity in mouse plasma for the study of Example 4; specifically, using data from weeks 5 and 8, individual values ​​for C1-INH activity were plotted against each animal's respective C1-INH antigen levels; values ​​were log transformed and a linear fit was generated using regression analysis; for visualization purposes, values ​​below quantification level (BQL) were excluded and values ​​above quantification level (AQL) were included; I7 = SEQ ID NO:20. [Figure 17]FIG. 17 is a graph showing vector genome concentrations from liver tissue at endpoint for the study of Example 4, specifically showing vector genome copy numbers normalized to mouse Foxp1 after administration of one of three doses of AAV-encapsidated SEQ ID NO:20; values ​​are expressed as mean ± standard deviation; I7 = SEQ ID NO:20. [Figure 18] FIG. 18 shows a graph representing normalized SERPING1 mRNA expression from liver tissue at endpoint, specifically, showing SERPING1 mRNA expression levels normalized to mouse Ppie in mice administered one of three doses of AAV-encapsidated SEQ ID NO:20; endpoint is defined as week 8; values ​​are expressed as mean ± standard deviation; I7 = SEQ ID NO:20. [Figure 19] FIG. 19 shows graphs representing a comparison of vector dose, normalized vector genome concentration, normalized mRNA expression levels, and terminal C1-INH antigen levels for the study of Example 4, specifically plotting values ​​for male (FIG. 19A) and female (FIG. 19B) Serping1− / − mice administered various doses of AAV-encapsidated SEQ ID NO:20; endpoint was defined as week 8; VG copy numbers were normalized to mouse Foxp1; SERPING1 mRNA expression levels were normalized to mouse Ppie; C1-INH antigen levels were measured by ELISA; individual values ​​were log-transformed and fitted to a nonlinear 4PL sigmoidal curve; I7 = SEQ ID NO:20. [Figure 20] Figure 20 shows data characterizing Serping1 deficiency in the B6.SJL mouse background. Figure 20A shows differences in paw volume. Figure 20B shows differences in plasma C1-INH. Figure 20C shows differences in bradykinin. Figure 20D shows differences in complement C4a (C4a). Figure 20E shows differences in tissue plasminogen activator (tPA). [Figure 21]Figure 21 shows that I21 (SEQ ID NO: 22) can sustain dose-dependent hC1-INH antigen levels in plasma using HAE model mice. Plasma hC1-INH levels in B6 / SJL Serping1+ / + (Figure 21A), B6 / SJL Serping1+ / - (Figure 21B), and B6 / SJL Serping1- / - (Figure 21C) male mice were measured in 1 / 4 log increments after injection with one of three doses of AAV-encapsidated I21 ranging from 1.0x1012 to 3.16x1012 vg / kg. [Figure 22] Figure 22 shows C1-INH expression and pharmacodynamics of I21 (SEQ ID NO:22). Figure 22A provides data comparing hC1-INH expression in different Serping1 genotypes. Figure 22B shows hC1-INH dose response. Figure 22C shows Bradykinin dose response. Figure 22D shows tPA activity dose response. [Diagram 23] Figure 23 shows I21 (SEQ ID NO:22) dose-dependent sustained hC1-INH antigen levels in plasma of HAE model mice. B6 / SJL Serping1- / - male mice were injected with one of five doses of I21 ranging from 9.5x1011 to 3.0x1013 vg / kg. Figure 23A shows plasma C1-INH levels as a function of time measured by ELISA. Values ​​are in IU / mL and are shown as mean ± standard deviation. Figure 23B shows steady-state C1-INH antigen levels in B6 / SJL Serping1- / - male mice as a function of vector dose. All values ​​were log-transformed and linear fits were generated using regression analysis (R2 = 0.88). [Figure 24] Figure 24 shows the reduction of bradykinin mediated by I21 (SEQ ID NO: 22) using HAE disease model mice. Figure 24A shows individual plasma bradykinin levels and mean ± standard deviation plasma bradykinin levels in different dose cohorts. Statistical comparisons compared to vehicle were performed by one-way ANOVA with post-hoc Dunnett's multiple comparison test (**, p<0.01; ***, p<0.001). Figure 24B shows plasma C1-INH levels. [Diagram 25]Figure 25 shows I21 (SEQ ID NO:22) mediated C1-INH restored C4 levels in HAE mouse model. Plasma C4 was analyzed by automated capillary-based immunoassay system (WesTM, ProteinSimple) at the end of the study. Figure 25A shows individual and mean ± standard deviation relative C4 expression in each dose cohort. Figure 25B shows individual relative plasma C4 values ​​plotted against each animal's respective C1-INH antigen level at week 31. All values ​​were log transformed and linear fits were generated using regression analysis (R2 = 0.87). Figure 25C shows the levels of C4 as a function of plasma C1-INH. BQL refers to below quantification level. AQL refers to above quantification level. [Figure 26] Figure 26 shows the C1-INH levels produced in mice administered AAV-encapsidated I21 (SEQ ID NO:22) to assess toxicity. Male mice were administered 2.5x1012 vg / kg, 5.0x1012 vg / kg, or 1.0x1013 AAV-encapsidated I21 (Figure 26A). Female mice were administered 1.0x1013 vg / kg, 5.0x1013 vg / kg, or 9.9x1013 AAV-encapsidated I21 (Figure 26B). [Figure 27] Figure 27 shows an I21 (SEQ ID NO:22) dosing study in male (Figure 27A) and female (Figure 27B) cynomolgus monkeys, where the percent of normal C1-INH activity was determined at different time points after AAV-encapsidated I21 dosing. Cynomolgus monkeys were administered 1.0x1013 vg / kg (low), 3.2x1013 vg / kg (medium) or 1.0x1014 vg / kg (high) AAV-encapsidated I21. [Figure 28] Figure 28 shows the hC1-INH antigen levels (Figure 28A) and peak percent normal C1-INH activity (Figure 28B) produced by different doses of AAV-encapsidated I21. 1.0x1013 vg / kg (low), 3.2x1013 vg / kg (medium) or 1.0x1014 vg / kg (high) of AAV-encapsidated I21 were administered to female and male cynomolgus monkeys. [Figure 29]Figure 29 shows the hC1-INH antigen levels in the supernatant of transfected Huh7 cells. Huh7 cells were transfected in vitro with different SERPING1 expression plasmids and the hC1-INH antigen levels were measured. Results are the average of n=3 biological replicates. Error bars indicate standard deviation. [Diagram 30] Figure 30 shows the effect of adding miR-142-3p target to expression plasmid. Huh7 cells were transfected in vitro with nucleic acid of SEQ ID NO:28 (pSwap-SERPING1_I21) or SEQ ID NO:55 (mir 142-3p), or plasmid containing pCAG#GFP, and hC1-INH antigen was assayed. Results are the average of biological replicates n=3. Error bars indicate standard deviation. Detailed Description of the Invention

[0051] Various publications, articles and patents are cited or described in the Background and throughout this specification, and each of these references is incorporated herein by reference in its entirety. Discussion of documents, acts, materials, devices, articles and the like which is included in the present specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items form part of the prior art with respect to the invention disclosed or claimed.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, certain terms cited herein have the meanings set forth herein.

[0053] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0054] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise", as well as variations such as "comprises" and "comprising", are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including", or, in some cases, with the term "having".

[0055] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element, where such element, step, or ingredient is relevant to the claimed invention. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the above terms "containing," "including," and "having," whenever used herein in the context of aspects or embodiments of the invention, may be replaced with the terms "consisting of" or "consisting essentially of" to modify the scope of the disclosure.

[0056] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more options is also understood to be within the meaning and thus meets the requirements of the term "and / or".

[0057] All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, a disclosed feature is an example of a genus of equivalent or similar features.

[0058] The term "about" as used herein refers to a value within 10% (i.e., ±10%) of the underlying parameter. For example, "about 1:10" means 1.1:10.1 or 0.9:9.9, about 5 hours means 4.5 hours or 5.5 hours, etc. The word "about" at the beginning of a value string modifies each of the values ​​by up to 10%.

[0059] All numerical values ​​or numerical ranges include integers within such ranges and fractions of integers or values ​​within such ranges unless the context clearly indicates otherwise. Thus, by way of example, a reference to a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, etc., as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. Thus, also by way of example, a reference to a numerical range such as "1 to 4" includes 2, 3, as well as 1.1, 1.2, 1.3, 1.4, etc. For example, "1 to 4 weeks" includes 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0060] Furthermore, a numerical range such as "0.01 to 10" includes 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, a dosage of "about 0.01 to about 10 mg / kg" per subject's body weight includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc.

[0061] Reference to more (greater) or smaller integers includes any number greater or less than the referenced numerical value, respectively. Thus, for example, greater than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. For example, "two or more" administrations of a non-viral vector and / or immune cell modulator include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more administrations.

[0062] Additionally, references to numerical ranges such as "1 to 90" include 1.1, 1.2, 1.3, 1.4, 1.5, etc., as well as 81, 82, 83, 84, 85, etc. For example, "between about 1 minute and about 90 days" includes 1.1 minutes, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as 1 day, 2 days, 3 days, 4 days, 5 days, ..., 81 days, 82 days, 83 days, 84 days, 85 days, etc.

[0063] In an attempt to aid the reader of this application, the description is separated into various paragraphs or sections or directed to various embodiments of the present invention. These separations should not be considered as separating the substance of a paragraph or section or embodiment from the substance of another paragraph or section or embodiment. On the contrary, those skilled in the art will understand that the description has broad application and encompasses all combinations of the various sections, paragraphs, and sentences that may be contemplated. The description of any embodiment is merely exemplary and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples.

[0064] Provided herein are modified nucleic acids encoding C1 inhibitors, expression cassettes comprising modified nucleic acids encoding C1 inhibitors, viral vectors comprising modified nucleic acids encoding C1 inhibitors, and non-viral vectors comprising modified nucleic acids encoding C1 inhibitors. The invention also provides recombinant AAV particles comprising modified nucleic acids encoding C1 inhibitors, non-viral particles comprising modified nucleic acids encoding C1 inhibitors, pharmaceutical compositions comprising modified nucleic acids encoding C1 inhibitors, and methods of treating hereditary angioedema (HAE).

[0065] nucleic acid The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotide, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).Nucleic acid includes genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA, (polynucleotide and oligonucleotide), chromosomal DNA, spliced ​​or unspliced ​​mRNA, rRNA, tRNA inhibitor DNA or RNA (RNAi, e.g., small or small hairpin (sh) RNA, microRNA (miRNA), small or small interfering (si) RNA, trans-splicing RNA, or antisense RNA), locked nucleic acid analog (LNA), oligonucleotide DNA (ODN) single-stranded and double-stranded, immune stimulating sequence (ISS), riboswitch and ribozyme.

[0066] Nucleic acid includes naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acid can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length. When describing nucleic acid, the sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0067] According to some embodiments, the polynucleotide is a single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule. According to some embodiments, the polynucleotide is for therapeutic use, for example, ssDNA or dsDNA encoding a therapeutic transgene. According to some embodiments, the dsDNA molecule is a minicircle, nanoplasmid, open linear double-stranded DNA or closed-end linear double-stranded DNA (CELiD / ceDNA / doggybone DNA). According to some embodiments, the ssDNA molecule is closed circular DNA or open linear DNA.

[0068] "Transgene" is used herein to conveniently refer to a nucleic acid that is introduced into a cell or organism or a nucleic acid that has been introduced.Transgene includes any nucleic acid, such as a modified nucleic acid that codes for C1 inhibitor, or a heterogeneous polynucleotide sequence, such as a heterogeneous nucleic acid that codes for a protein or peptide or a nucleic acid (e.g., miRNA, etc.).The terms transgene and heterogeneous nucleic acid / polynucleotide sequence are used interchangeably herein.

[0069] As used herein, the terms "C1 inhibitor" or "C1 esterase inhibitor" or "C1-INH" or "C1EI" or "SERPING1" are all used interchangeably and refer to any nucleic acid or protein of SERPING1 or C1 inhibitor. In an embodiment, the nucleic acid encoding C1 inhibitor encodes human C1 inhibitor protein. The complete DNA sequence of SERPING1, including introns and exons, is available at GenBank Accession No. NG_009625.1. Human C1 inhibitor consists of 500 amino acids and is available at GenBank Accession No. NP_000053.2. Examples of C1 inhibitors include any naturally occurring C1 inhibitor, and variants thereof. As used herein, "nucleic acid encoding C1 inhibitor" refers to a recombinant nucleic acid molecule that encodes a protein having at least a portion of the function or activity of the wild-type C1 inhibitor protein. Examples of such nucleic acids include modified nucleic acids that encode C1 inhibitor.

[0070] As used herein, the term "mutant protein" refers to any protein that contains one or more mutations in the amino acid sequence compared to the wild type. The term "mutant protein" includes, for example, amino acid insertions, additions, substitutions and deletions. The term "mutant" also includes, for example, fusion proteins.

[0071] According to an embodiment, the mutant C1 inhibitor exhibits the function of the native protein. In an embodiment, the mutant C1 inhibitor exhibits at least 50%, optionally at least 55%, optionally at least 60%, optionally at least 65%, optionally at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95% of the function of the native protein. The functional activity of the mutant C1 inhibitor protein can be measured, for example, by assessing the inhibition of the esterase activity of complement component C1. In view of the present disclosure, any suitable method known to those skilled in the art can be used in the present invention. Examples of methods that can be used to determine the functional activity of mutant C1 inhibitor proteins include spectrophotometric assays such as those described in Drouet et al., Clin Chim Acta, 1988, 174(2):121-130, enzyme immunoassays such as those described in the Mayo Clinic (website: www.mayocliniclabs.com / test-catalog / Performance / 81493), colorimetric assays such as those described in Munkvad et al., Clin Chem., 1990, 36(5):737-41, each of the references incorporated herein by reference in their entireties, and commercially available methods such as those described in the present Examples.

[0072] As used herein, the term "fusion protein" or "chimeric protein" refers to a protein made by the joining of two or more originally separate proteins or portions thereof. In some embodiments, a linker or spacer is present between each protein.

[0073] As used herein, the term "modify" and grammatical variations thereof mean that a nucleic acid or protein deviates from a reference or parent sequence. A modified nucleic acid encoding a C1 inhibitor is altered compared to a reference nucleic acid (e.g., wild type) or parent nucleic acid. Thus, a modified nucleic acid may have substantially the same, greater, or less activity or function as a reference or parent nucleic acid, but retain at least partial activity, function, and / or sequence identity with the reference or parent nucleic acid. A modified nucleic acid may be genetically modified to encode a modified or mutant C1 inhibitor.

[0074] "Modified nucleic acid encoding C1 inhibitor" means that the C1 inhibitor nucleic acid has a modification compared to the parent unmodified nucleic acid encoding C1 inhibitor. A specific example of a modification is a nucleotide substitution. The nucleotide substitution can be a silent mutation that encodes the same amino acid, or a missense mutation that encodes a different amino acid. The missense mutation can be a conservative or non-conservative mutation. Other examples of modifications include, for example, truncation and insertion. Modified nucleic acid can also include codon-optimized nucleic acid that encodes the same protein as a wild-type protein or a nucleic acid that is not codon-optimized. Codon optimization can be used in a broader sense, including, for example, removal of CpG. The term "modification" in this specification does not have to appear in every instance of a reference made to a nucleic acid encoding C1 inhibitor.

[0075] In certain embodiments, for modified nucleic acids encoding C1 inhibitor, the C1 inhibitor protein retains at least some of the function or activity of the wild-type C1 inhibitor.

[0076] As described herein, modified nucleic acids encoding C1 inhibitors may exhibit different properties or characteristics compared to the reference or parent nucleic acid. For example, modified nucleic acids include sequences that have 100% identity to the reference nucleic acids encoding C1 inhibitors described herein, as well as sequences that have less than 100% identity to the reference nucleic acids encoding C1 inhibitors.

[0077] The terms "identity", "homology" and their grammatical variants mean that two or more referenced entities are the same when they are "aligned" sequences. Thus, for example, when two nucleic acids are identical, they have the same sequence, at least within the referenced region or portion. Identity can be over a defined region (region or domain) of the sequence.

[0078] An "area" or "region" of identity refers to a portion of two or more referenced entities that are the same. Thus, if two protein or nucleic acid sequences are identical over one or more sequence areas or regions, they share identity within that region. An "aligned" sequence refers to multiple protein (amino acid) or nucleic acid sequences, often including corrections for deleted or added bases or amino acids (gaps) compared to a reference sequence.

[0079] The identity can span the entire length or a portion of the sequence. In some embodiments, the length of the sequences that share percent identity is 2, 3, 4, 5 or more consecutive amino acids or nucleic acids, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. consecutive amino acids or nucleic acids. In some embodiments, the length of the sequences that share identity is 21 or more consecutive amino acids or nucleic acids, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. consecutive amino acids or nucleic acids. In one embodiment, the length of the sequence sharing identity is 41 or more consecutive amino acids or nucleic acids, for example, 42, 43, 44, 45, 46, 47, 48, 49, 50, etc. In one embodiment, the length of the sequence sharing identity is 50 or more consecutive amino acids or nucleic acids, for example, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 500, 500 to 1,000, etc.

[0080] As described herein, a modified nucleic acid encoding a C1 inhibitor can have 100% identity or less than 100% identity to a reference nucleic acid encoding a C1 inhibitor.

[0081] According to one embodiment, the nucleic acid encoding the C1 inhibitor is selected from the group consisting of: (1) 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity to the sequence of SEQ ID NO: 105. (2) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO: 105, such as 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, etc. to the sequence of SEQ ID NO: 106; (3) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO: 106, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (3) a polynucleotide having at least 80% sequence identity, 81% or more sequence identity, 82% or more sequence identity, or 83% or more sequence identity to the sequence of SEQ ID NO: 107, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity. 3% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.(4) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 107, such as 5% or more sequence identity; (5) a polynucleotide having 80% or more sequence identity, 81% or more sequence identity, 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, or 91% or more sequence identity to the sequence of SEQ ID NO: 108. (5) polynucleotides having at least 80% sequence identity to the sequence of SEQ ID NO: 108, such as 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (6) polynucleotides having at least 80% sequence identity to the sequence of SEQ ID NO: 108, such as 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, or more than 87% sequence identity, or more than 88% sequence identity, or more than 89% sequence identity, or more than 90% sequence identity, or more than 90% sequence identity, or more than 91% sequence identity, or more than 92% sequence identity, or more than 93% sequence identity, or more than 94% sequence identity, or more than 95% sequence identity, or more than 96% sequence identity, or more than 97% sequence identity, or more than 98% sequence identity, or more than 99% sequence identity, or more than 99.5 ...2% sequence identity, or more than 93% sequence identity, or more than 94% sequence identity, or more than 95% sequence identity, or more than 96% sequence identity, or more than 97% sequence identity, or more than 98% sequence identity, or more than 9 A polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO: 109, such as 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity. (6) 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.(7) a polynucleotide having at least 84% sequence identity to the sequence of SEQ ID NO: 110, such as 5% or more sequence identity; (8) a polynucleotide having 80% or more sequence identity, 81% or more sequence identity, 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity to the sequence of SEQ ID NO: 111. (8) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 111, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO: 112; (9) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO: 112, such as 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (10) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO: 113, such as 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; For 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.(10) a polynucleotide having at least 82% sequence identity to the sequence of SEQ ID NO: 113, such as 5% or more sequence identity; (11) a polynucleotide having 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity to the sequence of SEQ ID NO: 114. (11) a polynucleotide having at least 82% sequence identity to the sequence of SEQ ID NO: 114, such as 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (12) a polynucleotide having at least 82% sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, or 88% or more sequence identity to the sequence of SEQ ID NO: 115, (12) a polynucleotide having at least 82% sequence identity to the sequence of SEQ ID NO: 115, such as 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (13) a polynucleotide having at least 82% sequence identity to the sequence of SEQ ID NO: 115, such as 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; sequence identity, 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.(13) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 116, such as 5% or more sequence identity; (14) a polynucleotide having 80% or more sequence identity, 81% or more sequence identity, 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, or 92% or more sequence identity to the sequence of SEQ ID NO: 117. (13) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 117, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (14) a polynucleotide having at least 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, or the like, to the sequence of SEQ ID NO: 118; (14) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO: 118, such as 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (15) a polynucleotide having at least 80% or more sequence identity to the sequence of SEQ ID NO: 119, such as 81% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; % or more sequence identity, 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.(16) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 119, such as 5% or more sequence identity; (17) a polynucleotide having 80% or more sequence identity, 81% or more sequence identity, 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, or 92% or more sequence identity to the sequence of SEQ ID NO: 120. (17) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 120, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (18) a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 121, such as 80% or more sequence identity, 81% or more sequence identity, 82% or more sequence identity, 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, and / or a polynucleotide having at least 80% sequence identity to the sequence of SEQ ID NO: 121, such as 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity. (18) 83% or more sequence identity, 84% or more sequence identity, 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.(19) a polynucleotide having at least 83% sequence identity to the sequence of SEQ ID NO:122, such as 5% or more sequence identity; (20) a polynucleotide having 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO:123; (21) a polynucleotide having 92% or more sequence identity to the sequence of SEQ ID NO:124. (21) a polynucleotide having 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO: 125; (22) a polynucleotide having 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, or 95% or more sequence identity to the sequence of SEQ ID NO: 125. (21) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 125, such as 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (22) a polynucleotide having at least 86% sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, or the like, to the sequence of SEQ ID NO: 126; (23) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 126, such as 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (24) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 126, such as 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, or 96% or more sequence identity to the sequence of SEQ ID NO: 127, A polynucleotide having 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO: 128; (24) a polynucleotide having 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.(25) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 128, such as 5% or more sequence identity; (26) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 129, such as 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity. A polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 129; (26) a polynucleotide having at least 91% sequence identity to the sequence of SEQ ID NO: 130, such as 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity. (27) a polynucleotide having at least 92% sequence identity to the sequence of SEQ ID NO: 131, such as 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO: 131; (28) a polynucleotide having at least 93% sequence identity, 94% or more sequence identity, or 95% or more sequence identity, or 96% or more sequence identity, or 97% or more sequence identity, or 98% or more sequence identity, or 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO: 132; (29) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO:132, such as 5% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.5% or more sequence identity; (30) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO:133, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.5% or more sequence identity.(30) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO: 133, such as 5% or more sequence identity; (31) a polynucleotide having at least 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity, 99.5% sequence identity, etc., to the sequence of SEQ ID NO: 134. (31) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 135, such as 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO: 135; (32) A polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO: 136, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity to the sequence of SEQ ID NO: 136; (33) A polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO: 137, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, or 98% or more sequence identity. (33) a polynucleotide having at least 93% sequence identity to the sequence of SEQ ID NO:137, such as 99% or more sequence identity, 99.5% or more sequence identity, or 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.(35) a polynucleotide having at least 87% sequence identity to the sequence of SEQ ID NO: 138, such as 5% or more sequence identity; (36) a polynucleotide having 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, (36) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 139, such as 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity; (37) a polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 140, such as 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, or 92% or more sequence identity. (37) polynucleotides having at least 86% sequence identity to the sequence of SEQ ID NO: 140, such as 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, 99.5% or more sequence identity to the sequence of SEQ ID NO: 141; and (38) polynucleotides having at least 86% sequence identity, 87% or more sequence identity, 88% or more sequence identity, 99.5% or more sequence identity to the sequence of SEQ ID NO: 142. A polynucleotide having at least 86% sequence identity to the sequence of SEQ ID NO: 141, such as 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity.

[0082] According to one embodiment, the nucleic acid of the invention encodes a C1 inhibitor having the amino acid sequence of SEQ ID NO:181 or 192.

[0083] In some embodiments, the polynucleotide comprises a nucleic acid encoding a mutant C1 inhibitor, examples of which include, but are not limited to, a truncated C1 inhibitor, a fusion of two or more C1 inhibitors, or a fusion of a C1 inhibitor with a stabilizing moiety, such as an Fc region or domain.

[0084] As used herein, the term "Fc region" or "Fc domain" refers to the carboxyl-terminal portion of an immunoglobulin heavy chain constant region, or an analog or portion thereof. That is, an immunoglobulin Fc region of an Ig, preferably an IgG, which may include, for example, at least a portion of the hinge region, CH2 domain, and CH3 domain. The Fc region may be a native sequence Fc region or a variant Fc region. A native sequence Fc region comprises an amino acid sequence identical to that of an Fc region found in nature. A variant Fc region, as understood by those skilled in the art, comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification. According to certain embodiments, the Fc region is one of those described in Dall'Acqua et al., J Biol Chem., 281(33): 23514-24 (2006), Ishino et al., J Biol Chem., 288(23): 16529-37 (2013), Ying et al., J Biol Chem., 287(23): 19399-19408 (2012), or Zalevsky et al., Nat Biotechnol., 28(2): 157-159 (2010), which are incorporated herein by reference.

[0085] According to one embodiment, the Fc region of the present invention has the amino acid sequence of any of SEQ ID NOs: 219 to 224. According to one embodiment, the nucleic acid encoding the Fc region of the present invention has the sequence of any of SEQ ID NOs: 159 to 164.

[0086] In certain embodiments, the nucleic acid encoding the mutant C1 inhibitor has the sequence of any of SEQ ID NOs:143-144, 158, and 165-170.

[0087] According to one embodiment, the nucleic acid of the present invention encodes a mutant C1 inhibitor having an amino acid sequence of any of SEQ ID NOs:193-201.

[0088] According to an embodiment, the polynucleotide comprises a nucleic acid encoding a fusion of two or more C1 inhibitor proteins. In an embodiment, a first C1 inhibitor protein or variant thereof is fused to a second C1 inhibitor protein or variant thereof via an autoprotease peptide, such as the peptide sequence of porcine teschovirus-1 2A (P2A). In an embodiment, the nucleic acid has the sequence of SEQ ID NO: 158. According to an embodiment, the polynucleotide comprises a nucleic acid encoding a fusion of a first and a second C1 inhibitor protein having the amino acid sequence of SEQ ID NO: 195.

[0089] Modified nucleic acids encoding C1 inhibitors that exhibit different characteristics or properties compared to a reference or parent nucleic acid include nucleotide substitutions. For example, modified nucleic acids encoding C1 inhibitors include nucleic acids that have a reduced number of CpG dinucleotides compared to the reference nucleic acid encoding C1 inhibitor, referred to as CpG-reduced nucleic acids.

[0090] As used herein, the phrase "CpG reduction" or "CpG depletion" refers to a nucleic acid sequence that is generated synthetically or by mutation of a nucleic acid sequence, such that one or more CpG dinucleotides are removed from the nucleic acid sequence. In an embodiment, all CpG motifs are removed, providing what is referred to herein as modified CpG-free sequences. CpG motifs are suitably reduced or removed not only in coding sequences (e.g., transgenes), but also in non-coding sequences, including, for example, 5' untranslated regions (UTRs) and 3' UTRs, promoters, enhancers, signal peptides, polyA, ITRs, introns, and any other sequences present in a polynucleotide molecule.

[0091] According to certain embodiments, the nucleic acid encoding the C1 inhibitor comprises fewer than 24 CpG dinucleotides, e.g., 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 CpG dinucleotides.

[0092] The phrase "consisting essentially of," when referring to a particular nucleotide or amino acid sequence, means a sequence having the characteristics of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel characteristics of the sequence.

[0093] Nucleic acids, expression vectors, AAV vector genomes, non-viral vectors, plasmids containing modified nucleic acids encoding the C1 inhibitors of the present invention can be prepared using recombinant DNA technology techniques. The availability of nucleotide sequence information allows for the preparation of isolated nucleic acid molecules of the present invention by various means. Nucleic acids encoding C1 inhibitors can be produced using a variety of standard cloning, recombinant DNA techniques, through cell expression or in vitro translation and chemical synthesis techniques. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides with shared structural features, e.g., antibody screening with expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers that can anneal to the nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of differential nucleic acid libraries.

[0094] The nucleic acid may be maintained as DNA in any convenient cloning vector. In certain embodiments, the clone is maintained in a plasmid cloning / expression vector, such as pBluescript (Stratagene, La Jolla, Calif.), propagated in a suitable E. coli host cell. Alternatively, the nucleic acid may be maintained in a vector suitable for expression in mammalian cells, for example, an AAV vector. Nucleic acid molecules may be expressed in mammalian cells if post-translational modifications affect protein function.

[0095] Expression cassette The present invention also provides an expression cassette comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor as described herein, operably linked to an expression control element.

[0096] In one embodiment, the expression cassette comprises a polynucleotide comprising a nucleic acid encoding a C1 inhibitor, the nucleic acid comprising: (1) A polynucleotide having at least 83% sequence identity (e.g., 83 to 100% identity) to the sequence of SEQ ID NO: 105; (2) A polynucleotide having at least 83% sequence identity (e.g., 83 to 100% identity) to the sequence of SEQ ID NO: 106; (3) A polynucleotide having at least 80% sequence identity (e.g., 80 to 100% identity) to the sequence of SEQ ID NO: 107; (4) A polynucleotide having at least 80% sequence identity (e.g., 80 to 100% identity) to the sequence of SEQ ID NO: 108; ( (5) A polynucleotide having at least 83% sequence identity (e.g., 83 to 100% identity) to the sequence of SEQ ID NO: 109; (6) A polynucleotide having at least 84% sequence identity (e.g., 84 to 100% identity) to the sequence of SEQ ID NO: 110; (7) A polynucleotide having at least 80% sequence identity (e.g., 80 to 100% identity) to the sequence of SEQ ID NO: 111; (8) A polynucleotide having at least 83% sequence identity (e.g., 83 to 100% identity) to the sequence of SEQ ID NO: 112; (9) (10) a polynucleotide having at least 82% sequence identity (e.g., 82 to 100% identity) to the sequence of SEQ ID NO: 113; (11) a polynucleotide having at least 82% sequence identity (e.g., 82 to 100% identity) to the sequence of SEQ ID NO: 115; (12) a polynucleotide having at least 80% sequence identity (e.g., 80 to 100% identity) to the sequence of SEQ ID NO: 116; (1 (3) a polynucleotide having at least 80% sequence identity (e.g., 80 to 100% identity) to the sequence of SEQ ID NO: 117; (14) a polynucleotide having at least 83% sequence identity (e.g., 83 to 100% identity) to the sequence of SEQ ID NO: 118; (15) a polynucleotide having at least 80% sequence identity (e.g., 80 to 100% identity) to the sequence of SEQ ID NO: 119; (16) a polynucleotide having at least 80% sequence identity (e.g., 80 to 100% identity) to the sequence of SEQ ID NO: 120;(17) A polynucleotide having at least 80% sequence identity (e.g., 80 to 100% identity) to the sequence of SEQ ID NO: 121; (18) A polynucleotide having at least 83% sequence identity (e.g., 83 to 100% identity) to the sequence of SEQ ID NO: 122; (19) A polynucleotide having at least 93% sequence identity (e.g., 93 to 100% identity) to the sequence of SEQ ID NO: 123; (20) A polynucleotide having at least 92% sequence identity (e.g., 92 to 100% identity) to the sequence of SEQ ID NO: 124. (21) a polynucleotide having at least 89% sequence identity (e.g., 89 to 100% identity) to the sequence of SEQ ID NO: 125; (22) a polynucleotide having at least 86% sequence identity (e.g., 86 to 100% identity) to the sequence of SEQ ID NO: 126; (23) a polynucleotide having at least 92% sequence identity (e.g., 92 to 100% identity) to the sequence of SEQ ID NO: 127; (24) a polynucleotide having at least 89% sequence identity to the sequence of SEQ ID NO: 128. (25) a polynucleotide having at least 89% sequence identity (e.g., 89 to 100% identity) to the sequence of SEQ ID NO: 129; (26) a polynucleotide having at least 91% sequence identity (e.g., 91 to 100% identity) to the sequence of SEQ ID NO: 130; (27) a polynucleotide having at least 92% sequence identity (e.g., 92 to 100% identity) to the sequence of SEQ ID NO: 131; (28) a polynucleotide having at least 92% sequence identity (e.g., 92 to 100% identity) to the sequence of SEQ ID NO: 132. (29) a polynucleotide having at least 93% sequence identity (e.g., 93 to 100% identity) to the sequence of SEQ ID NO: 133; (30) a polynucleotide having at least 87% sequence identity (e.g., 87 to 100% identity) to the sequence of SEQ ID NO: 134; (31) a polynucleotide having at least 89% sequence identity (e.g., 89 to 100% identity) to the sequence of SEQ ID NO: 135;(32) a polynucleotide having at least 93% sequence identity (e.g., 93 to 100% identity) to the sequence of SEQ ID NO: 136; (33) a polynucleotide having at least 93% sequence identity (e.g., 93 to 100% identity) to the sequence of SEQ ID NO: 137; (34) a polynucleotide having at least 87% sequence identity (e.g., 87 to 100% identity) to the sequence of SEQ ID NO: 138; (35) a polynucleotide having at least 86% sequence identity (e.g., 86 to 100% identity) to the sequence of SEQ ID NO: 139; (36) a polynucleotide having at least 86% sequence identity (e.g., 86 to 100% identity) to the sequence of SEQ ID NO: 140; and (37) a polynucleotide having at least 86% sequence identity (e.g., 86 to 100% identity) to the sequence of SEQ ID NO: 141.

[0097] In certain embodiments, the C1 inhibitor comprises the amino acid sequence of SEQ ID NO:181 or 192.

[0098] According to one embodiment, the expression cassette comprises a polynucleotide comprising a nucleic acid encoding a mutant C1 inhibitor, the mutant C1 inhibitor comprising a truncated C1 inhibitor, a fusion of two or more C1 inhibitors, or a fusion of a C1 inhibitor with an Fc region or domain. According to one embodiment, the nucleic acid has a sequence of any of SEQ ID NOs: 143-144, 158, and 165-170.

[0099] According to one embodiment, the nucleic acid of the present invention encodes a mutant C1 inhibitor having an amino acid sequence of any of SEQ ID NOs:193-201.

[0100] In certain embodiments, the expression cassette includes a coding sequence for an appropriate secretory signal sequence or signal peptide that allows for secretion of the polypeptide encoded by the polynucleotide molecule of the present invention. As used herein, the term "secretory signal sequence" or "signal peptide" or variations thereof are intended to refer to an amino acid sequence that can function to drive secretion of an operably linked polypeptide from a cell. In certain embodiments, the secretory signal sequence or signal peptide can function to enhance secretion of an operably linked polypeptide from a cell compared to the level of secretion seen with a native polypeptide having a native or naturally occurring signal peptide. A signal peptide is a short peptide (typically 25-30 amino acids long) located at the N-terminus of a protein that carries the information for protein secretion. A signal peptide directs a protein to or through the endoplasmic reticulum secretory pathway. "Enhanced" secretion means that the relative proportion of polypeptides synthesized by a cell that are secreted from the cell is increased, and it is not necessary that the absolute amount of protein secreted is also increased. In certain embodiments, essentially all of the polypeptide (i.e., at least 95%, 97%, 98%, 99% or more) is secreted. However, essentially all or most of the polypeptide need not be secreted, so long as the secretion level is enhanced compared to a native polypeptide without a signal peptide. Generally, the secretory signal sequence is cleaved in the endoplasmic reticulum, and in certain embodiments, the secretory signal sequence is cleaved prior to secretion. However, so long as the secretion of the polypeptide from the cell is enhanced and the polypeptide is functional, the secretory signal sequence need not be cleaved. Thus, in certain embodiments, the secretory signal sequence is partially or completely retained. The secretory signal sequence may be derived in whole or in part from the secretory signal of the polypeptide to be secreted (i.e., from a precursor) and / or may be synthetic in whole or in part. The length of the secretory signal sequence is not critical, and generally, known secretory signal sequences are about 10-15 amino acids long to 50-60 amino acids long.Furthermore, known secretory signals from the polypeptide to be secreted can be altered or modified (e.g., by amino acid substitution, deletion, truncation, or insertion), so long as the resulting secretory signal sequence functions to enhance secretion of the operably linked polypeptide. The secretory signal sequence of the present invention can comprise, essentially consist of, or consist of a naturally occurring secretory signal sequence or a variant thereof. Numerous secretory proteins and sequences that direct secretion from a cell are known in the art, including those described in Owji et al., Eur. J. Cell Biol. 97:422-441 (2018). The secretory signal sequence of the present invention can further be wholly or partially synthetic or artificial. Synthetic or artificial secretory signal peptides are known in the art. See, for example, Barash et al., Biochem. Biophys. Res. Comm. 294:835-42 (2002).

[0101] Any suitable signal peptide known to one of skill in the art in light of the present disclosure can be used in the present invention. Examples of signal peptides include, but are not limited to, those found in the Signal Peptide Database (www.signalpeptide.de / ). Examples of signal peptides suitable for the present invention include wild-type C1 inhibitor peptide, human chymotrypsinogen B2 signal peptide ("Sp7"; an 18 amino acid signal peptide of NCBI reference sequence NP_001020371), albumin (ALB) signal peptide, orosomucoid 1 (ORM1) signal peptide, transferrin (TF) signal peptide, alpha 1-microglobulin / bikunin precursor (AMBP) signal peptide, lysosomal-associated membrane glycoprotein 1 (LAMP1) signal peptide, butyrophilin subfamily 2 member A2 (BTN2A2) signal peptide, CD300 signal peptide, Notch2 signal peptide, stereocillin (STRC) signal peptide, alpha 2-HS-glycoprotein (AHSG) signal peptide, SYN1 signal peptide (SEQ ID NO: 215), SYN2 signal peptide (SEQ ID NO: 216), SYN3 signal peptide (SEQ ID NO: 217), SYN4 signal peptide (SEQ ID NO: 218), secrecon (Barash signal peptide), and the like. Signal sequences that can be used in the present invention include, but are not limited to, the artificial signal peptide described in Sigma et al., Biochem Biophys Res Commun., 294: 835-842 (2002), mouse IgKVIII, human IgKVIII, CD33, tPA, a-1 antitrypsin signal peptide, and natural secreted alkaline phosphatase (SEAP). Any conventional signal sequence that directs a protein through the endoplasmic reticulum secretory pathway, including mutants of the above signal peptides, can be used in the present invention.

[0102] In certain embodiments, the invention relates to signal peptides that comply with any one, two, three, or all four of the following definitions: (1) an amino-terminal N-region of 2-5 amino acids with a net positive charge; (2) a hydrophobic H-region of 6-15 amino acids; (3) a carboxyl-terminal C-region of 5-10 amino acids with an uncharged amino acid at position -3 from the C-terminus of the signal peptide and an amino acid at position -1 from the C-terminus of the signal peptide including a short side chain; and (4) a leucine residue at position -10 from the C-terminus of the signal peptide.

[0103] In certain embodiments, the invention relates to a signal peptide comprising a sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 215. In certain embodiments, the invention relates to a signal peptide comprising a sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 216. In certain embodiments, the invention relates to a signal peptide comprising a sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 217. In certain embodiments, the invention relates to a signal peptide comprising a sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 218.

[0104] In one embodiment, the invention relates to a nucleic acid encoding a signal peptide, the nucleic acid comprising a sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 100. In one embodiment, the invention relates to a nucleic acid encoding a signal peptide, the nucleic acid comprising a sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 101. In one embodiment, the invention relates to a nucleic acid encoding a signal peptide, the nucleic acid comprising a sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 102. In one embodiment, the invention relates to a nucleic acid encoding a signal peptide, the nucleic acid comprising a sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 103. In a further embodiment, the encoded signal peptide has at least 95% or 100% sequence identity to any of SEQ ID NOs: 215-218.

[0105] In certain embodiments, the signal peptides of the invention are useful for causing or driving the secretion of any therapeutic protein expressed from any therapeutic transgene known in the art, in certain embodiments, the signal peptides of the invention are useful for causing or driving the secretion from the liver of any therapeutic protein expressed from any therapeutic transgene known in the art.

[0106] In certain embodiments, a signal peptide comprising one of SEQ ID NOs:215-218 is useful for causing or driving the secretion of any therapeutic protein expressed from any therapeutic transgene known in the art. In certain embodiments, a signal peptide comprising one of SEQ ID NOs:215-218 is useful for causing or driving the secretion of any therapeutic protein expressed from any therapeutic transgene known in the art.

[0107] Certain embodiments relate to a polypeptide comprising a sequence at least 95% or 100% identical to any of SEQ ID NOs:215, 216, 217, and 218; and a nucleic acid comprising a sequence encoding a polypeptide comprising a sequence at least 95% or 100% identical to any of SEQ ID NOs:215, 216, 217, and 218.

[0108] Examples of therapeutic transgenes include myelin oligodendrocyte glycoprotein (MOG), proteolipid protein 1 (PLP1), or myelin basic protein (MBP) for the treatment of multiple sclerosis, such as those disclosed in PCT / US2020 / 061356, filed November 19, 2020, which is incorporated by reference in its entirety; GAA (acidic alpha glucoprotein), for the treatment of Pompe's disease or another glycogen storage disease, such as those disclosed in WO2019 / 222411, which is incorporated by reference in its entirety. sidase); ATP7B (copper transporting ATPase 2) for the treatment of Wilson's disease; GLA (alpha galactosidase A) for the treatment of Fabry's disease; ASS1 (argininosuccinate synthase) for the treatment of citrullinemia type 1; beta glucocerebrosidase for the treatment of Gaucher disease type 1; beta hexosaminidase A for the treatment of Tay-Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for the treatment of hereditary angioedema (HAE), also known as C1 inhibitor deficiency types I and II; glycogen storage diseases Glucose-6-phosphatase for the treatment of type I (GSDI); insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epithelial growth factor (EGF), erythropoietin (EPO ... Growth factors (EGF), transforming growth factor alpha (TGFα), platelet-derived growth factor (PDF), insulin growth factor I or II (IGF-I or IGF-II), TGFβ, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 or NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 or netrin-2, hepatocyte growth factor (HGF), ephrin, noggin,sonic hedgehog or tyrosine hydroxylase; thrombopoietin (TPO), interleukins (IL-1 to IL-36, etc.), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor α or β, interferon α, β or γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD or IgE, chimeric immunoglobulin, humanized antibody, single-chain antibody, T cell receptor, chimeric T cell receptor, class I or class II MHC molecules; CFTR (cystic fibrosis transmembrane conductance regulator protein), blood coagulation (factor XIII, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), blood coagulation factors (factor XIII, factor IX (FIX), factor VIII (FVIII), factor X, factor VII, factor VIIa, protein C), gain of function of blood coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, beta globin, alpha globin, spectrin, alpha antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, lysosomal storage disease-associated enzyme (ARSA), hypoxanthine guanine phosphoribosyltransferase, beta-25 glucocerebromo sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, transforming growth factor alpha and beta, cytokines, alpha interferon, beta interferon, interferon gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)),Peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or tregitope proteins or hCDR1, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (choroideremia), LCA 5 (LCA-reversillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked recessive retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activating protein; erythropoietin (EPO) for the treatment of anemia; interferon-α, interferon-β, and interferon-γ for the treatment of various immune disorders, viral infections, and cancer; any of IL-1 through IL-36 for the treatment of various inflammatory diseases or immune deficiencies. interleukins (IL) and corresponding receptors, including chemokines (CXC motif) ligand 5 (CXCL5) for the treatment of immune disorders; granulocyte-colony stimulating factor (G-CSF) for the treatment of immune disorders such as Crohn's disease; granulocyte-macrophage colony stimulating factor (GM-CSF) for the treatment of various human inflammatory diseases; macrophage-colony stimulating factor (M-CSF) for the treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for the treatment of epithelial tissue damage; recurrent miscarriage, HIV-related complications,and chemokines such as monocyte chemoattractant protein-1 (MCP-1) for the treatment of insulin resistance; tumor necrosis factor (TNF) and receptors for the treatment of various immune disorders; α1-antitrypsin for the treatment of emphysema or chronic obstructive pulmonary disease (COPD); mucopolysaccharidosis I (MPS α-L-iduronidase for the treatment of ornithine transcarbamoylase (OTC) deficiency; OTC for the treatment of ornithine transcarbamoylase (OTC) deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia-lyase (PAL) for the treatment of phenylketonuria (PKU); lipoprotein lipase for the treatment of lipoprotein lipase deficiency; apolipoprotein (Apo) for the treatment of apolipoprotein (Apo) AI deficiency; low density lipoprotein receptor (LDL-R) for the treatment of familial hypercholesterolemia (FH); albumin for the treatment of hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate synthetase; argininosuccinate lyase; arginase, fumarylacetoacetate hydrolase, porphobilinogen deamidation enzymes; cystathionine beta synthase for the treatment of homocystinuria; branched-chain keto acid decarboxylase; isovaleryl CoA dehydrogenase; propionyl CoA carboxylase; methylmalonyl CoA mutase; glutaryl CoA dehydrogenase; insulin; pyruvate carboxylase; hepatic phosphorylase; phosphorylase kinase:glycine decarboxylase; H protein; T protein; cystic fibrosis transmembrane conductance regulator (CFTR); ATP-binding cassette, subfamily A (ABC1), member 4 (ABCA4) for the treatment of Stargardt disease; dystrophin; gene editing nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), functional type II CRISPR-Cas9, but are not limited to these.

[0109] In certain embodiments, the signal peptide is the endogenous or native C1 inhibitor signal peptide or a variant thereof.

[0110] In certain embodiments, the signal peptide is a heterologous signal peptide or a variant thereof.

[0111] In some embodiments, the expression cassette comprises a nucleic acid encoding a signal peptide sequence located at the 5' end of the nucleic acid encoding the C1 inhibitor. In some embodiments, the signal peptide has the sequence of any of SEQ ID NOs: 84-103. In some embodiments, the signal peptide has the amino acid sequence of any of SEQ ID NOs: 203-224.

[0112] In certain embodiments, the expression control element is located 5' to the nucleic acid encoding the C1 inhibitor.

[0113] The term "expression cassette" as used herein refers to a nucleic acid construct that contains sufficient nucleic acid elements for expression of a polynucleotide molecule of the invention. Typically, an expression cassette contains a polynucleotide molecule of the invention operably linked to a promoter sequence.

[0114] "Expression control element" refers to a nucleic acid sequence that affects the expression of an operably linked nucleic acid. Expression control elements described herein include promoters and enhancers. Vector sequences, including AAV vectors and non-viral vectors, may include one or more "expression control elements". Typically, such elements are included to facilitate proper heteropolynucleotide transcription and proper translation (e.g., promoters, enhancers, splicing signals of introns, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, and stop codons, etc.). Such elements typically act in cis and are referred to as "cis-acting" elements, but can also act in trans.

[0115] Expression control can be affected at the level of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., within an intron). Expression control elements can be located adjacent to the transcribed sequence or at a distance away from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100-500, or more nucleotides from the polynucleotide), even at a significant distance. Nevertheless, due to the length limitations of the AAV vector, expression control elements in the AAV vector are typically within 1-1000 nucleotides from the transcription start site of the heterologous nucleic acid.

[0116] Functionally, expression of an operably linked nucleic acid can be at least partially controlled by an element (e.g., a promoter) that regulates transcription of the nucleic acid and, optionally, translation of the transcript. Specific examples of expression control elements include promoters, which are usually located 5' of the transcribed nucleic acid sequence. Promoters typically increase the amount of expression from an operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0117] The term "operably linked" means that the regulatory sequences necessary for the expression of a nucleic acid sequence are arranged in a suitable position relative to the sequence so as to affect the expression of the nucleic acid sequence. This same definition may be applied to the arrangement of nucleic acid sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector, such as a recombinant AAV (rAAV) vector or a non-viral vector. The coding sequence may be operably linked to the regulatory sequence in a sense or antisense orientation. In an embodiment, the promoter is a heterologous promoter.

[0118] The term "heterologous promoter" as used herein refers to a promoter that is not found operably linked to a given coding sequence in nature. In certain embodiments, the expression cassette may contain additional elements, such as introns, enhancers, polyadenylation sites, woodchuck response elements (WREs), and / or other elements known to affect the expression levels of coding sequences.

[0119] As used herein, the term "promoter" refers to a nucleotide sequence that can control the expression of a coding sequence or functional RNA. In general, the nucleic acid molecule of the present invention is located 3' of the promoter sequence. In an embodiment, the promoter sequence is composed of proximal and more distal upstream elements and can include enhancer elements.

[0120] As used herein, "enhancer" may refer to a sequence located adjacent to a heterologous nucleic acid. Enhancer elements are typically located upstream of a promoter element, but may also function and be located downstream or within the sequence. Thus, enhancer elements may be located 10-50 base pairs, 50-100 base pairs, 100-200 base pairs, or 200-300 base pairs, or more base pairs upstream or downstream of a heterologous nucleic acid sequence. Enhancer elements typically increase expression of an operably linked nucleic acid over that provided by a promoter element.

[0121] An expression construct may contain a control element that serves to drive expression in a specific cell or tissue type. Expression control elements (e.g., promoters) include those that are active in a specific tissue or cell type, referred to herein as "tissue-specific expression control elements / promoters." Tissue-specific expression control elements are typically active in a specific cell or tissue type (e.g., liver). Expression control elements are typically active in a specific cell, tissue, or organ because they are recognized by a transcriptional activator protein or other regulator of transcription that is specific to that cell, tissue, or organ. Such control elements are known to those skilled in the art (see, e.g., Sambrook et al. (1989) and Ausubel et al. (1992)).

[0122] The incorporation of tissue-specific regulatory elements in the expression construct provides at least partial tissue tropism for expression of the heterologous nucleic acid encoding the protein or inhibitor RNA. Examples of promoters active in the liver include the transthyretin (TTR) gene promoter; human alpha1-antitrypsin (hAAT) promoter; lipoprotein AI promoter; albumin, Miyatake, et al., J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig, et al., Gene Ther. 3: 1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14 (1996); human factor IX promoter; thyroxine-binding globulin (TBG) promoter; TTR minimal enhancer / promoter, alpha antitrypsin promoter, LSP (845 nt) (requires intronless scAAV); LSP1 promoter, and the like.

[0123] Expression control elements also include ubiquitous or promiscuous promoters / enhancers that can drive expression of polynucleotides in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers active in various mammalian cell types, or synthetic elements that do not occur in nature (see, for example, Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerate kinase (PGK) promoter.

[0124] Expression control elements can also confer expression in a regulatable manner, i.e., a signal or stimulus increases or decreases the expression of an operably linked heteropolynucleotide. Regulatable elements that increase the expression of an operably linked polynucleotide in response to a signal or stimulus are also referred to as "inducible elements" (i.e., induced by a signal). Particular examples include, but are not limited to, hormone (e.g., steroid) inducible promoters. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimulus present, with the greater the amount of signal or stimulus, the greater the increase or decrease in expression. Specific, non-limiting examples include the zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 98 / 10088); the tetracycline repression system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551(1992)); the tetracycline inducible system (Gossen, et al., Science. 268: 1766-1769(1995); Harvey, et al., Curr. Opin. Chem. Biol. :512-518 (1998)); the RU486 inducible system (Wang, et al., Nat. Biotech. 15:239-243(1997) and Wang, et al., Gene Ther. 4:432-441(1998)). (1997); and the rapamycin inducible system (Magari, et al., J. Clin. Invest. 100:2865-2872(1997); Rivera, et al., Nat. Medicine. 2:1028-1032 (1996)). Other regulatable control elements that may be useful in this context are those that are regulated by specific physiological conditions, e.g., temperature, acute phase, development.

[0125] Other examples of promoters include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, the cytomegalovirus enhancer, CAG (a complex of the CMV enhancer, chicken β-actin promoter (CBA) and rabbit β-globin intron) and other constitutive promoters, the neuron-specific enolase (NSE) promoter, the SV40 early promoter, the mouse mammary tumor virus LTR promoter; the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the spleen focus forming virus (SFFV) promoter, the Rous sarcoma virus (RSV) promoter, the rat insulin promoter, the thyroxine-binding globulin (TBG) promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EF1-α promoter, synthetic promoters, hybrid promoters, promoters with multiple tissue specificities, and the like, all of which are well known and readily available to those skilled in the art. Other promoters may be of human origin or from other species, including mouse.

[0126] Expression control elements also include the natural elements of heteropolynucleotide.When it is desired that the expression of heteropolynucleotide should mimic natural expression, natural control elements (such as promoters) can be used.When the expression of heteropolynucleotide is regulated in a temporal or developmental manner, or in a tissue-specific manner, or in response to specific transcriptional stimulation, natural elements can be used.Other natural expression control elements, such as introns, polyadenylation sites, or Kozak consensus sequences, can also be used.

[0127] In the example of an expression control element operably linked to a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid. More specifically, for example, two DNA sequences that are operably linked means that the two DNA sequences are arranged in a relationship (either in cis or trans) such that at least one of the DNA sequences can exert a physiological effect on the other sequence.

[0128] Thus, additional elements for a vector include, but are not limited to, expression control (e.g., promoter / enhancer) elements, transcription termination signals or stop codons, 5' or 3' untranslated regions flanking sequences such as one or more copies of AAV ITR sequences (e.g., polyadenylation (polyA) sequences), or introns.

[0129] Additional elements include, for example, filler or stuffer polynucleotide sequences, e.g., to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically accept inserts of DNA that generally have a size range of about 4 kb to about 5.2 kb, or slightly larger. Thus, for shorter sequences, a stuffer or filler is included to adjust the length to near or at the normal size of the viral genome sequence that is acceptable for packaging the AAV vector into a viral particle. In an embodiment, the filler / stuffer nucleic acid sequence is a non-translated (non-protein-coding) segment of nucleic acid. For nucleic acid sequences less than 4.7 kb, the filler or stuffer polynucleotide sequence has a length that, when combined with the sequence (e.g., inserted into a vector), has a total length of about 3.0 to 5.5 kb, or about 4.0 to 5.0 kb, or about 4.3 to 4.8 kb.

[0130] In an embodiment, the expression cassette further comprises one or more tissue-specific elements. As used herein, "tissue-specific element" refers to any polynucleotide sequence that directs tissue-specific expression of a transgene. In an embodiment, the tissue-specific element is a promoter. In an embodiment, the promoter sequence has reduced CpGs compared to the wild-type promoter sequence. In an embodiment, the promoter is a hAAT promoter. In an embodiment, the hAAT promoter sequence has the polynucleotide sequence of SEQ ID NO: 79 or 80.

[0131] In an embodiment, the expression cassette further comprises one or more potency elements. As used herein, "potency element" refers to any polynucleotide sequence that enhances the stability of an mRNA molecule. In an embodiment, the potency element is an enhancer or polyadenylation (polyA) sequence. In an embodiment, the enhancer or polyA sequence has reduced CpG compared to a wild-type enhancer or polyA sequence. The one or more potency elements can be located anywhere in the expression cassette. In certain embodiments, the efficacy element is an enhancer, such as those described in, for example, Van Linthout et al., Hum Gene Ther. 2002 May 1;13(7):829-40, Donello et al., J Virol. 1998;72(6):5085-5092, Zufferey et al., J Virol. 1999;73(4):2886-2892, or Choi et al., Mol Brain 7, 17 (2014), the entireties of which are incorporated herein by reference. In an embodiment, the efficacy element is an enhancer selected from the group consisting of ApoE, 2xApoE, 4xApoE, hAAT enhancer, WPRE, WPRE3, and the intron is optionally a human hemoglobin beta (HBB) derived intron, such as that described in Ronzitti et al., Mol Ther Methods Clin Dev. 2016;3:160, which is incorporated herein by reference in its entirety. In an embodiment, the enhancer has a polynucleotide sequence selected from the group consisting of SEQ ID NOs:225, 74-76, 81-82, and 173-178. In an embodiment, the efficacy element is optionally a polyA sequence that is a bovine growth hormone (bGH) polyadenylation (polyA) sequence. In an embodiment, the polyA sequence has the polynucleotide of SEQ ID NO:83.

[0132] In some embodiments, the expression control element comprises an ApoE / hAAT enhancer / promoter sequence located 5' of a nucleic acid encoding a C1 inhibitor. In some embodiments, the ApoE / hAAT enhancer / promoter sequence has reduced CpGs compared to a wild-type ApoE / hAAT enhancer / promoter sequence. In some embodiments, the ApoE enhancer sequence has the sequence of any of SEQ ID NOs: 225 and 74-76. In some embodiments, the hAAT promoter sequence has the sequence of SEQ ID NO: 79 or 80.

[0133] In an embodiment, the expression cassette further comprises one or more response elements. As used herein, "response element" refers to a nucleic acid sequence that can regulate transcriptional activity when placed adjacent to or within a promoter. In an embodiment, the response element is a miRNA binding site, a regulated Ire1-dependent decay (RIDD) sequence, an acute phase response element (APRE), or a 5' UTR or 3' UTR sequence. In an embodiment, the miRNA binding site, the regulated Ire1-dependent decay (RIDD) sequence, the acute phase response element (APRE), or a 5' UTR or 3' UTR sequence has reduced CpG compared to wild-type miRNA binding site, the regulated Ire1-dependent decay (RIDD) sequence, the acute phase response element (APRE), or a 5' UTR or 3' UTR sequence. In an embodiment, the response element is a miRNA binding site, and optionally a miR-142-3p sequence, such as those described in Brown et al., Nat Med 12, 585-591 (2006). In an embodiment, the miR-142-3p sequence has a polynucleotide sequence of SEQ ID NO: 179. In an embodiment, the response element is a regulated Ire1-dependent decay (RIDD) sequence. In an embodiment, the response element is a RIDD sequence, such as those described in, for example, Oikawa et al., Nucleic Acids Res. 2010 Oct;38(18):6265-73 or Moore and Hollien, Molecular Biology of the Cell 2015 26:16, 2873-2884, which are incorporated herein by reference in their entirety. In an embodiment, the response element is a RIDD sequence selected from the group consisting of 1xRIDD, 3xRIDD, and RIDD1xBlos. In an embodiment, the RIDD sequence has a polynucleotide sequence selected from the group consisting of SEQ ID NO: 185-187. In an embodiment, the response element is an acute phase response element (APRE).In certain embodiments, the response element is an APRE sequence, such as those described in, for example, Longley et al., J Immunol. 1999 Oct 15;163(8):4537-45, Podvinec et al., PNAS, 2004;101(24):9127-9132, Prada et al., Immunobiology. 1998 Aug;199(2):377-88, Rygg et al., Scand J Immunol. 2001 Jun;53(6):588-95, or Serrano-Mendioroz et al., Hum Gene Ther. 2018;29(4):480-491, each of which is incorporated by reference in its entirety. In some embodiments, the response element is an APRE selected from the group consisting of SAA2 APRE, 2x ADRES, SERPING1 5' UTR, APRE 5' UTR, and SAA2 5' UTR. In some embodiments, the APRE has a polynucleotide sequence selected from the group consisting of SEQ ID NOs:77-78, 180, and 182-183. In some embodiments, the response element is a 5' UTR or 3' UTR sequence, optionally an SAA2 3' UTR sequence, such as, for example, those described in Longley et al., J Immunol. 1999 Oct 15;163(8):4537-45. In some embodiments, the SAA2 3' UTR sequence has the polynucleotide sequence of SEQ ID NO:184.

[0134] In certain embodiments, one or more tissue-specific elements, one or more efficacy elements, and / or one or more response elements are located 5' of the nucleic acid. In certain embodiments, one or more tissue-specific elements, one or more efficacy elements, and / or one or more response elements are located 3' of the nucleic acid.

[0135] In one embodiment, the expression cassette has a sequence of any of SEQ ID NOs: 1 to 69 and 227 to 229. In one embodiment, the expression cassette has a sequence of at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.5% sequence identity, or 100% sequence identity to any of SEQ ID NOs: 1 to 69 and 227 to 229.

[0136] Gene Delivery System Viral Vectors

[0137] The present invention further provides a viral vector, such as an adeno-associated viral (AAV) vector, comprising a polynucleotide that includes a nucleic acid encoding a C1 inhibitor as described herein.

[0138] The term "vector" or "gene transfer vector" as used herein refers to the nucleic acid molecule that contains a gene of interest.Examples of vectors include, but are not limited to, viral vectors delivered by virus particles or virus-like particles (VLPs) that are similar to virus particles, such as retroviruses, adenoviruses, adeno-associated viruses, and lentivirus particles or virus-like particles (VLPs), but are non-infectious; and non-viral vectors delivered by non-viral gene transfer systems, such as microinjection, electroporation, liposomes, large natural polymers, large synthetic polymers, and polymers that are composed of both natural and synthetic components.

[0139] A vector nucleic acid sequence generally contains at least one origin of replication for propagation in a cell, and optionally contains additional elements, such as heteropolynucleotide sequences, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), selection markers (e.g., antibiotic resistance), polyadenylation signals, etc.

[0140] As used herein, the term "gene transfer system" refers to any means of delivering a composition comprising nucleic acid sequence to cells or tissue.For example, the gene transfer system can be a viral gene transfer system, such as intact virus, modified virus and VLP, to facilitate the delivery of viral vector to desired cells or tissue.The gene transfer system can also be a non-viral delivery system that does not contain viral coat protein or does not form viral particles or VLPs, such as liposome-based system, polymer-based system, protein-based system, metal particle-based system, peptide cage system, etc.

[0141] Viral vectors are derived from or based on one or more nucleic acid elements comprising a viral genome. Particular viral vectors include, but are not limited to, lentiviruses and adeno-associated virus (AAV) vectors.

[0142] The term "recombinant" as a modifier of vectors, such as recombinant AAV (rAAV) vectors, and sequences, such as recombinant polynucleotides and polypeptides, means that the compositions are manipulated (i.e., altered) in a manner that is not generally found in nature. The term "recombinant" is not necessarily used herein with respect to AAV vectors, and sequences, such as polynucleotides, although recombinant forms that include polynucleotides are expressly included despite such omission.

[0143] "Recombinant AAV vector" or "rAAV" is derived from the wild-type genome of AAV by using molecular methods to remove the wild-type genome from the AAV genome and replace it with a non-natural nucleic acid sequence called heteronucleic acid. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. rAAV is distinguished from the AAV genome because all or part of the AAV genome is replaced with a non-natural sequence with respect to the AAV genome nucleic acid. Thus, the incorporation of the non-natural sequence defines the AAV vector as a "recombinant" vector, which can be called a "rAAV vector".

[0144] The rAAV sequence can be packaged for subsequent ex vivo, in vitro or in vivo infection (transduction) of cells, and is referred to herein as a "particle". When a recombinant AAV vector sequence is encapsidated or packaged in an AAV particle, the particle can also be referred to as a "rAAV vector" or "rAAV particle". Such rAAV particles contain proteins that encapsidate or package the vector genome, and in the case of AAV, they are referred to as capsid proteins.

[0145] Vector "genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., rAAV) particle. When a recombinant plasmid is used to construct or manufacture a recombinant vector, the vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid may be referred to as the "plasmid backbone" and is important for the cloning and amplification of the plasmid, a process that is necessary for propagation and recombinant virus production, but is not itself packaged or encapsidated into a viral (e.g., AAV) particle. Thus, vector "genome" refers to the polynucleotide that is packaged or encapsidated by the virus (e.g., AAV).

[0146] Host cells for producing recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients for heterologous rAAV vectors. Cells derived from a stable human cell line, HEK293 (e.g., readily available through the American Type Culture Collection under accession number ATCC CRL1573), can be used. In one embodiment, a modified human embryonic kidney cell line (e.g., HEK293) transformed with adenovirus type 5 DNA fragments and expressing adenovirus E1a and E1b genes is used to generate recombinant AAV particles. The modified HEK293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV particles. Other host cell lines suitable for recombinant AAV production are described in International Application PCT / 2017 / 024951, the disclosure of which is incorporated herein by reference in its entirety.

[0147] In certain embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct prior to or simultaneously with transfection of the AAV expression vector. Host cells with AAV helper functions may be referred to as "helper cells" or "packaging helper cells". Thus, AAV helper constructs may be used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions required for productive AAV transduction. AAV helper constructs often lack AAV ITRs and cannot replicate or package themselves. These constructs may be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products. Many other vectors are known that encode Rep and / or Cap expression products.

[0148] The method of making rAAV particles that can transduce mammalian cells is known in the art.For example, rAAV particles can be produced as described in U.S. Patent No. 9,408,904; and International Application No. PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are incorporated herein by reference in their entirety.

[0149] The present invention provides a cell comprising a nucleic acid encoding C1 inhibitor, a cell comprising an expression cassette comprising a polynucleotide comprising a nucleic acid encoding C1 inhibitor, a cell comprising a viral vector, such as an AAV vector, comprising a nucleic acid encoding C1 inhibitor, and a cell comprising a non-viral vector comprising a polynucleotide comprising a nucleic acid encoding C1 inhibitor.In some embodiments, the cell produces a viral vector.In some embodiments, the cell produces an AAV vector as described herein.

[0150] Also provided are methods for producing viral vectors, such as the AAV vectors described herein. In an embodiment, the method for producing an AAV vector comprises introducing an AAV vector genome comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor as described herein or an expression cassette comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor as described herein into a packaging helper cell; and culturing the helper cell under conditions for producing an AAV vector. In an embodiment, the method for producing an AAV vector comprises introducing a polynucleotide comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor as described herein or an expression cassette comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor as described herein into a packaging helper cell; and culturing the helper cell under conditions for producing an AAV vector.

[0151] In some embodiments, the cell is a mammalian cell.

[0152] In some embodiments, the cells for vector production provide helper functions, such as AAV helper functions, for packaging vectors into viral particles. In some embodiments, the helper functions are Rep and / or Cap proteins for AAV vector packaging. In some embodiments, the cells for vector production can be stably or transiently transfected with polynucleotides encoding Rep and / or Cap protein sequences. In some embodiments, the cells for vector production provide Rep78 and / or Rep68 proteins. In such cells, the cells can be stably or transiently transfected with polynucleotides encoding Rep78 and / or Rep68 protein sequences.

[0153] In some embodiments, the cells for vector production are human embryonic kidney cells. In some embodiments, the cells for vector production are HEK-293 cells.

[0154] The term "transduce" and its grammatical variants refer to the introduction of a molecule, such as a rAAV vector, into a cell or a host organism. The heterologous nucleic acid / transgene may or may not be integrated into the genomic nucleic acid of the recipient cell. The introduced heterologous nucleic acid may also be present in the recipient cell or host organism extrachromosomally or only transiently.

[0155] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell (e.g., in a mammal, such as a cell or tissue or organ cell) refers to, for example, a genetic change in a cell following incorporation of a nucleic acid (e.g., a transgene) into the cell. Thus, a "transduced" cell is a cell into which an exogenous nucleic acid has been introduced, or a progeny thereof. The cell may be propagated and express the introduced protein. For gene therapy uses and methods, the transduced cell may be present in a subject.

[0156] The term "isolated," when used as a modifier of a composition, means that the composition is made by the hand of man or is completely or at least partially separated from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they are normally associated in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0157] The term "isolated" does not exclude combinations produced by the hand of man, such as rAAV sequences that package or encapsidate an AAV vector genome and a pharmaceutical formulation, or rAAV particles. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrids / chimers, multimers / oligomers, modified (e.g., phosphorylated, glycosylated, lipidated) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0158] The term "substantially pure" refers to a preparation that contains at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation may contain at least 75%, or at least 85%, or about 90-99% by weight of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0159] The recombinant AAV vector and its method and use include any virus strain or serotype.As a non-limiting example, the recombinant AAV vector can be based on any AAV genome, such as LK03, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B or AAV-2i8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or different from each other.As a non-limiting example, the recombinant AAV vector based on a specific serotype genome can be the same as the serotype of the capsid protein that packages the vector.In addition, the recombinant AAV vector genome can be based on an AAV serotype genome that is different from the serotype of the AAV capsid protein that packages the vector. For example, the AAV vector genome can be based on AAV2, while at least one of the three capsid proteins can be LK03, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B or AAV-2i8, or a variant thereof.

[0160] In certain embodiments, adeno-associated virus (AAV) vectors include LK03, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B, and AAV-2i8, as well as those described in, for example, WO 2013 / 158879 (disclosing RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6; International Application PCT / US2013 / 037170), WO No. 2015 / 013313 (International Application PCT / US2014 / 047670), US2013 / 0059732 (U.S. Patent No. 9,169,299 disclosing LK01, LK02, LK03, etc.), and variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions and deletions) as described in WO 2016 / 210170, the disclosures of which are incorporated by reference in their entireties.

[0161] As used herein, the term "serotype" is a distinction used to refer to AAVs that have a capsid that is serologically different from other AAV serotypes. Serological characteristics are determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). AAV variants, including capsid variants, may not be serologically distinct from a reference AAV or other AAV serotypes, but they differ in at least one nucleotide or amino acid residue compared to a reference or other AAV serotypes.

[0162] In the traditional definition, serotype means that the virus of interest has been characterized for neutralizing activity and tested against all existing serotype-specific sera, and no antibody has been found to neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, serological differences with any of the existing serotypes may or may not exist. Thus, if a new virus (e.g., AAV) does not have serological differences, this new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed to determine whether a mutant virus with capsid sequence modification is of another serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid duplication, the term "serotype" refers broadly to both serologically distinct viruses (e.g., AAV), as well as serologically indistinguishable viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0163] As described herein, the AAV capsid proteins are 100% identical to reference or parent AAV serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:188), AAV3B, LK03 (SEQ ID NO:191), AAV-2i8, SEQ ID NO:226, SEQ ID NO:189, and / or SEQ ID NO:190. % sequence identity but is not identical to or distinct from known AAV genes or proteins such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:188), AAV3B, LK03 (SEQ ID NO:191), AAV-2i8, SEQ ID NO:226, SEQ ID NO:189, and / or SEQ ID NO:190. In certain embodiments, the modified / mutated AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO: 188), AAV3B, LK03 (SEQ ID NO: 191), AAV-2i8, SEQ ID NO: 226, SEQ ID NO: 189, and / or or comprises or consists of a sequence at least 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 99.9% identical to a reference or parent AAV capsid protein, such as SEQ ID NO:190.

[0164] In certain embodiments, a viral vector, such as an adeno-associated viral (AAV) vector, comprises any of the polynucleotides comprising a nucleic acid encoding a C1 inhibitor described herein operably linked to an expression control element.

[0165] In certain embodiments, a viral vector, such as an adeno-associated viral (AAV) vector, comprises any of the expression cassettes comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor described herein.

[0166] In certain embodiments, an AAV vector comprises one or more AAV capsids and one or more AAV inverted terminal repeats (ITRs), where the AAV ITRs flank the 5' or 3' end of the polynucleotide or expression cassette.

[0167] In one embodiment, the AAV vector further comprises an intron located 5' or 3' to one or more ITRs.

[0168] In one embodiment, the AAV vector comprising at least one or more ITRs or introns has one or more ITRs or introns modified to have reduced CpGs.

[0169] In one embodiment, the present invention provides (1) a 5' AAV ITR, optionally an AAV2 5' ITR, optionally a 5' ITR comprising the polynucleotide sequence of SEQ ID NO: 70 or 72; (2) one or more enhancers, optionally one or more enhancers having a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 225, 74-78, and 173-178; (3) one or more 5' UTRs, optionally one or more 5' UTRs having a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 180 and 183; (4) optionally, an intron, optionally having the polynucleotide sequence of SEQ ID NO: 81 or 82; (5) a nucleic acid encoding a signal peptide, optionally encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 203 to 218, optionally encoding a signal peptide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 84 to 103; (6) A nucleic acid encoding at least one of C1 inhibitor, mutant C1 inhibitor, and fusions or combinations thereof, a. optionally a C1 inhibitor having the amino acid sequence of SEQ ID NO:181 or 192, and optionally a polynucleotide sequence selected from the group consisting of SEQ ID NOs:104-142, 145-147, 156, and 171-172; b. a nucleic acid that is optionally a mutant C1 inhibitor having an amino acid sequence selected from the group consisting of SEQ ID NOs: 193-201, and optionally a polynucleotide selected from the group consisting of SEQ ID NOs: 143-144, 158, and 165-170; (7) one or more 3' UTRs, optionally one or more 3' UTRs having the polynucleotide sequence of SEQ ID NO: 83 or 184; (8) optionally, an miRNA binding site, optionally having the polynucleotide sequence of SEQ ID NO: 179; (9) Optionally, a regulated Ire1-dependent decay (RIDD) sequence, optionally a RIDD having a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 185-187; and (10) A 3' AAV ITR, optionally an AAV2 3' ITR, optionally comprising the polynucleotide sequence of SEQ ID NO: 71 or 73. The present invention relates to an AAV vector comprising the

[0170] In some embodiments, the AAV vector comprises a polynucleotide comprising a nucleic acid encoding a C1 inhibitor, the nucleic acid having reduced CpGs compared to a wild-type coding sequence of the C1 inhibitor, and the polynucleotide is encapsidated by a capsid comprising a VP1 of SEQ ID NO:226. In some embodiments, the capsid comprises a VP1 of SEQ ID NO:226, a VP2 of SEQ ID NO:189, and a VP3 of SEQ ID NO:190. In some embodiments, the polynucleotide comprises a nucleic acid sequence at least 99% identical to SEQ ID NO:22, with the proviso that the nucleic acid sequence encodes SEQ ID NO:192. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:22. In some embodiments, the polynucleotide comprises a nucleic acid sequence at least 99% or 100% identical to SEQ ID NO:22, and the capsid comprises a VP1 of SEQ ID NO:226. In one embodiment, the polynucleotide comprises a nucleic acid sequence that is at least 99% or 100% identical to SEQ ID NO:22, and the capsid comprises a VP1 of SEQ ID NO:226, a VP2 of SEQ ID NO:189, and a VP3 of SEQ ID NO:190.

[0171] In one embodiment, the AAV vectors of the invention are delivered via non-viral delivery systems, including, for example, those encapsulated in lipid nanoparticles (LNPs).

[0172] Non-viral methods In certain embodiments, the polynucleotides and expression cassettes of the invention are delivered or administered with non-viral delivery systems, including, for example, chemical methods such as non-viral vectors or extracellular vesicles, and physical methods such as gene guns, electroporation, particle bombardment, ultrasound application, and magnetic cleavage.

[0173] Recombinant cells capable of expressing the C1 inhibitor sequences of the present invention can be used for delivery or administration.

[0174] Naked DNA such as minicircles and transposons can be used for administration or delivery or lentiviral vectors.Furthermore, gene editing techniques such as zinc finger nucleases, meganucleases, TALENs, and CRISPRs can also be used to deliver the coding sequences of the present invention.

[0175] In certain embodiments, the polynucleotides and expression cassettes of the invention are delivered as naked DNA, minicircles, transposons, closed-end linear double-stranded DNA.

[0176] In certain embodiments, the polynucleotides and expression cassettes of the invention are delivered or administered in AAV vector particles or other viral particles that are further encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles, or extracellular vesicles.

[0177] In certain embodiments, the polynucleotides and expression cassettes of the present invention are delivered or administered with a non-viral vector.

[0178] As used herein, "non-viral vector" refers to a vector that is not delivered by a virus particle or a virus-like particle (VLP).According to an embodiment, a non-viral vector is a vector that is not delivered by a capsid.Vector can be encapsulated, mixed, or otherwise associated with non-viral delivery particle or nanoparticle.

[0179] Any suitable non-viral delivery system known to those of skill in the art in view of the present disclosure can be used in the present invention. Non-viral delivery particles or nanoparticles can be, for example, lipid-based nanoparticles, polymer-based nanoparticles, protein-based nanoparticles, microparticles, microcapsules, metal particle-based nanoparticles, peptide cage nanoparticles, etc.

[0180] The non-viral delivery particles or nanoparticles of the invention may be constructed by any method known in the art, and the non-viral vectors of the invention comprising nucleic acid molecules that include therapeutic transgenes may be constructed by any method known in the art.

[0181] Lipid-Based Delivery Systems Lipid-based delivery systems are well known in the art, and any suitable lipid-based delivery system known to one of skill in the art in light of the present disclosure can be used in the present invention. Examples of lipid-based delivery systems include, for example, liposomes, lipid nanoparticles, micelles, or extracellular vesicles.

[0182] "Lipid nanoparticles" or "LNPs" refer to lipid-based vesicles useful for delivery of AAV and non-viral vectors having a size on the nanoscale, i.e., about 10 to about 1000 nm, or about 50 to about 500 nm, or about 75 to about 127 nm. Without being bound by theory, it is believed that LNPs provide the polynucleotide, expression cassette, AAV vector, or non-viral vector with partial or complete shielding from the immune system. Shielding allows for delivery of the polynucleotide, expression cassette, AAV vector, or non-viral vector to tissues or cells while avoiding inducing a substantial immune response to the polynucleotide, expression cassette, AAV vector, or non-viral vector in vivo. Shielding may also allow for repeated administration without inducing a substantial immune response to the polynucleotide, expression vector, AAV vector, or non-viral vector in vivo (e.g., in a subject such as a human). Shielding may also improve or increase polynucleotide, expression cassette, AAV vector, or non-viral vector delivery efficiency in vivo.

[0183] The isoelectric point (pI) of AAV is in the pH range of about 6 to about 6.5. Thus, the AAV surface has a slight negative charge. Thus, for example, it may be beneficial for the LNP to include a cationic lipid, such as an amino lipid. Exemplary amino lipids are described in U.S. Patent Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, 7,745,651, and U.S. Patent Publication No. 201 Nos. 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411, and 2010 / 0117125, the disclosures of which are incorporated herein by reference in their entireties.

[0184] The terms "cationic lipid" and "amino lipid" are used interchangeably herein and include lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino group (e.g., alkylamino or dialkylamino group). Cationic lipids are typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and are substantially neutral at a pH above its pKa. The cationic lipid may be a titratable cationic lipid. In certain embodiments, the cationic lipid comprises a protonatable tertiary amino group (e.g., pH-titratable); a C18 alkyl chain, each alkyl chain independently having 0-3 (e.g., 0, 1, 2, or 3) double bonds; and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0185] Cationic lipids include l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (g-DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (g-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA, also known as DLin-C2K-DMA, XTC2, and C2K), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (M-C2-DMA, also known as MC2), (6Z,9Z,28Z,3l Z)-heptatriaconta-6,9,28,3l-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA, also known as MC3), salts thereof, and mixtures thereof. Other cationic lipids also include, but are not limited to, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, y-DLen-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).

[0186] Further cationic lipids include 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), l,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), l,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-D-DAC), and 1,2-dilinoleyloxy-3-morpholinopropane (DLin-D-DAC). 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-Linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-l, 2-propanediol (DLinAP), l,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-Dioleyl-N,N-dimethylammonium chloride (DODAC), N-(l-(2,3-Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-Distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2, 3-Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propionyl Panaminium trifluoroacetate (DOSPA), 2,3-dioleyloxy-N-propanamido-1-propanamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-4-oxy)-l-(cis,9,l2-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-βoxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CLinDMA), 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS) and disubstituted spermine (D2S) or mixtures thereof.

[0187] Many commercially available preparations of cationic lipids can be used, such as LIPOFECTIN® (containing DOTMA and DOPE available from GIBCO / BRL) and LIPOFECT AMINE® (containing DOSPA and DOPE available from GIBCO / BRL).

[0188] In certain embodiments, the cationic lipid may be present in an amount from about 10% by weight of the LNP to about 85% by weight of the lipid nanoparticle, or from about 50% by weight of the LNP to about 75% by weight of the LNP.

[0189] Sterols can provide fluidity to the LNP. As used herein, "sterol" refers to any naturally occurring sterol of plant origin (phytosterol) or animal origin (zoosterol), as well as non-naturally occurring synthetic sterols, all of which are characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. The sterol can be any sterol conventionally used in the field of liposome, lipid vesicle or lipid particle preparations, most commonly cholesterol. Phytosterols can include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication No. 2011 / 0177156, the disclosure of which is incorporated herein by reference in its entirety. In certain embodiments, the sterol can be present in an amount of about 5% by weight of the LNP to about 50% by weight of the lipid nanoparticle, or about 10% by weight of the LNP to about 25% by weight of the LNP.

[0190] LNPs may include neutral lipids. Neutral lipids may include any lipid species that exists in either uncharged or neutral zwitterionic form at physiological pH. Such lipids include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside. The selection of neutral lipids is generally guided by, among other things, considerations of particle size and required stability. In certain embodiments, the neutral lipid component may be a lipid with two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0191] Lipids with various acyl chain groups of various chain lengths and degrees of saturation are available or can be isolated or synthesized by well-known techniques. In certain embodiments, lipids containing saturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. In certain embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. Additionally, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include, but are not limited to, l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or any related phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups such as serine and inositol.

[0192] In certain embodiments, the neutral lipid may be present in an amount between about 0.1% by weight of the lipid nanoparticle and about 75% by weight of the LNP, or between about 5% by weight of the LNP and about 15% by weight of the LNP.

[0193] The LNP-encapsulated nucleic acid, expression cassette, AAV vector, and non-viral vector can be incorporated into a pharmaceutical composition, such as a pharma- ceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful, inter alia, for the administration and delivery of the LNP-encapsulated nucleic acid, expression cassette, AAV vector, and non-viral vector to a subject in vivo or ex vivo.

[0194] Preparations of LNPs can be combined with additional components, non-limiting examples of which include polyethylene glycol (PEG) and sterols.

[0195] The term "PEG" refers to polyethylene glycol, a linear water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight, for example, PEG 2,000 has an average molecular weight of about 2,000 daltons, and PEG 5,000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies, and include, for example, the following functional PEGs: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol succinate (MePEG-S), monomethoxypolyethyleneglycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycolamine (MePEG-NH2), monomethoxypolyethyleneglycol tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol imidazolylcarbonyl (MePEG-IM).

[0196] In some embodiments, the PEG may be a polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons, optionally substituted with alkyl, alkoxy, acyl, or aryl. In some embodiments, the PEG may be substituted with methyl at the terminal hydroxyl position. In some embodiments, the PEG may have an average molecular weight of about 750 to about 5,000 daltons, or about 1,000 to about 5,000 daltons, or about 1,500 to about 3,000 daltons, or about 2,000 daltons, or about 750 daltons. The PEG may be substituted with alkyl, alkoxy, acyl, or aryl. In some embodiments, the terminal hydroxyl group may be substituted with a methoxy group or a methyl group.

[0197] PEG-modified lipids include PEG-dialkyloxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397, the disclosures of which are incorporated herein by reference in their entirety. Useful PEG-modified lipids (or lipid polyoxyethylene conjugates) can have various "anchor" lipid moieties for fixing PEG moieties to the surface of lipid vesicles. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide conjugates (e.g., PEG-CerCl4 or PEG-CerC20) (which are described in U.S. Patent No. 5,820,873, the disclosures of which are incorporated herein by reference in their entirety), PEG-modified dialkylamines and PEG-modified l,2-diacyloxypropan-3-amines. In some embodiments, the PEG-modified lipids can be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, PEG can be in an amount from about 0.5% by weight of the LNP to about 20% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP.

[0198] Furthermore, the LNP can be PEG-modified LNP and sterol-modified LNP. The LNP can be the same or different LNP combined with additional components. In other words, the same LNP can be PEG-modified or sterol-modified, or the first LNP can be PEG-modified and the second LNP can be sterol-modified. Optionally, the first and second modified LNP can be combined.

[0199] In certain embodiments, the LNP prior to encapsulation may have a size ranging from about 10 nm to 500 nm, or from about 50 nm to about 200 nm, or from 75 nm to about 125 nm. In certain embodiments, the LNP-encapsulated nucleic acid, expression vector, AAV vector, or non-viral vector may have a size ranging from about 10 nm to 500 nm.

[0200] Polymer-Based Systems Polymer-based delivery systems are well known in the art, and in view of the present disclosure, any suitable polymer-based delivery system or polymer nanoparticle known to those skilled in the art can be used in the present invention.DNA can be entrapped in the polymer matrix of polymer nanoparticles, or adsorbed or conjugated onto the surface of nanoparticles.Examples of polymers commonly used for gene delivery include, for example, poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI), chitosan, dendrimers, polyanhydrides, polycaprolactones, and polymethacrylates.

[0201] The polymer-based non-viral vectors may have a variety of sizes ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally from about 100 nm to about 150 nm, optionally less than about 150 nm.

[0202] Protein-based systems Protein-based delivery systems are well known in the art, and in light of the present disclosure, any suitable protein-based delivery system or cell penetrating peptide (CPP) known to the skilled artisan can be used in the present invention.

[0203] CPPs are short peptides (6-30 amino acid residues) that are potentially capable of intracellular penetration to deliver therapeutic molecules. The majority of CPPs consist mainly of arginine and lysine residues, making them cationic and hydrophilic, but CPPs can also be amphipathic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., Tat, HIV-1 protein) or obtained by synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018;25(1):1996-2006). Examples of CPPs include, for example, cationic CPPs (highly positively charged) (e.g., Tat peptide, penetratin, protamine, poly-L-lysine, polyarginine, etc.); amphipathic CPPs (chimeric or fusion peptides constructed from various sources containing both positively and negatively charged amino acid sequences) (e.g., transportan, VT5, bactenecin-7 (Bac7), proline-rich peptides (PPRs), SAP (VRLPP)3, TP10, pep-1, MPG, etc.); membrane-philic CPPs (exhibiting both hydrophobic and amphipathic properties simultaneously, containing both large aromatic and small residues) (e.g., gH625, SPION-PEG-CPP NP, etc.); and hydrophobic CPPs (containing only non-polar motifs or residues) (e.g., SG3, PFVYLI, pep-7, fibroblast growth factor (FGF), etc.).

[0204] Proteinaceous non-viral vectors may have a variety of sizes ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally from about 100 nm to about 150 nm, and optionally below about 150 nm.

[0205] Peptide Cage System Peptide cage-based delivery systems are well known in the art, and in light of the present disclosure, any suitable peptide cage-based delivery system known to those skilled in the art can be used in the present invention. In general, any proteinaceous material that can be assembled into a cage-like structure that forms a confined internal environment can be used. Several different types of protein "shells" can be assembled and loaded with different types of materials. For example, protein cages that include shells of viral coat proteins (e.g., from Cowpea Chlorotic Mottle Virus (CCMV) protein coats) that encapsulate non-viral materials, as well as protein cages formed from non-viral proteins have been described (see, e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Application No. 20040028694, and U.S. Patent Application No. 20090035389, the disclosures of which are incorporated herein by reference in their entirety). A peptide cage may comprise a proteinaceous shell that self-assembles to form a protein cage (e.g., a structure with an internal cavity that is naturally accessible to solvent or can be made so by altering solvent concentration, pH, equilibrium ratios).

[0206] Examples of protein cages derived from non-viral proteins include, for example, ferritins and apoferritins from both eukaryotic and prokaryotic species, e.g., 12 and 24 subunit ferritins; and heat shock proteins (HSPs), e.g., the class of 24 subunit heat shock proteins that form the inner core space, the small HSP of Methanococcus jannaschii, the dodecameric Dps HSP of Escherichia coli, protein cages formed from MrgA proteins, etc. As will be appreciated by those skilled in the art, the monomers of the protein cages can be naturally occurring or mutants containing amino acid substitutions, insertions and deletions (e.g., fragments) that can be created.

[0207] The protein cages may have different core sizes ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally from about 100 nm to about 150 nm, optionally up to about 150 nm.

[0208] Pharmaceutical Compositions The present invention further provides pharmaceutical compositions comprising any of a polynucleotide comprising a nucleic acid encoding a C1 inhibitor, an expression cassette comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor, a viral vector such as an AAV vector comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor, or a non-viral vector comprising a polynucleotide comprising a nucleic acid encoding a C1 inhibitor as described herein.

[0209] rAAV vectors and non-viral vectors can be administered to patients via injection into biologically compatible carriers, for example, via intravenous injection.rAAV vectors and non-viral vectors can be administered alone or in combination with other molecules.Thus, rAAV vectors and non-viral vectors and other compositions, agents, drugs, biologics (proteins) can be incorporated into pharmaceutical compositions.Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo.

[0210] In certain embodiments, the pharmaceutical composition also contains a pharma- ceutically acceptable carrier or excipient, including any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition and that may be administered without undue toxicity.

[0211] As used herein, the terms "pharmaceutical acceptable" and "physiologically acceptable" refer to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutical acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing substantial undesirable biological effects. Thus, such a pharmaceutical composition can be used, for example, to administer a polynucleotide, vector, viral particle, or protein to a subject.

[0212] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugar, and ethanol. Pharmaceutically acceptable salts can also include, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids such as acetate, propionate, malonate, benzoate, and the like. Excipients also include proteins such as albumin.

[0213] Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0214] Pharmaceutical compositions may be provided as salts, which may be formed with many acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous solutions or other protic solvents than the corresponding free base forms. In other cases, the preparation may be a lyophilized powder, which may contain any or all of the following: 1-50 mM histidine, 0.1-2% sucrose, and 2-7% mannitol, in a pH range of 4.5-5.5, to be combined with a buffer prior to use.

[0215] Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonicity agents, and absorption enhancers or retarders, and are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Such pharma-ceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) may also be incorporated into the compositions.

[0216] Pharmaceutical compositions can be formulated to suit a particular route of administration or delivery, as described herein or known to those skilled in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0217] Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of active compounds, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.Non-limiting examples include water, buffered saline, Hank's solution, Ringer's solution, dextrose, fructose, ethanol, animal oil, vegetable oil or synthetic oil.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

[0218] Furthermore, suspension of active compound can be prepared as suitable oil injection suspension.Suitable lipophilic solvent or vehicle includes fatty oil such as sesame oil, or synthetic fatty acid ester such as ethyl oleate or triglyceride, or liposome.In some cases, suspension can contain suitable stabilizer or agent that increases the solubility of compound, so that highly concentrated solution can be prepared.

[0219] Cosolvents and adjuvants can be added to the formulation.Non-limiting examples of cosolvents include alcohols with hydroxyl groups or other polar groups, such as isopropyl alcohol; glycols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.Adjuvants include surfactants such as soybean lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0220] In some embodiments, a pharmaceutical composition comprising any of the AAV vectors described herein further comprises an empty AAV capsid. In some embodiments, in a pharmaceutical composition comprising an AAV vector and an empty AAV capsid, the ratio of empty AAV capsid to AAV vector is within or between about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100. In some embodiments, in a pharmaceutical composition comprising an AAV vector and an empty AAV capsid, the ratio of empty AAV capsid to AAV vector is about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0221] In certain embodiments, the pharmaceutical composition comprises a surfactant.

[0222] After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment. Such labeling can include the amount, frequency, and method of administration.

[0223] Delivery systems suitable for the compositions, methods and uses of the present invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, pp. 253-315).

[0224] An "effective amount" or a "sufficient amount" refers to an amount, in single or multiple doses, that alone or in combination with one or more other compositions (therapeutic agents such as drugs or immunosuppressants), treatments, protocols, or therapeutic agents, provides a detectable response of any duration (long or short term), any measurable or detectable degree of expected or desired result in a subject, or a benefit to a subject of any duration (e.g., minutes, hours, days, months, years, or being cured).

[0225] Compositions, such as pharmaceutical compositions, can be delivered to a subject to allow for production of the encoded protein. In certain embodiments, the pharmaceutical composition contains sufficient genetic material to allow the recipient to produce a therapeutically effective amount of the protein in the subject.

[0226] "Therapeutically effective amount" refers to the amount of active ingredient or ingredients that induces a desired biological or medical response in a subject. Therapeutically effective amount can be determined empirically and in a routine manner in relation to the stated purpose. For example, in vitro assays can be used as necessary to help identify optimal dosage ranges. The selection of a specific effective dose can be determined by a person skilled in the art (e.g., through clinical trials) based on the consideration of several factors, including the disease to be treated or prevented, the symptoms involved, the patient's weight, the patient's immune status, and other factors known to those skilled in the art. The exact dose to be used in the formulation also depends on the route of administration, and the severity of the disease, and should be determined according to the physician's judgment and each patient's circumstances. The effective dose can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

[0227] The compositions may be formulated and / or administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be formulated and / or administered to a patient alone or in combination with other agents that affect hemostasis (e.g., cofactors).

[0228] Treatment method HAE treatment with C1-INH replacement therapy increases survival rates, reduces event frequency and severity, and is the current standard of care for patients with HAE disease. However, current treatment modalities have several drawbacks, such as potential breakthrough attacks, safety / tolerability, high patient burden, and potential limited compliance. HAE gene transfer treatment preferably overcomes one or more of these drawbacks to C1-INH replacement therapy. For example, in the HAE gene transfer treatment described herein, it is expected that less frequent dosing is required, and preferably a single dose is sufficient.

[0229] The present invention further provides a method for treating a subject in need of C1 inhibitor, comprising administering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, AAV vector, non-viral vector, or pharmaceutical composition of the invention, wherein the C1 inhibitor is expressed in the subject.

[0230] The methods and uses of the present invention include delivering (transducing) a polynucleotide (transgene) to a host cell, including dividing and / or non-dividing cells. The polynucleotides, expression cassettes, rAAV vectors, non-viral vectors, methods, uses, and pharmaceutical preparations of the present invention are further useful in a method of delivering, administering, or providing a sequence encoded by a heterogeneous nucleic acid to a subject in need thereof as a therapeutic method. In this way, the polynucleotide comprising the nucleic acid is transcribed, and a protein is produced in vivo in the subject. The subject may benefit from or require a protein because the subject has a deficiency of the protein, or because the production of the protein in the subject may provide some therapeutic effect as a therapeutic or other method.

[0231] The present invention is useful in medical applications in animals, including humans and veterinary medicine. Thus, suitable subjects include mammals, such as humans, as well as non-human mammals. The term "subject" refers to animals, typically mammals, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), livestock (poultry, such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, juvenile, and adult subjects. Subjects include animal disease models, such as mouse and other animal models of protein / enzyme deficiencies, such as HAE, and others known to those skilled in the art.

[0232] Subjects suitable for treatment according to the invention include those who have or are at risk of producing insufficient amounts of C1 inhibitor, or those who produce abnormal, partially functional, or non-functional C1 inhibitor. Subjects can be tested for C1 inhibitor activity to determine whether such subjects are suitable for treatment according to the methods of the invention. Subjects suitable for treatment according to the invention also include those who would benefit from C1 inhibitor. Such subjects who could benefit from C1 inhibitor include subjects with complement-mediated disorders. Treated subjects can be monitored periodically after treatment, for example, every 1-4 weeks, every 1-6 months, every 6-12 months, or every 1, 2, 3, 4, 5, or more years.

[0233] Subjects can be tested for immune response, for example, for antibodies against AAV. Thus, candidate subjects can be screened before treatment with the method of the present invention. Subjects can also be tested for antibodies against AAV after treatment, and optionally monitored for a period of time after treatment. Subjects with existing or developing AAV antibodies can be treated with immunosuppressants or other regimens described herein.

[0234] The subject suitable for treatment according to the present invention also includes a subject who has or is at risk of producing antibodies against AAV. Using several techniques, rAAV vectors can be administered or delivered to such subjects. For example, AAV empty capsid (i.e., AAV particles that lack modified nucleic acid encoding C1 inhibitor) can be delivered to bind to AAV antibodies in the subject, thereby allowing the rAAV vector containing heterogeneous nucleic acid to transduce the cells of the subject. The terms "AAV empty capsid", "AAV empty capsid particle", "empty capsid AAV", and "empty capsid AAV particle" are used interchangeably herein.

[0235] The modified nucleic acid, expression cassette, rAAV vector and non-viral vector of the present invention can be used to treat C1 inhibitor deficiency.Thus, in some embodiments, the modified nucleic acid encoding C1 inhibitor, the expression cassette comprising the modified nucleic acid encoding C1 inhibitor, rAAV vector and non-viral vector of the present invention can be used as a therapeutic and / or prophylactic agent.

[0236] In certain embodiments, the modified nucleic acid encoding C1 inhibitor, the expression cassette comprising the modified nucleic acid encoding C1 inhibitor, the rAAV vector, and the non-viral vector of the present invention can be used to treat HAE. Administration of the modified nucleic acid encoding C1 inhibitor, the expression cassette comprising the modified nucleic acid encoding C1 inhibitor, the rAAV vector, and the non-viral vector of the present invention to a patient with HAE results in expression of the C1 inhibitor protein.

[0237] In certain embodiments, methods according to the present invention may result in a level of C1 inhibitor expression or activity that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the normal expression of C1 inhibitor protein found in a subject not in need of C1 inhibitor.

[0238] Subjects, animals, or patients administered modified nucleic acid encoding C1 inhibitor, expression cassettes comprising modified nucleic acid encoding C1 inhibitor, rAAV vectors, and expression cassettes comprising non-viral vectors of the present invention can be evaluated by various tests, assays, and functional evaluations to demonstrate, measure, and / or evaluate the efficacy of modified nucleic acid encoding C1 inhibitor, expression cassettes comprising modified nucleic acid encoding C1 inhibitor, rAAV vectors, and non-viral vectors of the present invention as therapeutic and / or prophylactic agents. Such tests and assays include, but are not limited to, measuring C1 inhibitor activity (such as by using standard C1 inhibitor activity assays) and / or measuring C1 inhibitor amount (such as by Western blot with anti-C1 inhibitor antibodies) in biological samples such as blood or plasma; analyzing peak and steady-state vector-derived C1 inhibitor enzyme levels as assessed by total C1 inhibitor protein and activity in plasma; testing for immune response to AAV capsid; and testing for immune response to C1 inhibitor transgene protein products.

[0239] Furthermore, the modified nucleic acid encoding C1 inhibitor of the present invention, the expression cassette comprising the modified nucleic acid encoding C1 inhibitor, the rAAV vector, and the non-viral vector of the present invention can be used to treat complement-mediated disorders.According to an embodiment, the modified nucleic acid encoding C1 inhibitor, the expression cassette comprising the modified nucleic acid encoding C1 inhibitor, the rAAV vector, and the non-viral vector of the present invention can be used to treat patients who need C1 inhibitor.According to an embodiment, the modified nucleic acid encoding C1 inhibitor of the present invention, the expression cassette comprising the modified nucleic acid encoding C1 inhibitor, the rAAV vector, and the non-viral vector of the present invention can be used to treat hereditary angioedema (HAE).

[0240] As used herein, the term "hereditary angioedema" or "HAE" refers to a blood disorder characterized by unpredictable and recurrent bouts of inflammation. HAE is typically associated with C1-INH deficiency, which can be the result of impaired or reduced activity of C1-INH or low levels of C1-INH. Symptoms include, but are not limited to, swelling that can occur in any part of the body, such as the face, extremities, genitals, gastrointestinal tract, and upper respiratory tract. As used herein, HAE can be type I, type II, or type III.

[0241] As described herein, rAAV can penetrate cells and introduce nucleic acid / genetic material into cells, making it useful as a gene therapy vector.Since AAV is not associated with pathogenic disease in humans, rAAV vector can deliver heteropolynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without causing substantial AAV pathogenesis or disease.

[0242] rAAV vectors have many desirable features for such applications, including affinity for dividing and non-dividing cells. Early clinical experience with these vectors also shows no persistent toxicity, and immune responses are typically minimal or undetectable. AAV is known to infect a wide variety of cell types in vivo by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting many tissues, such as retinal epithelium, liver, skeletal muscle, airway, brain, joints, and hematopoietic stem cells.

[0243] For example, it may be desirable to introduce a rAAV vector that can provide multiple copies of C1 inhibitor, i.e., provide a higher amount of C1 inhibitor protein. Improved rAAV vectors and methods for producing these vectors are described in detail in many references, patents, and patent applications, including Wright JF (Hum. Gene Ther., 20:698-706, 2009).

[0244] Dosage may vary and depends on the type, onset, progression, severity, frequency, duration, or probability of the disease to be treated, the desired clinical endpoint, previous or concurrent treatment, the general health, age, sex, race, or immunological competence of the subject, and other factors understood by those skilled in the art. The dose, number, frequency, or duration may be increased or decreased proportionately as indicated by any adverse side effects, complications, or other risk factors of the treatment or treatment, and the condition of the subject. Those skilled in the art will understand the factors that may affect the dosage and timing required to provide an amount sufficient to provide therapeutic or prophylactic benefit.

[0245] Dosages to achieve a therapeutic effect, such as the dose of rAAV vector genome / kilogram of body weight (vg / kg), or the dose of a non-viral vector, will vary based on several factors, including, but not limited to, the route of administration, the level of heteropolynucleotide expression required to achieve a therapeutic effect, the particular disease being treated, any host immune response to the viral vector, the host immune response to the heteropolynucleotide or expression product (protein), and the stability of the expressed protein. Those skilled in the art will be able to determine the rAAV / vector genome or non-viral vector dosage range for treating a patient with a particular disease or disorder based on the above factors as well as other factors.

[0246] Generally, the dose of rAAV should be at least 1×10 per kilogram of subject body weight to achieve a therapeutic effect. 8 vector genome (vg / kg), or more, e.g., 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 Or 1×10 14 , or more, vector genomes per kilogram of subject body weight (vg / kg).

[0247] For example, about 2 × 10 11 Recombinant AAV vg / kg or approximately 2 × 10 11 Recombinant AAV vg / kg dose greater than 3×10 11 Recombinant AAV vg / kg or approximately 3 × 10 11 Recombinant AAV vg / kg dose greater than 4×10 11 Recombinant AAV vg / kg or approximately 4 × 10 11 Recombinant AAV vg / kg dose greater than 5×10 11 Recombinant AAV vg / kg or approximately 5 × 10 11 Recombinant AAV vg / kg dose greater than 1×10 12 Recombinant AAV vg / kg or approximately 1 × 10 12 Recombinant AAV vg / kg dose greater than 2×1012 Recombinant AAV vg / kg or approximately 2 × 10 12 Recombinant AAV vg / kg dose greater than 3×10 12 Recombinant AAV vg / kg or approximately 3 × 10 12 Recombinant AAV vg / kg dose greater than 4×10 12 Recombinant AAV vg / kg or approximately 4 × 10 12 Recombinant AAV vg / kg dose greater than 5×10 12 Recombinant AAV vg / kg or approximately 5 × 10 12 Recombinant AAV vg / kg dose greater than 1×10 13 Recombinant AAV vg / kg or approximately 1 × 10 13 Recombinant AAV vg / kg dose greater than 2×10 13 Recombinant AAV vg / kg or approximately 2 × 10 13 Recombinant AAV vg / kg dose greater than 3×10 13 Recombinant AAV vg / kg or approximately 3 × 10 13 Recombinant AAV vg / kg dose greater than 4×10 13 Recombinant AAV vg / kg or approximately 4 × 10 13 Recombinant AAV vg / kg dose greater than 5×10 13 Recombinant AAV vg / kg or approximately 5 × 10 13 This is a dose greater than recombinant AAV vg / kg.

[0248] An exemplary dose range of recombinant AAV vg / kg to be administered is about 1.5x10 11 ~about 5x10 13 of recombinant AAV vg / kg; approximately 1.5 x 10 11 ~Approx. 2×10 11 of recombinant AAV vg / kg; approximately 2 x 10 11 ~Approx. 2.5×10 11 of recombinant AAV vg / kg; approximately 2.5 x 10 11 ~Approx. 3×10 11 of recombinant AAV vg / kg; approximately 3 x 10 11 ~Approx. 3.5×10 11 of recombinant AAV vg / kg; approximately 3.5 x 10 11 ~Approx. 4×1011 of recombinant AAV vg / kg; approximately 4 × 10 11 ~Approx. 4.5×10 11 of recombinant AAV vg / kg; approximately 4.5 x 10 11 ~Approx. 5×10 11 of recombinant AAV vg / kg; approximately 5 × 10 11 ~Approx. 1×10 12 of recombinant AAV vg / kg; approximately 1 x 10 12 ~Approx. 1.5×10 12 of recombinant AAV vg / kg; approximately 1.5 x 10 12 ~Approx. 2×10 12 of recombinant AAV vg / kg; approximately 2 x 10 12 ~Approx. 2.5×10 12 of recombinant AAV vg / kg; approximately 2.5 x 10 12 ~Approx. 3×10 12 of recombinant AAV vg / kg; approximately 3 x 10 12 ~Approx. 3.5×10 12 of recombinant AAV vg / kg; approximately 3.5 x 10 12 ~Approx. 4×10 12 of recombinant AAV vg / kg; approximately 4 × 10 12 ~Approx. 4.5×10 12 of recombinant AAV vg / kg; approximately 4.5 x 10 12 ~Approx. 5×10 12 of recombinant AAV vg / kg; approximately 5 × 10 12 ~Approx. 1×10 13 of recombinant AAV vg / kg; approximately 1 x 10 13 ~Approx. 1.5×10 13 of recombinant AAV vg / kg; approximately 1.5 x 10 13 ~Approx. 2×10 13 of recombinant AAV vg / kg; approximately 2 x 10 13 ~Approx. 2.5×10 13 of recombinant AAV vg / kg; approximately 2.5 x 10 13 ~Approx. 3×10 13 of recombinant AAV vg / kg; approximately 3 x 10 13 ~Approx. 3.5×10 13 of recombinant AAV vg / kg; approximately 3.5 x 10 13 ~Approx. 4×10 13 of recombinant AAV vg / kg; approximately 4 × 10 13 ~Approx. 4.5×1013 of recombinant AAV vg / kg; approximately 4.5 x 10 13 ~Approx. 5×10 13 recombinant AAV vg / kg; and approximately 5 × 10 13 ~Approx. 1×10 14 The dose range is 100 mg / kg of recombinant AAV vg / kg.

[0249] In one embodiment, the AAV vg / kg is about 1×10 11 vg / kg, approximately 2 × 10 11 vg / kg, approximately 3 × 10 11 vg / kg, approximately 4 × 10 11 vg / kg, approximately 5 × 10 11 vg / kg, approximately 6 × 10 11 vg / kg, approximately 7 × 10 11 vg / kg, approximately 8 × 10 11 vg / kg, approximately 9 × 10 11 vg / kg, approximately 1 × 10 12 vg / kg, approximately 2 × 10 12 vg / kg, approximately 3 × 10 12 vg / kg, approximately 4 × 10 12 vg / kg, approximately 5 × 10 11 vg / kg, approximately 6 × 10 12 vg / kg, approximately 7 × 10 12 vg / kg, approximately 8 × 10 12 vg / kg, approximately 9 × 10 12 vg / kg, approximately 1 × 10 13 vg / kg, approximately 2 × 10 13 vg / kg, approximately 3 × 10 13 vg / kg, approximately 4 × 10 13 vg / kg, approximately 5 × 10 13 vg / kg, approximately 6 × 10 13 vg / kg, approximately 7 × 10 13vg / kg, approximately 8 × 10 13 vg / kg, approximately 9 × 10 13 The drug is administered at a dose of 100 mg / kg.

[0250] As used herein, a "unit dosage form" refers to a physically discrete unit suitable as a unitary dose for a subject to be treated, each unit containing a predetermined amount, optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filler), calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms can be, for example, in ampoules and vials that can contain liquid compositions, or compositions in lyophilized or lyophilized form; for example, a sterile liquid carrier can be added prior to administration or in vivo delivery. Individual unit dosage forms can be included in multi-dose kits or containers, and the rAAV particles, non-viral vectors, and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0251] An "effective amount" or "sufficient amount" of a therapeutic dose (e.g., to improve or provide a therapeutic benefit or improvement) is typically effective to provide a measurable response to one, more than one, or all adverse symptoms, consequences or complications of a disease, e.g., one or more adverse symptoms, disorders, diseases, conditions, or complications caused by or associated with a disease, while a satisfactory result would be to reduce, decrease, inhibit, suppress, limit, or control the progression or worsening of the disease.

[0252] In certain embodiments, methods according to the invention reduce, decrease, or inhibit the need for C1 inhibitor or one or more symptoms of HAE; or prevent or reduce the progression or worsening of the need for C1 inhibitor or one or more symptoms of HAE; or stabilize the need for C1 inhibitor or one or more symptoms of HAE; or ameliorate the need for C1 inhibitor or one or more symptoms of HAE.

[0253] An effective or sufficient amount can be provided in a single dose, but does not necessarily have to be, and can require multiple doses, and can be administered alone or in combination with another composition (e.g., drug), treatment, protocol, or treatment regimen, but does not necessarily have to be. For example, the amount can be increased proportionately as indicated by the subject's needs, the type, condition, and severity of the disease being treated, or the side effects of the treatment (if any). In addition, an effective or sufficient amount does not have to be effective or sufficient when given in a single or multiple doses without a second composition (e.g., another drug or drug), because additional doses, amounts, or durations beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols, or treatment regimens may be included to be considered effective or sufficient in a given subject. Amounts considered effective also include amounts that result in a reduction in the use of another treatment, treatment plan, or protocol, such as the administration of modified nucleic acids encoding C1 inhibitor for the treatment of C1 inhibitor deficiency (e.g., HAE).

[0254] Therefore, the method and use of the present invention also includes, among others, the method and use that results in the reduction of the need or use of another compound, agent, drug, treatment regimen, treatment protocol, process, or treatment.For example, for C1 inhibitor deficiency, the method or use of the present invention has therapeutic benefit in a given subject when less frequent or reduced dose or elimination of recombinant C1 inhibitor dose is required to compensate for the deficiency or deficiency of C1 inhibitor in the subject.Therefore, the present invention provides a method and use that reduces the need or use of another treatment or therapy.

[0255] Effective amount or sufficient amount does not have to be effective in each and every subject that is treated, nor does it have to be effective in the majority of subjects that are treated in a given group or population.Effective amount or effectiveness or sufficient amount means effectiveness or sufficiency in a specific subject, not in a group or general population.As is typical for such methods, some subjects show greater response, or little or no response to a given treatment method or use.

[0256] Administration or in vivo delivery to a subject may occur prior to the onset of adverse symptoms, conditions, complications, etc., caused by or associated with a disease. For example, screening (e.g., genetic) may be used to identify such subjects as candidates for the compositions, methods, and uses of the present invention. Thus, such subjects include screened subjects who are positive for insufficient amounts or deficiencies of functional gene products (e.g., deficiencies of C1 inhibitor or proteins that result in HAE) or who produce abnormal, partially functional, or non-functional gene products (e.g., C1 inhibitor or proteins involved in HAE).

[0257] Administration or in vivo delivery to a subject according to the methods and uses of the invention disclosed herein may be performed within 1-2 hours, 2-4 hours, 4-12 hours, 12-24 hours, or 24-72 hours after the subject has been identified as having a disease targeted for treatment, identified as having one or more symptoms of the disease, or identified as having one or more symptoms of the disease, and identified as positive as described herein. Of course, the methods and uses of the invention may be performed 1-7 days, 7-14 days, 14-24 days, 24-48 days, 48-64 days, or more days, months, or years after the subject has been identified as having a disease targeted for treatment, identified as having one or more symptoms of the disease, or identified as having one or more symptoms of the disease, and identified as positive as described herein.

[0258] The term "ameliorate" refers to a detectable or measurable improvement in a subject's disease or symptoms thereof, or an underlying cellular response. Detectable or measurable improvement includes a subjective or objective reduction, decrease, inhibition, suppression, inhibition, capping or control in the occurrence, frequency, severity, progression or persistence of a disease, or a complication caused by or associated with a disease, or an improvement in the symptoms or underlying causes or consequences of a disease, or a reversal of a disease.

[0259] For HAE disease, an effective amount would be an amount that improves markers of HAE disease such as subcutaneous edema and swelling in any part of the body, such as the face, extremities, genitals, gastrointestinal tract, and upper respiratory tract, as well as biomarkers such as levels of C1 inhibitor antigen and activity, levels of complement C4 (95% of patients with HAE have C4 levels below normal, even when asymptomatic), and levels of bradykinin.

[0260] Improved biomarkers for HAE include increased levels of C1 inhibitor antigen and activity, increased or stabilized levels of complement C4 (low circulating levels of complement C4 are indicative of HAE), and decreased levels of bradykinin (indicating restoration of C1-INH function / activity). Many assays for measuring or quantifying levels of complement C4 in plasma, serum or tissue are known in the art, including multiplex assays (Lai et al., J. Pharm. Biomed. Anal., 195: 113844 (2020) doi: 10.1016 / j.jpba.2020.113844) and radial immunodiffusion (Koelle et al., J. Clin. Microbio., 16: 271-275 (1982)) and ELISA (Kaplan et al., Front. Med., 4: 206 (2017).

[0261] In some embodiments, the method and use of the present invention increases the level of C1 inhibitor antigen and activity in the blood, plasma, serum and / or tissue of a subject.In some embodiments, the method and use of the present invention reduces the bradykinin level in the blood, plasma, serum and / or tissue of a subject.In some embodiments, the method and use of the present invention increases or stabilizes the level of complement C4 in the plasma, serum and / or tissue of a subject.

[0262] The therapeutic dose depends, among other factors, on the age and general condition of the subject, the severity of the disease or disorder. Thus, the therapeutically effective amount in humans falls within a relatively broad range that can be determined by a physician based on the response of the individual patient.

[0263] The methods and uses of the present invention include delivery and administration systemically, locally or regionally, or by any route, such as injection or infusion. Delivery of pharmaceutical compositions in vivo can generally be achieved via injection using a conventional syringe, but other delivery methods such as convection-enhanced delivery are also envisioned (see, for example, U.S. Pat. No. 5,720,720, the disclosure of which is incorporated herein by reference in its entirety). For example, the compositions can be delivered subcutaneously, intraepidermally, intradermally, intrathecally, intraorbitally, intramucosally, intranasally, intraperitoneally, intravenously, intrapleurally, intraarterially, intracavity, orally, intrahepatically, via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application. Clinicians who specialize in the treatment of patients with HAE can determine the optimal route for administration of AAV vectors and non-viral vectors based on a number of criteria, including but not limited to the patient's condition and the goal of treatment (e.g., enhancing or reducing C1 inhibitor levels).

[0264] The composition may be administered alone. In some embodiments, the rAAV particles or non-viral vectors provide a therapeutic effect without an immunosuppressant. The therapeutic effect may be sustained, for example, for 2-4 days, 4-6 days, 6-8 days, 8-10 days, 10-14 days, 14-20 days, 20-25 days, 25-30 days, or 30-50 days or more, for example, 50-75 days, 75-100 days, 100-150 days, 150-200 days or more, in some cases, without the administration of an immunosuppressant. Thus, the therapeutic effect is provided for a period of time.

[0265] The rAAV vectors, non-viral vectors, methods, and uses of the present invention can be combined with any compound, agent, drug, treatment, or other therapeutic regimen or protocol that has a desired therapeutic, beneficial, additive, synergistic, or complementary activity or effect. Exemplary combination compositions and treatments include a second active agent, such as a biologic (protein), a drug (e.g., an immunosuppressant), and a drug. Such biologics (proteins), agents, drugs, treatments, and therapies can be administered or performed before, substantially simultaneously with, or after any other method or use of the present invention.

[0266] The compound, agent, drug, treatment or other treatment regimen or protocol can be administered as a combination composition, or can be administered separately, such as simultaneously, or in series, or consecutively (before or after) with the delivery or administration of the polynucleotide, expression cassette, rAAV particle, or non-viral vector. Thus, the present invention provides a combination in which the method or use of the present invention is combined with any compound, agent, drug, treatment regimen, treatment protocol, process, therapeutic agent, or composition described herein or known to those skilled in the art. The polynucleotide, expression cassette, non-viral vector, or rAAV particle of the present invention can be administered to a subject before, substantially simultaneously with, or after the compound, agent, drug, treatment regimen, treatment protocol, process, therapeutic agent, or composition is administered or performed.

[0267] In certain embodiments, a nucleic acid, expression vector, non-viral vector, or rAAV particle of the invention is administered to a patient in combination with an immunosuppressant agent or regimen in which the patient has or is at risk of developing an immune response to the rAAV particle and / or C1 inhibitor protein. Such an immunosuppressant agent or regimen may be administered prior to, substantially simultaneously with, or after administration of a polynucleotide, expression cassette, non-viral vector, or rAAV vector of the invention.

[0268] In some embodiments, a subject or patient, such as a human patient with HAE, has developed inhibitors to C1 inhibitor protein (including anti-C1 inhibitor antibodies and / or anti-C1 inhibitor T cells), which may occur after treatment with conventional enzyme replacement therapy (e.g., after administration of recombinantly produced C1 inhibitor protein). Such development of C1 inhibitors may occur in patients undergoing enzyme replacement therapy, particularly when the patient has undetectable C1 inhibitor levels, and the replacement C1 inhibitor protein appears to be "exogenous" to the patient's immune system. In some embodiments, the HAE patient with C1 inhibitor is administered one or more regimens aimed at achieving immune tolerance or mitigating immune responses to the C1 inhibitor protein in the patient prior to, substantially simultaneously with, or after administration of the rAAV vector or non-viral vector of the invention. Such regimens for achieving immune tolerance or mitigating immune responses to C1 inhibitor protein may include administration of one or more immunosuppressive agents, including, but not limited to, methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, and synthetic vaccine particle (SVP™)-rapamycin (rapamycin encapsulated in a biodegradable nanoparticle), and / or administration of one or more immunosuppressive protocols or procedures, such as B cell depletion, immunoadsorption, and plasma exchange.

[0269] In some embodiments, the rAAV vector or non-viral vector is administered in conjunction with one or more immunosuppressive agents prior to, substantially simultaneously with, or after administration of the rAAV vector or non-viral vector, In some embodiments, the one or more immunosuppressive agents are administered, e.g., 1-12 hours, 12-24 hours, or 24-48 hours, or 2-4 days, 4-6 days, 6-8 days, 8-10 days, 10-14 days, 14-20 days, 20-25 days, 25-30 days, 30-50 days, or more than 50 days, after administration of the rAAV vector or non-viral vector. Such administration of an immunosuppressant after a period of time after administration of the rAAV vector or non-viral vector can occur when there is a decrease in the encoded protein or inhibitor nucleic acid for a period of time after the initial expression level, e.g., 20-25 days, 25-30 days, 30-50 days, 50-75 days, 75-100 days, 100-150 days, 150-200 days or more than 200 days after administration of the rAAV vector or non-viral vector.

[0270] In certain embodiments, the immunosuppressant is an anti-inflammatory agent.

[0271] In certain embodiments, the immunosuppressant is a steroid, for example a corticosteroid. In certain embodiments, the immunosuppressant is prednisone, prednisolone, a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), a CD52 inhibitor (e.g., alemtuzumab), a CTLA4-Ig (e.g., abatacept, belatacept), an anti-CD3 monoclonal antibody, an anti-LFA-1 monoclonal antibody (e.g., efalizumab), an anti-CD40 monoclonal antibody (e.g., ASKP1240), an anti-CD22 monoclonal antibody (e.g., epratuzumab), an anti-CD20 monoclonal antibody (e.g., rituximab, orelizumab, ofatuzumab, veltuzumab), proteasome inhibitors (e.g. bortezomib), TACI-Ig (e.g. atacicept), anti-C5 monoclonal antibodies (e.g. eculizumab), mycophenolate, azathioprine, sirolimus, everolimus, TNFR-Ig (e.g. etanercept (Enbrel®), anti-TNF monoclonal antibodies (e.g. adalimumab (Humira®), infliximab (Remicade®); Absora®)), tofacitinib, anti-I L-2R (e.g., basiliximab), anti-IL-17 monoclonal antibodies (e.g., secukinumab), anti-IL-6 monoclonal antibodies (e.g., anti-IL-6 antibody sirukumab, anti-IL-6 receptor antibody tocilizumab (Actemra®)), IL-10 inhibitors, TGF-β inhibitors, B cell targeting antibodies (e.g., rituximab), mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin), synthetic vaccine particles (SVP™)-rapamycin (biodegradable nanoparticle-encapsulated rapamycin), intravenous gamma globulin (IVI G), omalizumab, methotrexate, tyrosine kinase inhibitors (e.g. ibrutinib), cyclophosphamide, fingolimod, inhibitors of B-cell activating factor (BAFF) (e.g. anti-BAFF monoclonal antibodies, e.g. belimumab), inhibitors of proliferation-inducing ligand (APRIL), anti-IL-1b monoclonal antibodies (e.g. canakinumab (Haris®)), C3a inhibitors, trezitopes (see, e.g., US 10,213,496), or combinations and / or derivatives thereof.

[0272] In certain embodiments, the rAAV vector or non-viral vector is administered in conjunction with one or more immunosuppressive agents prior to, substantially simultaneously with, or after administration of the rAAV vector or non-viral vector, and / or in conjunction with the administration of one or more immunosuppressive protocols or procedures, such as B cell depletion, immunoadsorption, and plasma exchange.

[0273] Immunosuppressive protocols including the use of rapamycin, alone or in combination with IL-10, can be used to reduce, decrease, inhibit, prevent, or block humoral and cellular immune responses to C1 inhibitor protein. Liver gene transfer using the AAV vectors of the invention can be used to induce immune tolerance to C1 inhibitor protein through the induction of regulatory T cells (Treg) and other mechanisms. Strategies to overcome or avoid humoral immunity to AAV in systemic gene transfer include administering high vector doses, using empty AAV capsids as decoys to adsorb anti-AAV antibodies, administering immunosuppressants to reduce, decrease, inhibit, prevent, or block humoral immune responses to AAV, changing the AAV capsid serotype or engineering the AAV capsid to be less susceptible to neutralizing antibodies (Nab), using plasma exchange cycles to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titers, and using delivery techniques such as balloon catheters followed by saline lavage. Such strategies are described in Mingozzi et al., 2013, Blood, 122:23-36. Further strategies include using an AAV-specific plasmapheresis column to selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma, as described in Bertin et al., 2020, Sci. Rep. 10:864. Apheresis strategies for removing, depleting, capturing, and / or inactivating AAV antibodies in subjects are described in WO2019018439.

[0274] The ratio of empty AAV capsid to rAAV vector can be within or between about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100. The ratio can also be about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0275] The amount of empty AAV capsid administered can be adjusted based on the amount (titer) of AAV antibodies produced in a particular subject. The AAV empty capsid can be of any serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:188), AAV3B, AAV-2i8, SEQ ID NO:226, SEQ ID NO:189, SEQ ID NO:190, and / or SEQ ID NO:191.

[0276] Alternatively, or in addition, rAAV vectors or non-viral vectors can be delivered by direct intramuscular injection (e.g., into one or more slow-twitch fibers of muscle). In another alternative, a catheter introduced into the femoral artery can be used to deliver rAAV vectors or non-viral vectors to the liver via the hepatic artery. Non-surgical means, such as endoscopic retrograde cholangiopancreatography (ER-P), can also be used to deliver rAAV vectors or non-viral vectors directly to the liver, thereby bypassing the bloodstream and AAV antibodies. Other ductal systems, such as the ducts of the submandibular gland, can also be used as portal veins to deliver rAAV vectors or non-viral vectors to subjects who develop or have existing anti-AAV antibodies.

[0277] Further strategies to reduce humoral immunity to AAV include methods to remove, deplete, capture, and / or inactivate AAV antibodies, commonly referred to as apheresis, and more specifically, plasmapheresis involving blood products. Apheresis or plasmapheresis is a process in which a human subject's plasma is circulated ex vivo through a device that modifies the plasma through the addition, removal, and / or replacement of components before returning to the patient. Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from blood products (e.g., plasma). This procedure depletes, captures, inactivates, reduces, or removes immunoglobulins (antibodies) that bind to AAV, thereby reducing the titer of AAV antibodies in the treated subject that may contribute to AAV vector neutralization. One example is a device that is composed of an AAV capsid affinity matrix column. Passing a blood product (eg, plasma) through an AAV capsid affinity matrix will result in only AAV antibodies and all isotypes (including IgG, IgM, etc.) binding.

[0278] A sufficient amount of plasmapheresis using an AAV capsid affinity matrix is ​​expected to substantially remove AAV capsid antibodies and reduce AAV capsid antibody titers (load) in humans. In an embodiment, the titers in treated subjects are substantially reduced to low levels (<1:4, or <1:3, or <1:2, or <1:1, etc., <1:5, or less). The reduction in antibody titers is expected to be temporary, since B lymphocytes producing AAV capsid antibodies are expected to gradually restore AAV capsid antibody titers to steady-state levels prior to plasma exchange.

[0279] When pre-existing AAV antibody titers are reduced from 1:100 to 1:1, the AAV antibody titer rebound is about 0.15% (corresponding to a titer of 1:1.2), 0.43% (1:1.4), 0.9% (1:1.9), 1.7% (1:2.7), and 3.4% (1:4.4), occurring 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours, respectively, after the end of plasma exchange. Such temporary clearance of AAV antibodies from a subject corresponds to a time window (e.g., about 24 hours or less, such as 12 hours or less, or 6 hours or less, or 3 hours or less, or 2 hours or less, or 1 hour or less) during which an AAV vector may be administered to a subject and expected to efficiently transduce target tissues without substantial neutralization of the AAV vector by AAV antibodies.

[0280] When pre-existing AAV antibody titers were reduced from 1:1000 to 1:1, the AAV antibody titer rebound was approximately 0.15% (corresponding to a titer of 1:2.5), 0.4% (1:5.3), 0.9% (1:9.7), 1.7% (1:18), and 3.4% (1:35), occurring 1 h, 3 h, 6 h, 12 h, and 24 h after the end of plasma exchange, respectively. Thus, the window for administration of AAV vectors becomes relatively short.

[0281] AAV antibodies can be pre-existing and can be present at a level that reduces or blocks the therapeutic C1 inhibitor gene transfer vector transduction of target cells.Alternatively, AAV antibodies can be developed after exposure to AAV or administration of AAV vector.When such antibodies develop after administration of AAV vector, these subjects can also be treated through apheresis, more specifically through plasma exchange.

[0282] In some embodiments, the polynucleotides, expression cassettes, AAV vectors, and non-viral vectors of the present invention can be used in combination with methods for reducing antibody (e.g., IgG) levels in human plasma.In some embodiments, the polynucleotides, expression cassettes, AAV vectors, and non-viral vectors of the present invention can be used in combination with agents that block, inhibit, or reduce the interaction between IgG and fetal Fc receptor (FcRn), such as anti-FcRn antibodies, agents that reduce IgG recycling in vivo and enhance IgG clearance, and / or agents that reduce circulating antibody binding to viral vectors, such as agents that bind to the nucleic acid or polypeptide, protein, or peptide encoded by the recombinant viral vector, or therapeutic heteropolynucleotide packaged by the recombinant viral vector, or agents that bind to therapeutic heteropolynucleotide.

[0283] In certain embodiments, antibody binding to the viral vector is reduced or inhibited by agents that reduce the interaction of IgG with FcRn, proteases, or glycosidases.

[0284] In certain embodiments, the polypeptides, expression cassettes, AAV vectors, or non-viral vectors of the invention may be used in combination with an endopeptidase (e.g., IdeS from Streptococcus pyogenes) or modified variants thereof, or an endoglycosidase (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof. In certain embodiments, the polypeptides, expression cassettes, AAV vectors, or non-viral vectors of the invention are administered to a subject in combination with an endopeptidase (e.g., IdeS from Streptococcus pyogenes) or modified variants thereof, or an endoglycosidase (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof to reduce or reveal neutralizing antibodies against the AAV capsid, allowing treatment of patients previously deemed ineligible for gene therapy or who develop AAV antibodies following AAV gene therapy. Such a strategy is described in Leborgne et al., C., Barbon, E., Alexander, JM et al., 2020, Nat. Med., 26:1096-1101 (2020), doi.org / 10.1038 / s41591-020-0911-7.

[0285] In certain embodiments, the nucleic acids, expression cassettes, AAV vectors, and non-viral vectors of the invention are available from, for example, Verinert®, Synlyse®, Oradeo®, TM Symptomatic and supportive care and medications including (berotralstat), Ruconest® (recombinant C1-INH), Hegarder® (plasma-derived C1-INH concentrate), lanadelumab (Taxyro®), androgens (danazol), ecaled, icatant, and tranexamic acid; immunosuppressive regimens including, for example, rapamycin, prednisolone, tacrolimus, and tocilizumab; or may be used in combination with FDA-approved drugs such as barbiturates, sulfonamides, and estrogens.

[0286] In certain embodiments, the polynucleotides, expression cassettes, and AAV vectors of the invention can be used in combination with pharmacological chaperone treatment (also known as enzyme enhancement treatment), where one or more pharmacological chaperones are administered prior to, simultaneously with, or following administration of the polynucleotides, expression cassettes, AAV vectors, or non-viral vectors of the invention for the treatment of complement-mediated disorders such as HAE.

[0287] In some embodiments, the polynucleotide and expression cassette of the present invention can be delivered or administered via AAV vector particle.In some embodiments, the polynucleotide and expression cassette of the present invention can be delivered or administered via other types of virus particles, including retrovirus, adenovirus, helper-dependent adenovirus, hybrid adenovirus, herpes simplex virus, lentivirus, poxvirus, Epstein-Barr virus, vaccinia virus, and human cytomegalovirus particle.In some embodiments, the polynucleotide and expression cassette of the present invention can be delivered or administered via non-viral vector.

[0288] kit The present invention provides a kit having packaging material and one or more components therein.The kit typically includes a label or packaging insert that includes a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein.The kit may include a combination of such components, such as rAAV particles or non-viral vectors, and optionally a second active component, such as another compound, agent, drug, or composition.

[0289] A kit refers to a physical structure that contains one or more components of the kit.

[0290] The packaging material can maintain the components sterile and can be made from materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0291] The label or insert may include identification information of one or more of the components therein, dosage, mechanism of action, clinical pharmacology of the active components, including pharmacokinetics and pharmacodynamics. The label or insert may include information identifying manufacturer information, lot number, manufacturing location and date, and expiration date. The label or insert may include information regarding the disease for which the kit components can be used. The label or insert may include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or treatment regimen. The instructions may include dosage, frequency or duration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0292] The label or insert may include information regarding any benefit that the component may provide, such as preventive or therapeutic benefit. The label or insert may include information regarding potentially adverse side effects, complications, or reactions, such as warnings to the subject or clinician regarding situations in which it is not appropriate to use a particular composition. Adverse side effects or complications may also occur when the subject has, is taking, or is using one or more other drugs that may be incompatible with the composition, or the subject has, is taking, is taking, or is using another treatment protocol or treatment regimen that may be incompatible with the composition, and thus the instructions may include information regarding such incompatibilities.

[0293] Labels or inserts include "printed matter", such as paper or cardboard, or "printed matter" separate or attached to a component, kit or packaging material (e.g., a box), or attached to an ampoule, tube or vial containing a kit component. Labels or inserts may also include computer readable media, such as a printed label with a bar code, optical disks such as a disk, CD- or DVD ROM / RAM, DVD, MP3, magnetic tape, or electronic storage media such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage media, FLASH media, or memory type cards.

[0294] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.

[0295] The present invention is generally disclosed herein using positive language to describe a number of embodiments of the present invention. The present invention also specifically includes embodiments in which certain subjects are completely or partially excluded, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments of the present invention, materials and / or method steps are excluded. Thus, the present invention is generally not expressed herein in terms of what the present invention does not include, but aspects that are not expressly excluded in the present invention are nevertheless disclosed herein.

[0296] Specific embodiments of the present invention have been described. Nevertheless, those skilled in the art can make various changes and modifications to the invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Thus, the following examples are intended to illustrate, but not to limit in any way, the scope of the invention as claimed.

[0297] Sequence Listing [Table 1] TIFF2024506266000002.tif232170TIFF2024506266000003.tif216170TIFF2024506266000004.tif214170TIFF2024506266000005.tif223170TIFF2024506266000006.tif205170TIFF2024506266000007.tif219170TIFF2024506266000008.tif218170TIFF2024506266000009.tif232170TIFF2024506266000010.tif222170TIFF2024506266000011.tif219170TIFF2024506266000012.tif214170TIFF2024506266000013.tif211170TIFF2024506266000014.tif217170TIFF2024506266000015.tif213170TIFF2024506266000016.tif212170TIFF2024506266000017.tif214170TIFF2024506266000018.tif203170TIFF2024506266000019.tif216170TIFF2024506266000020.tif222170TIFF2024506266000021.tif217170TIFF2024506266000022.tif218170TIFF2024506266000023.tif218170TIFF2024506266000024.tif213170TIFF2024506266000025.tif214170TIFF2024506266000026.tif215170TIFF2024506266000027.tif229170TIFF2024506266000028.tif212170TIFF2024506266000029.tif215170TIFF2024506266000030.tif219170TIFF2024506266000031.tif216170TIFF2024506266000032.tif214170TIFF2024506266000033.tif214170TIFF2024506266000034.tif214170TIFF2024506266000035.tif217170TIFF2024506266000036.tif219170TIFF2024506266000037.tif213170TIFF2024506266000038.tif219170TIFF2024506266000039.tif217170TIFF2024506266000040.tif228170TIFF2024506266000041.tif217170TIFF2024506266000042.tif220170TIFF2024506266000043.tif215170TIFF2024506266000044.tif218170TIFF2024506266000045.tif218170TIFF2024506266000046.tif209170TIFF2024506266000047.tif221170TIFF2024506266000048.tif220170TIFF2024506266000049.tif209170TIFF2024506266000050.tif221170TIFF2024506266000051.tif221170TIFF2024506266000052.tif221170TIFF2024506266000053.tif220170TIFF2024506266000054.tif220170TIFF2024506266000055.tif218170TIFF2024506266000056.tif222170TIFF2024506266000057.tif213170TIFF2024506266000058.tif221170TIFF2024506266000059.tif215170TIFF2024506266000060.tif216170TIFF2024506266000061.tif223170TIFF2024506266000062.tif218170TIFF2024506266000063.tif218170TIFF2024506266000064.tif225170TIFF2024506266000065.tif222170TIFF2024506266000066.tif220170TIFF2024506266000067.tif218170TIFF2024506266000068.tif218170TIFF2024506266000069.tif106170.

[0298] Further embodiments relate to the C1-INH coding sequence provided in any of SEQ ID NOs:232-258 and 269. In certain embodiments, the polynucleotide comprises a sequence that is at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the C1-INH coding sequence provided in any of SEQ ID NOs:232-258 and 269; independently, the C1-INH has at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:181. When used independently, it is indicated that any of the provided sequence identities to the C1-INH coding sequence may be combined with any of the provided sequence identities of SEQ ID NO:181.

[0299] Independently exemplified examples of the set include a nucleic acid encoding C1-INH having at least 90% sequence identity to bases 1 to 1500 of SEQ ID NO: 236 or SEQ ID NO: 236 and at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 181; a nucleic acid encoding C1-INH having at least 95% sequence identity to bases 1 to 1500 of SEQ ID NO: 236 or SEQ ID NO: 236 and at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 181; and a nucleic acid encoding C1-INH having at least 99% sequence identity to bases 1 to 1500 of SEQ ID NO: 236 or SEQ ID NO: 236 and at least 99% sequence identity to the sequence of SEQ ID NO: 181.

[0300] Independently exemplified examples of another set include a nucleic acid encoding C1-INH having at least 90% sequence identity to bases 1 to 1500 of SEQ ID NO: 238 or SEQ ID NO: 238 and at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 181; a nucleic acid encoding C1-INH having at least 95% sequence identity to bases 1 to 1500 of SEQ ID NO: 238 or SEQ ID NO: 238 and at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 181; and a nucleic acid encoding C1-INH having at least 99% sequence identity to bases 1 to 1500 of SEQ ID NO: 238 or SEQ ID NO: 238 and at least 99% sequence identity to the sequence of SEQ ID NO: 181.

[0301] Independently exemplified examples of another set include a nucleic acid encoding C1-INH having at least 90% sequence identity to bases 1 to 1540 of SEQ ID NO: 243 or SEQ ID NO: 243 and at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 181; a nucleic acid encoding C1-INH having at least 93%, at least 95% sequence identity to bases 1 to 1540 of SEQ ID NO: 243 or SEQ ID NO: 243 and at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 181; and a nucleic acid encoding C1-INH having at least 99% sequence identity to bases 1 to 1540 of SEQ ID NO: 243 or SEQ ID NO: 243 and at least 99% sequence identity to SEQ ID NO: 181.

[0302] In one embodiment, the encoded C1-INH further comprises a sequence encoding a signal peptide that is at least 95%, at least 97%, or 100% identical to any of the sequences of SEQ ID NOs:84-103 and 259-268, and independently encodes a C1-INH having at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:192.

[0303] Human C1-INH protein is well characterized and sequences from various species, including primates, are well known in the art. Various sequences show the sequence diversity of C1-INH. Examples of various C1-INH proteins are shown in the table below. The percent identity in the table below is determined using BLAST with default settings, and the numbers listed are % pairwise identities. In different embodiments, the percent identity provided throughout the application is determined using BLAST with default settings to obtain % pairwise identities.

[0304] [Table 2]

[0305] [Table 3] EXAMPLES

[0306] Example 1 - Overview of expression cassettes C1 inhibitor expression cassettes were designed as shown in Table 1. See also Figure 1 for a schematic diagram of sample expression cassettes. The expression cassettes contained 5' and 3' flanking AAV inverted terminal repeats (ITRs), liver-specific ApoE / hAAT enhancer / promoter sequences, signal peptides, human C1 inhibitor coding sequences, and bovine growth hormone (bGH) polyadenylation (polyA) sequences, with or without a P2A linker or Fc domain. Some of the expression cassettes further contained a human hemoglobin subunit beta (HBB2) intron, additional enhancer sequences, miRNA binding sites, and / or RIDD sequences.

[0307] [Table 4] TIFF2024506266000073.tif218170TIFF2024506266000074.tif217170TIFF2024506266 000075.tif199170TIFF2024506266000076.tif221170TIFF2024506266000077.tif25170

[0308] The expression cassette is packaged into an AAV viral particle by encapsidation into an AAV capsid, such as the AAV-4-1 capsid mutant described in International Patent Application Publication WO 2016 / 210170, the contents of which are incorporated herein by reference in their entirety, or the LK03 capsid mutant, the contents of which are incorporated herein by reference in their entirety. Viral particles are generally produced using triple transfection protocols well known in the art.

[0309] Example 2 - Evaluation of vector efficacy of a SERPING1-expressing vector encapsidated in AAV in C57BL / 6J mice at two doses the purpose In an effort to develop a SERPING1 transgene with improved secretion from the liver, the endogenous SERPING1 signal peptide was replaced with a panel of natural and synthetic peptides selected from initial in vitro screening studies. The naturally occurring signal peptides corresponded to those of alpha-2-HS-glycoprotein (AHSG) and chymotrypsinogen B2 (sp7), and the synthetic peptides, designated Synthetic 1 (Synthetic 1) and Synthetic 4 (Synthetic 4), were derived by rational design. A non-GLP study was performed in C57BL / 6J mice to evaluate the efficacy of five SERPING1 expression vectors with different signal peptide sequences at two doses by intravenous injection (vector SEQ ID NO: 1, 13, 16, 12, and 2).

[0310] The study included 100 male C57BL / 6J wild-type mice aged 11–12 weeks. Four candidate cassettes containing heterologous signal peptides were benchmarked against the native human SERPING1 signal peptide. All expression cassettes contained the native SERPING1 cDNA sequence. Other regulatory elements in the cassettes that were common to the five vectors included the apolipoprotein E hepatic control region 1 (ApoE HCR-1) enhancer, human alpha-1 antitrypsin (hAAT) promoter, modified human hemoglobin beta (HBB)-derived synthetic intron (HBB2), and a CpG-reduced bovine growth hormone (bGH) polyadenylation (polyA) signal sequence. All cassettes were packaged into bioengineered AAV capsids. The vectors utilized in this study are detailed in Table 2 below. Low dose (1.0 × 10 12 vg / kg) or high dose (4.0 × 10 12 Mice (n = 10–11 / group) were injected with 100 mg (vg / kg) of the specific vector (Table 3). Animals were monitored over a period of 18 weeks for groups 7–11 (low-dose cohorts) or 28 weeks for groups 1–5 (high-dose cohorts). Human C1-INH antigen levels, C1-INH activity, and liver transduction were assessed as detailed in Table 4.

[0311] [Table 5]

[0312] [Table 6]

[0313] [Table 7]

[0314] result Human C1-INH antigen levels in mouse plasma To evaluate the production and secretion of protein encoded by the SERPING1 expression cassette, mice were treated with a low dose (1.0 × 10) starting at week 3 until the end of the study, either at week 18 (low-dose cohort, groups 7–11) or at week 28 (high-dose cohort, groups 1–5). 12 vg / kg) or high dose (4.0 × 10 12 C1-INH antigen levels were assessed in the plasma of animals administered one of the five vectors at 100 mg / kg (vg / kg).

[0315] Antigen levels were quantified using a C1-INH kit-based ELISA kit (Molecular Innovations, #HC1INHKIT-TOT) and a custom sandwich-type ELISA capture assay using plates coated with anti-human C1-INH IgG (Affinity Biologicals, #GACINH-AP) and anti-human C1-INH HRP-conjugated IgG (Affinity Biologicals, #GACINH-HRP) for detection.

[0316] Regardless of dose, mice administered AAV-encapsidated SEQ ID NO:1 (Group 7, low dose cohort, or Group 1, high dose cohort) consistently expressed the highest mean levels of detectable human C1-INH antigen compared to all other groups (Figure 2). In the low dose cohort, only mice treated with AAV-encapsidated SEQ ID NO:1 had C1-INH levels consistently higher than pooled normal human plasma. Conversely, administration of all signal peptide variants at high vector doses resulted in plasma C1-INH levels above physiological levels that were sustained for at least 28 weeks after vector administration. Over the 6-month experiment, expression levels were 9-fold higher than normal, and no morbidity or mortality was observed.

[0317] Steady-state C1-INH antigen levels (linear regression analysis, not shown), defined as no significant difference in mean antigen levels across the time points evaluated, were significantly higher in animals administered AAV-encapsidated SEQ ID NO:1 compared to animals administered AAV-encapsidated SEQ ID NO:13, 16, and 12 at both vector doses (Figure 3). In animals treated with AAV-encapsidated SEQ ID NO:1 at low and high doses, mean ± standard deviation human C1-INH levels in plasma remained stable at 315.01 ± 32.98 μg / mL and 1321.79 ± 302.97 μg / mL, respectively; these steady-state levels corresponded to 1.58-3.27-fold higher levels of plasma C1-INH antigen compared to all other constructs across both dose cohorts.

[0318] In summary, the vector cassette containing the native SERPING1 signal peptide (AAV-encapsidated SEQ ID NO:1) was superior to the other four vectors in vivo with respect to production and secretion of C1-INH antigen, regardless of dose.

[0319] Human C1-INH activity in mouse plasma To assess human C1-INH function in plasma, samples from the high dose cohort (groups 1-5) were tested for activity by chromogenic assay at weeks 22, 25, and 27 using a modified version of the Technochrom C1-INH kit (Diapharma, 5345003). For quantification, plasma C1-INH was titrated against excess C1-esterase and residual C1-esterase activity was measured. Activity levels were reported as a percentage of activity relative to an agglutination reference standard.

[0320] Steady-state human C1-INH activity levels were significantly elevated in animals administered AAV-encapsidated SEQ ID NO:1 compared to other signal peptide variants (p<0.0001) (Figure 4). 12 Activity levels in animals administered AAV-encapsidated SEQ ID NO:1 at 100 vg / kg reached a steady state of 766.30% activity. Furthermore, human C1-INH activity was positively correlated with antigen levels, showing a direct linear relationship, suggesting that functional C1-INH was produced (Pearson r = 0.8778, p < 0.0001, R 2 = 0.771) (Figure 5).

[0321] Normalized vector genome concentration in liver tissue - qPCR To assess target tissue transduction following intravenous AAV vector administration, end-point liver samples were analyzed for vector genome concentration using a real-time quantitative PCR (qPCR) assay. DNA was extracted from end-point liver samples using a modified DNA extraction method using the QIAamp Fast DNA Tissue Kit (Qiagen, Cat. No. 51404).

[0322] The bGH polyA signal was selected as the AAV target since it was a component of all candidate expression cassettes. Mouse forkhead box P1 (Foxp1) was targeted to measure genomic DNA concentrations between samples and to remove non-biological variation. Final copy numbers of bGH polyA were normalized to Foxp1 and reported as bGH polyA copies per Foxp1 copy (Figure 6). As expected, vector genome copies in the distal liver were greater in animals receiving higher vector doses compared to lower vector doses (Figure 5). In both dose cohorts, animals receiving AAV-encapsidated SEQ ID NO:1 consistently demonstrated the highest mean vector genome concentrations in target tissues, with significantly higher levels compared to animals receiving AAV-encapsidated SEQ ID NO:16 and 12. Furthermore, C1-INH antigen levels were positively correlated with relative vector genome copies across both dose cohorts, demonstrating the expected direct relationship between these two parameters (Pearson r = 0.9306, p < 0.0001, R 2 =0.866) (Figure 7).

[0323] Vector potency of signal peptide mutants To compare the potency of the signal peptide variants, steady-state C1-INH antigen levels were normalized to endpoint liver vector genome copies in both low and high vector dose cohorts (Figure 8). No differences in vector potency were observed between animals in the low vector dose group, but animals receiving high vector doses of AAV-encapsidated SEQ ID NO:16 showed increased normalized steady-state C1-INH antigen levels compared to AAV-encapsidated SEQ ID NO:1; variability in vector genome copies for AAV-encapsidated SEQ ID NO:1 may contribute to the observed differences in potency.

[0324] conclusion In this study, we evaluated C1-INH antigen levels, C1-INH activity, and vector genome copies derived from five candidate expression cassettes containing unique signal peptide coding sequences. Of the five vectors tested, the vectors containing the native human SERPING1 signal peptide coding sequence in their transgene cassette (AAV-encapsidated SEQ ID NO:1; Groups 7 and 1) showed the highest steady-state C1-INH antigen, C1-INH activity levels, and end-point vector genome concentrations, regardless of dose. Since the difference in efficacy was minimal, the results indicate that in the context of AAV capsids in wild-type mice, AAV-encapsidated SEQ ID NO:1 had the strongest response compared to vectors containing other signal peptides.

[0325] Example 3 - Evaluation of vector efficacy in C57BL / 6J mice at a single dose of an optimized SERPING1 expression vector encapsidated in AAV the purpose Two non-GLP studies (A and B) were performed in C57BL / 6J mice to evaluate the vector potency of 13 unique human SERPING1 expression vectors by intravenous injection. SERPING1 transgene variants were generated using codon optimization (Integrated DNA Technologies (IDT) Codon Optimization Tool, Codon Harmonizer, and Best Codon scripts) and truncation strategies (Table 5). Selected variants previously demonstrated high C1-INH secretion in in vitro screening studies.

[0326] The two studies included a total of 80 male C57BL / 6J wild-type mice aged 9–10 weeks. Twelve candidate cassettes were evaluated against the native human SERPING1 cDNA sequence. Other control elements of the cassettes maintained in all vectors included the apolipoprotein E hepatic control region 1 (ApoE HCR-1) enhancer, human alpha-1 antitrypsin (hAAT) promoter, modified human hemoglobin beta (HBB)-derived synthetic intron (HBB2), and a CpG-reduced bovine growth hormone (bGH) polyadenylation (polyA) signal sequence. All cassettes were packaged into bioengineered AAV capsids. Five animals per group were injected with 1.0 × 10 12 vg / kg of the specified vector (Tables 6 and 7) and monitored for 7 weeks in Study A and 6 weeks in Study B. Human C1-INH antigen, bradykinin antigen, and vector genome concentrations were assessed as detailed in Table 8.

[0327] [Table 8]

[0328] [Table 9]

[0329] [Table 10]

[0330] [Table 11]

[0331] result Plasma C1-INH antigen To assess in vivo production of C1-INH antigen from the modified (codon-optimized or truncated) SERPING1 expression cassette, circulating C1-INH antigen levels were assessed by ELISA (Molecular Innovations, #HC1INHKIT-TOT). C1-INH antigen assessment began 1 week after vector administration and continued weekly until the end of the study.

[0332] Candidate vectors in Study A generally produced higher levels of plasma C1-INH antigen compared to Study B, the focus of Example 3. Following vector administration, mean plasma C1-INH levels peaked around week 3 (±1 week) in most groups and then declined (Figure 9). The highest peak antigen level at week 3 (±1 week) reached a mean ± standard deviation of 722.31 ± 242.03 μg / mL in mice administered AAV-encapsidated SEQ ID NO:22. Mice administered AAV-encapsidated SEQ ID NO:22, 23, 20, and 18 showed similar or higher levels of plasma C1-INH throughout the course of the study compared to the parental cassette (AAV-encapsidated SEQ ID NO:1); the vectors also exceeded the mean ± standard deviation levels of pooled normal human plasma (179.2 ± 20.88 μg / mL) by week 2 and persisted for the duration of the study.

[0333] In Study A, relative steady-state (i.e., no significant differences in mean antigen levels across all time points tested, one-way ANOVA not shown) C1-INH antigen levels in animals administered either AAV-encapsidated SEQ ID NO:22, or AAV-encapsidated SEQ ID NO:23 were significantly greater (1.75- and 1.3-fold greater, respectively) than in animals administered AAV-encapsidated SEQ ID NO:1 (Figure 9).

[0334] In Study B, steady-state C1-INH antigen levels were similar in animals administered AAV vectors containing codon-optimized SERPING1 mutants compared to AAV-encapsidated SEQ ID NO:1 (FIG. 9). However, animals administered AAV-encapsidated SEQ ID NO:24 showed a 50% decrease in steady-state C1-INH antigen levels compared to AAV-encapsidated SEQ ID NO:1 (119.89±69.29 μg / mL versus 237.10±55.88 μg / mL, respectively). An initial peak in C1-INH levels was observed around weeks 3 and 4 in most groups, followed by a decline in antigen levels (FIG. 10). The highest peak antigen levels at weeks 3 and 4 reached a mean ± standard deviation of 299.19 ± 110.92 μg / mL in mice administered AAV-encapsidated SEQ ID NO:17, followed by 289.45 ± 56.12 μg / mL in the AAV-encapsidated SEQ ID NO:25 group. Both mutants showed higher mean ± standard deviation peak levels of C1-INH antigen compared to the parent cassette (287.32 ± 76.21 μg / mL). As in study A, an increase in circulating C1-INH levels was observed in several groups after an initial drop in antigen levels. At the end of the study, the highest mean ± standard deviation levels of C1-INH antigen were 347.69 ± 139.64 μg / mL in the AAV-encapsidated SEQ ID NO:17 and 297.57 ± 56.74 μg / mL in the AAV-encapsidated SEQ ID NO:25 group. Peak C1-INH levels in these groups exceeded both the parental cassette and PNP values, but the highest antigen levels observed in study B were lower than those exhibited by the third highest C1-INH expressing mutant in study A. In animals administered AAV vectors containing truncated SERPING1 mutants (Δ76 and Δ98), levels of C1-INH antigen were reduced and remained below the limit of quantification from week 4 through the end of the study.

[0335] In summary, administration of several codon-optimized SERPING1 expression variants resulted in steady-state C1-INH antigen levels 1.13-1.75-fold higher than the parental cassette across both studies as well as the pooled normal human plasma range. Across both studies, AAV-encapsidated SEQ ID NO:22, AAV-encapsidated SEQ ID NO:23, and AAV-encapsidated SEQ ID NO:20 from Study A showed the highest levels of plasma C1-INH antigen.

[0336] Plasma bradykinin concentration To assess downstream effects of C1-INH, plasma bradykinin was assessed by ELISA (Enzo Life Sciences, product #ADI-900-206, room temperature) at the end of the study. At week 7, bradykinin was measured in the two variants of AAV-encapsidated SEQ ID NO:22 and 20 (Study A) that have higher levels of C1-INH antigen.

[0337] At the end of the study, circulating bradykinin levels were significantly reduced in mice administered either AAV-encapsidated SEQ ID NO:22 or 20, reaching levels less than 3% of plasma bradykinin in vehicle-treated mice (FIG. 11). As expected, plasma bradykinin concentrations were inversely correlated with plasma C1-INH, with a significantly higher mean titer in the AAV-encapsidated SEQ ID NO:22 group (Pearson r = -0.9417, p<0.0168, R 2 = 0.8867) in the AAV-encapsidated SEQ ID NO:20 group (Pearson r = -0.6383, p = ns, R 2 = 0.4074). In summary, sustained supraphysiological levels of human C1-INH were associated with a significant reduction in plasma bradykinin in wild-type mice.

[0338] Normalized vector genome concentration in liver tissue To assess target tissue transduction following intravenous AAV vector administration, a real-time quantitative PCR (qPCR) assay was used to evaluate vector genome concentrations in end-point liver samples. Copy numbers of the shared polyadenylation signal (bGH polyA) were normalized to copies of the mouse Foxp1 gene.

[0339] There was no statistical difference in normalized vector genome concentration at the termini between AAV-encapsidated SEQ ID NO:1 and the six codon-optimized variants in study A (Figure 12A).

[0340] In study B, the mean end point liver vector genome copies between AAV-encapsidated SEQ ID NO:1 and the six codon-optimized or truncated SERPING1 variants ranged from 0.0388 to 0.0936 BGHpA copies per Foxp1 copy in the AAV-encapsidated SEQ ID NO:24 and 17 groups, respectively. Following the AAV-encapsidated SEQ ID NO:17 group, vector genome concentrations were highest in animals administered AAV-encapsidated SEQ ID NO:1 and 26 (0.0829 and 0.0785 BGHpA copies per Foxp1 copy, respectively). There were no statistical differences in normalized vector genome concentrations at the end point among all SERPING1-expressing vectors (Figure 12B). End point vector genome concentrations in animals administered AAV-encapsidated SEQ ID NO:30 and 31 (0.0417 and 0.0606, respectively) indicated that liver transduction occurred and was comparable to the remaining vectors in the study. AAV-encapsidated SEQ ID NOs:30 and 31 provided lower levels of circulating C1EI (data not shown).

[0341] A positive correlation between C1-INH antigen and relative vector genome copies was observed for AAV-encapsidated SEQ ID NO:1 and the codon-optimized variants (SEQ ID NO:20, 22, and 23) with the highest C1-INH antigen expression (Pearson r = 0.8510, p < 0.0001, R 2 = 0.7243), demonstrating a direct relationship between these two parameters (Figure 13).

[0342] Vector potency of codon-optimized mutants To assess the effect of codon optimization on vector potency, steady-state C1-INH antigen levels were normalized to vector genome copies in end-point liver samples ( FIG. 14 ). No differences in vector potency were observed between the parent cassette (AAV-encapsidated SEQ ID NO:1) and the codon-optimized variants in Study A, except for mice administered AAV-encapsidated SEQ ID NO:21; mice administered AAV-encapsidated SEQ ID NO:21 showed reduced vector potency.

[0343] conclusion In summary, AAV-mediated delivery of a codon-optimized SERPING1 transgene resulted in elevated and sustained levels of functional C1-INH in wild-type mice.

[0344] Example 4 - Dose range evaluation of codon-optimized SERPING1 vectors encapsidated in AAV in Serping1 knockout mice the purpose 129 / S5x C57BL / 6J-Tyrc-Brd(Serping1 - / - A non-GLP study was performed in human C1-INH knockout or C1-INH null mice to evaluate vector efficacy of a codon-optimized human SERPING1 expression vector (AAV encapsidated SEQ ID NO:20) at three doses in the disease model by intravenous injection.

[0345] The study included 20 male and 19 female C1-INH knockout mice aged 13-18 weeks. Up to five rats per group were administered three doses (1.0 × 10 12 vg / kg, 4.0×10 12 vg / kg, or 1.0 × 10 13Animals were injected with one AAV-encapsidated SEQ ID NO:20 at 100 mg / kg (Table 9). In addition to the codon-optimized transgene, the control elements of the cassette included the apolipoprotein E hepatic control region 1 (ApoE HCR-1) enhancer, human alpha-1 antitrypsin (hAAT) promoter, modified human hemoglobin beta (HBB)-derived synthetic intron (HBB2), and a CpG-reduced bovine growth hormone (bGH) polyadenylation (polyA) signal sequence. The cassette was packaged into an AAV-4-1 capsid. Animals were monitored for 8 weeks. Plasma C1-INH antigen levels, C1-INH activity, liver vector genome concentration, and SERPING1 mRNA expression were assessed as detailed in Table 10.

[0346] [Table 12]

[0347] [Table 13]

[0348] result Plasma C1-INH antigen C1-INH antigen levels were quantified using a kit-based ELISA and a custom sandwich-type ELISA capture assay, as described in Example 2.

[0349] Circulating C1-INH antigen was assessed by ELISA beginning 1 week after injection and monitored weekly from week 4 to week 8 (end of study). A dose-dependent increase in mean plasma C1-INH antigen levels was observed in male and female mice from week 4 to week 8 (Figure 15). 12 vg / kg) and high (1.0 × 10 13At the 100-300 mg / kg (1000 mg / kg) vector dose, all animals showed detectable levels of C1-INH by week 4. Regardless of sex, mice receiving medium or high doses of vector showed mean plasma C1-INH levels equal to or greater than pooled normal human plasma (PNP; mean ± SD = 175.4 ± 41.3 μg / mL) by week 4, which persisted until the end of the study. Male mice showed higher mean antigen levels compared to female mice at the medium and high vector doses, which may be due to higher liver transduction following AAV administration in male mice compared to female mice (Davidoff et al., Blood. 2003; 102(2): 480-488).

[0350] In summary, administration of AAV-encapsidated SEQ ID NO:20 resulted in a dose-dependent increase in C1-INH antigen levels, with higher absolute antigen levels observed in males than females.

[0351] An orthogonal approach was used to compare methods for human C1-INH antigen detection. At week 5, C1-INH antigen was assessed by ELISA as well as an additional capillary electrophoresis immunoassay (Wes) in male mice receiving low or medium vector doses.

[0352] Low vector dose (1.0 × 10 12 At the mid-vector dose (4.0 × 10 vg / kg), mean C1-INH values ​​and variability (standard deviation) were comparable for the ELISA and Wes approaches, but the relative concentrations of C1-INH antigen were not consistent between the quantification methods. 12 At the mid-vector dose (vg / kg), an increase in C1-INH was observed using both techniques compared to lower vector doses. However, the mean C1-INH values ​​detected by ELISA were nearly twice the levels quantified by Wes (1063.99 µg / mL vs. 547.25 µg / mL); sample variability using Wes was also greater than ELISA at the mid-vector dose.

[0353] The correlation analysis between the activity of C1-INH antigen and two types of antigen detection methods was performed by detecting C1-INH antigen using ELISA (R 2 = 0.995), Wes(R 2 = 0.993) (data not shown). Because the sample size for this comparison was relatively small (n = 9 total samples) and limited by vector dose and animal sex, a larger, more comprehensive sample population may provide further insight into the utility of each antigen detection method.

[0354] Antigen and gender correlation of C1-INH in mouse plasma To determine the biological activity of the transgene product encoded by SEQ ID NO:20, C1-INH activity was assessed by a chromogenic assay, titrating C1-INH against excess C1-esterase and measuring residual C1-esterase activity. C1-INH activity was assessed in male and female mice of all dose cohorts at 5 and 8 weeks after vector administration. Activity, reported as a percentage of activity relative to a C1-INH agglutinating reference standard, was compared to C1-INH antigen levels.

[0355] A positive correlation was observed between C1-INH activity and C1-INH antigen across all SEQ ID NO:20 treatment groups (R 2 = 0.981; Figure 16). Consistent with antigen levels, male mice receiving the high vector dose had the greatest levels of C1-INH activity. In addition, male mice in the high and medium vector dose cohorts often showed higher levels of C1-INH activity compared to dose-matched females.

[0356] Normalized Vector Genome Concentration in Liver Tissue - qPCR To assess target tissue transduction following vector administration, endpoint (week 8) liver samples were analyzed for vector genome concentration as previously described.

[0357] A dose-dependent increase in vector genome concentration was observed in the livers of male and female mice, confirming functional transduction of the target tissue (Figure 17). Higher levels of liver transduction were observed in male mice administered AAV-encapsidated SEQ ID NO:20 compared to dose-matched female mice, consistent with previous reports of sex affecting AAV transduction of mouse liver (Davidoff et al., Blood. 2003; 102(2): 480-488). The average liver vector genome concentration in male mice administered high and medium doses of AAV-encapsidated SEQ ID NO:20 was approximately twice that observed in dose-matched females.

[0358] Normalized mRNA expression levels in liver tissue SERPING1 mRNA expression was quantified using qRT-PCR in end-point (week 8) liver samples. SERPING1 mRNA was normalized to mouse peptidyl prolyl cis-trans isomerase E (Ppie) and reported as SERPING1 copies per Ppie copy.

[0359] Consistent with vector genome concentration, a dose-dependent increase in transgene expression was observed in male and female mice (Figure 18). Furthermore, the average transgene expression in males was greater than in females receiving the same vector dose.

[0360] Comparison of vector dose with vector is stabilized in null mouse (C1-INH knockout) studies.

[0361] conclusion 4.0×10 12 vg / kg (medium dose) and 1.0 × 10 13 Administration of AAV-encapsidated SEQ ID NO:20 at a dose of 10000 vg / kg (high dose) was able to restore circulating levels of C1-INH antigen to physiological or supraphysiological levels in C1-INH knockout male and female mice. Consistent with previous reports in mice, sex-specific responses were observed after AAV-mediated liver-directed gene delivery.

[0362] Example 5 - B6. Characterization of Serping1 deletion in the SJL mouse background (Figure 20) Serping1 in C57BL / 6xSJL background (Molecular Innovations) - / - Mice were used as a model of HAE. Experiments performed to characterize a commercially available mouse model showed that Serping1+ / - mice were significantly higher in both the sham and sham-treated groups than strain-matched wild-type controls as assessed by Evans blue dye extravasation (data not shown and FIG. 20A) and limb volume plethysmography. - / - No differences in vascular permeability were observed between mice. Clear differences were observed in plasma C1-INH (Figure 20B), bradykinin (Figure 20C), C4a (Figure 20D), and tissue plasminogen activator (tPA) (Figure 20E). C1-INH, C4a, and tPA followed the expected relationship with C1-INH, but bradykinin levels appeared to increase rather than decrease in the presence of endogenous mC1-INH.

[0363] Example 6-I21 (SEQ ID NO: 22) sustains dose-dependent hC1-INH antigen levels in plasma of HAE model mice (Figure 21) Figure 21A) B6 / SJL Serping1+ / + , Fig. 21B) B6 / SJL Serping1+ / - , and Fig. 21C) B6 / SJL Serping1- / - The plasma hC1-INH level in male mice was 1.0 × 10 12 ~3.16×10 12 Three doses of AAV-encapsidated I21 (SEQ ID NO:22) ranging from 1 / 4 log unit to 1 / 4 vg / kg were administered in mice injected at 1 / 4 log units. These doses were intended to span a threshold of functional C1-INH in the presence of various levels of endogenous mC1-INH, below which constitutive low-level complement activation could theoretically lead to accelerated C1-INH catabolism. No time lag was observed for the peak of circulating C1-INH, but Serping1 - / -Animals showed delayed kinetics to threshold. Differences in peak expression were found to result from variations in dosing regimen rather than being genotype driven (data not shown).

[0364] Example 7 - C1-INH Expression and Pharmacodynamics (Figure 22) Data comparing hC1-INH expression in different Serping1 genotypes indicated a potential effect of endogenous mC1-INH on hC1-INH dose response (Figures 22A and 22B), but no clear relationship was established. Circulating plasma hC1-INH had a clear and consistent negative correlation with plasma bradykinin (Figure 22C) and tPA activity (Figure 22D), indicating functional regulation of elements of the contact system and hemostasis.

[0365] Example 8-I21 (SEQ ID NO:22) sustains dose-dependent hC1-INH antigen levels in plasma of HAE model mice (Figure 23) B6 / SJL Serping1- / - Male mice were given 9.5 × 10 11 ~3.0×10 13 1 of 5 doses of I21 (SEQ ID NO:22) ranging from 100 to 1500 mg / kg were injected. (FIG. 23A) Plasma C1-INH levels as a function of time measured by ELISA. Values ​​are in IU / mL and are presented as mean±standard deviation. (FIG. 23B) B6 / SJL Serping1- / - Steady-state C1-INH antigen levels as a function of vector dose in male mice. All values ​​were log-transformed and analyzed by regression (R 2 = 0.88) was used to generate a linear fit. Activity as measured by C1-INH C1-esterase inhibition showed a linear dose response and correlated with antigen levels (data not shown).

[0366] Example 9 - I21 (SEQ ID NO:22)-mediated reduction of bradykinin, a key driver of neovascular attacks in HAE disease model mice (Figure 24) FIG. 24A) Individual and mean ± SD plasma bradykinin levels in each dose cohort. With the exception of the lowest vector dose cohort, animals administered the I21 vector showed a significant decrease in mean plasma bradykinin compared to vehicle-treated mice. Post-hoc Dunnett's multiple comparison test ( ** , p < 0.01; *** Statistical comparisons compared to vehicle were performed by one-way ANOVA using the ANOVA with p<0.001). Figure 24B) Plasma C1-INH levels were inversely correlated with plasma bradykinin levels and showed a linear relationship with respect to C1-INH antigen (R 2 = 0.81).

[0367] Example 10 - I21 (SEQ ID NO:22) mediated C1-INH rescues C4 levels in a mouse model of HAE (Figure 25) Plasma C4 was analyzed at the end of the study by an automated capillary-based immunoassay system (Wes™, ProteinSimple). Figure 25A) Individual and mean ± standard deviation relative C4 expression in each dose cohort. Figure 25B) Individual relative plasma C4 values ​​plotted against each animal's respective C1-INH antigen level at week 31. All values ​​were log-transformed and analyzed by regression (R 2 = 0.87) was used to generate a linear fit. The levels of C4 as a function of plasma C1-INH were also examined (Figure 25C).

[0368] Example 11 - Toxicity Testing AAV-encapsidated I21 (SEQ ID NO:22) toxicity was evaluated in C57BL / 6J male and female mice. Male mice were administered 2.5×10 12 vg / kg, 5.0×10 12 vg / kg, or 1.0 × 10 13 Female mice were administered 1.0 × 10 vg / kg of AAV-encapsidated I21. 13 vg / kg, 5.0×10 13 vg / kg, or 9.9 × 10 13vg / kg of AAV-encapsidated I21. All treated mice survived to 30 days, and microscopic findings in histopathological evaluation did not appear to be directly related to AAV-encapsidated I21 administration. C1-INH levels in treated mice were >30-60 fold, exceeding 100% of normal (Figures 26A and 26B).

[0369] Example 12 - Non-human primate (NHP) dose study The objectives of this NHP dose estimation study were to evaluate the safety of AAV-encapsidated I21 vectors, elucidate the dose range to achieve therapeutic levels of human C1-INH, and inform the starting clinical dose. 13 vg / kg, 3.2 × 10 13 vg / kg, and 1.0 × 10 14 Three ascending vector doses in vg / kg were aimed at targeting 20%, 70%, and 200% of normal circulating C1-INH in both male and female cynomolgus monkeys.

[0370] [Table 14]

[0371] Human C1-INH antigen levels in NHP plasma: Human C1-INH was quantified using an LC-MS / MS method. Plasma-derived human C1-INH (Molecular Innovations, #HC1INH-1.0MG) was used to prepare standards ranging from 10 μg / mL to 0.1 μg / mL, and QC samples targeting 30 μg / mL and 300 μg / mL after 50-fold dilution. Samples were diluted up to 5-fold prior to quantification.

[0372] Activity of human C1-INH in NHP plasma: C1-INH activity was quantified with a chromogenic C1-esterase inhibitor assay that titrated C1-INH against excess C1-esterase and measured residual C1-esterase activity using a modified version of the Technochrom C1-INH kit (Diapharma, 5345003). Provided vials of C1-esterase and substrate were reconstituted with the amount of nuclease-free water indicated on each vial, and National Institute for Biological Standards and Control (NIBSC) concentrate (08 / 256) was reconstituted with cell culture grade water to give a stock concentration of 19.2 IU / mL (1920% normal activity). Provided buffer B was placed in a 37°C water bath and equilibrated to the appropriate temperature before use. Samples were diluted 1:10 in provided buffer A in a 96-well tissue culture plate. A standard curve was prepared using NIBSC concentrate (08 / 256) in buffer A with an end point consisting of buffer alone (0% of standard C1-INH activity) for a total of eight reference points ranging from 200% standard activity to 17.3% standard activity. 1:6 dilutions of substrate were prepared in buffer B and QC samples were prepared sequentially in Cyno Normal Pooled Plasma and buffer A to achieve 75% and 3-0% normal activity. 20 μL of human C1-esterase was added to a fresh 96-well tissue culture plate. 20 μL of standards, QCs and samples were added in duplicate to all wells containing human C1-esterase and the plate was incubated at 37 °C for 5 min 30 s, after which 120 μL of diluted pre-warmed substrate was added to each well and the plate was incubated at 37 °C for an additional 20 min. After a 20 min incubation, plates were immediately read every 54 s for 5 min for optical density at 405 nm on a SpectraMax i3x plate reader (Molecular Devices) set at 37° C. SoftMax Pro software was used to plot standards using a Log-Log transformation fit and calculations were performed automatically.Results reported are back-calculated dilutions obtained from interpolation using this standard curve and are reported as percentages of normal C1-INH activity after normalization to the % normal C1-INH activity obtained in the pre-dose day 1 plasma sample.

[0373] Figures 27A and 27B show the results of administration studies at different time points. AAV-encapsidated I21 (SEQ ID NO:22) produced a sustained increase in both hC1-INH antigen and activity. Furthermore, C1-INH expression spanned the therapeutic range.

[0374] Figure 28A and Figure 28B show the hC1-INH antigen levels and peak percent normal C1-INH activity produced by different doses of AAV-encapsidated I21. For both analyses, two-way ANOVA was performed on log-transformed data. Antigen and activity correlated well across all doses tested, regardless of gender. No statistical difference in expression was observed between males and females.

[0375] Example 13 - Expression of hC1-INH from a plasmid in vitro Huh7 cells were transfected in vitro with different SERPING1 expression plasmids and the hC1-INH antigen levels in the supernatant were measured. The different expression plasmids contained AAV expression cassettes as shown in Table 12.

[0376] [Table 15] TIFF2024506266000089.tif83170

[0377] The results are shown in Figure 29. Results are the average of n = 3 biological replicates. Error bars indicate standard deviation. Nearly all plasmids showed increased expression levels of secreted human C1-INH compared to the control plasmid with green fluorescent protein (GFP) driven by CAG (pCAG_GFP), indicating that these plasmids lead to expression of the SERPING1 transgene product.

[0378] Huh7 cells were transfected in vitro with different SERPING1 expression plasmids containing SEQ ID NO: 49, 50, 51, 52, 53, or 54; C1-INH antigen levels in the supernatant were undetectable (data not shown).

[0379] Example 14 - SERPING1 expression plasmid with miR-142-3p target site Huh7 cells were transfected in vitro with plasmids containing the nucleic acid of SEQ ID NO:28 (pSwap-SERPING1_I21), SEQ ID NO:55 (mir 142-3p), or the control plasmid pCAG_GFP, and the secreted hC1-INH antigen in the supernatant was assayed. The results are shown in Figure 30. Results are the average of n = 3 biological replicates. Error bars indicate standard deviation.

[0380] The miR-142-3p target site plasmid showed increased expression levels of secreted human C1-INH compared to the green fluorescent protein (pCAG_GFP) control plasmid, indicating that it resulted in expression of the SERPING1 transgene product. Furthermore, the miR-142-3p plasmid produced relatively similar levels of human C1-INH to those of pSwap-SERPING1_I21.

[0381] The present invention is generally disclosed herein using positive language to describe a number of embodiments of the present invention. The present invention also specifically includes embodiments in which certain subjects are completely or partially excluded, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in some embodiments of the present invention, materials and / or method steps are excluded. Thus, the present invention is generally not expressed herein in terms of what the present invention does not include, but aspects that are not expressly excluded in the present invention are nevertheless disclosed herein.

Claims

1. A polynucleotide comprising a sequence encoding a C1 inhibitor, said polynucleotide comprising a nucleic acid sequence at least 85% identical to SEQ ID NO:238, 236 or 243, and said C1 inhibitor comprising a sequence at least 93% identical to SEQ ID NO:

181.

2. The polynucleotide of claim 1, wherein the polynucleotide comprises a nucleic acid sequence that is at least 85% identical or at least 95% identical to SEQ ID NO: 238 and the C1 inhibitor comprises a sequence that is at least 95% identical to SEQ ID NO: 181, preferably, the polynucleotide comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 238 or comprises SEQ ID NO: 238 and the C1 inhibitor comprises the sequence of SEQ ID NO:

181.

3. The polynucleotide of any one of claims 1 to 2, wherein the polynucleotide further comprises a signal peptide sequence at the 5' end of the nucleic acid sequence, and the signal peptide sequence is at least 95% identical to or comprises the nucleic acid of SEQ ID NO:

264.

4. The polynucleotide of any one of claims 1 to 3, wherein the nucleic acid comprises less than 24 CpG dinucleotides, optionally comprising 0 CpG dinucleotides.

5. The polynucleotide described in claim 1, wherein the nucleic acid comprises the sequence of SEQ ID NO:119 and the C1 inhibitor comprises the amino acid sequence of SEQ ID NO:

192.

6. 5. The polynucleotide of claim 1, 2, or 4, comprising a nucleic acid encoding a signal peptide sequence operably linked to the 5' end of a polynucleotide sequence encoding a C1 inhibitor or a mutant C1 inhibitor, preferably the signal peptide is selected from the group consisting of C1 inhibitor signal peptide, human chymotrypsinogen B2 signal peptide, ALB signal peptide, ORM1 signal peptide, TF signal peptide, AMBP signal peptide, LAMP1 signal peptide, BTN2A2 signal peptide, CD300 signal peptide, NOTCH2 signal peptide, STRC signal peptide, AHSG signal peptide, SYN1 signal peptide, SYN2 signal peptide, SYN3 signal peptide, and SYN4 signal peptide, more preferably the signal peptide has a polynucleotide sequence of one of SEQ ID NOs: 84-103.

7. An expression cassette comprising the polynucleotide of any one of claims 1 to 6, operably linked to an expression control element, preferably further comprising a polyadenylation sequence operably linked to the 3' end of the nucleic acid encoding C1 inhibitor, more preferably the polyadenylation sequence comprises the bovine growth hormone (bGH) polyadenylation sequence, even more preferably the bGH polyadenylation sequence comprises the polynucleotide sequence of SEQ ID NO:

83.

8. 8. The expression cassette of claim 7, wherein the expression control element or polyadenylation sequence has reduced CpGs compared to the wild-type expression control element or polyadenylation sequence.

9. 9. The expression cassette of claim 7 or 8, wherein the expression control element comprises an ApoE / hAAT enhancer / promoter sequence, preferably the ApoE enhancer sequence comprises one of the polynucleotide sequences of SEQ ID NOs: 225 and 74-76, and / or the hAAT promoter sequence comprises the polynucleotide sequence of SEQ ID NO: 79 or 80.

10. 10. The expression cassette of claim 9, wherein the ApoE / hAAT enhancer / promoter sequence or the bGH polyadenylation sequence has reduced CpGs compared to the wild-type ApoE / hAAT enhancer / promoter sequence or the bGH polyadenylation sequence.

11. 11. The expression cassette of any one of claims 7 to 10, further comprising an intron, preferably said intron comprising a human hemoglobin beta (HBB) derived intron, more preferably said human hemoglobin beta (HBB) derived intron sequence comprising the polynucleotide sequence of SEQ ID NO: 81 or 82.

12. 12. The expression cassette of any one of claims 7 to 11, further comprising one or more enhancers, preferably the enhancers are selected from the group consisting of ApoE, 2xApoE, 4xApoE, hAAT, WPRE3 and WPRE, preferably ApoE, 2xApoE, 4xApoE, hAAT, WPRE3 and WPRE are codon optimized, more preferably the enhancer comprises one of the polynucleotide sequences of SEQ ID NOs: 225 and 74-78 and 173-178.

13. or one or more response elements selected from the group consisting of a miRNA binding site, a regulated Ire1-dependent decay (RIDD) sequence, an acute phase response element (APRE), a 5' UTR, and a 3' UTR, preferably wherein the expression cassette further comprises a miRNA binding site comprising the polynucleotide sequence of SEQ ID NO: 179; or The expression cassette further comprises a RIDD sequence comprising one of the polynucleotide sequences of SEQ ID NOs: 185-187; or The expression cassette further comprises an APRE sequence comprising one of the polynucleotide sequences of SEQ ID NOs: 77, 78, 180, 182 and 183; or The expression cassette according to any one of claims 7 to 12, further comprising a 3' UTR comprising the polynucleotide sequence of SEQ ID NO:

184.

14. An adeno-associated virus (AAV) vector comprising a polynucleotide or an expression cassette according to any one of claims 1 to 13.

15. The AAV vector of claim 14, wherein the AAV vector comprises (a) one or more AAV capsids and (b) one or more AAV inverted terminal repeats (ITRs), the AAV ITRs flanking the 5' or 3' ends of the polynucleotide or expression cassette, and preferably at least one or more of the ITRs are modified to have reduced CpGs.

16. AAV vector capsid having 90% or more sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:188), AAV3B, AAV-2i8, SEQ ID NO:226, SEQ ID NO:189, SEQ ID NO:190, and / or SEQ ID NO:

191. VP1, VP2 and / or VP3 capsids; or capsids having 95% or greater sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO:188), AAV3B, AAV-2i8, SEQ ID NO:226, SEQ ID NO:189, SEQ ID NO:190, and / or SEQ ID NO:

191. or a capsid having 100% sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 (SEQ ID NO: 188), AAV3B, AAV-2i8, SEQ ID NO: 226, SEQ ID NO: 189, SEQ ID NO: 190, and / or SEQ ID NO:

191.

17. 17. The AAV vector of any one of claims 14 to 16, wherein the ITRs include one or more ITRs of any of the AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B, AAV serotypes, or combinations thereof.

18. The AAV vector of claim 14, comprising a polynucleotide sequence having at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 21, 27, 28, 32, 34-48, 55-69 and 227-229, or comprising a sequence selected from the group consisting of SEQ ID NOs: 21, 27, 28, 32, 34-48, 55-69 and 227-229.

19. The AAV vector of any one of claims 14 to 18, wherein the polynucleotide is encapsidated by a capsid comprising VP1 of SEQ ID NO: 226, VP2 of SEQ ID NO: 189 and VP3 of SEQ ID NO:

190.

20. The AAV vector of claim 19, wherein the polynucleotide comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO:22, provided that the nucleic acid sequence encodes the sequence of SEQ ID NO:192, and preferably, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

22.

21. 21. A pharmaceutical composition comprising one or more AAV vectors according to any one of claims 14 to 20 in a biologically compatible carrier or excipient, preferably further comprising a surfactant.

22. 22. A therapeutically effective amount of the polynucleotide or expression cassette, or AAV vector, or pharmaceutical composition according to any one of claims 1 to 21 for use in treating a subject in need of C1 inhibitor, wherein C1 inhibitor is expressed in the subject, preferably the subject has hereditary angioedema (HAE), preferably the subject is a human, and wherein the method reduces, reduces or inhibits the need for C1 inhibitor or one or more symptoms of HAE; or prevents or reduces the progression or worsening of the need for C1 inhibitor or one or more symptoms of HAE; or stabilizes the need for C1 inhibitor or one or more symptoms of HAE; or ameliorates the need for C1 inhibitor or one or more symptoms of HAE.

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