Adeno-associated virus vector delivery of antibodies for the treatment of diseases mediated by dysregulation of plasma kallikrein - Patent Application 20070122999

Abstract

The present disclosure provides, inter alia, a recombinant adeno-associated virus (rAAV) vector comprising a codon-optimized nucleotide sequence encoding an agent that inhibits the proteolytic activity of plasma kallikrein. The present disclosure also provides a recombinant adeno-associated virus (rAAV) vector encoding an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Application No. 63 / 126,300, filed December 16, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on December 13, 2021, is named SHR-2020US1_SL.txt and is 63,250 bytes in size. [Background technology]

[0003] Dysregulation of plasma kallikrein activity can lead to excessive production of the inflammatory and vasoactive peptide bradykinin. An example of such a disease is hereditary angioedema (HAE), a rare but potentially life-threatening disorder characterized by unpredictable and recurrent attacks of vasodilation manifesting as subcutaneous and submucosal angioedema. In some cases, HAE is associated with low plasma levels of C1-inhibitor (type I), whereas in other cases, the protein circulates in normal or elevated amounts but is dysfunctional (type II). C1 inhibitor is the primary regulator of plasma kallikrein activity. Symptoms of an HAE attack include swelling of the face, mouth, and / or airways, either spontaneously or induced by minor trauma. Edema attacks affecting the airways can be fatal. In addition to acute inflammatory attacks, excess plasma kallikrein activity is also associated with chronic conditions, such as autoimmune diseases, including lupus erythematosus.

[0004] Various strategies for the treatment of C1-INH deficiency or dysfunction have been conceived and developed, including, for example, inhibiting members of the contact system, such as lanadelumab, a fully human monoclonal antibody inhibitor of plasma kallikrein approved for the treatment of HAE.

[0005] The use of vectors to produce proteins, including antibodies, in vivo is desirable for the treatment of disease but is limited by a variety of factors, including insufficient antibody production after delivery to a subject. Summary of the Invention

[0006] The present invention provides efficient and robust recombinant adeno-associated virus (rAAV) vectors encoding anti-plasma kallikrein antibodies. The present invention is based, in part, on the surprising discovery that certain recombinant AAV vectors containing codon-optimized nucleotide sequences encoding anti-plasma kallikrein antibody heavy chains and anti-plasma kallikrein antibody light chains result in high-level in vivo production of functional anti-plasma kallikrein antibodies. In some embodiments, vector constructs were codon-optimized to reduce CpG content and repeat sequences. In some embodiments, vector constructs were engineered to normalize GC content percentages to those found in native, unmodified AAV8. In some embodiments, each vector construct was evaluated for CpG content, codon adaptation index (CAI), codon context (CC), GC content, and repeat motifs. Thus, in some embodiments, vector constructs were evaluated for CpG content. In some embodiments, vector constructs were evaluated for codon adaptation index (CAI). In some embodiments, vector constructs were evaluated for GC content. In some embodiments, vector constructs were evaluated for the amount of repeat motifs. Specifically, rAAV resulted in robust and sustained production of anti-plasma kallikrein mAbs in vivo, and vector-mediated expressed anti-plasma kallikrein antibodies retained targeting activity comparable to antibody proteins produced by conventional recombinant expression methods (e.g., CHO cells). Prior to the present invention, delivery of anti-plasma kallikrein antibodies via administration of rAAV vectors carrying a desired payload resulted in unknown amounts of active antibody production. Therefore, prior to the present invention, it was neither predictable nor feasible to use rAAV vectors encoding anti-plasma kallikrein for the treatment of C1-INH deficiency or disorders, including, for example, hereditary angioedema.

[0007] In some aspects, a recombinant adeno-associated virus (rAAV) vector comprises a codon-optimized nucleotide sequence, wherein the rAAV vector encodes a full-length antibody comprising an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain, and the codon-optimized nucleotide sequence has at least about 75%, 80%, 85%, 90%, 95%, or greater than 95% identity to any one of SEQ ID NOs: 23-36. Thus, in some embodiments, the codon-optimized nucleotide sequence has at least about 75% identity to any one of SEQ ID NOs: 23-36. In some embodiments, the codon-optimized nucleotide sequence has at least about 80% identity to any one of SEQ ID NOs: 23-36. In some embodiments, the codon-optimized nucleotide sequence has at least about 85% identity to any one of SEQ ID NOs: 23-36. In some embodiments, the codon-optimized nucleotide sequence has at least about 90% identity to any one of SEQ ID NOs: 23-36. In some embodiments, the codon-optimized nucleotide sequence has at least about 95% identity to any one of SEQ ID NOs: 23-36. In some embodiments, the codon-optimized nucleotide sequence has more than 95% identity to any one of SEQ ID NOs: 23-36. In some embodiments, the codon-optimized nucleotide sequence is identical to any one of SEQ ID NOs: 23-36. SEQ ID NOs: 23-36 include any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36. Each of SEQ ID NOs: 23-36 is listed in Table 3.

[0008] In some embodiments, the codon-optimized nucleotide sequence is selected from any one of SEQ ID NOs: 23-36. Thus, in some embodiments, the codon-optimized sequence is SEQ ID NO: 23. In some embodiments, the codon-optimized sequence is SEQ ID NO: 24. In some embodiments, the codon-optimized sequence is SEQ ID NO: 25. In some embodiments, the codon-optimized sequence is SEQ ID NO: 26. In some embodiments, the codon-optimized sequence is SEQ ID NO: 27. In some embodiments, the codon-optimized sequence is SEQ ID NO: 28. In some embodiments, the codon-optimized sequence is SEQ ID NO: 29. In some embodiments, the codon-optimized sequence is SEQ ID NO: 30. In some embodiments, the codon-optimized sequence is SEQ ID NO: 31. In some embodiments, the codon-optimized sequence is SEQ ID NO: 32. In some embodiments, the codon-optimized sequence is SEQ ID NO: 33. In some embodiments, the codon-optimized sequence is SEQ ID NO: 34. In some embodiments, the codon-optimized sequence is SEQ ID NO: 35. In some embodiments, the codon-optimized sequence is SEQ ID NO: 36.

[0009] In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 50 CpG sites, less than about 40 CpG sites, less than about 35 CpG sites, less than about 30 CpG sites, less than about 25 CpG sites, less than about 20 CpG sites, less than about 15 CpG sites, or less than about 10 CpG sites. Thus, in some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 50 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 45 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 40 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 35 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 30 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 25 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 20 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 15 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 10 CpG sites. In some embodiments, the codon-optimized nucleotide sequence has a CpG content of less than about 5 CpG sites.

[0010] In some embodiments, the codon-optimized nucleotide sequence has about 5 CpG sites.

[0011] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are linked via a linker. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the linker comprises a non-cleavable linker.

[0012] In some embodiments, the codon-optimized nucleotide sequence comprises a linker. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the linker comprises a non-cleavable linker.

[0013] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are controlled by a single promoter.

[0014] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are controlled by separate promoters.

[0015] In some embodiments, the single promoter or the separate promoters are selected from ubiquitous promoters, tissue-specific promoters, or controllable promoters. Thus, in some embodiments, the single promoter or the separate promoters are ubiquitous promoters. In some embodiments, the single promoter or the separate promoters are tissue-specific promoters. In some embodiments, the single promoter or the separate promoters are controllable promoters.

[0016] In some embodiments, the tissue-specific promoter is a liver-specific promoter.

[0017] In some embodiments, the liver-specific promoter comprises a promoter selected from the human transthyretin promoter (TTR), modified hTTR (hTTR mod.), α-antitrypsin promoter, liver promoter 1 (LP1), TRM promoter, human factor IX pro / liver transcription factor-responsive oligomer, LSP, CMV / CBA promoter (1.1 kb), CAG promoter (1.7 kb), mTTR, modified mTTR, mTTR pro, mTTR enhancer, or basic albumin promoter. Thus, in some embodiments, the liver-specific promoter comprises the human transthyretin promoter (TTR). In some embodiments, the liver-specific promoter comprises modified hTTR (hTTR mod.). In some embodiments, the liver-specific promoter comprises the α-antitrypsin promoter. In some embodiments, the liver-specific promoter comprises liver promoter 1 (LP1). In some embodiments, the liver-specific promoter comprises the TRM promoter. In some embodiments, the liver-specific promoter comprises human factor IX pro / liver transcription factor-responsive oligomer. In some embodiments, the liver-specific promoter comprises LSP. In some embodiments, the liver-specific promoter comprises a CMV / CBA promoter (1.1 kb). In some embodiments, the liver-specific promoter comprises a CAG promoter (1.7 kb). In some embodiments, the liver-specific promoter comprises mTTR. In some embodiments, the liver-specific promoter comprises an mTTR pro. In some embodiments, the liver-specific promoter comprises an mTTR enhancer. In some embodiments, the liver-specific promoter comprises a basic albumin promoter.

[0018] In some embodiments, the liver-specific promoter is the human transthyretin promoter (TTR).

[0019] In some embodiments, the controllable promoter is an inducible or repressible promoter. Thus, in some embodiments, the controllable promoter is an inducible promoter. In some embodiments, the controllable promoter is a repressible promoter.

[0020] In some embodiments, the vector further comprises one or more of the following: 5' and 3' inverted repeats, an intron upstream of the sequence, and a cis-acting regulatory module (CRM). Thus, in some embodiments, the vector further comprises 5' and 3' inverted repeats. In some embodiments, the vector further comprises an intron upstream of the sequence. In some embodiments, the vector further comprises a cis-acting regulatory module (CRM).

[0021] In some embodiments, the vector further comprises a WPRE sequence.

[0022] In some embodiments, the WPRE sequence is modified.

[0023] In some embodiments, the WPRE contains a mut6delATG modification.

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

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

[0026] In some embodiments, the vector comprises at least three CRMs. In some embodiments, the vector comprises three CRMs. In some embodiments, the vector comprises four CRMs. In some embodiments, the vector comprises at least five CRMs. In some embodiments, the vector comprises more than five CRMs.

[0027] In some embodiments, the vector comprises three CRM8s.

[0028] In some embodiments, the rAAV vector comprises an IRES sequence.

[0029] In some embodiments, the anti-plasma kallikrein antibody light chain and / or heavy chain comprises one or more mutations that increase the half-life and / or reduce the effector function of the antibody.

[0030] In some embodiments, the one or more mutations comprise a LALA mutation (L234A and L235A) and / or an NHance mutation (H433K and N434F).

[0031] In some embodiments, the one or more mutations comprise a LALA mutation (L234A and L235A). In some embodiments, the one or more mutations comprise an NHance mutation (H433K and N434F).

[0032] The rAAV vector of any one of the preceding claims, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAVrh.10. Accordingly, in some embodiments, the AAV vector is AAV1. In some embodiments, the AAV vector is AAV2. In some embodiments, the AAV vector is AAV3. In some embodiments, the AAV vector is AAV4. In some embodiments, the AAV vector is AAV5. In some embodiments, the AAV vector is AAV6. In some embodiments, the AAV vector is AAV7. In some embodiments, the AAV vector is AAV8. In some embodiments, the AAV vector is AAV9. In some embodiments, the AAV vector is AAV10. In some embodiments, the AAV vector is AAV11. In some embodiments, the AAV vector is AAVrh.10.

[0033] In some embodiments, the rAAV vector has a GC content engineered to have approximately the same CC content as naturally occurring AAV. In some embodiments, the rAAV vector has a GC content engineered to have approximately the same CC content as naturally occurring AAV8.

[0034] In some embodiments, the rAAV vector capsid is engineered.

[0035] In some embodiments, the engineered rAAV vector comprises an AAV capsid sequence with a modified amino acid sequence.

[0036] In some embodiments, the modified amino acid sequence comprises an insertion, deletion, or substitution of an amino acid sequence. Thus, in some embodiments, the modified amino acid sequence comprises one or more insertions. In some embodiments, the modified amino acid sequence comprises one or more deletions. In some embodiments, the modified amino acid sequence comprises one or more amino acid substitutions.

[0037] In some embodiments, the rAAV capsid is naturally derived.

[0038] In some embodiments, the rAAV vector capsid is AAV8.

[0039] In some embodiments, the cleavable sequence is a furin-cleavable sequence.

[0040] In some embodiments, the furin-cleavable sequence is followed by a linker and a 2A sequence.

[0041] In some embodiments, the linker is a GSG linker.

[0042] In some embodiments, the 2A sequence is a T2A, P2A, E2A, or F2A sequence. Thus, in some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is an E2A sequence. In some embodiments, the 2A sequence is an F2A sequence.

[0043] In some embodiments, the vector further encodes a secretion signal.

[0044] In some embodiments, the secretion signal is a naturally occurring signal peptide.

[0045] In some embodiments, the secretion signal is an artificial signal peptide.

[0046] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain form a functional anti-plasma kallikrein antibody capable of binding to plasma kallikrein.

[0047] In some embodiments, the anti-plasma kallikrein antibody inhibits the proteolytic activity of plasma kallikrein.

[0048] In some embodiments, the anti-plasma kallikrein antibody binds to the plasma kallikrein active site.

[0049] In some embodiments, the binding occludes the active site of plasma kallikrein.

[0050] In some embodiments, the binding inhibits the activity of plasma kallikrein.

[0051] In some embodiments, the antibody does not bind to prekallikrein.

[0052] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are expressed from the same vector.

[0053] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are expressed from separate rAAV vectors.

[0054] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are expressed from separate rAAV vectors.

[0055] In some embodiments, the vector further comprises 5' and 3' inverted repeats (ITRs), one or more enhancer elements, and / or a poly(A) tail. Thus, in some embodiments, the vector further comprises 5' and 3' inverted repeats (ITRs). In some embodiments, the vector further comprises one or more enhancer elements. In some embodiments, the vector further comprises a poly(A) tail.

[0056] In some embodiments, the one or more enhancer elements are selected from a cluster of transcription factor binding sites and / or a WPRE sequence. Thus, in some embodiments, the one or more enhancer elements are a cluster of transcription factor binding sites. In some embodiments, the one or more enhancer elements are a WPRE sequence.

[0057] In some aspects, a recombinant adeno-associated virus (rAAV) is provided, comprising an AAV8 capsid and a rAAV vector comprising a codon-optimized nucleotide sequence having at least about 75%, 80%, 85%, 90%, 95%, or greater than 95% identity to any one of SEQ ID NOs:23-36, wherein the vector comprises: a. a 5' inverted repeat (ITR); b. a cis-acting regulatory module (CRM); c. a liver-specific promoter; e. a codon-optimized anti-plasma kallikrein antibody heavy chain sequence and an anti-plasma kallikrein antibody light chain sequence; f. a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and g. a 3' ITR.

[0058] In some embodiments, the liver-specific promoter comprises a promoter selected from human transthyretin promoter (TTR), modified hTTR (hTTR mod.), alpha-antitrypsin promoter, liver promoter 1 (LP1), TRM promoter, human factor IX pro / liver transcription factor responsive oligomer, LSP, CMV / CBA promoter (1.1 kb), CAG promoter (1.7 kb), mTTR, modified mTTR, mTTR pro, mTTR enhancer, or basic albumin promoter.

[0059] In some embodiments, the liver-specific promoter comprises the human transthyretin promoter (hTTR).

[0060] In some embodiments, the CRM is a liver-specific CRM.

[0061] In some embodiments, the vector comprises at least three CRMs.

[0062] In some embodiments, the vector comprises three CRM8s.

[0063] In some embodiments, the WPRE sequence is modified.

[0064] In some embodiments, the WPRE sequence is WPRE mut6delATG.

[0065] In some aspects, methods are provided for treating a disease or disorder associated with a deficiency or dysregulation of the activated kallikrein-kinin pathway in a subject in need thereof, the method comprising administering a recombinant adeno-associated viral vector (rAAV) described herein.

[0066] In some embodiments, the deficiency or dysregulation of the activated kallikrein-kinin pathway is a disease or disorder associated with a deficiency in C1 esterase inhibitor.

[0067] In some embodiments, the rAAV vector is administered intravenously, subcutaneously, or transdermally.Thus, in some embodiments, the rAAV vector is administered intravenously.In some embodiments, the rAAV vector is administered subcutaneously.In some embodiments, the rAAV vector is administered transdermally.

[0068] In some embodiments, the transdermal administration is by gene gun.

[0069] In some embodiments, the disorder associated with a deficiency or dysregulation of the activated kallikrein-kinin pathway or a deficiency in C1 esterase inhibitor is hereditary angioedema (HAE), acquired angioedema (AAE), angioedema with normal C1 inhibitor, diabetic macular edema, migraine, tumor, neurodegenerative disease, rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent-induced thrombus formation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event, restenosis, systemic lupus erythematosus nephritis / vasculitis, or burn injury.

[0070] In some embodiments, the disorder associated with a deficiency in C1 esterase inhibitor is HAE.

[0071] In some embodiments, the HAE is type I, II, or III. Thus, in some embodiments, the HAE is type I HAE. In some embodiments, the HAE is type II HAE. In some embodiments, the HAE is type III HAE.

[0072] In some embodiments, the rAAV vector is episomal after administration.

[0073] In some embodiments, after administration, the anti-plasma kallikrein antibody heavy and light chains assemble into functional antibodies.

[0074] In some embodiments, the antibody is an IgG.

[0075] In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the subject's plasma about 2 to 6 weeks after administration of the rAAV vector. For example, in some embodiments, anti-plasma kallikrein antibodies are detectable in the subject's plasma about 2 weeks after administration of the rAAV vector. In some embodiments, anti-plasma kallikrein antibodies are detectable in the subject's plasma about 3 weeks after administration of the rAAV vector. In some embodiments, anti-plasma kallikrein antibodies are detectable in the subject's plasma about 4 weeks after administration of the rAAV vector. In some embodiments, anti-plasma kallikrein antibodies are detectable in the subject's plasma about 5 weeks after administration of the rAAV vector. In some embodiments, anti-plasma kallikrein antibodies are detectable in the subject's plasma about 6 weeks after administration of the rAAV vector. In some embodiments, anti-plasma kallikrein antibodies are detectable in the subject's plasma more than about 6 weeks after administration of the rAAV vector.

[0076] In some aspects, a DNA expression cassette is provided comprising a codon-optimized nucleotide sequence encoding a full-length antibody comprising an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain, wherein the DNA expression cassette comprises a sequence having at least about 75%, 80%, 85%, 90%, 95%, or greater than 95% identity to any one of SEQ ID NOs: 23-36.

[0077] In some embodiments, the DNA expression cassette is contained within a delivery vehicle.

[0078] In some embodiments, the delivery vehicle is selected from a viral vector, a lipid nanoparticle, or an extracellular vesicle. Thus, in some embodiments, the delivery vehicle is a viral vector. In some embodiments, the delivery vehicle is selected from a lipid nanoparticle. In some embodiments, the delivery vehicle is selected from an extracellular vesicle. [Brief explanation of the drawings]

[0079] [Figure 1]

[0023] Figure 1 is a schematic diagram showing an exemplary gene therapy approach using an rAAV vector encoding an anti-plasma kallikrein antibody. Figure 1 shows an AAV vector encoding an anti-plasma kallikrein antibody that is administered intravenously (IV) to a subject in need; the vector is translated into a functional anti-plasma kallikrein antibody, which is secreted into the subject's circulation; the antibody results in binding and inhibition of plasma kallikrein in the subject. IV = intravenous; HC = heavy chain; LC = light chain. [Figure 2] Figure 1 shows a schematic diagram of the expression construct encoding anti-PKa IgG-2930-LALA. ITR: inverted terminal repeat; hTTR: human transthyretin promoter; CRM: cis-acting regulatory module; intron may be MVM intron (minute virus of mouse intron); WPRE: liver-specific promoter; SS: secretion signal; polyA is an upstream enhancer. [Figure 3] Figure 1 shows the levels of active IgG in HepG2 culture medium after transfection with plasmids encoding the Round 1 codon-optimized 2930-LALA constructs. HepG2 cells were transfected for either 48 or 72 hours. Six different rAAV Round 1 codon-optimized constructs (A010, A013, A014, A015, A016, and A017) were used. [Figure 4]This graph shows active IgG levels in HepG2 culture media after transfection with plasmids encoding Round 2 gene-optimized (GO) vectorized 2930-LALA constructs. HepG2 cells were transfected for either 48 or 72 hours. Fifteen different rAAV GO vectorized constructs (B041, B042, B043, B044, B045, B046, B047, B048, B049, B050, B051, B063, B065, B011, B021) were used. [Figure 5] 1 is a graph showing active IgG levels in mouse plasma 0 and 2 weeks after intravenous administration of the indicated vectors. C57B6 mice were injected with AAV vectors at a dose of 5x10 or 5x10 vg / kg, plasma was collected on day 0 and 2 weeks after rAAV injection, and active anti-PKa antibodies in the plasma were determined by an MSD assay that detects anti-PKa IgG1 molecules using an immobilized PKa surface. [Figure 6] Figure 1 shows the ex vivo bioactivity of anti-PKa antibodies generated in mice treated with rAAV8. Plasma samples were collected 14 days after intravenous administration of 5x10 vg / kg of the B041 vector construct. The efficacy of in vivo rAAV8-generated anti-PKa antibodies in inhibiting the kallikrein-kinin pathway in treated mouse plasma samples was compared to that of the commercially available inhibitor, Takhzyro™ (lanadelumab, a fully human monoclonal antibody inhibitor of plasma kallikrein), in inhibiting the same pathway in which the Takhzyro drug product was injected into control mouse plasma. Bioactivity was measured in terms of percent inhibition of plasma kallikrein activity as a function of anti-PKa antibody concentration. [Figure 7]Figure 1 shows the ex vivo bioactivity of anti-PKa antibodies generated in mice treated with rAAV8. Plasma samples were collected 14 days after intravenous administration of 5x10 vg / kg of the B048 vector construct. The efficacy of in vivo rAAV8-generated anti-PKa antibodies in inhibiting the kallikrein-kinin pathway in treated mouse plasma samples was compared to that of the commercially available inhibitor, Takhzyro™ (lanadelumab, a fully human monoclonal antibody inhibitor of plasma kallikrein), in inhibiting the same pathway when the Takhzyro drug product was injected into control mouse plasma. Bioactivity was measured in terms of percent inhibition of plasma kallikrein activity as a function of anti-PKa antibody concentration.

[0080] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0081] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context indicates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0082] 2A Sequence: As used herein, "2A" or "2A sequence" or "2A peptide" refers to a class of self-cleaving peptides. Examples of 2A peptides include T2A, P2A, E2A, and F2A. T2A has the sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 13); P2A has the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 14); E2A has the sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 15); and F2A has the sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 16). Cleavage-efficient 2A peptides suitable for the rAAV vectors described herein are described in Chng J. et al. Mabs. 2015;7(2):403-412 and Kim et al. PLoS One 2011;6(4), the contents of each of which are incorporated herein by reference in their entirety.

[0083] Adeno-associated virus (AAV): As used herein, the term "adeno-associated virus" or "AAV" or recombinant AAV ("rAAV") includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV (see Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers); Gao et al., J. Virology 78:6381-6388 (2004); Mori et al., Virology 330:375-383 (2004)). Typically, AAV can infect both dividing and non-dividing cells and can exist in an extrachromosomal state without being integrated into the genome of the host cell. AAV vectors are commonly used in gene therapy. In some embodiments, AAV is engineered. AAV can be engineered by any method known in the art. For example, in some embodiments, AAV capsid is engineered by protein engineering methods.

[0084] Administering: As used herein, the terms "administering" and "introducing" are used interchangeably in the context of delivering an antibody-encoding rAAV vector to a subject by a method or route that results in efficient delivery of the rAAV vector. Various methods for administering rAAV vectors are known in the art, including, for example, intravenous, subcutaneous, or transdermal administration. Transdermal administration of rAAV vectors can be performed by using a "gene gun" or a biolistec particle delivery system. In some embodiments, the rAAV vector is administered via non-viral lipid nanoparticles.

[0085] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0086] Antibody: As used herein, the term "antibody" or "Ab" or "Abs" or "mAbs" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" means that the antibody reacts with one or more regions of the desired antigen. Antibodies include antibody fragments. Antibodies also include polyclonal, monoclonal, chimeric dAbs (domain antibodies), single chain, F ab , F ab’ , F (ab’)2 Fragments, scFvs, and F abThese include, but are not limited to, expression libraries. The antibody can be a complete antibody, or an immunoglobulin, or an antibody fragment.

[0087] Recognized immunoglobulin polypeptides include kappa and lambda light chains and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu heavy chains, or equivalents in other species. A full-length immunoglobulin "light chain" (about 25 kDa or about 214 amino acids) contains a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. A full-length immunoglobulin "heavy chain" (about 50 kDa or about 446 amino acids) similarly contains a variable region (about 116 amino acids) and one of the previously described heavy chain constant regions, e.g., gamma (about 330 amino acids).

[0088] Antigen-binding site: As used herein, the term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that participates in antigen binding. The antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, termed "hypervariable regions," are interposed between more conserved adjacent stretches known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to be bound, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs."

[0089] Approximately or about: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (greater or less), unless otherwise specified or clear from the context (except when such number exceeds 100% of possible values).

[0090] Biologically active: As used herein, the phrase "biologically active" refers to the characteristic of any agent that has activity in a biological system, particularly an organism. For example, an agent that, when administered to an organism, has a biological effect on that organism is considered biologically active. In certain embodiments, if a peptide is biologically active, a portion of the peptide that shares at least one biological activity of the peptide is typically referred to as a "biologically active" portion.

[0091] C1-esterase deficiency or C1-esterase disorder: As used herein, "C1-esterase deficiency" or "C1-esterase disorder" means a reduced amount of functional C1-esterase inhibitor present in a subject compared to a healthy individual.

[0092] GC Content: GC content (or guanine-cytosine content) is the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C).

[0093] Codon Adaptation Index (CAI): CAI is the most widely used technique for analyzing codon usage bias, which refers to the difference in frequency of occurrence of synonymous codons in coding DNA.

[0094] Codon optimization: As used herein, the term "codon optimization" refers to a method for improving the codon composition of a recombinant gene based on various criteria without modifying the amino acid sequence. Various methods of codon optimization are known in the art, including, for example, a web-based multi-objective optimization platform for synthetic gene design (e.g., called COOL (Codon Optimization Online)). Various publications, such as Bioinformatics, 2014 Aug 1; 30(15)2210-2; BMC Syst Biol., 2012 Oct 20; 6:134; Methods, 2016 Jun 1; 102:26-35; and Enzyme Microb Technol., Jul-Aug 2015; 75-76:57-63, relate to codon optimization strategies. Each of these publications is incorporated herein by reference.

[0095] Cleavable linker: As used herein, the term "cleavable linker" includes any polypeptide linker that can be cleaved by a compound. For example, the cleavable linker can be an enzymatically cleavable polypeptide linker. Various enzymatically cleavable linkers are suitable for the present invention, including, for example, a furin-cleavable linker or a thrombin-cleavable linker.

[0096] CpG site: A CpG site or CG site is a region of DNA in which a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' to 3' direction.

[0097] Bound, linked, connected, or fused: As used herein, the terms "bound," "linked," "connected," "fused," and "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components together by any means, including chemical conjugation or recombinant means.

[0098] Epitope: As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment. Epitopic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide.

[0099] Functional equivalent or derivative: As used herein, the term "functional equivalent" or "functional derivative," in the context of a functional derivative of an amino acid sequence, refers to a molecule that retains a biological activity (either function or structure) substantially similar to that of the original sequence. Functional derivatives or equivalents may be naturally occurring or synthetically prepared. Exemplary functional derivatives include amino acid sequences with one or more amino acid substitutions, deletions, or additions, provided that the biological activity of the protein is preserved. It is desirable that the substituted amino acid have similar chemical and physical properties to the substituted amino acid. Desirable similar chemical and physical properties include similarities in charge, bulk, hydrophobicity, hydrophilicity, etc.

[0100] Hereditary angioedema or HAE: As used herein, the term "hereditary angioedema" or "HAE" refers to a blood disorder characterized by unpredictable and recurrent attacks of inflammation. HAE is typically associated with C1-INH deficiency, which can result in low levels of C1-INH or C1-INH with ineffective or reduced activity. HAE is also associated with other genetic mutations, such as mutations in FXII, among others. Symptoms include, but are not limited to, swelling that can occur in any part of the body, such as the face, limbs, genitals, digestive tract, and upper respiratory tract.

[0101] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, cell culture, etc.

[0102] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0103] IRES: As used herein, the term "IRES" refers to any suitable internal ribosome entry site sequence.

[0104] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that (1) has been separated from at least some of the components with which it was associated when originally produced (whether in nature and / or in an experimental setting), and / or (2) has been produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of other components with which they were originally associated. In some embodiments, an isolated material is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, greater than about 99%, substantially 100%, or 100% pure. As used herein, a material is "pure" if it is substantially free of other components. As used herein, the term "isolated cell" refers to a cell that is not contained in a multicellular organism.

[0105] Immunological binding: The term "immunological binding" refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ) and smaller K d represents a higher affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art.

[0106] Linker or Peptide Linker: The term "linker" or "peptide linker," as used herein, refers to an amino acid sequence that connects two polypeptide domains. For example, a "linker" or "peptide linker" can separate an antibody heavy chain amino acid sequence and an antibody light chain amino acid sequence. Various types of linkers are suitable for the present invention, including, for example, linkers having a Gly-Ser-Gly (GSG) motif.

[0107] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked via peptide bonds. The term is used to refer to an amino acid chain of any length, although those skilled in the art will understand that the term is not limited to long chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As known to those skilled in the art, polypeptides can be processed and / or modified.

[0108] Prevent: As used herein, the terms "prevent" or "prevention," when used in relation to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition.

[0109] Protein: The term "protein," as used herein, refers to one or more polypeptides that function as a separate unit. When a single polypeptide is a separate functional unit and does not require permanent or temporary physical association with other polypeptides to form the separate functional unit, the terms "polypeptide" and "protein" may be used interchangeably. When a separate functional unit is composed of multiple polypeptides that are physically associated with each other, the term "protein" refers to the multiple polypeptides that are physically associated and function together as a separate unit.

[0110] Repeat Sequences: Repeat sequences are patterns of nucleic acids (DNA or RNA) that occur in multiple copies throughout the genome.

[0111] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, a subject is a human. A subject may be a patient, which refers to a person who visits a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0112] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, reach perfection and / or progress to perfection or achieve or avoid certain results. The term "substantially" is therefore used to express the potential lack of perfection inherent in many biological and chemical phenomena.

[0113] Substantial homology: The phrase "substantial homology" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues that have, as appropriate, similar structural and / or functional characteristics. For example, as is well known to those skilled in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid with another amino acid of the same type can often be considered a "homologous" substitution.

[0114] As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., "Basic Local Alignment Search Tool," J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., "Methods in Enzymology;" Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., "Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins," Wiley, 1998; and Misener, et al., (eds.), "Bioinformatics Methods and Protocols" (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above-mentioned programs typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over the relevant stretch of residues, which in some embodiments is the complete sequence.In some embodiments, the relevant extension is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0115] Substantial identity: The phrase "substantial identity" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above-mentioned programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues, which in some embodiments is the complete sequence.In some embodiments, the relevant extension is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0116] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0117] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of the disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. It will be understood by those skilled in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0118] Treating: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, injury, and / or condition. Treatment may be administered to subjects who do not show signs of the disease and / or who show only early signs of the disease, with the intent of reducing the risk of developing pathology associated with the disease.

[0119] The recitation herein of numerical ranges by endpoints includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4, and 5). It is also understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0120] Various aspects of the present invention are described in detail in the following sections. The use of the sections is not intended to limit the present invention. Each section may be applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless stated otherwise. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0121] Various aspects of the invention are described in detail in the following sections. The use of the sections is not intended to limit the invention. Each section may be applicable to any aspect of the invention. In this application, the use of "or" means "and / or" unless stated otherwise. DETAILED DESCRIPTION OF THE INVENTION

[0122] The present invention provides vectors encoding anti-plasma kallikrein antibodies and methods for the delivery of such vectors to subjects diagnosed with diseases or conditions indicated for treatment with these therapeutic antibodies. Delivery of such vectors can be achieved via gene therapy, for example, by administering a viral vector or other DNA expression construct encoding a therapeutic antibody or antigen-binding fragment thereof to a subject diagnosed with a condition for which treatment with such a therapeutic antibody is indicated, to generate a depot in a tissue or organ of the subject that continuously supplies the antibody or antigen-binding fragment of the therapeutic antibody to the target tissue where the antibody or antigen-binding fragment exerts its therapeutic effect.

[0123] In some embodiments, the present disclosure describes an efficient and robust recombinant adeno-associated virus (rAAV) vector containing a codon-optimized nucleic acid sequence encoding an anti-plasma kallikrein antibody for the treatment of plasma kallikrein-mediated disorders, such as HAE-associated C1 INH deficiency. Human C1-INH is an important anti-inflammatory plasma protein with a wide range of inhibitory and non-inhibitory biological activities. Based on sequence homology, the structure of its C-terminal domain, and the mechanism of protease inhibition, it belongs to the serpin superfamily, the largest class of plasma protease inhibitors, which also includes antithrombin, α1-proteinase inhibitor, plasminogen activator inhibitor, and many other structurally similar proteins that regulate diverse physiological systems. C1-INH is an inhibitor of proteases in the complement system, the contact system for kinin production, and the intrinsic coagulation pathway.

[0124] Low plasma content of C1-INH or its dysfunction can result in activation of both the complement and contact plasma cascades and may also affect other systems. It has been shown that a decrease in plasma C1-INH content below 55 μg / mL (approximately 25% of normal) induces spontaneous activation of C1. There are other ways in which the kallikrein-kinin system can be overactivated even in the presence of normal C1-INH activity. For example, there are known mutations in factor XII (FXII) that make it more easily activated and therefore more prone to subsequently activating prekallikrein to plasma kallikrein. In some embodiments, the rAAV vectors described herein are used to treat subjects with diseases or dysfunctions mediated by excessive plasma kallikrein activity.

[0125] A schematic diagram illustrating the rAAV vector approach for delivery of antibodies that bind to plasma kallikrein is shown in Figure 1. As shown in Figure 1, an rAAV vector containing a recombinant anti-plasma kallikrein antibody sequence is administered to a subject, resulting in the production of fused heavy and light chain mRNA transcripts. During translation of this transcript, separate heavy and light chain polypeptides are made, resulting in the production of functional anti-plasma kallikrein antibodies that are secreted into the circulation. Figure 2 shows an embodiment of the rAAV vector described herein.

[0126] Thus, the present disclosure provides, inter alia, rAAV vectors comprising codon-optimized nucleic acid sequences encoding antibodies useful for treating diseases, e.g., diseases associated with a malfunction of the kallikrein-kinin system. rAAV vectors can be constructed to encode antibodies that target selected protein members of the kallikrein-kinin system, such as, for example, plasma kallikrein.

[0127] In some embodiments, the rAAV vector encodes an anti-plasma kallikrein antibody. In some embodiments, the rAAV vector encodes an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain.

[0128] The present disclosure further provides, inter alia, methods of treating diseases using the rAAV vectors described herein. In some embodiments, the disease is a disease associated with excessive activity of the kallikrein-kinin cascade, such as a C1-INH deficiency or disorder.

[0129] In some embodiments, the C1-INH deficiency or disorder is HAE.

[0130] Vector Design Provided herein are vectors encoding anti-plasma kallikrein antibodies or antigen-binding fragments thereof. Vectors encoding anti-plasma kallikrein antibodies or antigen-binding fragments include viral and non-viral vectors. The viral vectors and other DNA expression vectors provided herein include any suitable method for transgene delivery to target cells, such as viral vectors and / or extracellular vesicles. Transgene delivery vehicles include viral vectors, liposomes, other lipid-containing complexes including lipid nanoparticles (LNPs), other macromolecular complexes, inorganic nanoparticles, synthetic modified mRNA, unmodified mRNA, small molecules, biologically inactive molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a targeted vector, such as a vector targeted to retinal pigment epithelial cells, CNS cells, muscle cells, or liver cells.

[0131] Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV8), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, Japanese hemagglutinin virus (HVJ), alphavirus, vaccinia virus, and retroviral vectors. Retroviral vectors include murine leukemia virus (MLV) and human immunodeficiency virus (HIV)-based vectors. Alphavirus vectors include Semliki Forest virus (SFV) and Sindbis virus (SIN). In some embodiments, the viral vectors provided herein are recombinant viral vectors. In some embodiments, the viral vectors provided herein are modified to be replication-deficient in humans. In some embodiments, the viral vector is a hybrid vector, e.g., an AAV vector disposed within a "helpless" adenoviral vector. In some embodiments, provided herein are viral vectors comprising a viral capsid from a first virus and a viral envelope protein from a second virus. In certain embodiments, the second virus is vesicular stomatitis virus (VSV). In some embodiments, the envelope protein is a VSV-G protein.

[0132] In some embodiments, the viral vector is an HIV-based viral vector, hi some embodiments, the HIV-based vector comprises at least two polynucleotides, the gag and pol genes from the HIV genome and the env gene from another virus.

[0133] In certain embodiments, the viral vector is a herpes simplex virus-based viral vector. In certain embodiments, the herpes simplex virus-based vectors are modified to not contain one or more immediate early (IE) genes, rendering them non-cytotoxic.

[0134] In certain embodiments, the viral vector is an MLV-based viral vector. In certain embodiments, the MLV-based vector contains up to 8 kb of heterologous DNA in place of viral genes.

[0135] In some embodiments, the viral vector is a lentiviral-based viral vector. In some embodiments, the lentiviral vector is derived from a human lentivirus. In some embodiments, the lentiviral vector is derived from a non-human lentivirus. In some embodiments, the lentiviral vector is packaged into a lentiviral capsid. In some embodiments, the lentiviral vector comprises one or more of the following elements: a long terminal repeat, a primer binding site, a polypurine tract, an att site, and an encapsidation site.

[0136] In certain embodiments, the viral vector is an alphavirus-based viral vector. In certain embodiments, the alphavirus vector is a recombinant replication-deficient alphavirus. In certain embodiments, the alphavirus replicons in the alphavirus vector are targeted to specific cell types by displaying functional heterologous ligands on their virion surface.

[0137] In some embodiments, the viral vector is an AAV-based viral vector. In some embodiments, the AAV-based vector does not encode the AAV rep gene (required for replication) and / or the AAV cap gene (required for synthesis of capsid proteins) (the rep and cap proteins can be provided in trans by the packaging cell). Multiple AAV serotypes have been identified. In some embodiments, the AAV-based vectors provided herein comprise components from one or more serotypes of AAV. In some embodiments, the viral vectors provided herein are recombinant adeno-associated virus (rAAV) vectors.

[0138] In some aspects, provided herein are recombinant adeno-associated virus (rAAV) vectors comprising codon-optimized nucleic acid sequences encoding an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain.

[0139] In some embodiments, the rAAV vectors described herein generate fused anti-plasma kallikrein antibody heavy chains and anti-plasma kallikrein antibody light chains in an mRNA transcript. The fused heavy and light chain transcripts are then cleaved to generate functional anti-plasma kallikrein antibodies, which are secreted into the circulation. Thus, in some embodiments, the rAAV vectors described herein provide a single gene cassette containing both anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain sequences. In some embodiments, the liver acts as a depot after administration of the rAAV vector.

[0140] In some embodiments, the rAAV vector produces one or more mRNA transcripts linked together by an mRNA linkage. Thus, in some embodiments, the rAAV vector produces one mRNA transcript comprising heavy and light chain nucleotides linked together by an mRNA linkage. In some embodiments, the rAAV vector produces multiple mRNA transcripts comprising heavy and light chain nucleotides linked together by an mRNA linkage. In some embodiments, the mRNA linkage is subsequently cleaved, and the heavy and light chain polypeptides are expressed as separate entities during translation. In some embodiments, the mRNA linkage remains intact, and the heavy and light chain polypeptides are expressed as separate entities during translation.

[0141] In some embodiments, a linker connects the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain. Various types of linkers can be used in rAAV vectors. In some embodiments, the linker is a glycine / serine linker, i.e., a peptide linker consisting essentially of glycine and serine. In exemplary embodiments, the linker comprises GS or GSG. In some embodiments, the linker is GSG. In another embodiment, the linker comprises a Gly-Ser-Gly (GSG) motif, e.g., GGSG (SEQ ID NO: 7), (GS)x3 (SEQ ID NO: 12), (GGSG)x2 (SEQ ID NO: 8), SGGSGGSGG (SEQ ID NO: 9), GGSGGGSGGGSG (SEQ ID NO: 10), or (GGGGS)x3 (SEQ ID NO: 11).

[0142] In some embodiments, the linker is a cleavable linker. Many types of cleavable linkers are known in the art, for example, those that are enzymatically cleavable. In some embodiments, the linker is a furin- or thrombin-cleavable linker. In some embodiments, the linker is a furin-cleavable linker.

[0143] In some embodiments, the furin-cleavable linker is followed by a 2A sequence. Various types of 2A sequences are known in the art, including, for example, T2A, P2A, E2A, or F2A. In some embodiments, the 2A sequence is T2A. In some embodiments, the 2A sequence is P2A. In some embodiments, the 2A is E2A. In some embodiments, the 2A is F2A.

[0144] In some embodiments, the AAV vector has an IRES sequence. In some embodiments, the linker comprises an IRES sequence.

[0145] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are controlled by a single promoter. Such a configuration will result in the production of a transcription product containing one fused heavy and light chain, which, after cleavage of the fused heavy and light chain sequences, will result in two polypeptide products.

[0146] In some embodiments, the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are controlled by separate promoters.

[0147] Various types of promoters can be used in the rAAV vectors described herein. These include, for example, ubiquitous, tissue-specific, and regulatable (e.g., inducible or repressible) promoters. In some embodiments, the promoter is modified. Various types of modified promoters are known in the art, including, for example, truncated minimal promoters, among others. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is a chicken beta-actin promoter. In some embodiments, the promoter is a liver-specific promoter. Examples of suitable liver-specific promoters include the human transthyretin promoter (TTR), modified hTTR (hTTR mod.), α-antitrypsin promoter, liver promoter 1 (LP1), TRM promoter, human factor IX pro / liver transcription factor responsive oligomer, LSP, CMV / CBA promoter (1.1 kb), CAG promoter (1.7 kb), mTTR, modified mTTR, mTTR pro, mTTR enhancer, and basic albumin promoter. Liver-specific promoters are described, for example, in Zhijian Wu et al., Molecular Therapy vol. 16, no. 2, February 2008, the contents of which are incorporated herein by reference.

[0148] rAAV vectors may contain additional enhancer or regulatory elements (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, IRES, etc.) to facilitate mRNA transcription and / or translation. In some embodiments, the vector comprises 5' and 3' inverted repeats (ITRs). In some embodiments, the vector comprises one or more enhancer elements. In some embodiments, the vector comprises a poly(A) tail. In some embodiments, the rAAV vector comprises a liver-specific regulatory element / region (HCR). In some embodiments, the rAAV vector comprises an ApoE enhancer. In some embodiments, the rAAV vector comprises a liver-specific nucleic acid regulatory element, such as a cis-regulatory element (CRE). CREs are described in EP18202888, the contents of which are incorporated herein by reference in their entirety. Exemplary CREs include, for example, CRE4 and CRE6. In some embodiments, CRE4 is used in combination with the apolipoprotein A-II gene. In some embodiments, CRE6 is used in combination with the apolipoprotein CI gene.

[0149] In some embodiments, the rAAV vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Various optimized or variant forms of WPRE can be used with the vectors described herein, including, for example, WPRE wild-type, WPRE3, and WPREmut6delATG, among others. WPRE and related WPRE variants are described in U.S. Patent No. 10,179,918; U.S. Patent No. 7,419,829; U.S. Patent No. 9,731,033; U.S. Patent No. 8,748,169; U.S. Patent No. 7,816,131; U.S. Patent No. 8,865,881; U.S. Patent No. 6,287,814; U.S. Patent Publication No. 2016 / 0199412; U.S. Patent Publication No. 2017 / 0114363; U.S. Patent Publication No. 2017 / 0360961; U.S. Patent Publication No. 2019 / 0032078; U.S. Patent Publication No. 2018 / 0353621; International Publication No. WO2017201527; International Publication No. WO2018152451; International Publication No. WO2013153361; International Publication No. WO2014144756; European Patent No. EP1017785; and European Patent Publication No. 3440191. Each of the foregoing publications is incorporated herein by reference in its entirety.

[0150] In some embodiments, the rAAV vector comprises a WPRE element and / or a cluster of transcription factor binding sites. Thus, in some embodiments, the rAAV vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the rAAV vector comprises a cluster of transcription factor binding sites.

[0151] In some embodiments, the rAAV vector comprises a cis-regulatory module (CRM). Various types of CRMs are suitable for use in the vectors described herein, including, for example, a liver-specific CRM, a neuron-specific CRM, and / or CRM8. Thus, in some embodiments, the CRM is a liver-specific CRM. In some embodiments, the CRM is a neuron-specific CRM. In some embodiments, the CRM is CRM8. In some embodiments, the vector comprises multiple CRMs. For example, in some embodiments, the vector comprises 2, 3, 4, 5, or 6 CRMs. In some embodiments, the vector comprises three CRMs, e.g., three CRM8s.

[0152] The rAAV vector comprises a secretion signal that is a naturally occurring and / or artificial signal peptide (e.g., recombinantly engineered). In some embodiments, the secretion signal is a naturally occurring signal peptide. In some embodiments, the secretion signal is an artificial signal peptide (e.g., recombinantly engineered). In some embodiments, the secretion signal is a human secretion signal. In some embodiments, the secretion signal is a murine secretion signal.

[0153] In some embodiments, the rAAV vector is sequence optimized to increase transcript stability, for more efficient translation, and for reduced immunogenicity. In some embodiments, the rAAV vector comprising the anti-plasma kallikrein heavy and light chains is sequence optimized to increase transcript stability, for more efficient translation, and for reduced immunogenicity. In some embodiments, the anti-plasma kallikrein heavy and light chains are codon optimized.

[0154] In some embodiments, the rAAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, the rAAV vector is AAV1. In some embodiments, the rAAV vector is AAV2. In some embodiments, the rAAV vector is AAV3. In some embodiments, the rAAV vector is AAV4. In some embodiments, the rAAV vector is AAV5. In some embodiments, the rAAV vector is AAV6. In some embodiments, the rAAV vector is AAV7. In some embodiments, the rAAV vector is AAV8. In some embodiments, the rAAV vector is AAV9. In some embodiments, the rAAV vector is AAV10. In some embodiments, the rAAV vector is AAV11.

[0155] In some aspects, provided herein are nucleic acids comprising nucleotide sequences encoding an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is a combination of DNA or RNA. In some embodiments, provided herein are vectors comprising nucleotide sequences encoding an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain.

[0156] In some embodiments, the nucleotide sequence is operably linked to a promoter. In some embodiments, the promoter is a liver-specific promoter. Examples of liver-specific promoters include the human transthyretin promoter (TTR) and modified hTTR (hTTR mod.). Various suitable promoters that can be used in various embodiments are described above.

[0157] In some embodiments, the nucleotide sequence is operably linked to a cis-actin regulatory module (CRM). In some embodiments, the CRM comprises a liver-specific CRM. In some embodiments, three CRMs are included, for example, three CRMs. Various types of suitable CRMs that can be used in various embodiments are described herein.

[0158] In some embodiments, the nucleotide sequence is operably linked to a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE is WPREmut6. Various optimized or variant forms of WPRE are known in the art and are described herein.

[0159] In some embodiments, the nucleotide sequence is operably linked to a secretion signal that is a naturally occurring or artificial signal peptide (e.g., recombinantly engineered). In some embodiments, the secretion signal is a naturally occurring signal peptide. In some embodiments, the secretion signal is an artificial signal peptide (e.g., recombinantly engineered). In some embodiments, the secretion signal is a human secretion signal. In some embodiments, the secretion signal is a murine secretion signal.

[0160] Anti-plasma kallikrein antibodies Exemplary heavy and light chain anti-plasma kallikrein amino acid sequences encoded by rAAV vectors are shown in Tables 1-2 below.

[0161] In some embodiments, the anti-plasma kallikrein antibody is engineered to have an extended half-life. To this end, in some embodiments, the anti-plasma kallikrein antibody comprises NHance mutations (i.e., H433K and N434F). In some embodiments, the anti-plasma kallikrein antibody comprises YTE mutations (i.e., M252Y / S254T / T256E).

[0162] In some embodiments, anti-plasma kallikrein antibodies are engineered to have reduced interaction with Fc receptors. To this end, in some embodiments, the anti-plasma kallikrein antibody comprises the LALA mutation (i.e., L234A and L235A).

[0163] In some embodiments, the anti-plasma kallikrein antibody is engineered to have reduced CpG and repeat sequences. In some embodiments, the anti-plasma kallikrein antibody is engineered to normalize to the GC content percentage of native AAV8. To this end, in some embodiments, the anti-plasma kallikrein antibody comprises the 2930-LALA mutation.

[0164] In some embodiments, the anti-plasma kallikrein antibody is fused to albumin or an FcRn-interacting peptide.

[0165] In some embodiments, the heavy and light chain sequences are about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequences set forth in the tables below. In some embodiments, the heavy and light chain sequences are about 50% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 55% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 60% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 65% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 70% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 75% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 80% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 85% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 90% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 95% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are about 100% identical to the sequences set forth in Tables 1-2. In some embodiments, the heavy and light chain sequences are identical to the sequences set forth in Tables 1-2.

[0166] [Table 1]

[0167] [Table 2]

[0168] Exemplary codon-optimized nucleotide sequences encoding anti-plasma kallikrein antibodies are shown in Table 3 below.

[0169] In some embodiments, the heavy and light chain sequences are about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequences set forth in the tables below. In some embodiments, the heavy and light chain sequences are about 50% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 55% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 60% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 65% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 70% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 75% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 80% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 85% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 90% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 95% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are about 100% identical to the sequences set forth in Table 3. In some embodiments, the heavy and light chain sequences are identical to the sequences set forth in Table 3.

[0170] [Table 3-1]

[0171] [Table 3-2]

[0172] [Table 3-3]

[0173] [Table 3-4]

[0174] Table 3-5

[0175] Table 3-6

[0176] Table 3-7

[0177] Table 3-8

[0178] Table 3-9

[0179] Table 3-10

[0180] Table 3-11

[0181] Table 3-12

[0182] Table 3-13

[0183] Table 3-14

[0184] In some embodiments, an exemplary anti-plasma kallikrein antibody has a heavy chain CDR1 comprising FTFSHYIMM (SEQ ID NO: 17). In some embodiments, an exemplary anti-plasma kallikrein antibody has a heavy chain CDR2 comprising GIYSSGGITVYADSVKGRFTI (SEQ ID NO: 18). In some embodiments, an exemplary anti-plasma kallikrein antibody has a heavy chain CDR3 comprising RRIGVPRRDEFDI (SEQ ID NO: 19). In some embodiments, an exemplary anti-plasma kallikrein antibody has a heavy chain CDR1 comprising FTFSHYIMM (SEQ ID NO: 17), a CDR2 comprising GIYSSGGITVYADSVKGRFTI (SEQ ID NO: 18), and a CDR3 comprising RRIGVPRRDEFDI (SEQ ID NO: 19).

[0185] In some embodiments, an exemplary anti-plasma kallikrein antibody has a light chain CDR1 comprising RASQSISSWLA (SEQ ID NO: 20). In some embodiments, an exemplary anti-plasma kallikrein antibody has a light chain CDR2 comprising YKASTLESGVPSRF (SEQ ID NO: 21). In some embodiments, an exemplary anti-plasma kallikrein antibody has a light chain CDR3 comprising QQYNTYWT (SEQ ID NO: 22). In some embodiments, an exemplary anti-plasma kallikrein antibody has a light chain CDR1 comprising RASQSISSWLA (SEQ ID NO: 20), a light chain CDR2 comprising (SEQ ID NO: 21), and a light chain CDR3 comprising QQYNTYWT (SEQ ID NO: 22).

[0186] In some embodiments, an exemplary anti-plasma kallikrein antibody has a heavy chain CDR1 comprising FTFSHYIMM (SEQ ID NO: 17), a CDR2 comprising GIYSSGGITVYADSVKGRFTI (SEQ ID NO: 18), and a CDR3 comprising RRIGVPRRDEFDI (SEQ ID NO: 19). In some embodiments, an exemplary anti-plasma kallikrein antibody has a light chain CDR1 comprising RASQSISSWLA (SEQ ID NO: 20), a light chain CDR2 comprising YKASTLESGVPSRF (SEQ ID NO: 21), and a light chain CDR3 comprising QQYNTYWT (SEQ ID NO: 22).

[0187] In some embodiments, the CDRs disclosed herein have 1, 2, 3, or 4 amino acid substitutions, deletions, or insertions relative to the CDRs described herein. In some embodiments, the CDRs disclosed herein contain no more than 3, 2, or 1 amino acid substitutions, deletions, or insertions compared to the described CDR sequences. In some embodiments, affinity matured variants are obtained with desired binding properties. Various affinity matured CDR sequences are provided in WO2014152232, the contents of which are incorporated herein by reference in their entirety.

[0188] Exemplary anti-plasma kallikrein antibodies of the present disclosure include, but are not limited to, IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, and IgAsec), IgD, IgE, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, scFv, scFv-Fc, and SMIP binding sites. In certain embodiments, the anti-plasma kallikrein antibody encodes the heavy and light chain sequences of lanadelumab. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is not an antibody fragment. In some embodiments, the antibody is not a Fab.

[0189] In certain embodiments, the antibody is an scFv. The scFv can include, for example, a flexible linker that allows the scFv to orient in different directions to enable antigen binding. In various embodiments, the antibody can be a cytosol-stable scFv or intrabody, which retains its structure and function in the reducing environment within the cell (see, e.g., Fisher and DeLisa, J. Mol. Biol. 385(1):299-311, 2009, incorporated herein by reference). In certain embodiments, the scFv is converted into an IgG or chimeric antigen receptor according to methods described herein. In embodiments, the antibody binds to both denatured and native protein targets. In embodiments, the antibody binds to either denatured or native proteins. In some embodiments, the antibody binds to select members of the complement system. In some embodiments, the antibody binds to plasma kallikrein.

[0190] In most mammals, including humans, intact antibodies have at least two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3) and a hinge region between CH1 and CH2. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0191] Antibodies include all known forms of antibodies and other protein scaffolds with antibody-like properties. For example, antibodies can be monoclonal, polyclonal, human, humanized, bispecific, monovalent, chimeric, or protein scaffolds with antibody-like properties, such as fibronectin or ankyrin repeats. Antibodies can have any of the following isotypes: IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, and IgAsec), IgD, or IgE.

[0192] An antibody fragment may include one or more segments derived from an antibody. Segments derived from an antibody may retain the ability to specifically bind to a particular antigen. An antibody fragment may be, for example, a Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, scFv, or SMIP. An antibody fragment may be, for example, a diabody, triabody, affibody, nanobody, aptamer, domain antibody, linear antibody, single-chain antibody, or any of a variety of multispecific antibodies that can be formed from antibody fragments.

[0193] Examples of antibody fragments include: (i) Fab fragment: a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment: a fragment consisting of the VH and CH1 domains; (iv) Fv fragment: a fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragment: a fragment comprising the VH and VL domains; (vi) dAb fragment: a fragment which is a VH domain; (vii) dAb fragment: a fragment which is a VL domain; (viii) an isolated complementarity-determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be connected by one or more synthetic linkers. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods, for example, by a synthetic linker that allows them to be expressed as a single protein in which the VL and VH regions pair to form a monovalent binding site (known as a single-chain Fv (scFv)). Antibody fragments can be obtained using conventional techniques known to those skilled in the art and, in some instances, can be used in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibody fragments can further include any of the antibody fragments described above with the addition of additional C-terminal amino acids, N-terminal amino acids, or amino acids separating the individual fragments.

[0194] An antibody may be called chimeric if it contains one or more antigenic determining regions or constant regions from a first species and one or more antigenic determining regions or constant regions from a second species. Chimeric antibodies can be constructed, for example, by genetic modification. Chimeric antibodies may contain immunoglobulin gene segments from different species (e.g., from mouse and human).

[0195] Use of rAAV vectors encoding anti-plasma kallikrein antibodies for the treatment of disease Described herein are methods for treating a disease associated with unregulated plasma kallikrein activity, such as a C1 esterase inhibitor deficiency or disorder, in a subject in need thereof, comprising administering an AAV vector encoding an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain. After administration of the rAAV vector described herein, the anti-plasma kallikrein antibody heavy and light chains assemble into functional antibodies. The functional antibodies are secreted into the circulation and bind to plasma kallikrein.

[0196] The rAAV vectors described herein can be used to treat any C1 esterase inhibitor deficiency or disorder and / or disorder mediated by dysregulation of plasma kallikrein activity. In some embodiments, the disorder is hereditary angioedema (HAE), acquired angioedema (AAE), rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, thrombus formation induced by a ventricular assist device or stent, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event, restenosis, systemic lupus erythematosus nephritis / vasculitis, diabetic macular edema, or burn injury. In some embodiments, the C1 esterase inhibitor deficiency or disorder is HAE. The HAE can be any type of HAE, including HAE types I, II, or III.

[0197] In some embodiments, the rAAV vector maintains episomal status after administration to a subject in need thereof. In some embodiments, the rAAV vector does not maintain episomal status after administration to a subject in need thereof. For example, in some embodiments, the rAAV vector integrates into the subject's genome. Such integration can be achieved, for example, by using various gene editing techniques, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENS), ARCUS genome editing, and / or CRISPR-Cas systems.

[0198] In some embodiments, pharmaceutical compositions containing the rAAV vectors described herein are used to treat subjects in need thereof. Pharmaceutical compositions containing the rAAV vectors or particles of the present invention contain a pharmaceutically acceptable excipient, diluent, or carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solution, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and administered to subjects in therapeutically effective amounts.

[0199] The rAAV vector is administered to a subject in need thereof via a suitable route. In embodiments, the rAAV vector is administered intravenously, intraperitoneally, subcutaneously, or intradermally. In embodiments, the rAAV vector is administered intravenously. In embodiments, intradermal administration includes administration using a "gene gun" or a biolistec particle delivery system. In some embodiments, the rAAV vector is administered via a non-viral lipid nanoparticle. For example, a composition comprising an rAAV vector may include one or more diluents, buffers, liposomes, lipids, or lipid complexes. In some embodiments, the rAAV vector is contained within a microsphere or nanoparticle, for example, a lipid nanoparticle.

[0200] In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the subject's plasma about 2-6 weeks after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the subject's plasma at about 2 weeks. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the subject's plasma at about 3 weeks. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the subject's plasma at about 4 weeks. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the subject's plasma at about 5 weeks. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the subject's plasma at about 6 weeks. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the subject's liver cells about 2-6 weeks after administration of the rAAV vector.

[0201] In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 3 months, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years after administration of the rAAV vector. Thus, in some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 3 months after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 6 months after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 12 months after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 2 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 3 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 4 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 5 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 6 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 7 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 8 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 9 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject at least 10 years after administration of the rAAV vector. In some embodiments, functional anti-plasma kallikrein antibodies are detectable in the plasma of a subject for the remainder of the subject's life after administration of the rAAV vector.In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of active anti-PKa antibodies to the same extent as that seen after administration of purified anti-PKa IgG delivered intravenously. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of greater amounts of active anti-PKa antibodies compared to administration of purified anti-PKa IgG delivered intravenously.

[0202] In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 60% active anti-PKa antibodies. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 65% active anti-PKa antibodies. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 70% active anti-PKa antibodies. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 75% active anti-PKa antibodies. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 80% active anti-PKa antibodies. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 85% active anti-PKa antibodies. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 90% active anti-PKa antibodies. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 95% active anti-PKa antibodies. In some embodiments, administered rAAV comprising anti-plasma kallikrein antibody heavy chain and anti-plasma kallikrein antibody light chain antibodies results in the production of at least 99% active anti-PKa antibodies.

[0203] In some embodiments, after administration of an AAV vector to a subject, the level of plasma kallikrein IgG detectable in the circulation is about 4-10 times higher than the level of IgG detectable after direct administration of purified plasma kallikrein antibody to the subject. In some embodiments, after administration of an AAV vector to a subject, the level of detectable active plasma kallikrein IgG meets or exceeds human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration of an rAAV vector is about 2-35 times the human therapeutic level. In some embodiments, the level of active plasma kallikrein IgG after administration is about 2-fold the human therapeutic level. In some embodiments, the level of active plasma kallikrein IgG after administration is about 3-fold the human therapeutic level. In some embodiments, the level of active plasma kallikrein IgG after administration is about 4-fold the human therapeutic level. In some embodiments, the level of active plasma kallikrein IgG after administration is about 5-fold the human therapeutic level. In some embodiments, the level of active plasma kallikrein IgG after administration is about 6-fold the human therapeutic level. In some embodiments, the level of active plasma kallikrein IgG after administration is about 6-fold higher than human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration is about 7-fold higher than human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration is about 8-fold higher than human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration is about 9-fold higher than human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration is about 10-fold higher than human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration is about 15-fold higher than human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration is about 20-fold higher than human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration is about 25-fold higher than human therapeutic levels. In some embodiments, the level of active plasma kallikrein IgG after administration is about 30-fold higher than human therapeutic levels.In some embodiments, the level of active plasma kallikrein IgG following administration is about 35-fold the human therapeutic level.

[0204] Thus, administration of an rAAV vector comprising an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain results in sustained and robust expression compared to a single administration of purified anti-plasma kallikrein antibody to a subject in need.

[0205] In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 50-95%. Thus, in some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 50%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 55%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 60%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 65%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 70%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 75%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 75%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 80%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 85%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 90%. In some embodiments, the administered rAAV vector produces anti-plasma kallikrein antibodies capable of inhibiting plasma kallikrein activity by about 95%. [Example]

[0206] Other features, objects, and advantages of the present invention will become apparent in the following examples. However, it should be understood that the examples, while illustrating embodiments of the present invention, are given by way of illustration only, and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the examples.

[0207] Example 1. Vector design This example provides an exemplary method and design for producing rAAV expression constructs (rAAV vectors) containing codon-optimized nucleic acid sequences encoding anti-kallikrein antibodies and their variants. A recombinant AAV vector (rAAV8) was used in this study. The basic design of an rAAV vector comprises an expression cassette flanked by inverted terminal repeats (ITRs): the 5'-ITR and the 3'-ITR. These ITRs mediate replication and packaging of the vector genome by the AAV replication protein Rep and associated factors in vector producer cells. Typically, the expression cassette contains a promoter, coding sequence, poly(A) tail, and / or tag. An expression construct encoding a codon-optimized vectorized anti-plasma kallikrein (PKa)-IgG antibody was designed and prepared using standard molecular biology techniques. The coding sequence for the anti-PKa antibody heavy chain (HC) and the coding sequence for the anti-PKa antibody light chain (LC) were inserted downstream of the chicken B-actin promoter (CB). In another exemplary method and design, the promoter (+ / - enhancer) is a liver-specific promoter containing 3xCRM8 / hTTR. In some embodiments, the expression cassette also contains a WPRE element and a human secretory signal (SS). In some embodiments, the expression cassette also contains an intron. A short linker containing an oligonucleotide encoding a furin-cleavable site (F / 2A) was inserted between the HC and LC. A 168-bp SV40 pol A sequence and a DNA titer sequence were inserted downstream of the IgG LC. Figure 2 illustrates a schematic diagram of the expression construct. The expression construct was then ligated into an AAV vector and tested by sequencing. The vector was packaged into viral particles and stored.

[0208] Any number of variations of the above scheme can be implemented. Alternative constructs can be obtained by replacing the coding sequences for the HC and LC with coding sequences for fragment antigen-binding (Fab); by replacing the anti-PKa coding sequence with a variant containing a leucine-to-alanine mutation (LALA) that prevents interaction with Fc receptors. Another alternative construct can be obtained by replacing the anti-PKa coding sequence with a variant containing a specific leucine-to-alanine mutation, 2930-LALA, that reduces CpG dinucleotides and repeat sequences and normalizes to the guanine-cytosine content (GC content) percentage of native AAV8. Multiple promoters can also be used, and / or an IRES sequence can be introduced upstream of the LC.

[0209] Additionally, vector constructs were designed with the goal of reducing CpG content and repeat sequences. The vector constructs were also engineered to normalize the GC content percentage to that found in native, unmodified AAV8. Each of the designed vector constructs was evaluated for CpG content, codon adaptation index (CAI), codon context (CC), GC content, and repeat motifs. Data from these studies are shown in Tables 4 and 5 below.

[0210] [Table 4]

[0211] [Table 5]

[0212] Example 2. Expression of active IgG antibodies in vitro by rAAV-directed POOR codon-optimized anti-PKa2930-LALA constructs Figure 3 shows the expression of active IgG levels in HepG2 cell culture media after 48 and 72 hours of transfection of HepG2 cells with a plasmid containing the POOR codon-optimized anti-PKa2930-LALA construct. Constructs A010 and A013 contain the same codon optimization as construct B021. Construct B021 is a GO-vectored anti-PKa IgG+LALA construct variant.

[0213] HepG2 cells transfected with the A013 construct expressed high levels of active IgG despite having a large CpG repeat. In contrast, the A017 construct, which has a reduced CpG repeat, did not express any active IgG levels in HepG2 cells. Among all the POOR codon-optimized constructs, only the A016 construct expressed active IgG levels in HepG2 cells. However, the level of active IgG expression in the A016 construct was only about one-tenth that of the A013 construct. The A010 construct expressed active IgG similar to that of the A013 construct. Figure 3 and Table 4 show that there is no direct correlation between CpG amount and active IgG expression.

[0214] Example 3. Expression of active IgG antibodies in vitro by rAAV-directed GO-vectored anti-PKa2930-LALA constructs To evaluate the expression of GO vectored constructs in vitro, HepG2 cells (1.6 x 10 6 Cells / well (12-well plate) were transfected with a plasmid (pAAV) encoding the GO-vectored anti-PKa2930-LALA construct. A green fluorescent protein (GFP) plasmid was used as a control. Media was collected after 72 and 96 hours of transfection, and an MSD assay was performed as described below to determine the levels of active anti-PKa antibodies.

[0215] For the MSD assay, MSD standard 96-well plates were coated with 4 μg / ml plasma kallikrein (Enzyme Research Labs #HPKa1303) diluted in pH 9.4 carbonate-bicarbonate buffer to a final volume of 30 μl / well. Plates were then incubated overnight at 4°C. The following day, plates were washed five times with 300 μl of wash buffer (PBS + 0.05% Tween-20) and blocked for 1 hour in 150 μl of 5% BSA / PBS. Anti-PKa-LALA-IgG (#W28593, in-house) titrations were prepared in 2% BSA, starting at a top concentration of 100 ng / ml. Seven 3-fold titrations were made. For the eighth point, plasma alone was added as a no-anti-PKa-LALA control. Cell culture supernatant was diluted 1:30 in 2% BSA, followed by serial 1:3 dilutions. After five washes, 30 μl of 0.5 μg / ml sulfotagromosa anti-human IgG antibody (Jackson 709-006-149) was added to all wells. The plate was incubated at room temperature with shaking for 1 hour. After the final wash, 150 μl of 0.5x MSD read buffer was added to the wells, which were then read on the MSD Reader within 5 minutes.

[0216] All treatment groups, except for the B011 construct, expressed some levels of active IgG, as shown in Figure 4. However, cells transfected with B041, B044, B048, B050, B063, and B021 (new construct) expressed higher levels of active IgG compared to the other constructs. The B050 construct expressed the highest level of active IgG. However, as shown in Table 5, the B050 construct also contains a higher amount of CpG content. High CpG content may be associated with increased immunogenicity. The other constructs, B041, B044, B048, B063, and B021 (new), showed higher expression of active IgG, but they also contain low CpG content. B011 is a negative control plasmid and does not express any protein.

[0217] Example 4. Expression of active IgG antibodies in vivo by rAAV-directed GO-vectored anti-PKa2930-LALA constructs The exemplary study described below was designed to test rAAV-induced expression of anti-PKA antibodies. C57B6 mice were injected with rAAV vectors expressing (a) the anti-PKa B041 construct, (b) the anti-PKa B048 construct, and (c) the anti-PKa B021 construct, as described in Table 6.

[0218] [Table 6]

[0219] C57B6 mice were injected with AAV vectors on day 0 (5x10 11 or 5x10 12 Plasma was collected 14 days (2 weeks) after intravenous injection of rAAV (vg / kg), and active anti-PKa antibodies in the plasma were determined by MSD assay. Briefly, PKa protein was coated onto an MSD plate to capture anti-PKa mAb present in the plasma. Then, an anti-human IgG detection antibody was used to quantitate the expressed active IgG in the plasma.

[0220] 5x10 rAAV8-PKa-B041 construct 12 Mice injected with a dose of 5x10 vg / kg expressed high levels of active IgG as shown in Figure 5. 12 Similar results were obtained with the rAAV8-PKa-B021 construct at a dose of 1000 vg / kg, although the level of active IgG expression was less reproducible compared with that of the rAAV8-PKa-B041 construct.

[0221] Example 5: Evaluation of the ex vivo efficacy of anti-PKa antibodies generated in the plasma of mice treated with rAAV8 This study demonstrates the ex vivo bioactivity of anti-PKa antibodies generated in plasma samples from rAAV8-treated mice collected 28 days after intravenous administration of the rAAV8 construct. In this study, the kallikrein-kinin pathway was activated in control, untreated mouse plasma samples by adding ellagic acid in the presence of stepwise increases (titration) of the exogenous inhibitor Takhzyro™. Takhzyro™ (lanadelumab-furio) is an FDA-approved fully human monoclonal antibody drug for hereditary angioedema (HAE) attacks in patients aged 12 years and older. PKa activity in plasma was monitored via the addition of a PKa-specific fluorogenic substrate (PFR-AMC) and subsequent fluorescence measurements over time. To test the bioactivity of anti-PKa antibodies generated in rAAV8-treated mouse plasma samples, the kallikrein-kinin pathway was similarly activated by adding ellagic acid to plasma from these mice, and PKa activity was measured. Specifically, post-dose plasma from individual rAAV8-treated mice was serially diluted into pre-dose plasma samples from the same mice prior to the addition of ellagic acid and PFR-AMC to determine dose response.

[0222] Bioactivity was measured in terms of percent inhibition of plasma kallikrein activity as a function of anti-PKa antibody concentration from these dilution series, with higher antibody levels resulting in lower %PKa activity. Figures 6-7 show the results of this study. Figures 6-7 show the ex vivo bioactivity of anti-PKa antibodies generated in rAAV8-treated mouse plasma samples collected 14 days after intravenous administration of the rAAV8 B041 and rAAV8 B048 constructs, respectively. Day 14 plasma samples from individual mice were titrated to day 0 plasma from the same mice to maintain similar levels of kallikrein-kinin pathway components but dilute out the anti-PKa mAb transgene protein. The results demonstrate that the dose response of anti-PKa antibodies generated in rAAV8-treated mice is identical to that of the FDA-approved drug, Takhzyro™. This demonstrates that the anti-PKa antibodies generated in the plasma of rAAV8-treated mice were of extremely high overallity, indistinguishable from the Takhzyro™ drug product.

[0223] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

1. 1. A recombinant adeno-associated virus (rAAV) vector comprising a codon-optimized nucleotide sequence, the rAAV vector encoding a full-length antibody comprising an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain, the codon-optimized nucleotide sequence having at least 80% identity to any one of SEQ ID NOs:23-26, 28-32, or 34-36.

2. The rAAV vector of claim 1, wherein the codon-optimized nucleotide sequence is selected from any one of SEQ ID NOs:23-26, 28-32, or 34-36.

3. 2. The rAAV vector of claim 1, wherein the codon-optimized nucleotide sequence has a CpG content of less than 40 CpG sites.

4. The rAAV vector of claim 2, wherein the codon-optimized nucleotide sequence has about 5 CpG sites.

5. The rAAV vector of any one of claims 1 to 4, wherein the codon-optimized nucleotide sequence encodes a linker.

6. The rAAV vector of claim 5 , wherein the linker comprises a cleavable linker.

7. The rAAV vector of claim 5 , wherein the linker comprises a non-cleavable linker.

8. The rAAV vector of any one of claims 1 to 7, wherein the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are controlled by a single promoter.

9. The rAAV vector of any one of claims 1 to 7, wherein the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are controlled by separate promoters.

10. An rAAV vector described in claim 8 or 9, wherein the single promoter or the separate promoters are selected from a ubiquitous promoter, a tissue-specific promoter, or a controllable promoter.

11. The rAAV vector of claim 10, wherein the tissue-specific promoter is a liver-specific promoter.

12. 12. The rAAV vector of claim 11, wherein the liver-specific promoter comprises a promoter selected from human transthyretin promoter (TTR), modified hTTR (hTTR mod.), alpha-antitrypsin promoter, liver promoter 1 (LP1), TRM promoter, human factor IX pro / liver transcription factor responsive oligomer, LSP, CMV / CBA promoter (1.1 kb), CAG promoter (1.7 kb), mTTR, modified mTTR, mTTR pro, mTTR enhancer, or basic albumin promoter.

13. The rAAV vector of claim 12, wherein the liver-specific promoter is the human transthyretin promoter (TTR).

14. The rAAV vector of claim 10, wherein the controllable promoter is an inducible or repressible promoter.

15. The rAAV vector of claim 13, wherein the vector further comprises one or more of the following: 5' and 3' inverted repeats, an intron upstream of the sequence, and a cis-acting regulatory module (CRM).

16. The rAAV vector of claim 15, wherein the vector further comprises a WPRE sequence.

17. The rAAV vector of claim 16, wherein the WPRE sequence is modified.

18. The rAAV vector of claim 17, wherein the WPRE contains a mut6delATG modification.

19. The rAAV vector of claim 18, wherein the CRM is a liver-specific CRM.

20. The rAAV vector of claim 19, wherein the CRM is CRM8.

21. The rAAV vector of claim 20, wherein the vector comprises at least three CRMs.

22. 22. The rAAV vector of claim 21 , wherein the vector comprises three CRM8s.

23. The rAAV vector of claim 22, wherein the rAAV vector comprises an IRES sequence.

24. 24. The rAAV vector of claim 23, wherein the anti-plasma kallikrein antibody light chain and / or heavy chain comprises one or more mutations that increase the half-life and / or reduce the effector function of the antibody.

25. 25. The rAAV vector of claim 24, wherein the one or more mutations include a LALA mutation (L234A and L235A) and / or an NHance mutation (H433K and N434F).

26. 26. The rAAV vector of claim 25, wherein the one or more mutations include a LALA mutation (L234A and L235A).

27. 27. The rAAV vector of any one of claims 1 to 26, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAVrh.

10.

28. The rAAV vector of claim 27, wherein the AAV vector is AAV8.

29. The rAAV vector of claim 28, wherein the rAAV vector has a GC content engineered to have the same GC content as naturally occurring AAV8.

30. 29. The rAAV vector of claim 27 or 28, wherein the capsid of the rAAV vector is engineered.

31. The rAAV vector of claim 30, wherein the capsid engineered rAAV vector comprises an AAV capsid sequence having a modified amino acid sequence.

32. The rAAV vector of claim 31 , wherein the modified amino acid sequence comprises an insertion, deletion or substitution of an amino acid sequence.

33. The rAAV vector of claim 27, wherein the capsid of the rAAV vector is naturally derived.

34. The rAAV vector of claim 27, wherein the capsid of the rAAV vector is AAV8.

35. The rAAV vector of claim 6, wherein the cleavable linker sequence comprises a sequence cleavable by Furin.

36. The rAAV vector of claim 35, wherein the furin-cleavable sequence is followed by a linker and a 2A sequence.

37. 37. The rAAV vector of claim 36, wherein the linker is a GSG linker.

38. 38. The rAAV vector of claim 36 or 37, wherein the 2A sequence is a T2A, P2A, E2A or F2A sequence.

39. 39. The rAAV vector of claim 38, wherein the 2A sequence is a P2A sequence.

40. 40. The rAAV vector of claim 39, wherein the vector further encodes a secretion signal.

41. The rAAV vector of claim 40, wherein the secretion signal is a naturally occurring signal peptide.

42. The rAAV vector of claim 40, wherein the secretion signal is an artificial signal peptide.

43. The rAAV vector of claim 40, wherein the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain generate a functional anti-plasma kallikrein antibody capable of binding to plasma kallikrein.

44. The rAAV vector of claim 43, wherein the anti-plasma kallikrein antibody inhibits the proteolytic activity of plasma kallikrein.

45. The rAAV vector of claim 44, wherein the anti-plasma kallikrein antibody binds to the plasma kallikrein active site.

46. The rAAV vector of any one of claims 43 to 45, wherein the binding occludes the active site of plasma kallikrein.

47. The rAAV vector of claim 46, wherein the binding inhibits the activity of plasma kallikrein.

48. 48. The rAAV vector of claim 47, wherein the antibody does not bind to prekallikrein.

49. 49. The rAAV vector of claim 48, wherein the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are expressed from the same vector.

50. The rAAV vector of any one of claims 1 to 48, wherein the anti-plasma kallikrein antibody heavy chain and the anti-plasma kallikrein antibody light chain are expressed from separate rAAV vectors.

51. 51. The rAAV vector of claim 49 or 50, wherein the vector further comprises 5' and 3' inverted repeats (ITRs), one or more enhancer elements, and / or a poly(A) tail.

52. 52. The rAAV vector of claim 51 , wherein the one or more enhancer elements are selected from a cluster of transcription factor binding sites and / or a WPRE sequence.

53. 1. A recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a codon-optimized nucleotide sequence having at least 80% identity to any one of SEQ ID NOs:23-26, SEQ ID NOs:28-32, or SEQ ID NOs:34-36, the vector comprising: a. 5' terminal inverted repeat (ITR); b. cis-acting regulatory modules (CRMs); c. a liver-specific promoter; e. Codon-optimized anti-plasma kallikrein antibody heavy chain sequences and anti-plasma kallikrein antibody light chain sequences; f. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and g. 3' ITR The rAAV vector comprising:

54. 54. The rAAV vector of claim 53, wherein the liver-specific promoter comprises a promoter selected from human transthyretin promoter (TTR), modified hTTR (hTTR mod.), alpha-antitrypsin promoter, liver promoter 1 (LP1), TRM promoter, human factor IX pro / liver transcription factor responsive oligomer, LSP, CMV / CBA promoter (1.1 kb), CAG promoter (1.7 kb), mTTR, modified mTTR, mTTR pro, mTTR enhancer, or basic albumin promoter.

55. 55. The rAAV vector of claim 54, wherein the liver-specific promoter comprises the human transthyretin promoter.

56. 56. The rAAV vector of claim 55, wherein the CRM is a liver-specific CRM.

57. 57. The recombinant vector of claim 56, wherein the vector comprises at least three CRMs.

58. 58. The rAAV vector of claim 57, wherein the vector comprises three CRM8s.

59. 59. The rAAV vector of claim 58, wherein the WPRE sequence is modified.

60. 60. The rAAV vector of claim 59, wherein the WPRE sequence is WPRE mut6delATg.

61. 61. Use of the recombinant adeno-associated virus (rAAV) vector of claim 60 in the manufacture of a medicament for the treatment of a disease or disorder associated with a deficiency or dysregulation of the activated kallikrein-kinin pathway.

62. 62. The use of claim 61, wherein the deficiency or dysregulation of the activated kallikrein-kinin pathway is a disease or disorder associated with a deficiency of C1 esterase inhibitor.

63. The use of claim 62, wherein the rAAV vector is administered by intravenous, subcutaneous, or transdermal administration.

64. 64. The use of claim 63, wherein the transdermal administration is by gene gun.

65. 65. The use according to any one of claims 62 to 64, wherein the disorder associated with a deficiency or dysregulation of the activated kallikrein-kinin pathway or a deficiency of C1 esterase inhibitor is hereditary angioedema (HAE), acquired angioedema (AAE), angioedema with normal C1 inhibitor, diabetic macular edema, migraine, tumor, neurodegenerative disease, rheumatoid arthritis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema, cerebral edema, pulmonary embolism, stroke, ventricular assist device or stent induced thrombus formation, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event, restenosis, systemic lupus erythematosus nephritis / vasculitis, or burn injury.

66. 66. The use according to claim 65, wherein the disorder associated with a deficiency of C1 esterase inhibitor is HAE.

67. 67. The use of claim 66, wherein the HAE is type I, II, or III.

68. The use of claim 67, wherein the rAAV vector is episomal following administration.

69. 69. The use of claim 68, wherein after administration, the anti-plasma kallikrein antibody heavy and light chains assemble into a functional antibody.

70. 70. The use of claim 69, wherein the antibody is an IgG.

71. The use of claim 70, wherein the functional anti-plasma kallikrein antibodies are detectable in the plasma of the subject about 2 to 6 weeks after administration of the rAAV vector.

72. The use of claim 71, wherein the functional anti-plasma kallikrein antibodies are detectable in the plasma of the subject about 4 weeks after administration of the rAAV vector.

73. A DNA expression cassette comprising a codon-optimized nucleotide sequence encoding a full-length antibody comprising an anti-plasma kallikrein antibody heavy chain and an anti-plasma kallikrein antibody light chain, said DNA expression cassette having at least 80% identity to any one of SEQ ID NOs:23-26, 28-32, or 34-36.

74. A delivery vehicle comprising the DNA expression cassette of claim 73.

75. 75. The delivery vehicle of claim 74, selected from a viral vector, a lipid nanoparticle, or an extracellular vesicle.

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