Vector constructs for delivery of nucleic acids encoding therapeutic anti-IGF-1R antibodies and methods of using same - Patents.com

Abstract

The present disclosure provides gene therapy vectors, antibody expression cassettes, and vectors comprising a nucleic acid encoding a therapeutic anti-IGF-1R antibody (e.g., a monoclonal antibody) or an antigen-binding fragment thereof, a delivery vector (e.g., a viral vector) comprising a nucleic acid encoding a therapeutic anti-IGF-1R antibody (e.g., a monoclonal antibody) or an antigen-binding fragment thereof that is a gene therapy construct, a composition comprising the same, and a method of using the same. Some aspects of the present disclosure are directed to recombinant adenoviral vector (rAAV) particles for delivery of a nucleic acid encoding a therapeutic anti-IGF-1R antibody (e.g., a monoclonal antibody) or an antigen-binding fragment thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 297,787, filed January 9, 2022, and U.S. Provisional Application No. 63 / 374,878, filed September 7, 2022, each of which is incorporated by reference herein in its entirety.

[0002] Reference to Electronically Submitted Sequence Listings The contents of the electronically submitted Sequence Listing (Name: 4525_074PC02_Seqlisting_ST26.xml; Size: 214,621 bytes; and Creation Date: January 9, 2023) submitted with this application are hereby incorporated by reference in their entirety.

[0003] Field of Disclosure The present disclosure relates to the medical field, including AAV gene therapy. [Background technology]

[0004] background Autoimmune diseases are the third most common human pathology after cancer and cardiovascular disease. The pathogenesis of autoimmune diseases is multifactorial, with genetic and environmental triggers arising from a background of significant defects in immune regulation. Graves' disease and Hashimoto's thyroiditis are the most prevalent autoimmune conditions in Western countries. In Graves' disease, hyperthyroidism results from thyroid activation by agonistic antibodies against the thyroid-stimulating hormone receptor (TSHR). TSHR is also expressed by orbital fibroblasts, and binding of TSHR antibodies to orbital fibroblasts leads to hyaluronan production and differentiation into adipocytes and myofibroblasts (Eckstein et al. Endocrine 68: 265-70, 2020). The consequence is increased orbital fat and fibrosis of the orbital connective tissue, particularly in the extraocular muscles, resulting in thyroid eye disease (TED), also known as Graves' orbitopathy (GO) (Eckstein et al. Endocrine 68: 265-70, 2020). Overexpression of the insulin-like growth factor 1 receptor (IGF-1R) and its interaction with the thyrotropin receptor (TSH-R) are key pathogenic features of this disease.

[0005] Inhibition of IGF-1R can be achieved with anti-IGF-1R monoclonal antibodies, such as teprotumumab (also known as teprotumumab-trbw; RG-1507) (sold under the brand name Tepezza). Teprotumumab binding inhibits signaling through the IGF-1R / TSH-R complex and downstream pathways. Teprotumumab was first approved in the United States in 2020 for the treatment of acute and chronic TED. Teprotumumab is a fully human IgG1 monoclonal antibody administered by intravenous infusion. For most indications, maintenance treatment requires repeated infusions, e.g., a recommended initial dose of 10 mg / kg, followed by a dose doubling to 20 mg / kg for seven additional infusions, each spaced three weeks apart, for a total of eight infusions. Infusions are generally given over 60–90 minutes in clinical settings, imposing a significant treatment burden on patients. Gene therapy involves the delivery of nucleic acids into patient cells to treat disease. Advances in the field of gene therapy have been achieved using viruses to deliver therapeutic genetic materials. Although various physical and chemical methods have been developed to introduce exogenous DNA into eukaryotic cells, viruses have generally been shown to be more effective for this purpose. Several DNA-containing viruses, such as parvoviruses, adenoviruses, herpesviruses, and poxviruses, as well as RNA-containing viruses, such as retroviruses, have been used to develop eukaryotic cloning and expression vectors. Some challenges with viral vectors include low efficiency, DNA packaging capacity, and lack of target cell specificity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Eckstein et al. Endocrine 68: 265-70, 2020 Summary of the Invention [Means for solving the problem]

[0007] A brief overview Certain aspects of the present disclosure relate to the development of polynucleotides (e.g., antibody expression cassettes) encoding anti-insulin-like growth factor 1 receptor (anti-IGF-1R) antibodies or antigen-binding fragments thereof for use in gene therapy; vectors (e.g., viral vectors) comprising the same; recombinant adeno-associated virus (rAAV) particles comprising the same; compositions comprising the same, which are suitable for delivery of polynucleotides encoding anti-IGF-1R antibodies or antigen-binding fragments thereof to a desired target site; and methods of using the same. In some aspects, the present disclosure is directed to rAAV delivery of antibody expression cassettes encoding anti-IGF-1R antibodies or antigen-binding fragments thereof to a subject in need thereof (e.g., a subject suffering from Graves' orbitopathy).

[0008] Certain aspects of the present disclosure are directed to recombinant adeno-associated virus (rAAV) particles comprising a capsid and a vector genome, wherein the vector genome comprises inverted terminal repeats (ITRs) and an antibody expression cassette, and the antibody expression cassette comprises (a) a promoter, (b) a nucleic acid sequence encoding a heavy chain variable region (VH) of an anti-insulin-like growth factor 1 receptor (anti-IGF-1R) antibody or antigen-binding fragment thereof, and (c) a nucleic acid sequence encoding a light chain variable region (VL) of the anti-IGF-1R antibody or antigen-binding fragment thereof.

[0009] In some embodiments, the nucleic acid sequence encoding the VH of the anti-IGF-1R antibody or antigen-binding fragment thereof comprises: (i) a nucleic acid encoding a VH complementarity-determining region (CDR) 1 comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7 or 10 (or a VH CDR1-encoding sequence disclosed in Table 3 or Table 5); (ii) a nucleic acid encoding a VH comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, 11, or 14 (or a VH CDR2-encoding sequence disclosed in Table 3 or Table 5). (iii) a nucleic acid encoding a VH CDR3 comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9, 12, or 15 (or a VH CDR3-encoding sequence disclosed in Table 3 or Table 5);The nucleic acid sequence encoding the VL of the anti-IGF-1R antibody or antigen-binding fragment thereof may be (i) a nucleic acid sequence comprising a VL CDR1 comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16 (or a VL CDR1 coding sequence disclosed in Table 4 or Table 7); or (ii) a VL CDR2 comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17, 20, or 23 (or a VL CDR2 coding sequence disclosed in Table 4 or Table 7). (iii) a nucleic acid sequence comprising a VL CDR3 comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18, 21, or 24 (or a VL CDR3-encoding sequence disclosed in Table 4 or Table 7);

[0010] Certain aspects of the present disclosure are directed to vectors (e.g., vector genomes) comprising an antibody expression cassette comprising:

[0011] (a) promoter;

[0012] (b) (i) a nucleic acid encoding a VH complementarity-determining region (CDR) 1 comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7 or 10 (or a VH CDR1-encoding sequence disclosed in Table 3 or Table 5); (ii) a VH comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, 11, or 14 (or a VH CDR2-encoding sequence disclosed in Table 3 or Table 5). a nucleic acid encoding a CDR2, and (iii) a nucleic acid encoding a VH CDR3 comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9, 12, or 15 (or a VH CDR3-encoding sequence disclosed in Table 3 or Table 5);

[0013] (c) (i) a nucleic acid sequence comprising a VL CDR1 comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16 (or a VL CDR1-encoding sequence disclosed in Table 4 or Table 7); (ii) a VL comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17, 20, or 23 (or a VL CDR2-encoding sequence disclosed in Table 4 or Table 7). and (iii) a nucleic acid sequence encoding the light chain variable region (VL) of an anti-IGF-1R antibody or antigen-binding fragment thereof, comprising a nucleic acid sequence comprising a VL CDR3 that comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18, 21, or 24 (or a VL CDR3-encoding sequence disclosed in Table 4 or Table 7).

[0014] In some embodiments, the antibody expression cassette comprises a nucleic acid sequence encoding a signal peptide. In some embodiments, the antibody expression cassette comprises a nucleic acid sequence encoding a first signal peptide and a nucleic acid sequence encoding a second signal peptide. In some embodiments, the first and second signal peptides are the same. In some embodiments, the first and second signal peptides are different. In some embodiments, the first signal peptide (e.g., HC signal peptide) comprises a human IL-10 signal sequence. In some embodiments, the second signal peptide (e.g., LC signal peptide) comprises a human IL-2 signal sequence.

[0015] In some embodiments, the signal peptide (e.g., the first signal peptide or the second signal peptide) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 119 or 120.

[0016] In some aspects, the antibody expression cassette comprises a linker sequence selected from an internal ribosome entry site (IRES) sequence, a proteolytic cleavage site, or a combination thereof.

[0017] In some embodiments, the proteolytic cleavage site comprises a furin cleavage site, a 2A cleavage site, or a combination thereof.

[0018] In some embodiments, the antibody expression cassette comprises, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a VH, an IRES, and a nucleic acid sequence encoding a VL.

[0019] In some aspects, the antibody expression cassette includes, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a VH, an IRES, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a VL.

[0020] In some embodiments, the antibody expression cassette comprises, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a VL, an IRES, and a nucleic acid sequence encoding a VH.

[0021] In some embodiments, the antibody expression cassette includes, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a VL, an IRES, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a VH.

[0022] In some embodiments, the antibody expression cassette comprises, in 5' to 3' direction, a promoter, a nucleic acid sequence encoding a VH, a proteolytic cleavage site, and a nucleic acid sequence encoding a VL.

[0023] In some aspects, the antibody expression cassette includes, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a VH, a proteolytic cleavage site, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a VL.

[0024] In some embodiments, the antibody expression cassette comprises, in 5' to 3' direction, a promoter, a nucleic acid sequence encoding a VL, a proteolytic cleavage site, and a nucleic acid sequence encoding a VH.

[0025] In some aspects, the antibody expression cassette includes, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a VL, a proteolytic cleavage site, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a VH.

[0026] In some embodiments, the IRES comprises a nucleic acid having a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:43 or an IRES sequence disclosed in Table 15.

[0027] In some embodiments, the furin cleavage site comprises a nucleic acid having a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44 or a furin cleavage site sequence disclosed in Table 15.

[0028] In some embodiments, the 2A cleavage site comprises a nucleic acid having a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45 or a 2A cleavage site (e.g., F2A) sequence disclosed in Table 15.

[0029] In some embodiments, the signal peptide is an IL-2 signal peptide or an IL-10 signal peptide. In some embodiments, the encoded signal peptide comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either of SEQ ID NOs: 119 or 120. In some embodiments, the nucleic acid sequence encoding the signal peptide comprises a nucleic acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either of SEQ ID NOs: 121 or 122.

[0030] In some embodiments, the antibody expression cassette comprises a second promoter.

[0031] In some aspects, the antibody expression cassette comprises, in 5' to 3' direction, a promoter (a first promoter), a nucleic acid sequence encoding a VL, a second promoter, and a nucleic acid sequence encoding a VH.

[0032] In some aspects, the antibody expression cassette includes, in the 5' to 3' direction, a promoter (a first promoter), a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a VL, a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a VH.

[0033] In some aspects, the antibody expression cassette comprises, in 5' to 3' direction, a promoter (a first promoter), a nucleic acid sequence encoding a VH, a second promoter, and a nucleic acid sequence encoding a VL.

[0034] In some aspects, the antibody expression cassette includes, in the 5' to 3' direction, a promoter (a first promoter), a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a VH, a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a VL.

[0035] In some aspects, the antibody expression cassette comprises, in 5' to 3' direction, a nucleic acid sequence encoding a VH, a promoter (a first promoter), a second promoter, and a nucleic acid sequence encoding a VL.

[0036] In some aspects, the antibody expression cassette includes, from 5' to 3', a nucleic acid sequence encoding a first signal peptide, a VH, a promoter (first promoter), a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a VL.

[0037] In some aspects, the antibody expression cassette comprises, in 5' to 3' direction, a nucleic acid sequence encoding a VL, a promoter (a first promoter), a second promoter, and a nucleic acid sequence encoding a VH.

[0038] In some aspects, the antibody expression cassette includes, from 5' to 3', a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a VL, a promoter (first promoter), a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a VH.

[0039] In some embodiments, the promoter and / or second promoter is a constitutively active promoter, a cell type-specific promoter, a synthetic promoter, or an inducible promoter.

[0040] In some embodiments, the promoter and / or second promoter is a CBA promoter, a CMV promoter (optionally a human CMV promoter or a mouse CMV promoter), an EF1α promoter, a CAG promoter, or a tissue-specific promoter.

[0041] In some embodiments, the promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the promoter and / or enhancer sequences disclosed in SEQ ID NOs: 47-51, 83, or 93, or Table 15.

[0042] In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is selected from the group consisting of a desmin (DES) promoter, a human skeletal alpha-actin (HSA) promoter, a myosin creatine kinase (MCK) promoter, an alpha myosin heavy chain myosin creatine kinase 7 (HMCK7) promoter, a double MCK enhancer MCK (dMCK) promoter, a triple MCK enhancer MCK (tMCK) promoter, a double MCK enhancer muscle-type creatine kinase 8e (CK8e) promoter, a SPc5-12 promoter, a SP-301 promoter, an alpha myosin heavy chain (MHC) promoter, a Sk-CRM promoter, or a Sk-CRM4 promoter.

[0043] In some embodiments, the antibody expression cassette comprises an intron, hi some embodiments, the intron is a CAG intron, an SV40 intron, an MVM intron, or a human beta-globin intron, or any combination thereof.

[0044] In some embodiments, the intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46 or 82, or an intron sequence disclosed in Table 15.

[0045] In some embodiments, the promoter comprises different first and second promoters.

[0046] In some embodiments, the first and second promoters initiate transcription in the same direction.

[0047] In some embodiments, the first and second promoters initiate transcription in different directions.

[0048] In some embodiments, the antibody expression cassette comprises a pause element between the first and second promoters.

[0049] In some embodiments, the pause element comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54 or a pause element sequence disclosed in Table 15.

[0050] In some embodiments, the anti-IGF-1R antibody is a monoclonal antibody. In some embodiments, VH CDR1-3 correspond to the CDRs of teprotumumab, and / or VL CDR1-3 correspond to the CDRs of teprotumumab. In some embodiments, the anti-IGF-1R antibody is teprotumumab.

[0051] In some embodiments, the nucleic acid sequence encoding the VH comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs:25-27 (or a VH coding sequence disclosed in Table 5). In some embodiments, the encoded VH comprises SEQ ID NO:28 or SEQ ID NO:91 (or any of the VH amino acid sequences in Table 6).

[0052] In some embodiments, the nucleic acid sequence encoding the VL comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs:29-31 (or a VL coding sequence disclosed in Table 7). In some embodiments, the encoded VL comprises SEQ ID NO:32 or SEQ ID NO:92 (or any of the VL amino acid sequences in Table 8).

[0053] In some embodiments, the nucleic acid sequence encoding the heavy chain (HC) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 35-37 (or the HC coding sequences disclosed in Table 11). In some embodiments, the encoded HC comprises SEQ ID NO: 38 (or the HC amino acid sequence of Table 12).

[0054] In some embodiments, the nucleic acid sequence encoding the light chain (LC) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 39-41 (or the LC coding sequences disclosed in Table 13). In some embodiments, the encoded LC comprises SEQ ID NO: 42 (or the LC amino acid sequence of Table 14).

[0055] In some embodiments, the encoded anti-IGF-1R antibody is teprotumumab.

[0056] In some embodiments, the antibody expression cassette comprises a poly(A) sequence. In some embodiments, the poly(A) sequence is selected from bGHpA, hGHpA, SV40pA, or synthetic pA.

[0057] In some embodiments, the poly(A) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or 53, or a poly(A) sequence disclosed in Table 15.

[0058] In some embodiments, the antibody expression cassette comprises an open reading frame (ORF) comprising a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the ORF sequences disclosed in SEQ ID NOs: 57-67 and 94-97 or Table 16.

[0059] In some embodiments, the antibody expression cassette comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the antibody expression cassette sequences disclosed in SEQ ID NOs: 68-76 or Table 17.

[0060] In some embodiments, the AAV ITRs comprise a pair of ITRs flanking the antibody expression cassette. In some embodiments, the ITRs are of the same serotype as each other. In some embodiments, the ITRs are of the AAV2 serotype.

[0061] In some embodiments, the vector is packaged in an AAV capsid.

[0062] In some embodiments, the AAV capsid serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRh8, AAVrh9, AAV9, AAVrh10, AAV10, AAV11, AAV12, and modified versions thereof.

[0063] In some embodiments, the AAV capsid serotype is selected from the group consisting of AAV1, AAV2, AAV6, AAV8, AAV9, and modified versions thereof.

[0064] Certain aspects of the present disclosure are directed to host cells comprising the rAAV particles or vectors disclosed herein.

[0065] Certain aspects of the present disclosure are directed to compositions comprising an rAAV particle or vector disclosed herein and a carrier. In some aspects, the carrier is water or saline.

[0066] Certain aspects of the present disclosure are directed to methods for expressing an anti-IGF-1R antibody or antigen-binding fragment thereof in a cell, the method comprising administering to the cell an rAAV particle, vector, or composition disclosed herein, thereby expressing the anti-IGF-1R antibody or antigen-binding fragment thereof in the cell.

[0067] In some aspects, the cells are fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, or any combination thereof.

[0068] In some embodiments, the administration is in vitro.

[0069] In some aspects, the administering is in vivo.

[0070] Certain aspects of the present disclosure are directed to methods for expressing an anti-IGF-1R antibody or antigen-binding fragment thereof in a subject in need thereof, comprising administering to the subject an rAAV particle, vector, or composition disclosed herein, thereby expressing the anti-IGF-1R antibody or antigen-binding fragment thereof in the subject.

[0071] In some aspects, the subject has a thyroid eye disease selected from active Graves' orbital disease and chronic Graves' orbital disease.

[0072] Certain aspects of the present disclosure are directed to a method of treating thyroid eye disease in a patient in need thereof, comprising administering to the subject an rAAV particle, vector, or composition disclosed herein, thereby expressing an anti-IGF-1R antibody or antigen-binding fragment thereof in the subject and treating the thyroid eye disease.

[0073] In some aspects, the thyroid eye disease is selected from active Graves' orbital disease and chronic Graves' orbital disease.

[0074] In some aspects, administration is suitable for delivery of the rAAV particles or vectors to an ocular delivery site, a retro-orbital or periorbital delivery site, a retrobulbar delivery site, an extraocular muscle delivery site, a connective tissue delivery site, or any combination of these delivery sites.

[0075] In some aspects, administration is by injection or infusion.

[0076] In some aspects, administration is intramuscular (IM), intravenous (IV), intralymphatic, intraocular, retroorbital, periorbital, retrobulbar, or any combination thereof.

[0077] In some aspects, administration is suitable for delivery to retro-orbital or periorbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof.

[0078] In some aspects, administration is to an extraocular muscle.

[0079] In some aspects, the extraocular muscle is the levator muscle or the glabellar muscle.

[0080] In some aspects, administration is to connective tissue.

[0081] In some aspects, administration is transconjunctival to the periorbital space.

[0082] In some aspects, administration is intralymphatic to the preauricular or submandibular lymph nodes.

[0083] In some embodiments, the administration is a single dose. In some embodiments, the single dose is multiple injections and / or infusions.

[0084] In some embodiments, the administration is a single dose. In some embodiments, the single dose is multiple injections and / or infusions. [Brief explanation of the drawings]

[0085] [Figure 1] FIG. 1 shows a plasmid map of a vector containing the AAV2 ITRs flanked by the following ITR-to-ITR elements: from 5' to 3': a CMV enhancer, a CBA promoter, a CAG intron, a nucleic acid encoding a signal peptide, a nucleic acid encoding the teprotumumab heavy chain (Tepro HC), an F2A sequence, a nucleic acid encoding the signal peptide, a nucleic acid encoding the teprotumumab light chain (Tepro LC), and bovine polyadenylation (bGH pA).

[0086] [Figure 2]FIG. 2 shows a plasmid map of the vector containing the following ITR-to-ITR elements: from 5' to 3', a CMV enhancer, a CBA promoter, a CAG intron, a nucleic acid encoding a signal peptide, a nucleic acid encoding the teprotumumab light chain (Tepro LC), an IRES sequence (IRES2), a nucleic acid encoding the signal peptide, a nucleic acid encoding the teprotumumab heavy chain (Tepro HC), and an AAV2 ITR flanked by a synthetic polyadenylation site (syn pA).

[0087] [Figure 3] FIG. 3 shows a plasmid map of a vector containing the following ITR-to-ITR elements: from 5' to 3', a CMV enhancer, a CMV promoter, an SV40 intron, a nucleic acid encoding a signal peptide, a nucleic acid encoding the teprotumumab light chain (Tepro LC), a bovine polyadenylation site (bGH PA), a pause site, an EF-1a core promoter, a truncated 5' LTR, a chimeric human beta globin / immunoglobulin heavy chain intron, a nucleic acid encoding the signal peptide, a nucleic acid encoding the teprotumumab heavy chain (Tepro HC), and an AAV2 ITR flanked by a synthetic polyadenylation site (syn pA).

[0088] [Figure 4-1]Figures 4A-4D show protein and mRNA quantification of teprotumumab secreted from transfected HEK-293 cells. Figure 4A is a graph showing the amount of teprotumumab secreted from HEK-293 cells transfected with H-F2A-L (ORF #1), H-F2A-L (ORF #2), L-IRES-H (ORF #12), L-IRES-H (ORF #13), dual promoter (ORFs #4 and #5), or dual promoter (ORFs #6 and #7) teprotumumab AAV plasmid constructs. Figure 4B is a graph showing the amount of teprotumumab secreted from HEK-293 cells transfected with the H-F2A-L (ORF #1), H-F2A-L (ORF #2), L-IRES-H (ORF #12), or L-IRES-H (ORF #13) teprotumumab AAV plasmid constructs, or the H-F2A-L (ORF #1) (plasmid) or L-IRES-H (ORFs #4 and 5) (plasmid) teprotumumab non-AAV plasmid constructs. Figure 4C shows Western blot analysis of the secreted teprotumumab light and heavy chains performed under the indicated reducing and non-reducing conditions. Figure 4D is a graph showing teprotumumab mRNA levels in HEK-293 cells transfected with H-F2A-L (ORF #1), L-IRES-H (ORF #12), or dual promoter (ORFs #4 and 5) teprotumumab AAV plasmid constructs. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0089] [Figure 5]Figures 5A-B show the binding of vectored teprotumumab secreted from transfected HEK-293 cells to IGF-1R. Figure 5A is a graph showing the IGF-1R binding ability of teprotumumab secreted from HEK-293 cells transfected with H-F2A-L (ORF #1), H-F2A-L (ORF #2), L-IRES-H (ORF #12), or L-IRES-H (ORF #13) teprotumumab AAV plasmid constructs. Figure 5B is a graph showing the linearity of binding of vectored teprotumumab secreted from transfected HEK-293 cells to IGF-1R in duplicate binding experiments.

[0090] [Figure 6] FIG. 6 is a graph showing the reduction in IGF-1R phosphorylation levels upon treatment with recombinant teprotumumab or teprotumumab secreted from HEK-293 cells transfected with H-F2A-L (ORF #1) or H-F2A-L (ORF #2).

[0091] [Figure 7] Figures 7A-B show the reduction of total and phospho-IGF-1R expression in HT-1080 cells (Figure 7A) and Colo205 cells (Figure 7B) 48 hours after treatment with the indicated recombinant or vectorized teprotumumab. All wells in the MSD plate received the same total μg of either HT-1080 or Colo205 whole cell lysate. All values ​​shown are percentages of the treatment / mock-treated mean values. N = 3 technical replicates per sample; bars represent mean + / - SD. All statistics are one-way ANOVAs with post-hoc comparisons of each teprotumumab treatment to mock-treated controls, performed separately for total or phospho values. All "vectorized teprotumumab" data are HEK293T supernatants containing the respective ng of teprotumumab after transduction with an AAV expressing H-F2A-L (ORF #2).

[0092] [Figure 8]Figures 8A-C show the percentage reduction in expression of insulin growth factor 1 receptor (IGF-1R) protein (Figure 8A), insulin receptor (IR) protein (Figure 8B), and insulin receptor substrate 1 (IRS-1) protein (Figure 8C) in cultured primary fibrocytes isolated from peripheral blood of either normal donors (denoted normal) or Graves' disease donors (denoted Graves) after treatment with either vectorized teprotumumab (vTepro) or recombinant teprotumumab (rTepro) at a concentration of 500 ng / mL. All values ​​shown are percentages of (mock-treated mean value / mock-treated mean value × 100) - (treated value / mock-treated mean value × 100), where each mean value is n = 2 technical replicates. Normal sample values ​​are plotted as the mean of n = 2 biological replicates per sample, and bars represent the mean + / - SEM. Graves' disease sample values ​​are plotted as n = 1 biological replicate. All "vectorized teprotumumab" samples were HEK293T supernatants containing ng of teprotumumab after transduction with AAV expressing H-F2A-L (ORF #2). Treatment with vectorized teprotumumab resulted in a substantial reduction in IGF-1R, IR, and IRS-1 protein expression levels in Graves' disease fibrocytes.

[0093] [Figure 9] Figure 9 shows immunocytochemistry of Colo205 cells for IGF-1R with or without treatment with 500 ng / mL recombinant teprotumumab for 24 to 48 hours, demonstrating a reduction in IGF-1R expression. Panels A and E show untreated cell controls for 24 hours; panels B and F show cells treated with teprotumumab for 24 hours. Panels C and G show untreated cell controls for 48 hours; panels D and H show cells treated with teprotumumab for 48 hours. Panels A, B, C, and D show non-permeabilized cells; panels E, F, G, and H show cells permeabilized with 0.1% Triton® X-100.

[0094] [Figure 10]Figures 10A-C show tumor growth in Colo205 xenograft mice treated with an AAV1-delivered antibody expression cassette encoding teprotumumab (Figure 10A), an AAV2-delivered antibody expression cassette encoding teprotumumab (Figure 10B), or an AAV9-delivered antibody expression cassette encoding teprotumumab (Figure 10C), as indicated in the figure legends, compared to untreated control mice and mice treated with recombinant teprotumumab (6 mg / kg every 7 days).

[0095] [Figure 11] Figure 11 shows levels of teprotumumab in tumors obtained from animals treated with an AAV1-delivered antibody expression cassette encoding teprotumumab, an AAV2-delivered antibody expression cassette encoding teprotumumab, an AAV9-delivered antibody expression cassette encoding teprotumumab from days 7 to 31, and in tumors obtained from animals treated with recombinant teprotumumab (6 mg / kg every 7 days) on day 31. Bars represent the mean ± SEM. Days 7, 14, and 28 are the sampling arms, and day 31 is the efficacy arm.

[0096] [Figure 12] 12 shows serum levels of teprotumumab in the serum of animals treated with an AAV1-delivered antibody expression cassette encoding teprotumumab, an AAV2-delivered antibody expression cassette encoding teprotumumab, an AAV9-delivered antibody expression cassette encoding teprotumumab from days 7 to 31, and animals treated with recombinant teprotumumab (6 mg / kg every 7 days) on day 31. Bars represent the mean ± SEM. Days 7, 14, and 28 are the sampling arms, and day 31 is the efficacy arm.

[0097] [Figure 13]Figure 13 shows IGF-1R levels in tumors from animals treated with an AAV1-delivered antibody expression cassette encoding teprotumumab, an AAV2-delivered antibody expression cassette encoding teprotumumab, an AAV9-delivered antibody expression cassette encoding teprotumumab from days 7 to 31, and in tumors from animals treated with recombinant teprotumumab (6 mg / kg every 7 days) on day 31. Error bars represent the mean ± SEM. Days 7, 14, and 28 are the sampling arms, and day 31 is the efficacy arm. The reduction in IGF-1R levels compared to untreated controls was statistically significant at all time points. At day 31, the reduction in IGF-1R levels caused by AAV1-delivered teprotumumab and AAV9-delivered teprotumumab was not statistically different from that caused by recombinant teprotumumab (6 mg / kg every 7 days).

[0098] [Figure 14] FIG. 14 shows vector genome copies as copies / μg host genomic DNA in tumors from animals treated with an AAV1-delivered antibody expression cassette encoding teprotumumab, an AAV2-delivered antibody expression cassette encoding teprotumumab, an AAV9-delivered antibody expression cassette encoding teprotumumab, and untreated control animals at days 7, 14, and 28, as indicated in the figure legend.

[0099] [Figure 15] Figure 15 shows mRNA expression as single-stranded copies / μg host RNA in tumors from animals treated with an AAV1-delivered antibody expression cassette encoding teprotumumab, an AAV2-delivered antibody expression cassette encoding teprotumumab, an AAV9-delivered antibody expression cassette encoding teprotumumab, and untreated control animals at days 7, 14, and 28, as indicated in the figure legend.

[0100] [Figure 16]Figure 16 shows mRNA expression as vector genome copies as copies / μg host genomic DNA / and single-stranded copies / μg host RNA in tumors from animals treated with an AAV1-delivered antibody expression cassette encoding teprotumumab, an AAV2-delivered antibody expression cassette encoding teprotumumab, an AAV9-delivered antibody expression cassette encoding teprotumumab, and untreated control animals at days 7, 14, and 28, as indicated in the figure legend. DETAILED DESCRIPTION OF THE INVENTION

[0101] Detailed Description of Disclosure Certain aspects of the present disclosure relate to gene therapy vectors and expression constructs encoding anti-insulin-like growth factor 1 receptor (anti-IGF-1R) antibodies or antigen-binding fragments thereof; viral vectors (e.g., rAAV vectors) comprising same; compositions comprising same suitable for delivery (e.g., retrobulbar, periorbital, and / or intramuscular administration); and methods of using same. In some aspects, the present disclosure is directed to adeno-associated viral vector (AAV) delivery of an antibody expression cassette encoding an anti-IGF-1R antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof to a subject in need thereof.

[0102] Certain embodiments of the present disclosure are directed to a polynucleotide (e.g., an antibody expression cassette) comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds insulin-like growth factor 1 receptor (also referred to herein as IGF-1R or IGF-1R). In some embodiments, the polynucleotide (e.g., an antibody expression cassette) comprises an open reading frame (ORF) comprising a nucleic acid sequence encoding an anti-IGF-1R heavy chain and a nucleic acid sequence encoding an anti-IGF-1R light chain. In some embodiments, the ORF is operably linked to a promoter (e.g., a CBA promoter or a CMV promoter). In some embodiments, the ORF is operably linked to an enhancer (e.g., a CMV enhancer) and / or an intron sequence (e.g., a CAG intron or an SV40 intron sequence). In some embodiments, the ORF is operably linked to a polyadenylation (polyA) element (e.g., bGHpA, hGHpA, SV40pA, or synthetic pA).

[0103] In some embodiments, the ORF comprises a nucleic acid sequence encoding a signal peptide. In some embodiments, the signal peptide is an IL-2 signal peptide or an IL-10 signal peptide. In some embodiments, the ORF comprises a nucleic acid sequence encoding a first signal peptide and a nucleic acid sequence encoding a second signal peptide. In some embodiments, the first and second signal peptides are the same. In some embodiments, the first and second signal peptides are different.

[0104] In some embodiments, the ORF comprises a linker between the nucleic acid sequence encoding the anti-IGF-1R heavy chain and the nucleic acid sequence encoding the anti-IGF-1R light chain. In some embodiments, the linker is an internal ribosome entry sequence (IRES), a proteolytic cleavage site (e.g., furin and / or 2A cleavage site (e.g., F2A)), or a combination thereof. In some embodiments, the ORF further comprises a nucleic acid encoding a signal sequence. In some embodiments, the ORF is located between two inverted terminal repeats (ITRs).

[0105] In some embodiments, the polynucleotide (e.g., antibody expression cassette) comprises, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding an anti-IGF-1R heavy chain region, an IRES, and a nucleic acid sequence encoding an anti-IGF-1R light chain region.

[0106] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) includes, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding an anti-IGF-1R heavy chain region, an IRES, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding an anti-IGF-1R light chain region.

[0107] In some embodiments, the polynucleotide (e.g., antibody expression cassette) comprises, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding an anti-IGF-1R light chain region, an IRES, and a nucleic acid sequence encoding an anti-IGF-1R heavy chain region.

[0108] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) includes, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding an anti-IGF-1R light chain region, an IRES, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding an anti-IGF-1R heavy chain region.

[0109] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) comprises, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding an anti-IGF-1R heavy chain region, an F2A cleavage site, and a nucleic acid sequence encoding an anti-IGF-1R light chain region.

[0110] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) includes, from 5' to 3', a promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding an anti-IGF-1R heavy chain region, an F2A cleavage site, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding an anti-IGF-1R light chain region.

[0111] In some embodiments, the polynucleotide (e.g., antibody expression cassette) comprises, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding an anti-IGF-1R light chain region, an F2A cleavage site, and a nucleic acid sequence encoding an anti-IGF-1R heavy chain region sequence.

[0112] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) includes, in the 5' to 3' direction, a promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding an anti-IGF-1R light chain region, an F2A cleavage site, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding an anti-IGF-1R heavy chain region sequence.

[0113] In some aspects, the polynucleotide (eg, the antibody expression cassette) further comprises a second promoter.

[0114] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) comprises, in the 5' to 3' direction, a first promoter, a nucleic acid sequence encoding an anti-IGF-1R light chain, a second promoter, and a nucleic acid sequence encoding an anti-IGF-1R heavy chain.

[0115] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) comprises, in the 5' to 3' direction, a first promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding an anti-IGF-1R light chain, a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding an anti-IGF-1R heavy chain.

[0116] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) comprises, in the 5' to 3' direction, a first promoter, a nucleic acid sequence encoding an anti-IGF-1R heavy chain, a second promoter, and a nucleic acid sequence encoding an anti-IGF-1R light chain.

[0117] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) comprises, in the 5' to 3' direction, a first promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding an anti-IGF-1R heavy chain, a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding an anti-IGF-1R light chain.

[0118] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) comprises, in the 5' to 3' direction, a nucleic acid sequence encoding an anti-IGF-1R heavy chain, a first promoter, a second promoter, and a nucleic acid sequence encoding an anti-IGF-1R light chain.

[0119] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) includes, from 5' to 3', a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding an anti-IGF-1R heavy chain, a first promoter, a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding an anti-IGF-1R light chain.

[0120] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) comprises, in the 5' to 3' direction, a nucleic acid sequence encoding an anti-IGF-1R light chain, a first promoter, a second promoter, and a nucleic acid sequence encoding an anti-IGF-1R heavy chain.

[0121] In some embodiments, the polynucleotide (e.g., an antibody expression cassette) includes, from 5' to 3', a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding an anti-IGF-1R light chain, a first promoter, a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding an anti-IGF-1R heavy chain.

[0122] In some embodiments, the promoter is a constitutively active promoter, a cell type-specific promoter, a synthetic promoter, or an inducible promoter. In some embodiments, the promoter is selected from a CAG promoter, a CBA promoter, a human CMV promoter, a mouse CMV promoter, an EF1α promoter, an EF1α promoter and a CMV enhancer, a CMV promoter and a CMV enhancer (CMVe / p), a CMV promoter and an SV40 intron, or a tissue-specific promoter. In some embodiments, the cell type-specific promoter is a muscle-specific promoter, including a DES promoter, an HSA promoter, an MCK promoter, an HMCK7 promoter, a dMCK promoter, a tMCK promoter, a CK8e promoter, an SPc5-12 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM promoter, or an Sk-CRM4 promoter (see, e.g., Skopenkova et al., Acta Naturae 13: 47-58, 2012).

[0123] In some embodiments, the promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the promoter and / or enhancer sequences set forth in SEQ ID NOs: 47-51, 83, or 93, or Table 15. In some embodiments, a nucleic acid sequence comprising a promoter can comprise an intron. In some embodiments, the intron is selected from the group consisting of a CAG intron, an SV40 intron, an MVM intron, a human beta globin intron, or a chimeric human beta globin-human immunoglobulin chain intron. In some embodiments, the CAG intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 82. In some embodiments, the SV40 intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46. In some embodiments, the promoter comprises an intron sequence disclosed in Table 15.

[0124] In some embodiments, the promoter comprises a first and a second promoter. In some embodiments, the first and second promoters are different. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters initiate transcription in the same direction. In some embodiments, the first and second promoters initiate transcription in different directions. In some embodiments, the first and / or second promoter is a CMV promoter. In some embodiments, the first and / or second promoter is an EF-1α promoter. In some embodiments, the first and / or second promoter is a CBA promoter.

[0125] In some embodiments, the nucleic acid sequence encoding the first promoter and the nucleic acid sequence encoding the second promoter are operably linked. In some embodiments, the nucleic acid sequence encoding the first promoter and the nucleic acid sequence encoding the second promoter are operably linked by a pause element. In some embodiments, the pause element comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54 or the pause element sequence disclosed in Table 15.

[0126] In some aspects, the signal peptide is an endogenous signal peptide for HGH and its variants; an endogenous signal peptide for interferons and their variants, including the signal peptides of type I, II, and III interferons and their variants; or an endogenous signal peptide for known cytokines and their variants, such as erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β, and their variants. In some embodiments, the signal peptide is a modified signal peptide. In some aspects, the signal peptide is an IL-2 signal peptide. In some aspects, the signal peptide is an IL-10 signal peptide. In some aspects, the signal peptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 119 or 120.

[0127] In some embodiments, the anti-IGF-1R antibody is a monoclonal antibody. In some embodiments, the anti-IGF-1R antibody is teprotumumab.

[0128] In some embodiments, the anti-IGF-1R antibody heavy chain comprises a heavy chain variable region (VH) comprising complementarity determining region (CDR) 1, VH CDR2, and VH CDR3. In some embodiments, VH CDR1-3 correspond to the CDRs of teprotumumab. In some embodiments, the nucleic acid sequence encoding the VH CDR1 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7 or 10 (or a VH CDR1 coding sequence disclosed in Table 3 or Table 5); the nucleic acid sequence encoding the VH CDR2 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, 11 or 14 (or a VH CDR2 coding sequence disclosed in Table 3 or Table 5); The nucleic acid sequence encoding the CDR3 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9, 12 or 15 (or a VH CDR3-encoding sequence disclosed in Table 3 or Table 5).

[0129] In some embodiments, the anti-IGF-1R antibody light chain comprises a light chain variable region (VL) comprising complementarity-determining region (CDR) 1, VL CDR2, and VL CDR3. In some embodiments, VL CDR1-3 correspond to the CDRs of teprotumumab. In some embodiments, the nucleic acid sequence encoding the VL CDR1 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16 (or a VL CDR1 coding sequence disclosed in Table 4 or Table 7); the nucleic acid sequence encoding the VL CDR2 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17, 20, or 23 (or a VL CDR2 coding sequence disclosed in Table 4 or Table 7); Nucleic acid sequences encoding CDR3 include nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18, 21 or 24 (or a VL CDR3-encoding sequence disclosed in Table 4 or Table 7).

[0130] In some embodiments, the nucleic acid sequence encoding the heavy chain variable region (VH) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs:25-27 (or a VH-coding sequence disclosed in Table 5). In some embodiments, the encoded VH comprises SEQ ID NO:28 or SEQ ID NO:91 (or any of the VH amino acid sequences in Table 6). In some embodiments, the nucleic acid sequence encoding the light chain variable region (VL) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs:29-31 (or a VL-coding sequence disclosed in Table 7). In some embodiments, the encoded VL comprises SEQ ID NO: 32 or SEQ ID NO: 92 (or any of the VL amino acid sequences in Table 8).

[0131] In some embodiments, the nucleic acid sequence encoding the heavy chain (HC) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 35-37 (or the HC coding sequences disclosed in Table 11). In some embodiments, the encoded HC comprises SEQ ID NO: 38 (or the HC amino acid sequence in Table 12). In some embodiments, the nucleic acid sequence encoding the light chain (LC) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 39-41 (or the LC coding sequences disclosed in Table 13). In some embodiments, the encoded LC comprises SEQ ID NO: 42 (or the LC amino acid sequence in Table 14).

[0132] In some embodiments, the nucleic acid sequence encoding the heavy chain and the nucleic acid sequence encoding the light chain are operably linked. In some embodiments, the nucleic acid sequence encoding the heavy chain and the nucleic acid sequence encoding the light chain are operably linked by a linker sequence. In some embodiments, the linker sequence is selected from an IRES sequence, a proteolytic cleavage site (e.g., a furin and / or a 2A cleavage site, e.g., F2A), or a combination thereof. In some embodiments, the IRES comprises a nucleic acid having a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43 or an IRES sequence disclosed in Table 15. In some embodiments, a furin cleavage site comprises a nucleic acid having a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44 or a furin cleavage site sequence disclosed in Table 15. In some embodiments, a 2A cleavage site comprises a nucleic acid having a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a 2A cleavage site (e.g., F2A) sequence as SEQ ID NO: 45 or a furin cleavage site sequence disclosed in Table 15.

[0133] In some embodiments, the polynucleotide comprises an open reading frame (ORF) comprising a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the ORF sequences disclosed in SEQ ID NOs: 57-67 and 94-97 or Table 16.

[0134] In some embodiments, a polynucleotide (e.g., an antibody expression cassette) comprises poly(A). In some embodiments, the polyA sequence comprises a human growth hormone polyA signal sequence. In some embodiments, the polyA sequence comprises a bovine growth hormone polyA signal sequence. In some embodiments, the polyA sequence comprises a synthetic polyA sequence. In some embodiments, the polyA sequence comprises an SV40 polyA signal sequence (SV40pA). In some embodiments, a polynucleotide (e.g., an antibody expression cassette) comprises a poly(A) sequence comprising a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or 53, or any of the poly(A) sequences disclosed in Table 15.

[0135] In some embodiments, the antibody expression cassette comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 68-76, or any of the antibody expression cassette sequences disclosed in Table 17.

[0136] Certain aspects of the present disclosure are directed to methods for expressing and / or producing an anti-IGF-1R antibody or antigen-binding fragment thereof, comprising administering a polynucleotide (e.g., an antibody expression cassette), vector, or rAAV particle of the present disclosure to a cell, thereby expressing and / or producing the anti-IGF-1R antibody or antigen-binding fragment thereof in the cell. In some aspects, the cell is a fibroblast, an adipocyte, a myofibroblast, a myocyte, a muscle cell, or any combination thereof.

[0137] Certain aspects of the present disclosure are directed to compositions (e.g., gene therapy compositions) comprising the polynucleotides (e.g., antibody expression cassettes), vectors, or rAAV particles of the present disclosure.

[0138] In some aspects, a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition of the present disclosure is suitable for delivery to a subject in need thereof (e.g., a subject suffering from Graves' ophthalmopathy). In some aspects, a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition of the present disclosure is suitable for delivery to an ocular delivery site, a retroorbital or periorbital delivery site, a retrobulbar delivery site, an extraocular muscle delivery site, a connective tissue delivery site, or any combination thereof. In some aspects, delivery is by injection or infusion. In some aspects, delivery is by a route of administration selected from intramuscular (IM), intravenous (IV), intralymphatic, intraocular, retroorbital, periorbital, retrobulbar, or any combination thereof. In some aspects, administration is suitable for delivery to retroorbital or periorbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof. In some aspects, administration is to an extraocular muscle. In some embodiments, the extraocular muscle is the levator muscle or the glabellar muscle. In some embodiments, the administration is to connective tissue. In some embodiments, the administration is transconjunctival to the periorbital space. In some embodiments, the administration is intralymphatic to the preauricular or submandibular lymph node. In some embodiments, the polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition of the present disclosure is suitable for single-dose administration. In some embodiments, the single dose is multiple injections and / or infusions.

[0139] Certain embodiments of the present disclosure are directed to a method for expressing a therapeutic antibody or antigen-binding fragment thereof that binds IGF-1R in a subject in need thereof, comprising administering to the subject an effective amount of a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition of the present disclosure. In some embodiments, the administration is a single dose. In some embodiments, the single dose comprises multiple injections into the eye, retro-orbital or peri-orbital, retrobulbar, ocular muscle, or any other delivery site disclosed herein.

[0140] In some embodiments, delivery or administration can be intramuscular (IM), intravenous (IV), intraocular (IC), intralymphatic, periorbital, retrobulbar, or any combination thereof. In some embodiments, delivery or administration is to or near the eye (e.g., one or both eyes), e.g., intraocular, retroorbital or periorbital, retrobulbar, intramuscular near the eye (e.g., to the levator and / or glabellar muscles), to connective tissue near the eye, or any combination thereof. In some embodiments, delivery or administration is to retroorbital or periorbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by infusion. In some embodiments, delivery or administration is by injection and / or infusion as a single dose. In some embodiments, single dose administration comprises multiple injections or infusions.

[0141] Also provided herein is a method for expressing an anti-IGF-1R antibody or antigen-binding fragment thereof in a subject in need thereof, comprising administering to the subject an effective amount of a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition of the present disclosure, wherein the administration is intramuscular (IM), intravenous (IV), intraocular (IC), intralymphatic, periorbital, retrobulbar, or any combination thereof. In some embodiments, the gene therapy composition or AAV capsid is administered by periorbital, retrobulbar, and / or intramuscular injection (e.g., injection into the levator muscle and / or glabellar muscle). In some embodiments, administration is to an extraocular muscle. In some embodiments, the extraocular muscle is the levator muscle or glabellar muscle. In some embodiments, administration is to connective tissue. In some embodiments, administration is transconjunctival to the periorbital space. In some embodiments, administration is intralymphatic to a preauricular or submandibular lymph node. In some embodiments, the administration results in expression of the anti-IGF-1R antibody or antigen-binding fragment thereof in a cell type selected from the group consisting of fibroblasts, adipocytes, myofibroblasts, muscle cells, and any combination thereof.

[0142] In some embodiments, the subject has thyroid eye disease (TED), e.g., active or chronic Graves' ophthalmopathy.

[0143] Non-limiting examples of various embodiments are provided in this disclosure. I. Definition

[0144] In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed disclosure.

[0145] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleic acid sequence" is understood to refer to one or more nucleic acid sequences unless otherwise specified. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0146] Furthermore, when used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0147] Numerical values ​​given herein are in scientific notation, i.e., x x 10 y , or may be written or expressed in chemical E notation xEy.

[0148] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0149] The term "about" is used herein to mean approximately, roughly, roughly, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value by a variance above or below (higher or lower) the stated value, for example, by 10 percent.

[0150] The term "at least" preceding a number or a series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that can be logically included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each number in the series or range. "At least" is also not limited to integers (for example, "at least 5%" includes 5.0%, 5.1%, 5.18%, regardless of the number of significant digits).

[0151] As used herein, "no more than" or "less than" is understood as the value immediately adjacent to the phrase and the logically lower value or integer, logically from the context, up to zero. When "no more than" is present before a series of numbers or a range, it is understood that "no more than" can modify each of the numbers in the series or range.

[0152] As used herein, the term "intraocular" refers to a location within or throughout the eye.

[0153] As used herein, the term "intraorbital" refers to a location within the eye socket, which refers to the cavity in the skull that contains the eye and its associated muscles, glands, blood vessels, nerves, etc.

[0154] As used herein, the term "periorbital" as used herein refers to a location located around or surrounding the orbit (e.g., tissue surrounding or inside the orbit of the eye).

[0155] As used herein, the term "retroorbital" or "retrobulbar" refers to a location located or residing behind the eyeball.

[0156] As used herein, the term "retrobulbar" refers to a location located or present behind the eyeball.

[0157] As used herein, "intralymphatic" refers to a location associated with a lymph node or lymphatic vessel.

[0158] As used herein, the term "delivery vector" or "vector" refers to any vehicle for cloning and / or transferring nucleic acids into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. A vector may be a replicon to which another nucleic acid segment can be attached so as to cause replication of the attached segment. A "replicon" refers to any genetic element (e.g., a plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "delivery vector" or "vector" includes both viral and non-viral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses, are known and used in the art. In some embodiments, insertion of a polynucleotide into a suitable vector can be achieved by ligating an appropriate polynucleotide fragment into a selected vector with complementary cohesive termini. Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells into which the vector has been incorporated. The expression of the selectable marker or reporter allows for the identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc., and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc.A selectable marker can also be considered a reporter. In some embodiments, the delivery vector is selected from the group consisting of a viral vector (e.g., an AAV vector), a plasmid, a lipid, a protein particle, a bacterial vector, and a lysosome.

[0159] Some embodiments of the present disclosure are directed to biological vectors, which may include viruses, particularly attenuated and / or replication-defective viruses.

[0160] As used herein, the term "promoter" refers to a DNA sequence recognized by cellular or introduced synthetic machinery necessary to initiate specific transcription of a gene. The term "promoter" is also meant to encompass nucleic acid elements sufficient for promoter-dependent gene expression that can be controlled in a cell-type-specific, tissue-specific manner, or can be induced by external signals or agents; such elements can be located in the 5' or 3' region of the native gene. In some embodiments, the promoter is a constitutively active promoter, a cell-type-specific promoter, or an inducible promoter.

[0161] As used herein, the term "enhancer" refers to a cis-acting element that stimulates or inhibits the transcription of adjacent genes. Enhancers that inhibit transcription are also called "silencers." Enhancers can function in either direction (e.g., can associate with coding sequences) over distances of up to several kilobase pairs (kb) from the coding sequence and downstream of the transcribed region.

[0162] As used herein, the term "regulatable promoter" is any promoter whose activity is affected by cis- or trans-acting factors (e.g., an inducible promoter, e.g., an external signal or agent).

[0163] As used herein, the term "constitutive promoter" refers to any promoter that directs RNA production in most or all tissues / cell types, e.g., the human CMV immediate-early enhancer / promoter region that drives constitutive expression of a cloned DNA insert in mammalian cells.

[0164] The terms "transcriptional regulatory protein," "transcriptional regulator," and "transcription factor" are used interchangeably herein to refer to nuclear proteins that bind DNA response elements, thereby transcriptionally regulating expression of the associated gene(s). Transcriptional regulatory proteins generally bind directly to DNA response elements, although in some cases, binding to DNA can be indirect, via binding to another protein that in turn binds to or is bound to the DNA response element.

[0165] As used herein, the term "termination signal sequence" refers to any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence. A polyadenylation signal sequence is a recognition region required for endonuclease cleavage of an RNA transcript followed by the polyadenylation consensus sequence AATAAA. A polyadenylation signal sequence provides a "poly A site," i.e., a site on an RNA transcript where adenine residues are added by post-transcriptional polyadenylation.

[0166] As used herein, the term "signal peptide" refers to a polypeptide sequence or combination of sequences sufficient to mediate the translocation of a polypeptide to the cell surface. Without being bound by any particular theory, the translocation of a polypeptide to the cell surface can be mediated by the secretory pathway, including the translocation of the polypeptide from the cytosol to the endoplasmic reticulum, and the subsequent transport of the polypeptide through the Golgi to the cell membrane, where the protein can remain embedded in the cell membrane or be secreted from the cell. As used herein, "signal peptide" includes naturally occurring and synthetic signal sequences, signal "patches", etc. Examples of signal peptides include, but are not limited to, the endogenous signal peptides for HGH and its variants; the endogenous signal peptides for interferons and their variants, including the signal peptides of type I, II, and III interferons and their variants; and the endogenous signal peptides for known cytokines and their variants, such as erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β, and their variants. In some aspects, the signal peptide is an IL-2 signal peptide. In some aspects, the signal peptide is an IL-10 signal peptide. In some embodiments, the signal peptide is a modified signal peptide.

[0167] As used herein, the term "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into the start codon, e.g., ATG, of a cistron (protein-coding region), thereby resulting in cap-independent translation of the gene. See, for example, Jackson RJ et al., Trends Biochem Sci 15(12):477-83 (199); Jackson RJ and Kaminski, A. RNA 1(10):985-1000 (1995). As used herein, "under the translational control of an IRES" means that translation is associated with the IRES and proceeds in a cap-independent manner.

[0168] The term "self-processing cleavage site" or "self-processing cleavage sequence," as used herein, refers to a post-translational or co-translational processing cleavage site or sequence. Such a "self-processing cleavage" site or sequence refers herein to a DNA sequence or amino acid sequence exemplified by a 2A site, sequence, or domain, or a 2A-like site, sequence, or domain. The term "self-processing peptide" is defined herein as a peptide expression product of a DNA sequence encoding a self-processing cleavage site or sequence that, upon translation, mediates rapid intramolecular (cis) cleavage of a protein or polypeptide containing the self-processing cleavage site to yield a distinct mature protein or polypeptide product.

[0169] As used herein, the term "additional proteolytic cleavage site" refers to a sequence incorporated into an expression construct of the present disclosure adjacent to a self-processing cleavage site, e.g., a 2A sequence or a 2A-like sequence, to provide a means for removing additional amino acids remaining after cleavage by the self-processing cleavage sequence. Exemplary 2A peptides include, but are not limited to, P2A, E2A, F2A, and T2A. Exemplary "additional proteolytic cleavage sites" are described herein and include, but are not limited to, a furin cleavage site having the consensus sequence RXK(R)R. Such furin cleavage sites can be cleaved by endogenous subtilisin-like proteases, e.g., furin, and other serine proteases in the protein secretion pathway. In some embodiments, other exemplary "additional proteolytic cleavage sites" can be used, for example, as described in Lie et al., Sci Rep 7, 2193 (2017).

[0170] The terms "operably linked," "operably inserted," "operably located," "under control," or "under transcriptional control" mean that a promoter is in the correct location and orientation with respect to a nucleic acid to control RNA polymerase initiation and expression of a gene. The term "operably linked" means that the DNA sequence and regulatory sequence are connected in such a way that gene expression is possible when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence. The term "operably inserted" means that DNA of interest introduced into a cell is located adjacent to DNA sequences that direct the transcription and translation of the introduced DNA (i.e., facilitating, for example, the production of a polypeptide encoded by the DNA of interest).

[0171] The term "expression vector or construct" refers to any type of genetic construct containing a nucleic acid from which part or all of a nucleic acid coding sequence can be transcribed.

[0172] As used herein, the term "multicistronic" or "multicistronic vector" refers to a nucleic acid sequence having two or more open reading frames (e.g., genes). An open reading frame, in this context, is a sequence of codons translatable into a polypeptide or protein (e.g., a heavy chain or a light chain). A "bicistronic" or "bicistronic vector" refers to a nucleic acid sequence having two open reading frames (e.g., genes). An open reading frame, in this context, is a sequence of codons translatable into a polypeptide or protein (e.g., a heavy chain or a light chain). In some aspects, the constructs of the present disclosure are multicistronic (e.g., bicistronic) constructs (e.g., comprising a heavy chain and a light chain).

[0173] "Viral vector" refers to a vector made from at least a portion of a viral genome, which can be used to carry or deliver one or more polynucleotide regions that encode or contain a molecule of interest, such as a protein, peptide, and oligonucleotide, or a plurality thereof. Viral vectors can be used to deliver genetic material to cells. Viral vectors can be modified for specific applications. In some embodiments, the delivery vector of the present disclosure is a viral vector selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, or a retrovirus vector.

[0174] The term "adeno-associated virus vector" or "AAV vector" as used herein refers to any vector that contains or is derived from an adeno-associated vector component, and is suitable for infecting mammalian cells, preferably human cells. The term AAV vector typically designates a virus particle, i.e., a virion, of an AAV type that contains a payload. AAV vectors can be derived from various serotypes, including serotype combinations (i.e., "pseudotyped" AAV), or various genomes (e.g., single-stranded or self-complementary). In addition, AAV vectors can be replication-deficient and / or targeted. As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV1, AAV2, AAV3 (including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, the AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Morris et al. (Virol. 33:375 (2004)), and any other AAV now known or hereafter discovered. See, for example, FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV vector" includes derivatives of known AAV vectors. In some embodiments, "AAV vector" includes modified or artificial AAV vectors (e.g., muscle-enhanced AAV, e.g., AAVMYO (see Weinmann et al. Nat. Comm. 11: 5432, 2020)). The terms "AAV genome" and "AAV vector" can be used interchangeably. In some embodiments, the AAV vector is modified compared to a wild-type AAV serotype sequence.

[0175] As used herein, an "AAV particle" is an AAV virus comprising an AAV vector genome having at least one payload region (e.g., a polynucleotide encoding a therapeutic protein or peptide (e.g., an antibody expression cassette)) and at least one inverted terminal repeat (ITR) region. In some aspects, the term "AAV vector of the present disclosure" or "AAV vector" refers to an AAV vector comprising, for example, an antibody-encoding polynucleotide (e.g., an antibody expression cassette) packaged in an AAV particle.

[0176] A "coding sequence," or a sequence "encoding" a particular molecule (e.g., a therapeutic protein or peptide), is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of mRNA) into a polypeptide, either in vitro or in vivo, when operably linked to an appropriate regulatory sequence, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence is typically located 3' to the coding sequence.

[0177] The term "derived from," as used herein, refers to a component that is isolated from a specified molecule or organism, or that is created using a specified molecule or organism, or information (e.g., an amino acid sequence or a nucleic acid sequence) from a specified molecule or organism. For example, a nucleic acid sequence (e.g., an AVV vector) that is derived from a second nucleic acid sequence (e.g., another AVV vector) can contain a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence.

[0178] In the case of the polynucleotides disclosed herein, the derived species can be obtained, for example, by naturally occurring mutagenesis, artificially directed mutagenesis, or artificially random mutagenesis. The mutagenesis used to derive a polynucleotide can be intentionally directed, intentionally random, or a mixture of both. The mutagenesis of a polynucleotide to create a different polynucleotide from the original one can be a random event (e.g., caused by polymerase infidelity), and the identification of the derived polynucleotide can be performed by a suitable screening method.

[0179] As used herein, the term "mutation" refers to any change in the structure of a gene that results in a variant (also called a "mutant") form that can be transmitted to subsequent generations. Mutations in genes can be caused by the change of a single base in the DNA, or by the deletion, insertion, or rearrangement of larger sections of the gene or chromosome.

[0180] As used herein, the term "administration" refers to the administration of a composition of the present disclosure (e.g., a polynucleotide (e.g., an antibody expression cassette), AAV vector, rAAV particle, or composition disclosed herein) to a subject or system. Administration to an animal subject (e.g., a human) can be by any suitable route, including, but not limited to, periorbital, retrobulbar, intralymphatic, and / or intramuscular injection.

[0181] As used herein, the term "modified" refers to an altered state or structure of a molecule of the present disclosure. Molecules can be modified in many ways, including chemically, structurally, and functionally. In some aspects, the modification is relative to a reference wild-type molecule.

[0182] As used herein, the term "synthetic" means produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the disclosure can be chemical or enzymatic.

[0183] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably in this application. These terms refer only to the primary structure of the molecule. As such, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms "nucleic acid," "polynucleotide," and "oligonucleotide," as used herein, are defined as a molecule comprising two or more covalently linked nucleosides, as commonly understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as a nucleic acid molecule or oligomer. Polynucleotides can be produced recombinantly, enzymatically, or synthetically, for example, by solid-phase chemical synthesis followed by purification. Reference to the sequence of a polynucleotide or nucleic acid refers to the sequence or order of the nucleobase moieties or modifications thereof of the covalently linked nucleotides or nucleosides.

[0184] The term "mRNA," as used herein, refers to a single-stranded RNA that encodes the amino acid sequences of one or more polypeptide chains.

[0185] The term "antisense," as used herein, refers to a nucleic acid that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA so as to prevent expression of the endogenous gene. A "complementary" polynucleotide is one that can base-pair according to the standard Watson-Crick complementarity rules. Specifically, purines base-pair with pyrimidines to form combinations of guanine paired with cytosine (G:C), and adenine paired with thymine (A:T) in DNA and adenine paired with uracil (A:U) in RNA. It is understood that two polynucleotides can hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to each other.

[0186] The term " antisense strand " and " guide strand " refer to the strand of dsRNA, for example, shRNA, which comprises the region that is substantially complementary to target sequence, for example, mRNA.Antisense strand has a sequence that is sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, for example, has sufficient complementarity to trigger the destruction of desired target mRNA by RNAi mechanism or process.

[0187] The terms " sense strand " and " passenger strand " as used herein refer to the strand of dsRNA, for example, shRNA, that comprises the region that is substantially complementary to the region of antisense strand, as this term is defined herein.The antisense and sense strands of dsRNA, for example, shRNA, hybridize to form a double-stranded structure.

[0188] As used herein, the term "polypeptide" is intended to encompass not only the singular "polypeptide" but also the plural "polypeptides," including any chain or chains of two or more amino acids. Therefore, as used herein, "peptide," "peptide subunit," "protein," "amino acid chain," "amino acid sequence," or any other term used to refer to a chain or chains of two or more amino acids is included within the definition of "polypeptide," even though each of these terms may have a more specific meaning. The term "polypeptide" can be used in place of or interchangeably with any of these terms. The term further includes polypeptides that have undergone post-translational or post-synthetic modifications, such as conjugation of palmitoyl groups, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The term "peptide," as used herein, encompasses full-length peptides and fragments, variants, or derivatives thereof. As disclosed herein, a "peptide" may be part of a fusion polypeptide that includes additional components to increase half-life, such as an Fc domain or albumin domain. The peptides described herein can also be derivatized in several different ways. The peptides described herein can include modifications including, for example, the conjugation of a palmitoyl group.

[0189] The terms "antibody" and "antibodies" refer to an immunoglobulin molecule that recognizes and specifically binds to a target, e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibody exhibits the desired biological activity.

[0190] Antibodies can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, such as toxins, radioisotopes, etc.

[0191] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment may contain the antigen recognition site of the intact antibody (e.g., a complementarity-determining region (CDR) sufficient to bind the antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies (e.g., nanobodies). Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially produced.

[0192] The term "nanobody" or "nanobodies" or "single domain antibody" or "sdAb" refers to a class of antigen-binding fragments that are single-chain immunoglobulin molecules consisting of a monomeric variable antibody domain that recognizes and specifically binds to an antigen.

[0193] The term "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses both intact, full-length monoclonal antibodies and antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) mutants, fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments produced in any of several ways, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0194] The term "bispecific" or "bifunctional antibody" or antigen-binding fragment thereof refers to an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0195] The term "multispecific antibody" refers to an antibody having specificity for more than two different epitopes, typically non-overlapping epitopes or antibodies containing more than two separate antigen-binding sites.

[0196] The term "immunoglobulin" is used herein to include antibodies, functional fragments thereof, Fabs, scFvs, single-domain antibodies (e.g., nanobodies), DARTs, F(ab')2, BITEs, and immunoadhesins. These antibody fragments or artificial constructs may include single-chain antibodies, Fab fragments, monovalent antibodies, bivalent or multivalent antibodies, or immunoadhesins. Binding or neutralizing antibody constructs may be monoclonal antibodies, "humanized" antibodies, multivalent antibodies, or another suitable construct. An "immunoglobulin molecule" is a protein containing the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently coupled together and capable of specifically combining with an antigen. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The terms "antibody" and "immunoglobulin" are used interchangeably herein. An "immunoglobulin heavy chain" is a polypeptide containing at least a portion of an immunoglobulin's antigen-binding domain and at least a portion of an immunoglobulin heavy chain variable region. Therefore, immunoglobulin-derived heavy chains share significant regions of amino acid sequence homology with members of the immunoglobulin gene superfamily. For example, the heavy chain in a Fab fragment is an immunoglobulin-derived heavy chain. An "immunoglobulin light chain" is a polypeptide containing at least a portion of an immunoglobulin's antigen-binding domain and at least a portion of an immunoglobulin variable region. Therefore, immunoglobulin-derived light chains share significant regions of amino acid sequence homology with members of the immunoglobulin gene superfamily. An "immunoadhesin" is a binding protein, usually a functional domain of a receptor, ligand, or cell adhesion molecule, or a chimeric antibody-like molecule that combines one or two immunoglobulin variable domains with an immunoglobulin constant domain, usually including a hinge or GS linker and an Fc region. A "fragment antigen-binding (Fab) fragment" is the region of an antibody that binds to an antigen. It consists of one constant domain and one variable domain for each of the heavy and light chains.With respect to the immunoglobulins or antibodies described herein, each fragment of the immunoglobulin coding sequence can be derived from one or more sources or can be synthetic. Suitable fragments can include, for example, the coding region for a heavy chain, a light chain, and / or a fragment thereof, such as the coding region for one or more of the constant or variable regions of the heavy chain (CH1, CH2, and / or CH3) and / or the constant or variable region of the light chain. Alternatively, the variable region of the heavy or light chain can be utilized. Where appropriate, these sequences can be modified from the "native" sequences from which they are derived, as described herein. As used herein, the term "immunoglobulin construct" refers to any of the immunoglobulins described above or fragments thereof encoded by and contained in the expression cassettes and viral vectors described herein.

[0197] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically approximately the amino-terminal 110-120 or 110-125 amino acids in a mature heavy chain and approximately 90-115 amino acids in a mature light chain, which vary greatly in sequence within antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions in variable domains are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of an antibody with an antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0198] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0199] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0200] The VH and VL regions can be further divided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0201] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding fragment. In certain embodiments, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35, which may optionally include one or two additional amino acids after 35 (designated 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In some embodiments, the CDRs of the antibodies described herein were determined according to the Kabat numbering scheme.

[0202] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meanings in the art. The constant region is a portion of an antibody, e.g., the carboxyl-terminal portion of the light chain and / or heavy chain, that is not directly involved in binding the antibody to an antigen but may exhibit various effector functions, e.g., interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain. In certain embodiments, the antibody or antigen-binding fragment comprises a constant region or portion thereof that is sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0203] As used herein, the term "heavy chain" or "HC," when used in reference to an antibody, can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some embodiments, the heavy chain is a human heavy chain.

[0204] As used herein, the term "light chain" or "LC," when used in reference to an antibody, can refer to any distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0205] "Fc region" (Fragment Crystallizable Region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system.

[0206] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions; native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fc includes various Fc allotypes (see, e.g., Jefferis et al., (2009) mAbs 1:1; Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014)).

[0207] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include the FcγR family of receptors, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Human IgG1 binds to most human Fc receptors and induces the strongest Fc effector functions. This is considered to be equivalent to mouse IgG2a in terms of the types of activating Fc receptors it binds. Conversely, human IgG4 induces the lowest Fc effector functions. Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014).

[0208] The constant region can be engineered, for example, by recombinant technology, to eliminate one or more effector functions. "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or the biochemical events resulting therefrom. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions, such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and down-regulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain). Thus, the term "constant region without Fc function" includes a constant region with reduced or no one or more effector functions mediated by the Fc region.

[0209] The effector function of an antibody can be reduced or avoided by different approaches. The effector function of an antibody can be reduced or avoided by using an antibody fragment lacking the Fc region (e.g., Fab, F(ab')2, single-chain Fv (scFv), or sdAb consisting of a monomeric VH or VL domain). Alternatively, so-called aglycosylated antibodies can be created by removing sugars linked to specific residues in the Fc region, which reduces the effector function of the antibody, while retaining other valuable attributes of the Fc region (e.g., extended half-life and heterodimerization). Aglycosylated antibodies can be created, for example, by deleting or altering the residue to which the sugar is attached, enzymatically removing the sugar, producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or expressing the antibody in cells (e.g., bacterial host cells) that cannot glycosylate proteins. See, e.g., U.S. Patent Application Publication No. 20120100140. Another approach is to use Fc regions from IgG subclasses with reduced effector function. For example, IgG2 and IgG4 antibodies are characterized by lower levels of Fc effector function than IgG1 and IgG3. The residues closest to the hinge region in the CH2 domain of the Fc portion are responsible for the antibody's effector function, as they contain largely overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγRs) on effector cells of the innate immune system. Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014).Therefore, antibodies with reduced or no Fc effector function can be prepared by, for example, creating a chimeric Fc region comprising a CH2 domain derived from an IgG antibody of IgG4 isotype and a CH3 domain derived from an IgG antibody of IgG1 isotype, or a chimeric Fc region comprising a hinge region derived from IgG2 and a CH2 region derived from IgG4 (see, for example, Lau C. et al. J. Immunol. 191:4769-4777 (2013)), or an Fc region with a mutation that results in altered Fc effector function, for example, reduced or no Fc function. Such Fc regions with mutations are known in the art. See, for example, US Patent Application Publication No. 20120100140 and the US and PCT applications cited therein, and An et al., mAbs 1:6, 572-579 (2009); the disclosures of which are incorporated by reference in their entirety.

[0210] In some embodiments, an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof can be modified so that it does not bind to the Fc region. See, e.g., Saunders K., Front. Immunol., 10:1296 (2019).

[0211] The terms "hinge," "hinge domain," "hinge region," or "antibody hinge region" are used interchangeably and refer to the domain of the heavy chain constant region that joins the CH1 domain to the CH2 domain and includes the upper, middle, and lower segments of the hinge (Roux et al., J. Immunol. 1998 161:4083). The hinge provides varying levels of flexibility between the binding and effector regions of an antibody and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions.

[0212] As used herein, "isotype" refers to the class of antibody (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant region genes.

[0213] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0214] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies of different antigenicity. An isolated antibody that specifically binds to an epitope of a protein may, however, have cross-reactivity to other corresponding proteins from different species.

[0215] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to those of an antibody or antigen-binding fragment thereof from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to the sequences of an antibody or antigen-binding fragment thereof from another (usually human) species to avoid eliciting an immune response in that species.

[0216] The term "humanized" antibody or antigen-binding fragment thereof refers to a form of a non-human (e.g., murine) antibody or antigen-binding fragment thereof that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human (e.g., murine) sequence. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that has the desired specificity, affinity, and capacity ("CDR-grafted") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some embodiments, a humanized antibody or antigen-binding fragment thereof may comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to make humanized antibodies are described in U.S. Patent No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.

[0217] The term "human" antibody (HuMAb) or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, where such an antibody or antigen-binding fragment is produced using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof. In some aspects, a human antibody is a fully human antibody, i.e., the antibody has amino acid sequences derived from human immunoglobulin loci and contains no amino acid sequences derived from non-human immunoglobulin loci. In some aspects, a human antibody is a partially human antibody, i.e., the antibody has amino acid sequences derived from human immunoglobulin loci and amino acid sequences derived from non-human immunoglobulin loci. In some aspects, a human antibody is a fully human antibody, a partially human antibody, or a humanized antibody. In some aspects, a human antibody is a chimeric human antibody, i.e., the antibody has approximately equal amounts of amino acid sequences derived from human immunoglobulin loci and amino acid sequences derived from non-human immunoglobulin loci.

[0218] A "blocking" or "blocking" or "inhibitory" or "inhibiting" antibody is one that, when bound to a target protein, reduces or inhibits (partially or completely) binding of that target protein to one or more ligands and / or reduces or inhibits (partially or completely) one or more activities or functions of the target protein when bound to the target protein.

[0219] As used herein, "epitope" is a term of art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, joined together from two or more non-contiguous regions of a polypeptide(s) (conformational, non-linear, discontinuous, or discontinuous epitope). The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0220] The phrase "contacting a cell" (e.g., contacting a cell with a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition of the present disclosure), as used herein, includes directly or indirectly contacting a cell. In some aspects, contacting a cell with a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition includes contacting a cell with the polynucleotide (antibody expression cassette), vector, rAAV particle, or composition in vitro, or contacting a cell with the polynucleotide (antibody expression cassette), vector, rAAV particle, or composition in vivo. Thus, for example, the polynucleotide (antibody expression cassette), vector, rAAV particle, or composition can be physically contacted with the cell by the individual performing the method, or the polynucleotide (antibody expression cassette), vector, rAAV particle, or composition can be placed in conditions that allow or cause it to subsequently contact the cell.

[0221] In some embodiments, contacting cells in vitro can be performed, for example, by incubating cells with a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition. In some embodiments, contacting cells in vivo can be performed, for example, by injecting a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition of the present disclosure into or near the tissue where the cells are located (e.g., retrobulbar, periorbital, or ocular muscle), or by injecting a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition into another area, for example, the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue where the contacted cells are located. For example, the AAV vector genome can be encapsulated and / or coupled to a ligand that directs the AAV vector genome to the desired site. A combination of in vitro and in vivo contacting methods is also possible. For example, cells can be contacted in vitro with a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition of the present disclosure and then transplanted into a subject.

[0222] In some aspects, contacting a cell with a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition of the present disclosure includes "introducing" or "delivering" the polynucleotide (antibody expression cassette), vector, rAAV particle, or composition into a cell (directly or indirectly) by facilitating or causing uptake or absorption into the cell. Introducing a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the polynucleotide (antibody expression cassette), vector, rAAV particle, or composition can be injected into a specific tissue site (e.g., where a therapeutic effect is desired) or administered systemically (e.g., administering a polynucleotide (antibody expression cassette), vector, or rAAV particle targeted to where a therapeutic effect is desired). In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection.

[0223] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount," for example, of a polynucleotide, expression cassette, vector, rAAV particle, or composition of the present disclosure, refer to an amount sufficient to produce a beneficial or desired result, including a clinical result, when administered to a subject, including a human; thus, "effective amount" or its synonyms will depend on the context in which it is applied. In some aspects, a therapeutically effective amount of an agent (e.g., a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition disclosed herein) is an amount that produces a beneficial or desired result in a subject compared to a control.

[0224] The amount of a given agent (e.g., a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition of the present disclosure) corresponds to such amount, which will vary depending on a variety of factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject (e.g., age, sex, and / or weight) or host being treated, etc.

[0225] As used herein, the term "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., an antibody expression cassette comprising a promoter operably linked to a nucleic acid encoding a therapeutic molecule disclosed herein) into an individual's cells and / or tissues to treat, reduce the symptoms of, or reduce the likelihood of a disease. Gene therapy also includes the insertion of a transgene that is inhibitory in nature, i.e., inhibits, decreases, or reduces the expression, activity, or function of an endogenous gene or protein, e.g., an undesirable or abnormal (e.g., pathogenic) gene or protein. Such a transgene may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence that is not normally present in the cells, tissues, and / or individual being treated. Both acquired and congenital diseases are amenable to gene therapy.

[0226] The term "prophylactically effective amount," as used herein, includes the amount of an agent (e.g., a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition disclosed herein) when administered to a subject having or predisposed to a disease or disorder (e.g., Graves' orbitopathy). Ameliorating a disease or disorder includes slowing the course of the disease or disorder or reducing the severity of a later-occurring disease or disorder. A "prophylactically effective amount" can vary depending on the characteristics of the agent, e.g., a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition of the present disclosure, the method by which the agent is administered, the degree of risk for the disease, and medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the patient being treated.

[0227] As used herein, "off-target" refers to any unintended effect on any one or more targets, genes or cellular transcripts.

[0228] As used herein, the term "in vitro" refers to events that take place not within a living organism (e.g., an animal, plant, or microorganism), but in an artificial environment, such as a test tube or reaction vessel, cell culture, petri dish, etc.

[0229] As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0230] As used herein, the term " transfection " refers to the method of introducing exogenous nucleic acid into cells.Transfection methods include but are not limited to chemical methods, physical treatments, and cationic lipids or mixtures.The list of substances that can be transfected into cells is long, and includes, for example, siRNA, shRNA, sense and / or antisense sequences, expression plasmids, for example, the DNA that encodes one or more genes organized in vectors.

[0231] "Determining the level of a protein" refers to detecting the protein or the mRNA encoding the protein, either directly or indirectly, by methods known in the art. "Directly determining" means performing a process to obtain a physical entity or value (e.g., performing an assay or test on a sample, or "analyzing a sample," as that term is defined herein). "Indirectly determining" refers to receiving a physical entity or value from another person or source (e.g., a third-party laboratory that obtains the physical entity or value directly). The method for measuring protein level generally includes but is not limited to Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcytometry, microscopy, fluorescence-activated cell sorting (FACS) and flow cytometry, and assays based on the properties of protein, including but not limited to enzyme activity or interaction with other protein partners.Methods for measuring mRNA level are known in the art.

[0232] "Percent sequence identity (%)" to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be achieved by a variety of methods within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST.

[0233] "Level" refers to the level or activity of a protein or the mRNA encoding the protein, as appropriate, compared to a reference. The reference may be any useful reference as defined herein. A "decreased level" or "increased level" of a protein refers to a decrease or increase in the protein level compared to the reference. The protein level may be expressed as a mass / volume (e.g., g / dL, mg / mL, μg / mL, ng / mL) or percentage of the total protein or mRNA in the sample.

[0234] The term "pharmaceutical composition," as used herein, refers to a composition comprising a compound or molecule described herein, e.g., a polynucleotide (antibody expression cassette), vector, or rAAV particle disclosed herein, formulated with a pharmaceutically acceptable excipient and capable of being manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal.

[0235] "Pharmaceutically acceptable excipient," as used herein, refers to any ingredient other than the compounds described herein (e.g., a vehicle in which an active compound can be suspended or dissolved) that has substantially non-toxic and non-inflammatory properties in a patient.

[0236] "Reference" refers to any useful reference used to compare the level or activity of protein or mRNA. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample, or a reference standard or level. A "reference sample" can be, for example, a control, for example, a predetermined negative control value, such as a "normal control," or a previous sample obtained from the same subject; a sample from a normal healthy subject, for example, normal cells or normal tissue; a sample (e.g., cell or tissue) from a subject without a disease; a sample from a subject who has been diagnosed with a disease but has not yet been treated with the compounds described herein; a sample from a subject who has been treated with the compounds described herein; or a sample of purified protein with a known normal concentration (e.g., as described herein).

[0237] As used herein, the term "subject" refers to any organism to which a composition disclosed herein, e.g., a polynucleotide (antibody expression cassette), vector, rAAV particle, or composition of the present disclosure, can be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals, e.g., mice, rats, rabbits, non-human primates, and humans). A subject can be a human or animal seeking or requiring treatment, requiring treatment, undergoing treatment, will undergo treatment in the future, or receiving care from a trained professional for a particular disease or condition.

[0238] As used herein, the terms "treat," "treated," and "treating" refer to both therapeutic treatment and prophylactic or preventative measures, where the purpose is to prevent or slow (lessen) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. In some aspects, treating reduces or lessens the symptoms associated with a disease or disorder. In some aspects, treating produces a beneficial or desired clinical result.

[0239] As used herein, "Graves' orbital disease" (GO) refers to the active or chronic stage of a thyroid-related condition of autoimmune orbital inflammation. "Active" or "dynamic" GO can last approximately 6 months and up to 24 months, followed by "inactive" or "chronic" GO.

[0240] Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; attenuation of the extent of a condition, disorder, or disease; stabilization (i.e., not worsening) of the condition, disorder, or disease state; delaying or slowing the onset of condition, disorder, or disease progression; amelioration or remission (whether partial or complete) of a condition, disorder, or disease state, whether detectable or undetectable; amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or amelioration of a condition, disorder, or disease. In some embodiments, treatment involves eliciting a clinically significant response without excessive levels of side effects. In some embodiments, treatment involves prolonging survival compared to expected survival in the absence of treatment. As used herein, the term "amelioration" or "ameliorating" refers to a reduction in the severity of at least one indicator of a condition or disease. As used herein, the term "preventing" or "prevention" refers to delaying or forestalling the onset, development, or progression of a condition or disease for a period of time, including weeks, months, or years. II. Therapeutic antibodies

[0241] The present disclosure provides polynucleotides (e.g., antibody expression cassettes), vectors, and rAAV particles for delivery and expression of therapeutic anti-IGF-1R antibodies into cells or subjects. In some embodiments, the antibody expression cassette comprises a promoter operably linked to a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds insulin-like growth factor-1 receptor (also referred to herein interchangeably as an "anti-IGF receptor antibody," an "anti-IGF-1 receptor antibody," an "anti-IGF-1R antibody," and an "anti-IGF-1R antibody").

[0242] In some embodiments, the anti-IGF-1R antibody is an antibody or antigen-binding fragment thereof selected from a monoclonal antibody, a bispecific antibody, or a multispecific antibody, or an antigen-binding fragment thereof. In some embodiments, the therapeutic protein is an antibody fragment selected from a Fab, Fab', F(ab')2, Fv fragment, a linear antibody, or a single-chain antibody (e.g., a nanobody).

[0243] In some aspects, the antibody is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a nanobody, and a multispecific antibody.

[0244] In some aspects, the antibody is a monoclonal antibody.

[0245] In some aspects, the antibody expression cassettes disclosed herein comprise nucleic acid sequences encoding a heavy chain (HC) and / or a light chain (LC). In some aspects, the antibody expression cassettes disclosed herein comprise nucleic acid sequences encoding a variable heavy chain (VH) and / or a variable light chain (VL).

[0246] In some aspects, the antibody (e.g., monoclonal antibody) or antigen-binding fragment thereof is a chimeric antibody.

[0247] In some embodiments, the antibody (e.g., monoclonal antibody) or antigen-binding fragment thereof is a humanized antibody.

[0248] In some aspects, the antibody (e.g., monoclonal antibody) or antigen-binding fragment thereof is a human antibody. In some aspects, the human antibody is a fully human antibody that comprises amino acid sequences derived from human immunoglobulin loci and does not contain amino acid sequences derived from non-human immunoglobulin loci. In some aspects, the human antibody is a partially human antibody that comprises amino acid sequences derived from human immunoglobulin loci and amino acid sequences derived from non-human immunoglobulin loci. In some aspects, the human antibody is a fully human antibody, a partially human antibody, or a humanized antibody. In some aspects, the human antibody is a chimeric human antibody, i.e., the antibody has approximately equal amounts of amino acid sequences derived from human immunoglobulin loci and amino acid sequences derived from non-human immunoglobulin loci.

[0249] In some embodiments, the anti-IGF-1R antibody is selected from the group consisting of teprotumumab, VRDN-01100 (SEQ ID NO: 113), VRDN-02700 (SEQ ID NO: 116), ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L1 6H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29 , L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52, or a fragment, variant, or derivative thereof.

[0250] In some embodiments, the anti-IGF-1R antibody is VRDN-01100 or VRDN-02700, or a fragment, variant, or derivative thereof. In some embodiments, the anti-IGFR antibody comprises SEQ ID NO: 113 (corresponding to VRDN-01100). In some embodiments, the anti-IGFR antibody comprises SEQ ID NO: 116 (corresponding to VRDN-002700).

[0251] In some embodiments, the anti-IGF-1R antibody is teprotumumab, or a fragment, variant, or derivative thereof.

[0252] In some aspects, the antibody expression cassette comprises a nucleic acid encoding a signal peptide operably linked to a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds insulin-like growth factor-1 receptor. In some aspects, the signal peptide is an endogenous signal peptide for HGH and its variants; an endogenous signal peptide for interferon and its variants, including the signal peptides of type I, II, and III interferons and their variants; or an endogenous signal peptide for known cytokines and their variants, such as erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β, and their variants. In some embodiments, the signal peptide is a modified signal peptide. In some aspects, the signal peptide is an IL-2 signal peptide. In some aspects, the signal peptide is an IL-10 signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 119 or 120. In some embodiments, the nucleic acid sequence encoding the signal peptide comprises a nucleic acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 121 or 122.

[0253] In certain embodiments, compositions comprising a delivery vector, e.g., a viral vector, comprising a nucleic acid encoding an immunoglobulin (e.g., an anti-IGF-1R antibody) disclosed herein are suitable for delivery to a subject in need thereof.

[0254] In some embodiments, an antibody expression cassette comprising a nucleic acid sequence encoding an anti-IGF-1R antibody can be packaged into a viral vector (e.g., an AAV vector) disclosed herein, wherein the nucleic acid sequence encoding the anti-IGF-1R antibody is operably linked to a promoter. In some embodiments, the promoter can drive expression of the anti-IGF-1R antibody in host cells (e.g., fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, or any combination thereof). In some embodiments, a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody can be administered to the eye, retrobulbar, intralymphatic, periorbital, and / or muscular tissue (e.g., the levator muscle and / or glabellar muscle). In some embodiments, a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody can be administered intramuscularly, intralymphatic, intradermal, intravenously, intraocular, retrobulbar, periorbital, or any combination thereof. In some embodiments, a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody can be administered intramuscularly to an extraocular muscle. In some embodiments, the administration is to an extraocular muscle. In some embodiments, the extraocular muscle is the levator muscle or the glabellar muscle. In some embodiments, the administration is to connective tissue. In some embodiments, the administration is transconjunctival to the periorbital space. In some embodiments, the administration is intralymphatic to a preauricular or submandibular lymph node. In some embodiments, a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody can be administered intralymphatic to a preauricular and / or submandibular lymph node.

[0255] In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered to periorbital tissue. In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered to periorbital tissue. In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered intramuscularly to periorbital muscles. In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered intramuscularly to retrobulbar muscles. In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered intramuscularly to facial muscles. In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered to the connective tissue of the periorbital or retroorbital region. In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered intramuscularly to the levator muscle and / or glabellar muscle. In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered to fibroblasts in the connective tissue of the periorbital or retroorbital region. In some embodiments, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered to myofibroblasts in the connective tissue of the periorbital or retroorbital region. In some embodiments, a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered to adipocytes in the periorbital or retroorbital connective tissue.In some embodiments, a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition comprising a nucleic acid encoding an anti-IGF-1R antibody is administered to muscle cells in the periorbital or retroorbital connective tissue. In some embodiments, a nucleic acid encoding a protein or peptide disclosed herein is suitable for delivery to periorbital and / or retroorbital tissue. In some embodiments, a nucleic acid encoding a protein or peptide disclosed herein is suitable for delivery to periorbital tissue. In some embodiments, a nucleic acid encoding a protein or peptide disclosed herein is suitable for intramuscular delivery to periorbital or retroorbital muscles. In some embodiments, a nucleic acid encoding a protein or peptide disclosed herein is suitable for intramuscular delivery to facial muscles. In some embodiments, a nucleic acid encoding a protein or peptide disclosed herein is suitable for delivery to periorbital or retroorbital connective tissue. In some embodiments, administration is transconjunctival to the periorbital space. In some embodiments, administration is intralymphatic to the preauricular or submandibular lymph nodes.

[0256] In some aspects, nucleic acids encoding the proteins or peptides disclosed herein are suitable for delivery to other delivery sites disclosed herein. II.A.1 Antibodies

[0257] Certain aspects of the present disclosure are directed to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising nucleic acids encoding antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to insulin-like growth factor 1 receptor (IGF-1R), e.g., human IGF-1R. In some aspects, the encoded anti-IGF-1R antibody is an anti-insulin-like growth factor-1 receptor (anti-IGF-1R) antibody. In some embodiments, the encoded anti-IGF-1R antibody is selected from the group consisting of teprotumumab, VRDN-01100 (SEQ ID NO: 113), VRDN-02700 (SEQ ID NO: 116), ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H 16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30 H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52, or a fragment, variant, or derivative thereof.

[0258] In some embodiments, the encoded anti-IGF-1R antibody comprises the amino acid sequence of VRDN-01100 or VRDN-02700, or a fragment, variant, or derivative thereof.

[0259] In some embodiments, the encoded anti-IGF-1R antibody comprises the amino acid sequence of SEQ ID NO: 113 (corresponding to VRDN-01100). In some embodiments, the anti-IGFR antibody comprises SEQ ID NO: 116 (corresponding to VRDN-002700).

[0260] In some embodiments, the encoded anti-IGF-1R antibody comprises the amino acid sequence of teprotumumab, or a fragment, variant, or derivative thereof.

[0261] In some aspects, the present disclosure is directed to a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition of the present disclosure comprising a promoter operably linked to a nucleic acid encoding an immunoglobulin, e.g., an antibody or antigen-binding fragment thereof, that binds to IGF-1R.

[0262] In some embodiments, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions of the present disclosure used in the methods disclosed herein encode antibodies (e.g., monoclonal antibodies or antigen-binding fragments thereof) having CDR and / or variable region sequences of teprotumumab, or antibodies having at least 80% identity (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity) with those variable region or CDR sequences of teprotumumab. In some embodiments, the gene therapy construct encoding an anti-IGF-1R antibody (e.g., teprotumumab) is a multicistronic (e.g., bicistronic) construct (e.g., comprising a heavy chain and a light chain). In some embodiments, the multicistronic (e.g., bicistronic) construct further comprises an F2A or IRES element.

[0263] In some aspects, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition disclosed herein comprises a nucleic acid encoding an antibody comprising the heavy and light chains of teprotumumab or an antigen-binding fragment thereof. In some aspects, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition disclosed herein comprises a nucleic acid sequence that is modified relative to the wild-type (unmodified) teprotumumab coding sequence.

[0264] In some embodiments, the anti-IGF-1R antibody comprises a heavy chain and a light chain. In some embodiments, the nucleic acid sequence encoding the heavy chain comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 35-37 (or the HC coding sequences disclosed in Table 11). In some embodiments, the encoded HC comprises SEQ ID NO: 38 (or the HC amino acid sequence of Table 12). In some embodiments, the nucleic acid sequence encoding the light chain comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 39-41 (or the LC coding sequences disclosed in Table 13). In some embodiments, the encoded LC comprises SEQ ID NO: 42 (or the LC amino acid sequence of Table 14).

[0265] In some embodiments, the nucleic acid sequence encoding the heavy chain variable region comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs:25-27 (or a VH coding sequence disclosed in Table 5). In some embodiments, the encoded VH comprises SEQ ID NO:28 or SEQ ID NO:91 (or any of the VH amino acid sequences in Table 6). In some embodiments, the nucleic acid sequence encoding the light chain variable region comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs:29-31 (or a VL coding sequence disclosed in Table 7). In some embodiments, the encoded VL comprises SEQ ID NO: 32 or SEQ ID NO: 92 (or any of the VL amino acid sequences in Table 8).

[0266] In some embodiments, the heavy chain comprises a heavy chain variable region (VH) comprising complementarity determining region (CDR) 1, VH CDR2, and VH CDR3. In some embodiments, VH CDR1-3 correspond to the CDRs of teprotumumab. In some embodiments, the nucleic acid sequence encoding the VH CDR1 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7 or 10 (or a VH CDR1 coding sequence disclosed in Table 3 or Table 5); the nucleic acid sequence encoding the VH CDR2 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, 11 or 14 (or a VH CDR2 coding sequence disclosed in Table 3 or Table 5); The nucleic acid sequence encoding the CDR3 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9, 12 or 15 (or a VH CDR3-encoding sequence disclosed in Table 3 or Table 5).

[0267] In some embodiments, the light chain comprises a light chain variable region (VL) comprising complementarity determining region (CDR) 1, VL CDR2, and VL CDR3. In some embodiments, VL CDR1-3 correspond to the CDRs of teprotumumab. In some embodiments, the nucleic acid sequence encoding the VL CDR1 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16 (or a VL CDR1 coding sequence disclosed in Table 4 or Table 7); the nucleic acid sequence encoding the VL CDR2 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17, 20, or 23 (or a VL CDR2 coding sequence disclosed in Table 4 or Table 7); Nucleic acid sequences encoding CDR3 include nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18, 21 or 24 (or a VL CDR3-encoding sequence disclosed in Table 4 or Table 7).

[0268] In some aspects, the polynucleotides disclosed herein encode single domain antibodies (e.g., nanobodies) comprising either (i) a heavy chain variable region (VH) comprising complementarity determining region (CDR) 1, a VH CDR2 and / or a VH CDR3, or (ii) a light chain variable region (VL) comprising CDR1, a VL CDR2 and / or a VL CDR3. In some aspects, the encoded VH CDRs and / or VL CDRs are selected from the corresponding CDRs of teprotumumab.

[0269] In some aspects, the polynucleotides disclosed herein are selected from the group consisting of teprotumumab, VRDN-01100 (SEQ ID NO: 113), VRDN-02700 (SEQ ID NO: 116), ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H1 6, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H 30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52, or a fragment, variant, or derivative thereof.

[0270] In some aspects, the polynucleotides disclosed herein encode the amino acid sequence of VRDN-01100 or VRDN-02700, or a fragment, variant, or derivative thereof.

[0271] In some aspects, the polynucleotides disclosed herein encode the amino acid sequence of SEQ ID NO: 113 (corresponding to VRDN-01100). In some aspects, the anti-IGFR antibody comprises SEQ ID NO: 116 (corresponding to VRDN-002700).

[0272] In some aspects, the polynucleotides disclosed herein encode the amino acid sequence of teprotumumab, or a fragment, variant, or derivative thereof.

[0273] In some aspects, the polynucleotides disclosed herein encode antibodies or antigen-binding fragments thereof that comprise the six CDRs listed in Tables 1 and 2 (i.e., any set of three VH CDRs listed in Table 1 and any set of three VL CDRs listed in Table 2). In some aspects, the polynucleotides disclosed herein comprise the nucleotide sequences of the six CDRs listed in Tables 3 and 4 (i.e., nucleic acids encoding the three VH CDRs listed in Table 3 and nucleic acids encoding the three VL CDRs listed in Table 4). Table 1. Variable heavy chain CDR (VH CDR) amino acid sequences [Table 1] Table 2. Variable light chain CDR (VL CDR) amino acid sequences [Table 2] Table 3. Variable heavy chain CDR (VH CDR) nucleic acid sequences [Table 3] Table 4. Variable light chain CDR (VL CDR) nucleic acid sequences [Table 4]

[0274] In some aspects, the polynucleotides disclosed herein comprise a nucleic acid sequence listed in Table 5. In some aspects, the polynucleotides disclosed herein encode an antibody variable heavy chain (VH) sequence listed in Table 6, or an antigen-binding fragment thereof. Table 5. Variable heavy (VH) chain nucleic acid sequences [Table 5] Table 6. Variable heavy chain (VH) amino acid sequences [Table 6]

[0275] In some aspects, a polynucleotide disclosed herein comprises a nucleic acid sequence listed in Table 7. In some aspects, a polynucleotide disclosed herein encodes an antibody variable light chain (VL) or antigen-binding fragment thereof comprising a sequence listed in Table 8. Table 7. Variable light (VL) chain nucleic acid sequences [Table 7] Table 8. Variable light chain (VL) amino acid sequences [Table 8]

[0276] In some aspects, the polynucleotides disclosed herein include a nucleic acid selected from Table 5 (e.g., a nucleic acid encoding a VH of Table 6) and a nucleic acid from Table 7 (e.g., a nucleic acid encoding a VL of Table 8).

[0277] In some embodiments, the polynucleotides disclosed herein comprise a nucleic acid sequence shown in Table 9. Table 9. Light chain constant region nucleic acid sequence [Table 9]

[0278] In some aspects, the polynucleotides disclosed herein comprise a nucleic acid sequence shown in Table 10. Table 10. Heavy chain constant region nucleic acid sequence [Table 10-1] [Table 10-2] [Table 10-3]

[0279] In some aspects, a polynucleotide disclosed herein comprises a nucleic acid sequence listed in Table 11. In some aspects, a polynucleotide disclosed herein encodes an antibody, or antigen-binding fragment thereof, comprising a heavy chain (HC) of an antibody listed in Table 12. In some aspects, a polynucleotide disclosed herein encodes a signal peptide listed in Table 12. Table 11. Heavy chain (HC) nucleic acid sequence [Table 11-1] [Table 11-2] [Table 11-3] Table 12. Heavy chain (HC) amino acid sequence [Table 12]

[0280] In some aspects, a polynucleotide disclosed herein comprises a nucleic acid sequence listed in Table 13. In some aspects, a polynucleotide disclosed herein encodes an antibody, or antigen-binding fragment thereof, comprising a light chain (LC) of an antibody listed in Table 14. In some aspects, a polynucleotide disclosed herein encodes a signal peptide listed in Table 14. Table 13. Light chain (LC) nucleic acid sequences [Table 13-1] [Table 13-2] Table 14. Light chain (LC) amino acid sequences [Table 14]

[0281] In some aspects, the polynucleotides disclosed herein comprise a nucleic acid listed in Tables 11 and 13. In some aspects, the polynucleotides disclosed herein encode an antibody comprising the HC and LC of an antibody listed in Tables 12 and 14 (i.e., the HC of an antibody listed in Table 12 and the LC of the same antibody listed in Table 14).

[0282] In some embodiments, the therapeutic protein used in the methods disclosed herein is an antibody (e.g., a monoclonal antibody or antigen-binding fragment thereof) having the VH, VL, HC and / or LC sequences of teprotumumab, as well as antibodies having at least 80% identity, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the corresponding VH, VL, HC and / or LC sequences.

[0283] In some embodiments, the polynucleotide also comprises a linker sequence operably linked to the nucleic acid sequence encoding the heavy chain and / or the nucleic acid sequence encoding the light chain. In some embodiments, the polynucleotide also comprises a pause element sequence operably linked to the nucleic acid sequence encoding the heavy chain and / or the nucleic acid sequence encoding the light chain. In some embodiments, the pause element sequence has a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:54.

[0284] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof (e.g., teprotomumab), including a construct comprising: (i) a VH CDR1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) and a VL CDR1-3 (e.g., SEQ ID NOs: 16-18, 19-21, or 22-24); (ii) a VH (e.g., SEQ ID NO: 26 or 27) and a VL (e.g., SEQ ID NO: 30 or 31); (iii) a HC (e.g., SEQ ID NO: 36 or 37) and a LC (e.g., SEQ ID NO: 40 or 41); or (iv) any one of SEQ ID NOs: 68-76, further comprising one or more of an IRES, a furin cleavage site, a 2a site, or a dual promoter (e.g., promoter-VH-IRES-VL, etc.). II.A.2. Antigen-binding fragment

[0285] In some embodiments, antigen-binding fragments of the antibodies described herein, such as teprotumumab, are encoded by the polynucleotides disclosed herein. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and scFv, where the Fab, Fab', F(ab')2, or scFv comprises the heavy chain variable region sequence and light chain variable region sequence of teprotumumab described herein. The Fab, Fab', F(ab')2, or scFv can be produced by any technique known to those of skill in the art. In some embodiments, the antigen-binding fragment, e.g., Fab, Fab', F(ab')2, or scFv, further comprises a moiety that extends the half-life of the antibody in vivo. This moiety is also referred to as a "half-life extending moiety." Any moiety known to those of skill in the art for extending the half-life of an antigen-binding fragment, e.g., Fab, Fab', F(ab')2, or scFv in vivo, can be used. For example, the half-life extending moiety may comprise an Fc region, a polymer, albumin, or an albumin-binding protein or compound. The polymer may include a natural or synthetic, optionally substituted, linear or branched polyalkylene, polyalkenylene, polyoxylalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen, or derivatives thereof. The substituent may include one or more hydroxy, methyl, or methoxy groups. In some embodiments, the antigen-binding fragment, e.g., Fab, Fab', F(ab')2, or scFv, can be modified by the addition of one or more C-terminal amino acids for attachment of the half-life extending moiety. In some embodiments, the half-life extending moiety is polyethylene glycol or human serum albumin. In some embodiments, the antigen-binding fragment, e.g., Fab, Fab', F(ab')2, or scFv, is fused to an Fc region.

[0286] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to insulin-like growth factor-1 receptor (IGF-1R), eg, human IGF-1R.

[0287] In some embodiments, the encoded anti-IGF-1R antibody is selected from the group consisting of teprotumumab, VRDN-01100 (SEQ ID NO: 113), VRDN-02700 (SEQ ID NO: 116), ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L1 6H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29 , L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52, or an antigen-binding fragment thereof.

[0288] In some aspects, the encoded anti-IGF-1R antibody comprises the amino acid sequence of VRDN-01100 (SEQ ID NO: 113) or VRDN-02700 (SEQ ID NO: 116), or an antigen-binding fragment thereof.

[0289] In some embodiments, the encoded anti-IGF-1R antibody comprises the amino acid sequence of teprotumumab or an antigen-binding fragment thereof.

[0290] In some aspects, an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein is modified so that it has an enhanced half-life and / or reduced toxicity.

[0291] In some embodiments, the encoded antibody or antigen-binding fragment thereof is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody or antigen-binding fragment thereof can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody or antigen-binding fragment thereof is bispecific or multispecific. II.A.3. Antibody Expression Cassettes

[0292] In certain aspects, provided herein are antibody expression cassettes comprising nucleotide sequences encoding an antibody or antigen-binding fragment thereof described herein, or a domain thereof (e.g., a light chain, a heavy chain, a variable light chain region and / or a variable heavy chain region) that specifically binds to insulin-like growth factor receptor (IGFR), an antigen-binding fragment thereof, or any combination thereof, as well as vectors, e.g., vectors comprising such antibody expression cassettes for expression in a cell, e.g., a fibroblast cell.

[0293] In some aspects, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to insulin-like growth factor receptor (IGF-1R), an antigen-binding fragment thereof, or any combination thereof, and that comprise an amino acid sequence described herein, as well as antibody expression cassettes comprising nucleotide sequences encoding antibodies or antigen-binding fragments that compete with such antibodies or antigen-binding fragments for binding to IGF-1R, an antigen-binding fragment thereof, or any combination thereof (e.g., in a dose-dependent manner) or bind to the same epitope as such antibodies or antigen-binding fragments.

[0294] In some aspects, the antibody expression cassette is selected from the group consisting of teprotumumab, VRDN-01100 (SEQ ID NO: 113), VRDN-02700 (SEQ ID NO: 116), ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L 17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H 31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52, or an antigen-binding fragment thereof, and a nucleic acid sequence encoding an antibody that competes for binding to the same epitope as

[0295] In some aspects, the antibody expression cassette comprises a nucleic acid sequence encoding an antibody that competes for binding to the same epitope as VRDN-01100 (SEQ ID NO: 113) or VRDN-02700 (SEQ ID NO: 116), or an antigen-binding fragment thereof.

[0296] In some aspects, the antibody expression cassette comprises a nucleic acid sequence encoding an antibody that competes for binding to the same epitope as teprotumumab, or an antigen-binding fragment thereof.

[0297] Also provided herein is an antibody expression cassette comprising a nucleotide sequence encoding a polypeptide comprising the sequence of any one of SEQ ID NOs: 7-24, 25-27, 29-31, 33-37, 39-41, or 43-76. In some embodiments, an antibody or antigen-binding fragment thereof comprising the polypeptide specifically binds to insulin-like growth factor receptor (IGFR), an antigen-binding fragment thereof, or any combination thereof.

[0298] Also provided herein is a kit, vector, or host cell comprising: (i) a first antibody expression cassette comprising a nucleotide sequence encoding any of SEQ ID NOs: 68-76; and (ii) a delivery vector.

[0299] In some aspects, provided herein are antibody expression cassettes comprising a nucleotide sequence comprising three VH domain CDRs, e.g., a nucleotide sequence containing a VH CDR1, a VH CDR2, and a VH CDR3 (see, e.g., Table 3) of any one of the antibodies described herein, e.g., wherein the three VH domain CDRs are in a VH context. In some aspects, provided herein are polynucleotides comprising a nucleotide sequence comprising three VL domain CDRs, e.g., a nucleotide sequence containing a VL CDR1, a VL CDR2, and a VL CDR3 (see, e.g., Table 4) of any one of the antibodies described herein, e.g., wherein the three VL domain CDRs are in a VL context. In some aspects, provided herein is an antibody expression cassette (or combination of polynucleotides) comprising a nucleotide sequence comprising an antibody or antigen-binding fragment thereof, comprising: (i) a nucleotide sequence containing three VH domain CDRs, e.g., a VH CDR1, a VH CDR2, and a VH CDR3 of any one of the antibodies described herein (see, e.g., Table 3), e.g., where the three VH domain CDRs are in a VH context; and (ii) a nucleotide sequence containing three VL domain CDRs, e.g., a VL CDR1, a VL CDR2, and a VL CDR3 of any one of the antibodies described herein (see, e.g., Table 4), e.g., where the three VL domain CDRs are in a VL context.

[0300] In some aspects, provided herein are antibody expression cassettes comprising nucleotide sequences encoding three VH domain CDRs, e.g., a polypeptide containing a VH CDR1, a VH CDR2, and a VH CDR3 of any one of the antibodies described herein (see, e.g., Table 1), e.g., where the three VH domain CDRs are in a VH context. In some aspects, provided herein are antibody expression cassettes comprising nucleotide sequences encoding three VL domain CDRs, e.g., a polypeptide containing a VL CDR1, a VL CDR2, and a VL CDR3 of any one of the antibodies described herein (see, e.g., Table 2), e.g., where the three VL domain CDRs are in a VL context. In some aspects, provided herein is an antibody expression cassette (or combination of polynucleotides) comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof comprising: (i) a polypeptide containing three VH domain CDRs, e.g., a VH CDR1, a VH CDR2, and a VH CDR3 of any one of the antibodies described herein (see, e.g., Table 1), e.g., where the three VH domain CDRs are in a VH context; and (ii) a polypeptide containing three VL domain CDRs, e.g., a VL CDR1, a VL CDR2, and a VL CDR3 of any one of the antibodies described herein (see, e.g., Table 2), e.g., where the three VL domain CDRs are in a VL context.

[0301] In some embodiments, the heavy chain comprises a heavy chain variable region (VH) comprising complementarity determining region (CDR) 1, VH CDR2, and VH CDR3. In some embodiments, the modified VH CDR1-3 correspond to the CDRs of teprotumumab. In some embodiments, the nucleic acid sequence encoding VH CDR1 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7 or 10; the nucleic acid sequence encoding VH CDR2 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, 11 or 14; The nucleic acid sequence encoding the CDR3 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9, 12, or 15.

[0302] In some embodiments, the light chain comprises a light chain variable region (VL) comprising complementarity determining region (CDR) 1, VL CDR2, and VL CDR3. In some embodiments, VL CDR1-3 correspond to the CDRs of teprotumumab. In some embodiments, the nucleic acid sequence encoding the VL CDR1 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16; the nucleic acid sequence encoding the VL CDR2 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17, 21, or 23; The nucleic acid sequence encoding the CDR3 comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18, 21 or 24.

[0303] Also provided herein are antibody expression cassettes encoding the antibodies or antigen-binding fragments thereof, or domains thereof, described herein, which have been optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements.Methods for creating optimized nucleic acids encoding the antibodies or antigen-binding fragments thereof, or domains thereof (e.g., heavy chain, light chain, VH domain, or VL domain) described herein for recombinant expression by introducing codon changes (e.g., codon changes that encode the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in mRNA can therefore be achieved by adapting the optimization methods described in, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, each of which is incorporated herein by reference in its entirety.

[0304] Antibody expression cassettes containing nucleic acid sequences encoding the anti-IGF-1R antibodies or antigen-binding fragments thereof, or domains thereof, described herein can be generated from nucleic acids from a suitable source using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. In some embodiments, the hybridoma cell line has the accession number DSM ACC 2587 (deposited October 4, 2003). In some embodiments, the hybridoma cell line has the accession number DSM ACC 2594 (deposited September 5, 2003). Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the light chain and / or heavy chain of an antibody or antigen-binding fragment thereof. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the variable light chain region and / or variable heavy chain region of an antibody or antigen-binding fragment thereof. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning, eg, to create chimeric and humanized antibodies, or antigen-binding fragments thereof.

[0305] The antibody expression cassettes provided herein may be, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be the coding strand or the non-coding (antisense) strand. In some embodiments, the antibody expression cassette is a cDNA or DNA lacking one or more endogenous introns. In some embodiments, the antibody expression cassette is a non-naturally occurring antibody expression cassette. In some embodiments, the antibody expression cassette is recombinantly produced. In some embodiments, the antibody expression cassette is isolated. In some embodiments, the antibody expression cassette is substantially pure. In some embodiments, the antibody expression cassette is purified from natural components.

[0306] In some embodiments, the viral vectors disclosed herein comprise an antibody expression cassette comprising coding regions for two or more polypeptides, eg, a heavy chain and a light chain.

[0307] If an antibody expression cassette is desired to contain coding regions for two or more individual polypeptide chains, each additional coding region beyond the first is preferably linked to an element that facilitates coexpression of proteins in a host cell, such as an internal ribosome entry sequence (IRES) element (see, e.g., U.S. Pat. No. 4,937,190), a furin cleavage site, a 2A element, or a promoter. In some embodiments, an IRES, a furin cleavage site, or a 2A element can be used when a single vector contains sequences encoding each subunit of a multi-subunit protein. In cases where the protein of interest is an immunoglobulin with the desired specificity, for example, the first coding region (encoding either the heavy or light chain of the immunoglobulin) can be located downstream from the promoter. The second coding region (encoding the remaining chain of the immunoglobulin) can be located downstream from the first coding region, and the IRES, furin cleavage site, or 2A element can be located between the two coding regions, e.g., immediately preceding the second coding region. In some embodiments, incorporation of an IRES, a furin cleavage site, or a 2A element between the sequences of the first and second genes (encoding the heavy and light chains, respectively) may allow both chains to be expressed from the same promoter at approximately the same levels in a cell.

[0308] In some embodiments, the protein of interest comprises two or more subunits, such as immunoglobulin (Ig). In some embodiments, the delivery vector of the present disclosure can comprise a coding region for each of the subunits. For example, a viral vector can comprise both a coding region for an Ig heavy chain (or a variable region of an Ig heavy chain) and a coding region for an Ig light chain (or a variable region of an Ig light chain). In some embodiments, the vector comprises a first coding region for an antibody heavy chain variable region and a second coding region for an antibody light chain variable region. In some embodiments, the two coding regions can be separated, for example, by a 2A self-processing sequence, allowing multicistronic transcription of the two coding regions.

[0309] A viral vector can contain coding regions for two or more proteins of interest. For example, a viral vector can contain a coding region for a first protein of interest and a coding region for a second protein of interest. The first protein of interest and the second protein of interest can be the same or different.

[0310] Kozak consensus sequence, Kozak consensus, or Kozak sequence is known as a sequence present on eukaryotic mRNA and having the consensus (gcc)gccRccAUGG (where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G"). In some embodiments, a vector comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95%, or more sequence identity to a Kozak consensus sequence. In some embodiments, a vector comprises a Kozak consensus sequence after a polynucleotide encoding one or more proteins of interest is inserted into the vector, e.g., into a restriction site downstream of a promoter. For example, a vector can comprise the nucleotide sequence GCCGCCATG (SEQ ID NO: 77), where ATG is the start codon for the protein of interest. In some embodiments, the vector comprises the nucleotide sequence GCGGCCGCCATG (SEQ ID NO: 78), where ATG is the start codon for the protein of interest.

[0311] In certain aspects, provided herein are compositions comprising a delivery vector, eg, a viral vector, comprising a nucleic acid encoding an anti-IGF-1R antibody or antigen-binding fragment.

[0312] In some embodiments, a delivery vector, e.g., a viral vector, comprising a nucleic acid encoding an anti-IGF-1R antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein is secreted from the salivary gland and swallowed. In some embodiments, the therapeutic effect of the secreted anti-IGF-1R antibody or antigen-binding fragment thereof is local, systemic, or both.

[0313] In some embodiments, the delivery vector (e.g., a delivery vector comprising an antibody or antigen-binding fragment thereof that specifically binds to an insulin-like growth factor receptor (IGFR), e.g., human IGF-1R) is suitable for delivery to or near the eye (e.g., one or both eyes), e.g., intraocular, retro-orbital or periorbital, retrobulbar, intramuscularly near the eye, connective tissue near the eye, or any combination thereof. In some embodiments, administration is to an extraocular muscle. In some embodiments, the extraocular muscle is the levator muscle or the glabellar muscle. In some embodiments, administration is to connective tissue. In some embodiments, administration is transconjunctival to the periorbital space. In some embodiments, administration is intralymphatic to the preauricular or submandibular lymph nodes. In some embodiments, delivery or administration is to retro-orbital or periorbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by infusion. In some embodiments, delivery or administration is by injection and / or infusion as a single dose. In some embodiments, single dose administration comprises multiple injections or infusions.

[0314] In some embodiments, the antibody expression cassette comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof, comprising: (i) the VH CDR1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) and VL CDR1-3 (e.g., SEQ ID NOs: 16-18, 19-21, or 22-24) of modified teprotumumab; (ii) the VH (e.g., SEQ ID NO: 26 or 27) and VL (e.g., SEQ ID NO: 30 or 31) of modified teprotumumab; (iii) the HC (e.g., SEQ ID NO: 36 or 37) and LC (e.g., SEQ ID NO: 40 or 41) of modified teprotumumab; or (iv) a sequence comprising any one of SEQ ID NOs: 68-76, further comprising one or more of an IRES, a furin cleavage site, a 2a site, or a dual promoter (e.g., promoter-VH-IRES-VL, etc.). In some embodiments, the antibody expression cassette further comprises a sequence encoding a signal peptide (e.g., an IL-2 or IL-10 signal peptide). In some embodiments, the signal peptide is an IL-2 signal peptide or an IL-10 signal peptide. In some embodiments, the encoded signal peptide comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either of SEQ ID NOs: 119 or 120. In some embodiments, the nucleic acid sequence encoding the signal peptide comprises a nucleic acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either of SEQ ID NOs: 121 or 122. III. Vector Constructs

[0315] Some embodiments of the present disclosure are directed to vector constructs or expression constructs (e.g., antibody expression cassettes) having a eukaryotic promoter operably linked to a DNA of interest encoding an anti-IGF-1R antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein. In some embodiments, vector constructs or expression constructs containing a DNA sequence (or corresponding RNA sequence) that can be used in accordance with the present disclosure can be any eukaryotic expression construct containing a DNA or RNA sequence of interest. For example, a plasmid or viral construct (e.g., an AAV vector) can be cleaved to provide linear DNA with ligatable ends. These ends can be joined to exogenous DNA with complementary, ligatable ends to provide an intact replicon and a biologically functional recombinant DNA molecule with the desired phenotypic properties. In some embodiments, the vector constructs or expression constructs are capable of replicating in both eukaryotic and prokaryotic hosts, and the constructs are known in the art and commercially available.

[0316] In some embodiments, a vector construct or expression construct of the present disclosure encoding an anti-IGF-1R antibody (e.g., teprotumumab) is a multicistronic (e.g., bicistronic) construct (e.g., comprising a heavy chain and a light chain). In some embodiments, the multicistronic (e.g., bicistronic) construct further comprises an F2A or IRES element.

[0317] In some embodiments, the anti-IGF-1R antibody is selected from the group consisting of teprotumumab, VRDN-01100 (SEQ ID NO: 113), VRDN-02700 (SEQ ID NO: 116), ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L2 9H29, L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52, or an antigen-binding fragment thereof.

[0318] In some embodiments, the anti-IGF-1R antibody is VRDN-01100 (SEQ ID NO: 113) or VRDN-02700 (SEQ ID NO: 116), or an antigen-binding fragment thereof.

[0319] In some embodiments, the anti-IGF-1R antibody is teprotumumab or an antigen-binding fragment thereof.

[0320] The exogenous (i.e., donor) DNA used in the present disclosure is obtained from suitable cells, and vector constructs or expression constructs are prepared using techniques well known in the art.Similarly, techniques for obtaining the expression of exogenous DNA or RNA sequences in genetically modified host cells are known in the art (see, for example, Kormal et al., Proc. Natl. Acad. Sci. USA, 84:2150-2154 (1987); Sambrook et al. Molecular Cloning: a Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; each of these is incorporated herein by reference for the methods and compositions for eukaryotic expression of target DNA).

[0321] In some embodiments, the vector construct or expression construct contains a promoter that facilitates expression of the DNA of interest in secretory cells. In some embodiments, the promoter is a strong eukaryotic promoter, such as a promoter derived from human cytomegalovirus (CMV), mouse CMV promoter, mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), or adenovirus. Exemplary promoters include, but are not limited to, the promoter derived from the immediate early gene of human CMV (Boshart et al., Cell 41:521-530 (1985)) and the promoter derived from the long terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982)). In some embodiments, the promoter is a CMV early enhancer / chicken beta actin (CBA) promoter, a CAG promoter, CMV, EF1α, EF1α and a CMV enhancer, a CMV promoter and a CMV enhancer (CMVe / p), a CBA promoter and a CMV enhancer, a CMV promoter and an SV40 intron, a CBA promoter and a CMV enhancer and a CAG intron, an EF1α promoter and a truncated 5'LTR and a chimeric HBG and IgHC intron, or a tissue-specific promoter. In some embodiments, the tissue-specific promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is a DES promoter, an HSA promoter, an MCK promoter, an HMCK7 promoter, a dMCK promoter, a tMCK promoter, a CK8e promoter, a SPc5-12 promoter, a SP-301 promoter, a MH promoter, a Sk-CRM promoter, or a Sk-CRM4 promoter.

[0322] In some embodiments, the promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 34-38. In some embodiments, the nucleic acid sequence comprising the promoter can comprise an intron. In some embodiments, the intron is selected from the group consisting of an SV40 intron, an MVM intron, or a human beta globin intron. In some embodiments, the CMVp promoter is fused to an SV40 intron. In some embodiments, the SV40 intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46.

[0323] Alternatively, the promoter used may be a tissue-specific promoter. For example, if the cells are fibroblasts, the tissue-specific promoter may be the insulin-like growth factor binding protein 2 (IGFBP2) promoter, the fibroblast activation protein (FAP) promoter, or the fibroblast-specific protein 1 (FSP1) promoter. If the cells are muscle cells, the tissue-specific promoter may be the muscle creatine kinase (MCK) promoter, the troponin I (TNNI2) promoter, the skeletal alpha-actin (ASKA) promoter, the DES promoter, the HSA promoter, the MCK promoter, the HMCK7 promoter, the dMCK promoter, the tMCK promoter, the CK8e promoter, the SPc5-12 promoter, the SP-301 promoter, the MH promoter, the Sk-CRM promoter, or the Sk-CRM4 promoter. If the cells are adipocytes, the tissue-specific promoter may be the adiponectin promoter or the adipocyte fatty acid binding protein (AP2) promoter.

[0324] In some embodiments, the vector construct or expression construct contains a first promoter and a second promoter. In some embodiments, the first and second promoters are different. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters initiate transcription in the same direction. In some embodiments, the first and second promoters initiate transcription in different directions. In some embodiments, the first or second promoter is a CMV promoter. In some embodiments, the first or second promoter is an EF-1α promoter.

[0325] In some embodiments, the nucleic acid sequence encoding the first promoter and the nucleic acid sequence encoding the second promoter are operably linked. In some embodiments, the nucleic acid sequence encoding the first promoter and the nucleic acid sequence encoding the second promoter are operably linked by a pause element. In some embodiments, the pause element comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:54.

[0326] Given the genome size limitations of AAV, expression of large proteins in multicistronic vectors (multiple genes or multiple open reading frames) under the control of a single promoter can be a challenge.

[0327] In some embodiments, the multicistronic vector disclosed herein comprises an internal ribosome entry site (IRES) sequence or a 2A peptide. In some embodiments, the construct of the present disclosure can also comprise a proteolytic cleavage site. In some embodiments, the proteolytic cleavage site is a furin cleavage site and / or a 2A cleavage site.

[0328] In some embodiments, vector constructs or expression constructs of the present disclosure can also include other components, such as a marker to aid in selection of cells containing and / or expressing the construct (e.g., an antibiotic resistance gene (e.g., an ampicillin resistance gene) or β-galactosidase), an origin of replication (preferably a high copy number origin of replication) for stable replication of the construct in bacterial cells, a nuclear localization signal, or other elements that facilitate production of the DNA construct, the protein encoded thereby, or both. In some embodiments, vector constructs of the present disclosure can include antibiotic resistance genes, including but not limited to, neomycin, kanamycin, puromycin, and / or zeocin. In some embodiments, vector constructs of the present disclosure can include a ColE1, f1, pUC, p15A, or pMB1 origin of replication.

[0329] In some embodiments, the vector constructs of the present disclosure contain a backbone comprising the ColE1 origin of replication and / or the kanamycin resistance gene of SEQ ID NO:84.

[0330] In some embodiments, the vector construct contains a ColE1 origin of replication and kanamycin resistance, e.g., [ka] [ka] may include:

[0331] For eukaryotic expression, the vector construct or expression construct can include, at a minimum, a eukaryotic promoter operably linked to the DNA of interest, which is in turn operably linked to a polyadenylation sequence. The polyadenylation signal sequence can be selected from any of a variety of polyadenylation signal sequences known in the art. In some embodiments, the polyadenylation signal sequence is the SV40 early polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence is the bovine growth hormone polyadenylation signal sequence (bGHpA). In some embodiments, the polyadenylation signal sequence is the human growth hormone polyadenylation signal sequence (hGHpA). In some embodiments, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence (SV40pA). The construct can also include one or more introns, which can increase the level of expression of the DNA of interest, especially when the DNA of interest is cDNA (e.g., does not contain introns of the naturally occurring sequence). Any of a variety of introns known in the art can be used (e.g., a human β-globin intron, which is inserted into a vector construct or expression construct at a position 5' to the DNA of interest). In some embodiments, the intron is an SV40 intron. In some embodiments, the intron is derived from an immunoglobulin heavy chain. In some embodiments, the intron is chimeric between human β-globin and immunoglobulin heavy chain genes. In some embodiments, the intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 46, 56, or 82.

[0332] In some embodiments, the polynucleotide comprises poly(A). In some embodiments, the poly(A) is synthetic poly(A) or bovine growth hormone (BGH) poly(A). In some embodiments, the polynucleotide comprises a poly(A) sequence that comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 52-53.

[0333] When it is desired to include coding regions for two or more individual polypeptide chains or two or more subunits of a protein of interest in a single viral vector, each additional coding region beyond the first is preferably linked to an element that facilitates coexpression of proteins in host cells, such as an internal ribosome entry sequence (IRES) element (see, for example, U.S. Pat. No. 4,937,190) or a 2A element. In some embodiments, an IRES, a furin cleavage site, or a 2A element can be used when a single vector contains sequences encoding each subunit of a multi-subunit protein. For example, when the protein of interest is an immunoglobulin with a desired specificity, the first coding region (encoding either the heavy or light chain of the immunoglobulin) is located downstream from the promoter. The second coding region (encoding the remaining chain of the immunoglobulin) can be located downstream from the first coding region, and the IRES, furin cleavage site, or a 2A element can be located between the two coding regions, for example, immediately before the second coding region. In some embodiments, incorporation of an IRES, a furin cleavage site, or a 2A element between the sequences of the first and second genes (encoding the heavy and light chains, respectively) may allow both chains to be expressed from the same promoter at approximately the same levels in a cell.

[0334] In some embodiments, the nucleic acid sequence of the vector construct or expression construct comprises, in 5' to 3' direction, a promoter, a heavy chain, an IRES, and a light chain sequence. In some embodiments, the nucleic acid sequence of the construct comprises, in 5' to 3' direction, a promoter, a light chain variable, an IRES, and a heavy chain sequence.

[0335] In some embodiments, the nucleic acid sequence vector construct or expression construct includes a proteolytic cleavage site. For example, the nucleic acid sequence may include a self-processing cleavage site, e.g., a sequence incorporated into the vector construct or expression construct of the present disclosure adjacent to a 2A or 2A-like sequence, providing a means for removing additional amino acids remaining after cleavage by the self-processing cleavage sequence. Exemplary proteolytic cleavage sites are described herein and include, but are not limited to, a furin cleavage site having the consensus sequence RXK(R)R (SEQ ID NO: 79). Such furin cleavage sites can be cleaved by endogenous subtilisin-like proteases, e.g., furin, and other serine proteases in the protein secretion pathway. In some embodiments, other exemplary "additional proteolytic cleavage sites" can be used, for example, as described in Lie et al., Sci Rep 7, 2193 (2017).

[0336] In some aspects, the nucleic acid sequence vector construct or expression construct comprises a nucleic acid encoding a signal peptide operably linked to a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds insulin-like growth factor-1 receptor. In some aspects, the signal peptide is an endogenous signal peptide for HGH and its variants; an endogenous signal peptide for interferon and its variants, including the signal peptides of type I, II, and III interferons and their variants; or an endogenous signal peptide for known cytokines and their variants, such as erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β, and their variants. In some embodiments, the signal peptide is a modified signal peptide. In some aspects, the signal peptide is an IL-2 signal peptide. In some aspects, the signal peptide is an IL-10 signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 119 or 120. In some embodiments, the nucleic acid sequence encoding the signal peptide comprises a nucleic acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 121 or 122.

[0337] In some embodiments, the nucleic acid sequence vector construct or expression construct comprises, in 5' to 3' direction, a promoter, a heavy chain, a furin cleavage site, a 2A cleavage site, and a light chain sequence.

[0338] In some embodiments, the nucleic acid sequence vector construct or expression construct comprises, in the 5' to 3' direction, a promoter, a first signal peptide, a heavy chain, a furin cleavage site, a 2A cleavage site, a second peptide, and a light chain sequence.

[0339] In some embodiments, the nucleic acid sequence vector construct or expression construct comprises, in 5' to 3' direction, a promoter, a light chain, a furin cleavage site, a 2A cleavage site, and a heavy chain sequence.

[0340] In some embodiments, the nucleic acid sequence vector construct or expression construct comprises, in the 5' to 3' direction, a promoter, a first signal peptide, a light chain, a furin cleavage site, a 2A cleavage site, a second signal peptide, and a heavy chain sequence.

[0341] In some embodiments, the nucleic acid sequence vector construct or expression construct comprises, in the 5' to 3' direction, a promoter, a heavy chain sequence of an anti-IGF-1R antibody or antigen-binding fragment thereof, an IRES, and a light chain sequence of an anti-IGF-1R antibody or antigen-binding fragment thereof.

[0342] In some embodiments, the nucleic acid sequence vector construct or expression construct comprises, in the 5' to 3' direction, a promoter, a first signal peptide, a heavy chain sequence of an anti-IGF-1R antibody or antigen-binding fragment thereof, an IRES, a second signal peptide, and a light chain sequence of an anti-IGF-1R antibody or antigen-binding fragment thereof.

[0343] In some embodiments, the nucleic acid sequence vector construct or expression construct comprises, in the 5' to 3' direction, a promoter, a light chain sequence of an anti-IGF-1R antibody or antigen-binding fragment thereof, an IRES, and a heavy chain sequence of an anti-IGF-1R antibody or antigen-binding fragment thereof.

[0344] In some embodiments, the nucleic acid sequence vector construct or expression construct comprises, in the 5' to 3' direction, a promoter, a first signal peptide, a light chain sequence of an anti-IGF-1R antibody or antigen-binding fragment thereof, an IRES, a second signal peptide, and a heavy chain sequence of an anti-IGF-1R antibody or antigen-binding fragment thereof.

[0345] In some embodiments, the vector construct or expression construct comprises, in 5' to 3' direction, a first promoter, a nucleic acid sequence encoding a light chain, a second promoter, and a nucleic acid sequence encoding a heavy chain.

[0346] In some embodiments, the vector construct or expression construct comprises, in the 5' to 3' direction, a first promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a light chain, a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a heavy chain.

[0347] In some embodiments, the vector construct or expression construct comprises, in 5' to 3' direction, a first promoter, a nucleic acid sequence encoding a heavy chain, a second promoter, and a nucleic acid sequence encoding a light chain.

[0348] In some embodiments, the vector construct or expression construct comprises, in the 5' to 3' direction, a first promoter, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a heavy chain, a second promoter, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a light chain.

[0349] In some embodiments, the vector construct or expression construct comprises, in 5' to 3' direction, a nucleic acid sequence encoding a heavy chain, a first promoter sequence, a second promoter sequence, and a nucleic acid sequence encoding a light chain.

[0350] In some embodiments, the vector construct or expression construct comprises, in the 5' to 3' direction, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a heavy chain, a first promoter sequence, a second promoter sequence, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a light chain.

[0351] In some embodiments, the vector construct or expression construct comprises, in 5' to 3' direction, a nucleic acid sequence encoding a light chain, a first promoter sequence, a second promoter sequence, and a nucleic acid sequence encoding a heavy chain.

[0352] In some embodiments, the vector construct or expression construct comprises, in the 5' to 3' direction, a nucleic acid sequence encoding a first signal peptide, a nucleic acid sequence encoding a light chain, a first promoter sequence, a second promoter sequence, a nucleic acid sequence encoding a second signal peptide, and a nucleic acid sequence encoding a heavy chain.

[0353] In some embodiments, the promoter is selected from the group consisting of a CAG promoter, a CBA promoter, a CMV promoter, an EF1α promoter, an EF1α promoter and a CMV enhancer, a CMV promoter and a CMV enhancer (CMVe / p), a CMV promoter and an SV40 intron, or a tissue-specific promoter. In some embodiments, the tissue-specific promoter is selected from a DES promoter, an HSA promoter, an MCK promoter, an HMCK7 promoter, a dMCK promoter, a tMCK promoter, a CK8e promoter, an SPc5-12 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM promoter, and an Sk-CRM4 promoter.

[0354] In some embodiments, the promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 47-51, 83, or 93. In some embodiments, the nucleic acid sequence comprising the promoter can comprise an intron. In some embodiments, the intron is selected from the group consisting of an SV40 intron, an MVM intron, or a human beta-globin intron. In some embodiments, the CMVp is fused to the SV40 intron. In some embodiments, the CAG intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 82. In some embodiments, the SV40 intron comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:46.

[0355] In some embodiments, the first and second promoters are different. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters initiate transcription in the same direction. In some embodiments, the first and second promoters initiate transcription in different directions.

[0356] In some embodiments, the nucleic acid sequence encoding the first promoter and the nucleic acid sequence encoding the second promoter are operably linked. In some embodiments, the nucleic acid sequence encoding the first promoter and the nucleic acid sequence encoding the second promoter are operably linked by a pause element. In some embodiments, the pause element comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:54.

[0357] In some aspects, the signal is selected from the group consisting of endogenous signal peptides for HGH and its variants; endogenous signal peptides for interferons and their variants, including the signal peptides of type I, II, and III interferons and their variants; and endogenous signal peptides for known cytokines and their variants, such as erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β, and their variants. In some embodiments, the signal peptide is a modified signal peptide. In some aspects, the signal peptide is an IL-2 signal peptide. In some aspects, the signal peptide is an IL-10 signal peptide. In some aspects, the signal peptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 119 or 120. In some embodiments, the nucleic acid sequence encoding the signal peptide comprises a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 121 or 122.

[0358] The vector for delivery of the target DNA can be viral or non-viral, or can consist of naked DNA mixed with adjuvants such as viral particles (e.g., AAV particles) or cationic lipids or liposomes. An "adjuvant" is a substance that does not produce the desired effect by itself, but acts to enhance or otherwise improve the action of an active compound. The specific vector and vector formulation used depend on several factors, such as the secretory gland targeted for gene transfer.

[0359] In some embodiments, the present disclosure provides a composition comprising a delivery vector, for example, a viral vector, comprising a nucleic acid construct or expression construct comprising a nucleic acid encoding an anti-IGF-1R antibody (e.g., a monoclonal antibody) or its antigen-binding fragment. In some embodiments, the delivery vector is suitable for delivery to periorbital or retroorbital tissue. In some embodiments, the tissue is connective tissue, muscle tissue, or adipose tissue.

[0360] In some embodiments, the vector construct or expression construct comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof (e.g., teprotumumab), including a construct comprising: (i) a VH CDR1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) and a VL CDR1-3 (e.g., SEQ ID NOs: 16-18, 19-21, or 22-24); (ii) a VH (e.g., SEQ ID NO: 26 or 27) and a VL (e.g., SEQ ID NO: 30 or 31); (iii) a HC (e.g., SEQ ID NO: 36 or 37) and a LC (e.g., SEQ ID NO: 40 or 41); or (iv) any one of SEQ ID NOs: 68-76, further comprising one or more of an IRES, a furin cleavage site, a 2a site, or a dual promoter (e.g., promoter-VH-IRES-VL, etc.).

[0361] In some embodiments, the nucleic acid construct or expression construct comprises, in the 5' to 3' direction, a polynucleotide comprising a first promoter, a nucleic acid sequence encoding the heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, an F2A site, a light chain of the anti-IGF-1R antibody or antigen-binding fragment thereof, and poly(A).

[0362] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a first promoter, a nucleic acid sequence encoding a first signal peptide, a heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, an F2A site, a second signal peptide, a light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and poly(A).

[0363] In some embodiments, the nucleic acid construct or expression construct comprises, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CBA promoter, a CAG intron, a CBA exon, a nucleic acid encoding the heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, a furin cleavage site, a linker, a 2A peptide, a nucleic acid encoding the light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and BGHpA.

[0364] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CBA promoter, a CAG intron, a CBA exon, a nucleic acid encoding a first signal peptide, a heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, a furin cleavage site, a linker, a 2A peptide, a nucleic acid encoding a second signal peptide, a light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and BGHpA.

[0365] In some embodiments, the nucleic acid construct or expression construct comprises, from 5' to 3', a polynucleotide comprising a first promoter, a nucleic acid sequence encoding the heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, IRES2, a light chain of the anti-IGF-1R antibody or antigen-binding fragment thereof, and poly(A).

[0366] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a first promoter, a nucleic acid sequence encoding a first signal peptide, a heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, IRES2, a second signal peptide, a light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and poly(A).

[0367] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CBA promoter, a CAG intron, a CBA exon, a nucleic acid encoding the light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, an IRES2 element, a nucleic acid encoding the heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and SynpA.

[0368] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CBA promoter, a CAG intron, a CBA exon, a nucleic acid encoding a first signal peptide, a light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, an IRES2 element, a nucleic acid encoding a second signal peptide, a heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and SynpA.

[0369] In some embodiments, the nucleic acid construct or expression construct comprises, from 5' to 3', a polynucleotide comprising a first promoter, an intron, a nucleic acid sequence encoding the light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, poly(A), a pause element, a second promoter, a 5' LTR, a chimeric intron, a nucleic acid sequence encoding the heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and poly(A).

[0370] In some embodiments, the nucleic acid construct or expression construct comprises, from 5' to 3', a polynucleotide comprising a first promoter, an intron, a nucleic acid sequence encoding a first signal peptide, the light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, poly(A), a pause element, a second promoter, a 5' LTR, a chimeric intron, a nucleic acid sequence encoding a second signal peptide, the heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and poly(A).

[0371] In some embodiments, the nucleic acid construct or expression construct comprises, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CMV promoter, an SV40 intron, a nucleic acid encoding the light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, BGHpA, a pause element, an EF1α promoter, a 5' LTR, a chimeric human beta globin and immunoglobulin heavy chain intron, a nucleic acid encoding the heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and SynpA.

[0372] In some embodiments, the nucleic acid construct or expression construct comprises, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CMV promoter, an SV40 intron, a nucleic acid encoding a first signal peptide, a light chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, BGHpA, a pause element, an EF1α promoter, a 5' LTR, a chimeric human beta globin and immunoglobulin heavy chain intron, a nucleic acid encoding a second signal peptide, a heavy chain of an anti-IGF-1R antibody or antigen-binding fragment thereof, and SynpA.

[0373] In some embodiments, the nucleic acid construct or expression construct comprises, in the 5' to 3' direction, a polynucleotide comprising a first promoter, a nucleic acid sequence encoding a heavy chain, poly(A), a pause element, a second promoter, a 5' LTR, a nucleic acid sequence encoding a light chain, and poly(A).

[0374] In some embodiments, the nucleic acid construct or expression construct includes, in the 5' to 3' direction, a polynucleotide comprising a first promoter, a nucleic acid sequence encoding a first signal peptide, a heavy chain, poly(A), a pause element, a second promoter, a 5' LTR, a nucleic acid sequence encoding a second signal peptide, a light chain, and poly(A).

[0375] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CMV promoter, a nucleic acid encoding a heavy chain, BGHpA, a pause element, an EF1α promoter, a 5' LTR, a nucleic acid encoding a light chain, and SynpA.

[0376] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CMV promoter, a nucleic acid encoding a first signal peptide, a heavy chain, BGHpA, a pause element, an EF1α promoter, a 5' LTR, a nucleic acid encoding a second signal peptide, a light chain, and SynpA.

[0377] In some embodiments, the nucleic acid construct or expression construct comprises, in the 5' to 3' direction, a polynucleotide comprising a first promoter, a nucleic acid sequence encoding a light chain, poly(A), a pause element, a second promoter, a 5' LTR, a nucleic acid sequence encoding a heavy chain, and poly(A).

[0378] In some embodiments, the nucleic acid construct or expression construct includes, in the 5' to 3' direction, a polynucleotide comprising a first promoter, a nucleic acid sequence encoding a first signal peptide, a light chain, poly(A), a pause element, a second promoter, a 5' LTR, a nucleic acid sequence encoding a second signal peptide, a heavy chain, and poly(A).

[0379] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CMV promoter, a nucleic acid encoding a light chain, BGHpA, a pause element, an EF1α promoter, a 5' LTR, a nucleic acid encoding a heavy chain, and SynpA.

[0380] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising a CMV enhancer, a CMV promoter, a nucleic acid encoding a first signal peptide, a light chain, BGHpA, a pause element, an EF1α promoter, a 5' LTR, a nucleic acid encoding a second signal peptide, a heavy chain, and SynpA.

[0381] In some embodiments, the nucleic acid construct or expression construct comprises, from 5' to 3', a polynucleotide comprising poly(A), a nucleic acid sequence encoding a light chain, an intron, a 5' LTR, a first promoter, a second promoter, an intron, a nucleic acid sequence encoding a heavy chain, and poly(A).

[0382] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising poly(A), a nucleic acid sequence encoding a first signal peptide, a light chain, an intron, a 5' LTR, a first promoter, a second promoter, an intron, a nucleic acid sequence encoding a second signal peptide, a heavy chain, and poly(A).

[0383] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising SYNpA, a nucleic acid encoding a light chain, a chimera of a beta globin intron and an immunoglobulin heavy chain intron, a 5' LTR, an EF1α promoter fused to a CMV enhancer, a CMV promoter fused to an SV40 intron, a nucleic acid sequence encoding a heavy chain, and BGHpA.

[0384] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising SYNpA, a nucleic acid encoding a first signal peptide, a light chain, a chimera of a beta globin intron and an immunoglobulin heavy chain intron, a 5' LTR, an EF1α promoter fused to a CMV enhancer, a CMV promoter fused to an SV40 intron, a nucleic acid sequence encoding a second signal peptide, a heavy chain, and BGHpA.

[0385] In some aspects, the nucleic acid construct or expression construct comprises, from 5' to 3', a polynucleotide comprising poly(A), a nucleic acid sequence encoding a heavy chain, an intron, a 5' LTR, a first promoter, a second promoter, an intron, a nucleic acid sequence encoding a light chain, and poly(A).

[0386] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising poly(A), a nucleic acid sequence encoding a first signal peptide, a heavy chain, an intron, a 5' LTR, a first promoter, a second promoter, an intron, a nucleic acid sequence encoding a second signal peptide, a light chain, and poly(A).

[0387] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising SYNpA, a nucleic acid encoding a heavy chain, a chimera of a beta globin intron and an immunoglobulin heavy chain intron, a 5' LTR, an EF1α promoter fused to a CMV enhancer, a CMV promoter fused to an SV40 intron, a nucleic acid sequence encoding a light chain, and BGHpA.

[0388] In some embodiments, the nucleic acid construct or expression construct includes, from 5' to 3', a polynucleotide comprising SYNpA, a nucleic acid encoding a first signal peptide, a heavy chain, a chimera of a beta globin intron and an immunoglobulin heavy chain intron, a 5' LTR, an EF1α promoter fused to a CMV enhancer, a CMV promoter fused to an SV40 intron, a nucleic acid sequence encoding a second signal peptide, a light chain, and BGHpA.

[0389] In some embodiments, a vector construct or expression construct (e.g., an antibody expression cassette) disclosed herein comprises one or more of the elements listed in Table 15. Table 15. Nucleic acid sequences of exemplary construct elements. [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6] [Table 15-7] III.A. Delivery Vectors

[0390] In some aspects, the delivery vector is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome. In some aspects, the therapeutic effect of the antibody or antigen-binding fragment thereof is local, systemic, or both.

[0391] In certain aspects, disclosed herein are compositions comprising a delivery vector (e.g., a viral vector, a non-viral vector, a plasmid, a lipid, a protein particle, a bacterial vector, or a lysosome) comprising a nucleic acid encoding or comprising an antibody (e.g., a monoclonal antibody) or an antigen-binding fragment thereof.

[0392] In some embodiments, the delivery vector is suitable for delivery to or near the eye (e.g., one or both eyes), e.g., intraocular, retro-orbital or peri-orbital, retrobulbar, intramuscularly near the eye, connective tissue near the eye, or any combination thereof. In some embodiments, delivery or administration is to retro-orbital or peri-orbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by injection and / or infusion as a single dose. In some embodiments, single dose administration comprises multiple injections or infusions.

[0393] In some aspects, a composition comprising a delivery vector (e.g., a viral vector, a non-viral vector, a plasmid, a lipid, a protein particle, a bacterial vector, or a lysosome) comprising a nucleic acid encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein is produced in a cell. In some aspects, the cell is a fibroblast, an adipocyte, a myofibroblast, a muscle cell, or any combination thereof.

[0394] In some aspects, compositions comprising a delivery vector (e.g., a viral vector, a non-viral vector, a plasmid, a lipid, a protein particle, a bacterial vector, or a lysosome) comprising a nucleic acid encoding or comprising an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein are suitable for delivery to connective tissue, muscle tissue, and / or adipose tissue. In some aspects, administration is to an extraocular muscle. In some aspects, the extraocular muscle is the levator muscle or the glabellar muscle. In some aspects, administration is to connective tissue. In some aspects, administration is transconjunctival to the periorbital space. In some aspects, administration is intralymphatic to the preauricular or submandibular lymph nodes.

[0395] In some embodiments, the delivery vector comprises (i) a VH CDR1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) and a VL CDR2-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) (ii) CDR1-3 (e.g., SEQ ID NO: 16-18, 19-21, or 22-24); (ii) VH (e.g., SEQ ID NO: 26 or 27) and VL (e.g., SEQ ID NO: 30 or 31); (iii) HC (e.g., SEQ ID NO: 36 or 37) and LC (e.g., SEQ ID NO: 40 or 41); or (iv) any one of SEQ ID NOs: 68-76, further comprising one or more of an IRES, a furin cleavage site, a 2a site, a dual promoter (e.g., promoter-VH-IRES-VL, etc.), or a signal peptide (e.g., an IL-2 or IL-10 signal peptide), and the vector construct or expression construct (e.g., antibody expression cassette) comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof (e.g., teprotumumab). III.A.1 Non-viral vectors

[0396] The target DNA can be administered using a non-viral vector. As used herein, "non-viral vector" refers to naked DNA, chemical preparations containing naked DNA (e.g., a preparation of DNA and a cationic compound (e.g., dextran sulfate)), and naked DNA mixed with an adjuvant such as a viral particle (i.e., the target DNA is not contained within a viral particle, but the transformation preparation is composed of both naked DNA and a viral particle (e.g., AAV particle)) (see, for example, Curiel et al., Am. J. Respir. Cell Mol. Biol. 6:247-52 (1992)). Therefore, "non-viral vector" can include a vector composed of DNA and a viral particle, where the viral particle does not contain the target DNA within the viral genome.

[0397] In some embodiments, the non-viral vector is a bacterial vector. See, e.g., Baban et al., Bioeng Bugs., 1(6):385-394 (2010).

[0398] In some embodiments, the target DNA can be complexed with polycationic substances, such as poly-L-lysine or DEAC-dextran, targeting ligands, and / or DNA-binding proteins (e.g., histones). DNA- or RNA-liposome complex formulations contain a mixture of lipids that bind to genetic material (DNA or RNA) and facilitate the delivery of nucleic acids to cells. Liposomes that can be used according to the present disclosure include DOPE (dioleylphosphatidylethanolamine), CUDMEDA (N-(5-cholestrum-3-β-ol 3-urethanyl)-N',N'-dimethylethylenediamine).

[0399] Lipids that can be used in accordance with the present disclosure include, but are not limited to, DOPE (dioleoylphosphatidylethanolamine), cholesterol, and CUDMEDA (N-(5-cholestrum-3-ol-3-uretanyl)-N',N'-dimethylethylenediamine). As an example, DNA can be administered in a solution containing one of the following cationic liposome formulations: Lipofectin™ (LTI / BRL), Transfast™ (Promega Corp), Tfx50™ (Promega Corp), Tfx10™ (Promega Corp), or Tfx20™ (Promega Corp). The concentration of the liposome solution ranges from about 2.5% to 15% volume:volume, preferably about 6% to 12% volume:volume. Further exemplary methods and compositions for formulating nucleic acids (e.g., DNA, including DNA or RNA not contained within a viral particle) for delivery by the methods of the present disclosure are described in U.S. Patent Nos. 5,892,071; 5,744,625; 5,925,623; 5,527,928; 5,824,812; and 5,869,715.

[0400] Polymer particles can be used in accordance with the present disclosure for polymer-based gene delivery. See, e.g., Putnam et al., PNAS 98 (3): 1200-1205 (2001).

[0401] The target DNA can also be administered as a chemical formulation of DNA or RNA coupled to a carrier molecule (e.g., an antibody or receptor ligand) that facilitates delivery to host cells for the purpose of altering the biological properties of the host cells. The term "chemical formulation" refers to the modification of nucleic acids to allow the coupling of nucleic acid compounds to carrier molecules, such as proteins or lipids, or their derivatives. Exemplary protein carrier molecules include antibodies specific to target cells or receptor ligands, i.e., molecules that can interact with receptors associated with target cells.

[0402] In certain aspects, compositions comprising a non-viral delivery vector comprising a nucleic acid encoding or comprising an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein are suitable for delivery to or near the eye (e.g., one or both eyes), for example, intraocularly, retro-orbitally or periorbitally, retrobulbarly, intramuscularly near the eye, connective tissue near the eye, or any combination thereof. In some aspects, the periorbital or retro-orbital tissue is selected from muscle, connective tissue, and / or adipose tissue. In some aspects, administration is to an extraocular muscle. In some aspects, the extraocular muscle is the levator muscle or the glabellar muscle. In some aspects, administration is to connective tissue. In some aspects, administration is transconjunctival to the periorbital space. In some aspects, administration is intralymphatic to a preauricular or submandibular lymph node. In some aspects, delivery or administration is to retro-orbital or periorbital fibroblasts, adipocytes, myofibroblasts, myocytes, or any combination thereof. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by infusion. In some embodiments, delivery or administration is by injection and / or infusion as a single dose. In some embodiments, single dose administration comprises multiple injections or infusions.

[0403] In some embodiments, the composition comprising the non-viral delivery vector comprises an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, or a nucleic acid encoding a therapeutic peptide produced in target cells. In some embodiments, the therapeutic effect of the antibody or antigen-binding fragment thereof is local, systemic, or both.

[0404] In some embodiments, the non-viral vector comprises (i) a VH CDR1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) and a VL CDR2-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) (ii) CDR1-3 (e.g., SEQ ID NO: 16-18, 19-21, or 22-24); (ii) VH (e.g., SEQ ID NO: 26 or 27) and VL (e.g., SEQ ID NO: 30 or 31); (iii) HC (e.g., SEQ ID NO: 36 or 37) and LC (e.g., SEQ ID NO: 40 or 41); or (iv) any one of SEQ ID NOs: 68-76, further comprising one or more of an IRES, a furin cleavage site, a 2a site, a dual promoter (e.g., promoter-VH-IRES-VL, etc.), or a signal peptide (e.g., an IL-2 or IL-10 signal peptide), and the vector construct or expression construct (e.g., antibody expression cassette) comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof (e.g., teprotumumab). III.A.2 Viral vectors

[0405] Generally, viral vectors used in accordance with the present disclosure are composed of viral particles derived from naturally occurring viruses that have been genetically modified to render them replication-deficient and to express a recombinant gene of interest according to the present disclosure. When the virus delivers its genetic material to a cell, it does not give rise to additional infectious virus, but rather introduces the exogenous recombinant gene into the cell, preferably into the genome of the cell.

[0406] Many viral vectors are well known in the art, including, for example, retrovirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus vectors.Retrovirus requires cells to replicate, and secretory glands are mostly composed of slowly replicating and / or terminally differentiated cells, so retrovirus vectors are less preferred.Adenovirus and AAV are preferred viral vectors because they efficiently infect slowly replicating and / or terminally differentiated cells.In some embodiments, the delivery vector (e.g., viral vector) is selected from the group consisting of adeno-associated virus (AAV) vector, adenovirus vector, lentivirus vector, or retrovirus vector.

[0407] When replication-defective virus is used as a viral vector, the production of infectious viral particles containing either DNA or RNA corresponding to the target DNA can be generated by introducing the viral construct into a recombinant cell line that provides the missing components essential for viral replication.In some embodiments, the transformation of a recombinant cell line with a recombinant viral vector does not result in the production of replication-competent viruses, for example, by the homologous recombination of the viral sequence of the recombinant cell line into the introduced viral vector.Methods for producing replication-defective viral particles containing target nucleic acid are well known in the art, and are described, for example, in Rosenfeld et al., Science 252:431-434 (1991) and Rosenfeld et al., Cell 68:143-155 (1992) (adenovirus); U.S. Patent No. 5,139,941 (adeno-associated virus); U.S. Patent No. 4,861,719 (retrovirus); and U.S. Patent No. 5,356,806 (vaccinia virus).

[0408] In certain embodiments, viral delivery vectors comprising nucleic acids encoding or comprising the antibodies (e.g., monoclonal antibodies) or antigen-binding fragments thereof disclosed herein are suitable for delivery to or near the eye (e.g., one or both eyes), e.g., intraocular, retro-orbital or periorbital, retrobulbar, intramuscularly near the eye, connective tissue near the eye, or any combination thereof. In some embodiments, the periorbital or retro-orbital tissue is selected from muscle, connective tissue, and / or adipose tissue. In some embodiments, administration is to an extraocular muscle. In some embodiments, the extraocular muscle is the levator muscle or the glabellar muscle. In some embodiments, administration is to connective tissue. In some embodiments, administration is transconjunctival to the periorbital space. In some embodiments, administration is intralymphatic to the preauricular or submandibular lymph nodes. In some embodiments, delivery or administration is to retro-orbital or periorbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by injection and / or infusion as a single dose. In some embodiments, single dose administration comprises multiple injections or infusions.

[0409] In some embodiments, a viral delivery vector comprising a nucleic acid encoding a therapeutic protein, e.g., an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, is produced in the target cell. In some embodiments, the therapeutic effect of the therapeutic antibody or antigen-binding fragment thereof is local, systemic, or both.

[0410] In some embodiments, the viral vector comprises (i) a VH CDR1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) and a VL CDR2-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) (ii) CDR1-3 (e.g., SEQ ID NO: 16-18, 19-21, or 22-24); (ii) VH (e.g., SEQ ID NO: 26 or 27) and VL (e.g., SEQ ID NO: 30 or 31); (iii) HC (e.g., SEQ ID NO: 36 or 37) and LC (e.g., SEQ ID NO: 40 or 41); or (iv) any one of SEQ ID NOs: 68-76, further comprising one or more of an IRES, a furin cleavage site, a 2a site, a dual promoter (e.g., promoter-VH-IRES-VL, etc.), or a signal peptide (e.g., an IL-2 or IL-10 signal peptide), and the vector construct or expression construct (e.g., antibody expression cassette) comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof (e.g., teprotumumab). IV. Adeno-associated virus (AAV)-mediated gene therapy

[0411] AAV, a parvovirus belonging to the Dependovirus genus, possesses several attractive features not found in other viruses. For example, AAV can infect a wide range of host cells, including non-dividing cells. Furthermore, AAV can infect cells from different species. Importantly, AAV is not associated with any human or animal diseases and does not appear to alter the physiological properties of host cells upon integration. Finally, AAV is stable under a wide range of physical and chemical conditions, making it suitable for production, storage, and transportation requirements.

[0412] The AAV genome, a linear, single-stranded DNA molecule containing approximately 4700 nucleotides (the AAV-2 genome consists of 4681 nucleotides), generally contains an internal, non-repeated segment flanked at each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 nucleotides in length (AAV-1 has an ITR of 143 nucleotides) and have multiple functions, including serving as origins of replication and as packaging signals for the viral genome.

[0413] The internal, non-repeated portion of the genome contains two large open reading frames (ORFs) known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode the replication and capsid gene products, respectively, which enable the replication, assembly, and packaging of complete AAV virions. More specifically, at least four families of viral proteins are expressed from the AAV rep region: Rep 78, Rep 68, Rep 52, and Rep 40, all named for their apparent molecular weights. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.

[0414] AAV is a helper-dependent virus and requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) to form a functionally complete AAV virion. In the absence of co-infection with a helper virus, AAV establishes a latent state, in which the viral genome is inserted into the host cell chromosome or exists in an episomal form, but infectious virions are not produced. Subsequent infection with a helper virus "rescues" the integrated genome, allowing it to replicate and be packaged into viral capsids, thereby reconstituting infectious virions. AAV can infect cells from different species, but the helper virus must be of the same species as the host cell. Thus, for example, human AAV replicates in canine cells co-infected with canine adenovirus.

[0415] To produce recombinant AAV (rAAV) virions containing DNA, a suitable host cell line is transfected with an AAV vector containing DNA but lacking rep and cap. The host cells are then infected with wild-type (wt) AAV and a suitable helper virus to form rAAV virions. Alternatively, wt AAV genes (known as helper function genes, including rep and cap) and helper virus function genes (known as accessory function genes) can be provided in one or more plasmids, thereby eliminating the need for wt AAV and helper virus in the production of rAAV virions. The helper and accessory function gene products are expressed in the host cell, where they act in trans in the rAAV vector containing the heterologous gene. The heterologous gene is then replicated and packaged as if it were a wt AAV genome, forming recombinant AAV virions. When a patient's cells are transduced with the resulting rAAV virions, the DNA enters and is expressed in the patient's cells. Because the patient's cells lack the rep and cap genes and accessory function genes, the rAAV virion cannot further replicate and package its genome. Also, without the rep and cap genes, wt AAV virions cannot form in the patient's cells. See, e.g., U.S. Patent Application Publication No. 2003 / 0147853.

[0416] In some embodiments, the AAV vector of the present disclosure can comprise or be derived from any natural or recombinant AAV serotype.According to the present disclosure, the AAV serotype can be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, and AAV12.In some embodiments, the AAV serotype is AAV1, AAV2, AAV6, AAV8, or AAV9.In some embodiments, the AAV vector is modified compared to the wild-type AAV serotype sequence.In some embodiments, the AAV vector is modified compared to the wild-type AAV1, AAV2, AAV6, AAV8, or AAV9.In some embodiments, the AAV vector serotype is AAV1 or a modified AAV vector derived therefrom.In some embodiments, the AAV vector serotype is AAV2 or a modified AAV vector derived therefrom. In some embodiments, AAV vector serotype is AAV6 or modified AAV vector derived therefrom.In some embodiments, AAV vector serotype is AAV8 or modified AAV vector derived therefrom.In some embodiments, AAV vector serotype is AAV9 or modified AAV vector derived therefrom.In some embodiments, AAV vector serotype is AAVMYO (see Weinmann et al. Nat. Comm. 11: 5432, 2020).

[0417] In certain embodiments, compositions comprising an AAV delivery vector containing a nucleic acid encoding or comprising an antibody (e.g., a monoclonal antibody) or an antigen-binding fragment thereof are disclosed herein. In some embodiments, the AAV delivery vector containing a nucleic acid encoding or comprising the antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein is suitable for delivery to or near the eye (e.g., one or both eyes), for example, intraocular, retro-orbital or periorbital, retrobulbar, intramuscularly near the eye, connective tissue near the eye, or any combination thereof. In some embodiments, the periorbital or retro-orbital tissue is selected from muscle, connective tissue, and / or adipose tissue. In some embodiments, administration is to an extraocular muscle. In some embodiments, the extraocular muscle is the levator muscle or the glabellar muscle. In some embodiments, administration is to connective tissue. In some embodiments, administration is transconjunctival to the periorbital space. In some embodiments, administration is intralymphatic to a preauricular or submandibular lymph node. In some embodiments, the delivery or administration is to retro-orbital or periorbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof. In some embodiments, the delivery or administration is by injection. In some embodiments, the delivery or administration is by infusion. In some embodiments, the delivery or administration is by injection and / or infusion as a single dose. In some embodiments, single dose administration comprises multiple injections or infusions.

[0418] In some embodiments, an AAV delivery vector comprising a nucleic acid encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein is produced in fibroblasts, myofibroblasts, muscle cells, and / or adipocytes. In some embodiments, the therapeutic effect of the antibody or antigen-binding fragment thereof is local, systemic, or both.

[0419] In some embodiments, the AAV delivery vector comprises (i) VH CDRs 1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) and VL CDRs 1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15). (ii) CDR1-3 (e.g., SEQ ID NO: 16-18, 19-21, or 22-24); (ii) VH (e.g., SEQ ID NO: 26 or 27) and VL (e.g., SEQ ID NO: 30 or 31); (iii) HC (e.g., SEQ ID NO: 36 or 37) and LC (e.g., SEQ ID NO: 40 or 41); or (iv) any one of SEQ ID NOs: 68-76, further comprising one or more of an IRES, a furin cleavage site, a 2a site, a dual promoter (e.g., promoter-VH-IRES-VL, etc.), or a signal peptide (e.g., an IL-2 or IL-10 signal peptide), and the vector construct or expression construct (e.g., antibody expression cassette) comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof (e.g., teprotumumab). IV.A. AAV Vector Components IV.A.1 Inverted Terminal Repeats (ITRs)

[0420] The AAV vector of the present disclosure comprises a viral genome having at least one ITR region and a payload region, e.g., a therapeutic protein, e.g., an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, or a fusion protein (e.g., an Fc fusion protein), or a polynucleotide (e.g., an antibody expression cassette) comprising a nucleic acid encoding a therapeutic peptide. In some embodiments, the AAV vector has two ITRs. These two ITRs flank the payload region at the 5' and 3' ends. The ITRs function as origins of replication containing recognition sites for replication. The ITRs contain sequence regions that can be complementary and symmetrically arranged. The ITRs incorporated into the AAV vector of the present disclosure can be composed of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0421] The ITRs can be derived from the same serotype as the capsid, or can be derivatives thereof, selected from any of the serotypes listed herein. The ITRs can be of a different serotype from the capsid. In some embodiments, the AAV vector has two or more ITRs. In a non-limiting example, the AAV vector has a viral genome comprising two ITRs. In some embodiments, the ITRs are of the same serotype as each other. In some embodiments, the ITRs are of different serotypes. Non-limiting examples include zero, one, or both ITRs having the same serotype as the capsid. In some embodiments, both ITRs of the AAV vector are AAV2 ITRs.

[0422] Independently, each ITR can be about 75 to about 175 nucleotides in length. The ITRs can be about 100 to 105, about 106 to 110, about 111 to 115, about 116 to 120, about 121 to 125, about 126 to 130, about 131 to 135, about 136 to 140, about 141 to 145, or about 146 to 150 nucleotides in length. In some embodiments, the ITRs are about 140 to 142 nucleotides in length. Non-limiting examples of ITR lengths are about 102, about 140, about 141, about 142, and about 145 nucleotides, and those having at least 95% identity thereto.

[0423] In some embodiments, the AAV vector may be any of a variety of vectors, including, but not limited to, about 75-80, about 75-85, about 75-100, about 80-85, about 80-90, about 80-105, about 85-90, about 85-95, about 85-110, about 90-95, about 90-100, about 90-115, about 95-100, about 95-105, Approximately 95-120, approximately 100-105, approximately 100-110, approximately 100-125, approximately 105-110, approximately 105-115, approximately 105-130, approximately 110-115, approximately 110-120, approximately 110-135, approximately 115-120, approximately 115-125, approximately 115-140, approximately 120-125, approximately 120-130, approximately 120-145, approximately 125-130, approximately 125-135, approximately 125-150, approximately 130-135, approximately 130-140, approximately 130-155, approximately 135-140, approximately 135-145, approximately 135-160, approximately 140-145, approximately 140-150, approximately 140-165, approximately 145-150, approximately 145-155, approximately and at least one inverted terminal repeat sequence having a length of, for example, 145-170, about 150-155, about 150-160, about 150-175, about 155-160, about 155-165, about 160-165, about 160-170, about 165-170, about 165-175, or about 170-175 nucleotides.

[0424] In some embodiments, the length of the first and / or second ITR region for an AAV vector is about 75-80, about 75-85, about 75-100, about 80-85, about 80-90, about 80-105, about 85-90, about 85-95, about 85-110, about 90-95, about 90-100, about 90-115, about 95-100, about 95-105, about 95-120, about 100-105, about 100-110, about 100-125, about 105-110, about 105-115, about 105-130, about 110-115, about 110-120, about 110-135, about 115-120, about 115-125, about 115-140, about 120-125, approx. 120-130, approx. 120-145, approx. 125-130, approx. 125-135, approx. 125-150, approx. 130-135, approx. 130-140, approx. 130-155, approx. 135-140, approx. 135-145, approx. 135-160, approx. 140-145, approx. 140-150, approx. 140-160 5, about 145-150, about 145-155, about 145-170, about 150-155, about 150-160, about 150-175, about 155-160, about 155-165, about 160-165, about 160-170, about 165-170, about 165-175, and about 170-175 nucleotides.

[0425] In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located near the 5' end of the flipped ITR in the vector. In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located near the 3' end of the flipped ITR in the vector. In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located near the 5' end of the flipped ITR in the vector. In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located near the 3' end of the flipped ITR in the vector. In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located between the 5' end of the flip ITR and the 3' end of the flop ITR in the vector. In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located between the 3' end of the flip ITR and the 5' end of the flip ITR in the vector (e.g., between the 5' end of the flip ITR and the 3' end of the flop ITR, or midway between the 3' end of the flop ITR and the 5' end of the flop ITR).

[0426] In some aspects, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located within about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than about 30 nucleotides downstream or upstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in the vector.

[0427] As another non-limiting example, an AAV vector can include a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which can be located within about 1 to 5, about 1 to 10, about 1 to 15, about 1 to 20, about 1 to 25, about 1 to 30, about 5 to 10, about 5 to 15, about 5 to 20, about 5 to 25, about 5 to 30, about 10 to 15, about 10 to 20, about 10 to 25, about 10 to 30, about 15 to 20, about 15 to 25, about 15 to 30, about 20 to 25, about 20 to 30, or about 25 to 30 nucleotides downstream or upstream of the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in the vector.

[0428] In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located within about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, or more than about 25% of the first nucleotides upstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in the vector.

[0429] As another non-limiting example, an AAV vector can include a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which can be located within the first about 1-5%, about 1-10%, about 1-15%, about 1-20%, about 1-25%, about 5-10%, about 5-15%, about 5-20%, about 5-25%, about 10-15%, about 10-20%, about 10-25%, about 15-20%, about 15-25%, or about 20-25% downstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in the vector. IV.A.2 Promoters

[0430] In some embodiments, the payload region of the AAV vector comprises at least one element for enhancing nucleic acid specificity and / or expression. Non-limiting examples of elements for enhancing nucleic acid specificity and expression include, for example, a promoter, an endogenous miRNA, a post-transcriptional regulatory element (PRE), a polyadenylation (polyA) signal sequence and an upstream enhancer (USE), a CMV enhancer, and an intron. In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the CMV enhancer comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35.

[0431] Expression of the nucleic acids of the present disclosure after delivery to or integration into the genomic DNA of a target cell may require specific promoters, including, but not limited to, promoters that are species-specific, inducible, tissue-specific, or cell cycle-specific (Parr et al., Nat. Med. 3: 1145-9 (1997); the contents of which are incorporated herein by reference in their entirety).

[0432] In some aspects, a promoter is considered effective if it drives expression of an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein carried in the payload region of an AAV vector. In some aspects, a promoter is considered effective if it drives expression of a therapeutic molecule of the present disclosure in targeted cells (e.g., fibroblasts, muscle cells, adipocytes).

[0433] Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters and mammalian promoters. In some embodiments, the promoter may be a human promoter. In some embodiments, the promoter may be truncated. Promoters that drive or enhance expression in most tissues include, but are not limited to, human elongation factor 1a-subunit (EF1a), cytomegalovirus (CMV) immediate-early enhancer and / or promoter, chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC). In some embodiments, the promoter is a CMV early enhancer / chicken β-actin (CAG) promoter, CAG, CBA, CMV, EF1α, EF1α and CMV enhancer, CMV promoter and CMV enhancer (CMVe / p), CMV promoter and SV40 intron, or a tissue-specific promoter.

[0434] In some embodiments, tissue-specific expression elements can be used to restrict expression to certain cell types, such as, but not limited to, a muscle-specific promoter, a B-cell promoter, a monocyte promoter, a leukocyte promoter, a macrophage promoter, a pancreatic acinar cell promoter, an endothelial cell promoter, a lung tissue promoter, an astrocyte promoter, or a nervous system promoter that can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0435] Non-limiting examples of muscle-specific promoters include mammalian muscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, HSA promoter, HMCK7 promoter, dMCK promoter, tMCK promoter, CK8e promoter, SPc5-12 promoter, SP-301 promoter, MH promoter, Sk-CRM promoter, or Sk-CRM4 promoter, mammalian troponin I (TNNI2) promoter, and mammalian skeletal alpha-actin (ASKA) promoter (see, e.g., U.S. Patent Application Publication No. US20110212529, the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of tissue-specific expression elements for neurons include the neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl-CpG-binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin minigene ηβ2, preproenkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters. Non-limiting examples of tissue-specific expression elements for astrocytes include the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include the myelin basic protein (MBP) promoter. Non-limiting examples of tissue-specific expression elements for fibroblasts include the insulin-like growth factor binding protein 2 (IGFBP2) promoter, the fibroblast activation protein (FAP) promoter, and the fibroblast-specific protein 1 (FSP1) promoter.

[0436] Non-limiting examples of tissue-specific expression elements for adipocytes include the adiponectin promoter and the adipocyte fatty acid binding protein (AP2) promoter.

[0437] In some embodiments, the promoter may be less than 1 kb. In some embodiments, the promoter may be less than about 15-20, about 10-50, about 20-30, about 30-40, about 40-50, about 50-60, about 50-100, about 60-70, about 70-80, about 80-90, about 90-100, about 100-110, about 100-150, about 110-120, about 120-130, about 130-140, about 140-150, about 150-160, about 160-170, about 170-180, about 180-190, about 200-210, about 210-220, about 220-230, about 230-240, about 240-250, about 250-300, about 260-310, about 270-320, about 280-330, about 290-340, about 300-350, about 310-320, about 330-340, about 350-360, about 360-370, about 370-380, about 380-390, about 400-410, about 420-430, about 440-450, about 460-470, about 480-490, about 490-500, about 500-510, about 510-520, about 520-530, about 530-540, about 540-550, about 550-560, about 560- 0, approx. 130-140, approx. 140-150, approx. 150-160, approx. 150-200, approx. 160-170, approx. 170-180, approx. 180-190, approx. 190-200, approx. 200-210, approx. 200-250, approx. 210-220, approx. 220-230, approx. 230-240, approx. 240-250, approx. 2 50-260, approx. 250-300, approx. 260-270, approx. 270-280, approx. 280-290, approx. 290-300, approx. 200-300, approx. 200-400, approx. 200-500, approx. 200-600, approx. 200-700, approx. 200-800, approx. 300-400, approx. 300-500, approx. 300- It may have a length of between about 600, about 300-700, about 300-800, about 400-500, about 400-600, about 400-700, about 400-800, about 500-600, about 500-700, about 500-800, about 600-700, about 600-800 or about 700-800 nucleotides.

[0438] In some embodiments, the promoter can be a combination of two or more components of the same or different starting or parent promoters, for example, but not limited to, CMV, CAG, EF1α, and CBA. In some embodiments, the promoter is a CMV early enhancer / chicken β-actin (CAG) promoter, a CAG promoter, a CBA promoter, a human CMV promoter, a mouse CMV promoter, an EF1α promoter, an EF1α promoter and a CMV enhancer, a CMV promoter and a CMV enhancer (CMVe / p), or a CMV promoter and an SV40 intron. In some embodiments, the promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 47-51, 83, or 93.

[0439] In some embodiments, each component in the promoter can have a length of between about 200-300, about 200-400, about 200-500, about 200-600, about 200-700, about 200-800, about 300-400, about 300-500, about 300-600, about 300-700, about 300-800, about 400-500, about 400-600, about 400-700, about 400-800, about 500-600, about 500-700, about 500-800, about 600-700, about 600-800, or about 700-800 nucleotides. In some embodiments, the promoter is a combination of a 382-nucleotide CMV enhancer sequence and a 260-nucleotide CBA promoter sequence.

[0440] In some embodiments, the AAV vector comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include, for example, the human CMV promoter, the mouse CMV promoter, the CBA promoter (including derivatives CAG, CBh, etc.), the EF-la promoter, the PGK promoter, the UBC promoter, the GUSB promoter (hGBp), and the UCOE promoter (the promoter of HNRPA2B1-CBX3).

[0441] In some embodiments, the promoter is not cell-specific. In some embodiments, the promoter is a ubiquitin c (UBC) promoter. The UBC promoter can have a size of 300-350 nucleotides. In some embodiments, the UBC promoter is 332 nucleotides. In some embodiments, the promoter is a beta-glucuronidase (GUSB) promoter. The GUSB promoter can have a size of 350-400 nucleotides. In some embodiments, the GUSB promoter is 378 nucleotides. In some embodiments, the promoter is a neurofilament light chain (NFL) promoter. The NFL promoter can have a size of 600-700 nucleotides. In some embodiments, the NFL promoter is 650 nucleotides. In some embodiments, the construct can be AAV-promoter-CMV / globin intron-modulating polynucleotide-RBG, where AAV can be self-complementary and AAV can be a DJ serotype.

[0442] In some embodiments, the AAV vector comprises a Pol III promoter. In some embodiments, the AAV vector comprises a PI promoter. In some embodiments, the AAV vector comprises an FXN promoter. In some embodiments, the promoter is a phosphoglycerate kinase 1 (PGK) promoter. In some embodiments, the promoter is a chicken beta-actin (CBA) promoter. In some embodiments, the promoter is a CAG promoter, which is a construct comprising a cytomegalovirus (CMV) enhancer fused to a chicken beta-actin (CBA) promoter with a chimeric intron. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is a human cytomegalovirus (CMV) promoter. In some embodiments, the promoter is a murine cytomegalovirus (CMV) promoter. In some embodiments, the promoter is a CBA promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is an EF1α promoter fused to a CMV enhancer. In some embodiments, the promoter is a CMV promoter fused to a CMV enhancer. In some embodiments, the promoter is a CMV promoter fused to an SV40 intron. In some embodiments, the AAV vector comprises an HI promoter. In some embodiments, the AAV vector comprises a U6 promoter. In some embodiments, the AAV vector comprises an SP6 promoter. In some embodiments, the promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 34-38.

[0443] In some embodiments, the promoter is a liver or skeletal muscle promoter. Non-limiting examples of liver promoters include human α-1-antitrypsin (hAAT) and thyroxine-binding globulin (TBG). Non-limiting examples of skeletal muscle promoters include desmin, MCK, or synthetic C5-12. In some embodiments, the promoter is an RNA pol III promoter. In some embodiments, the RNA pol III promoter is U6. In some embodiments, the RNA pol III promoter is HI. In some embodiments, the AAV vector comprises two promoters. In some embodiments, the promoters are the EF1a promoter and the CMV promoter. Non-limiting examples of fibroblast promoters include insulin-like growth factor binding protein 2 (IGFBP2), fibroblast activation protein (FAP), and fibroblast-specific protein 1 (FSP1).

[0444] Non-limiting examples of adipocyte promoters include adiponectin and adipocyte fatty acid binding protein (AP2).

[0445] In some embodiments, the AAV vector comprises an enhancer element, a promoter, and / or a 5'UTR intron. The enhancer element, also referred to herein as an "enhancer," can be, but is not limited to, a CMV enhancer; the promoter can be, but is not limited to, an EF1α, CMV, CBA, UBC, GUSB, NSE, synapsin, MeCP2, and GFAP promoter; and the 5'UTR / intron can be, but is not limited to, an SV40, CBA-MVM (minute virus of mice), human β-globin, immunoglobulin heavy chain, or a chimera between a human β-globin and an immunoglobulin heavy chain gene. In some embodiments, the intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 46, 56, or 82. In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the CMV enhancer comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48. In some embodiments, the enhancers, promoters and / or introns used in combination may be: (1) a CMV enhancer, a CMV promoter, and an SV40 5'UTR intron; (2) a CMV enhancer, a CBA promoter, and an SV40 5'UTR intron; (3) a CMV enhancer, a CBA promoter, and a CBA-MVM 5'UTR intron; (4) a UBC promoter; (5) a GUSB promoter; (6) an NSE promoter; (7) a synapsin promoter; (8) an MeCP2 promoter, (9) a GFAP promoter, (10) an HI promoter; (11) a U6 promoter; (12) a CMV promoter and a CMV enhancer; (13) an EF1α promoter and a CMV enhancer; or (14) a CMV promoter and an SV40 intron; or (15) a human β-globin and immunoglobulin heavy chain chimera, an EF1α promoter, a CMV enhancer, a CMV promoter, and an SV40 intron.In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the intron is an SV40 intron, an MVM intron, or a human beta globin intron in the vector. In some embodiments, the promoter is a CBA promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is a CMV promoter fused to a CMV enhancer. In some embodiments, the promoter is a CMV enhancer fused to an EF1α promoter. In some embodiments, the promoter is a CMV promoter fused to an SV40 intron. In some embodiments, the AAV vector comprises an engineered promoter. In some embodiments, the AAV vector comprises a CMV early enhancer / chicken beta actin (CAG) promoter. In some embodiments, the AAV vector comprises a promoter derived from a naturally expressed protein. IV.A.3 Untranslated Regions (UTRs)

[0446] By definition, the wild-type untranslated region (UTR) of a gene is transcribed but not translated. Generally, the 5' UTR begins at the transcription start site and ends at the start codon, and the 3' UTR begins immediately after the stop codon and continues until the termination signal for transcription.

[0447] Features typically found in abundantly expressed genes of specific target organs can be engineered into UTRs to enhance transcript stability and production. In some embodiments, the 5'UTR from mRNA normally expressed in the liver (e.g., albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can be used in the AAV vectors of the present disclosure to enhance expression, for example, in brain tissue, particularly in neural cells.

[0448] Wild-type 5' untranslated regions (UTRs) contain features that play a role in translation initiation. A Kozak sequence, which is generally known to be involved in the process by which ribosomes initiate translation of many genes, is usually included in the 5' UTR. The Kozak sequence has the consensus CCR(A / G)CCAUGG (where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), followed by another 'G'). In some embodiments, the 5' UTR in the AAV vectors of the present disclosure contains a Kozak sequence. In some embodiments, the 5' UTR in the AAV vectors of the present disclosure does not contain a Kozak sequence.

[0449] Wild-type 3'UTRs are known to have stretches of adenosines and uridines embedded therein. These AU-rich signatures are particularly prevalent in genes with a high rate of metabolic turnover. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al., 1995, the contents of which are incorporated herein by reference in their entirety). Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of the AUUUA motif within the U-rich region. Class II AREs, such as, but not limited to, GM-CSF and IGFR-α, have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III AREs, such as, but not limited to, c-Jun and myogenin, are less well defined. These U-rich regions do not contain the AUUUA motif. While most proteins that bind to AREs are known to destabilize messengers, members of the ELAV family, most notably HuR, have been documented to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule results in HuR binding and, therefore, stabilization of the message in vivo.

[0450] The introduction, removal or modification of 3'UTR AU-rich element (ARE) can be used to modulate the stability of polynucleotide.When manipulating a specific polynucleotide, for example, the payload region of a viral genome, one or more copies of ARE can be introduced to reduce the stability of the polynucleotide, thereby reducing translation and reducing the production of the resulting protein.Similarly, ARE can be identified, removed or mutated to increase intracellular stability, thus increasing translation and the production of the resulting protein.

[0451] In some embodiments, the 3'UTR of the AAV vectors of the present disclosure can include an oligo(dT) sequence for addition of a polyA tail.

[0452] In some embodiments, the AAV vectors of the present disclosure can be engineered to include, alter, or remove at least one miRNA binding site, sequence, or seed region.

[0453] Any UTR from any gene known in the art can be incorporated into the AAV vector of the present disclosure. These UTRs or portions thereof can be positioned in the same direction as in the selected gene, or they can be changed in direction or location. In some embodiments, the UTRs used in the AAV vectors of the present disclosure can be inverted, shortened, extended, or created using one or more other 5'UTRs or 3'UTRs known in the art. As used herein, the term "modified," when referring to a UTR, means that the UTR has been altered in some way relative to the reference sequence. For example, the 3' or 5'UTR can be altered by changing the direction or location as taught above, or by including additional nucleotides, deleting nucleotides, exchanging nucleotides, or rearranging nucleotides, compared to the wild-type or native UTR. In some embodiments, the AAV vector of the present disclosure includes at least one artificial UTR that is not a variant of the wild-type UTR. In some embodiments, the AAV vector of the present disclosure includes a UTR selected from a family of transcripts whose proteins share a common function, structure, characteristic, or property. IV.A.4 Polyadenylation Sequences

[0454] In some embodiments, the AAV vectors of the present disclosure comprise at least one polyadenylation sequence. The AAV vectors of the present disclosure can comprise a polyadenylation sequence between the 3' end of the payload coding sequence and the 5' end of the 3' ITR.

[0455] In some embodiments, the polyadenylation sequence or "polyA sequence" can range from absent to about 500 nucleotides in length.

[0456] In some embodiments, the polyadenylation sequence may be about 10-100, about 10-90, about 10-80, about 10-70, about 10-60, about 10-55, about 10-50, about 20-100, about 20-90, about 20-80, about 20-70, about 20-60, about 20-55, about 20-50, about 30-100, about 30-90, about 30-80, The polyadenylation sequence is about 30 to 70, about 30 to 60, about 30 to 55, about 30 to 50, about 40 to 100, about 40 to 90, about 40 to 80, about 40 to 70, about 40 to 60, about 40 to 55, about 40 to 50, about 45 to 100, about 45 to 90, about 45 to 80, or about 45 to 70, about 45 to 60, about 45 to 55, or about 45 to 50 nucleotides in length. In some embodiments, the polyadenylation sequence is about 49 nucleotides in length.

[0457] In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located upstream of a polyadenylation sequence in the vector. In some embodiments, the AAV vector comprises a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located downstream of a promoter in the vector, for example, but not limited to, EF1α, CMV, U6, CAG, CBA EF1α and CMV enhancer, CMV promoter and SV40 intron, CMV promoter and CMV enhancer, or CBA promoter and SV40 intron, MVM intron, human beta globin intron, immunoglobulin heavy chain intron, or a chimera of human beta globin intron and immunoglobulin heavy chain intron.

[0458] In some aspects, the AAV vectors of the present disclosure comprise a nucleic acid sequence encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, which may be located within about 1-5, about 1-10, about 1-15, about 1-20, about 1-25, about 1-30, about 5-10, about 5-15, about 5-20, about 5-25, about 5-30, about 10-15, about 10-20, about 10-25, about 10-30, about 15-20, about 15-25, about 15-30, about 20-25, about 20-30, or about 25-30 nucleotides downstream from the promoter and / or upstream of the polyadenylation sequence in the vector.

[0459] In some embodiments, the AAV vector comprises a rabbit globin polyadenylation (polyA) signal sequence. In some embodiments, the AAV vector comprises a human growth hormone polyadenylation (polyA) signal sequence. In some embodiments, the AAV vector comprises a bovine growth hormone polyadenylation (polyA) signal sequence. In some embodiments, the polyA signal sequence has a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 52 or 53.

[0460] In some embodiments, the AAV vector comprises the SV40 polyadenylation signal sequence (SV40 pA), the bovine growth hormone polyadenylation signal sequence (bGHpA), or the human growth hormone polyadenylation signal sequence (hGHpA). IV.A.5 Introns

[0461] In some embodiments, the payload region of an AAV vector of the present disclosure comprises at least one element for enhancing expression, such as one or more introns or portions thereof. Non-limiting examples of introns include MVM (67-97 bp), F.IX truncated intron 1 (300 bp), β-globin SD / immunoglobulin heavy chain splice acceptor (250 bp), adenovirus splice donor / immunoglobulin splice acceptor (500 bp), SV40 late splice donor / splice acceptor (19S / 16S) (180 bp), and hybrid adenovirus splice donor / IgG splice acceptor (230 bp).

[0462] In some embodiments, an intron or portion of an intron can be between about 100 and about 500 nucleotides in length. In some embodiments, an intron can have a length of between about 80-100, about 80-120, about 80-140, about 80-160, about 80-180, about 80-200, about 80-250, about 80-300, about 80-350, about 80-400, about 80-450, about 80-500, about 200-300, about 200-400, about 200-500, about 300-400, about 300-500, or about 400-500 nucleotides.

[0463] In some embodiments, the AAV vector can include a promoter, for example, but not limited to, a CMV or U6. In some embodiments, the promoter for the AAV vector of the present disclosure is a CMV promoter. In some embodiments, the promoter for the AAV vector of the present disclosure is a CMV early enhancer / chicken beta actin (CAG) promoter. As another non-limiting example, the promoter for the AAV vector of the present disclosure is a U6 promoter. In some embodiments, the AAV vector can include a CMV and a U6 promoter. In some embodiments, the AAV vector can include an HI promoter. In some embodiments, the AAV vector can include a CBA promoter. In some embodiments, the AAV vector can include a chimeric intron. In some embodiments, the AAV vector can include an SV40 intron. In some embodiments, the AAV vector can include an immunoglobulin heavy chain intron. In some embodiments, the AAV vector can include a human beta globin intron. In some embodiments, the AAV vector can include a chimera of a human beta globin intron and an immunoglobulin heavy chain intron.

[0464] In some embodiments, the promoter is a CMV early enhancer / chicken beta actin (CAG) promoter, EF1α, human CMV, mouse CMV, an EF1α promoter fused to a CMV enhancer, a CMV promoter fused to an SV40 intron, a CMV promoter fused to a CMV enhancer, or a tissue-specific promoter. In some embodiments, the promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 47-51.

[0465] In some aspects, the encoded antibody (e.g., monoclonal antibody) or antigen-binding fragment thereof disclosed herein can be located downstream of a promoter in an expression vector, such as, but not limited to, a CMV, U6, HI, CBA, CAG, or CBA promoter, and an intron, such as an SV40, MVM intron, a human beta globin intron, a human immunoglobulin heavy chain intron, a chimera of a human beta globin intron and a human immunoglobulin heavy chain intron, or others known in the art. In some aspects, the intron is selected from the group consisting of an SV40 intron, an MVM intron, a human beta globin intron, a human immunoglobulin heavy chain intron, or a chimera of a human immunoglobulin heavy chain intron and a human beta globin intron. In some embodiments, the intron comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 46, 56, or 82.

[0466] Furthermore, the encoded antibody or antigen-binding fragment thereof can also be located upstream of a polyadenylation sequence in an expression vector. In some embodiments, the encoded therapeutic protein, e.g., an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, or a fusion protein (e.g., an Fc fusion protein), or therapeutic peptide, can be located within about 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 15 to 20, 15 to 25, 15 to 30, 20 to 25, 20 to 30, or 25 to 30 nucleotides downstream from the promoter in the vector and / or upstream of the polyadenylation sequence. IV.A.6 Filler Arrangement

[0467] In some embodiments, the AAV vector comprises one or more filler sequences (also referred to as "stuffer sequences"). In some embodiments, the AAV vector comprises one or more filler sequences to provide an AAV vector length that is optimal for packaging. In some embodiments, the AAV vector comprises at least one filler sequence to provide an AAV vector length of approximately 2.0 to 2.5 kb, e.g., approximately 2.3 kb. In some embodiments, the AAV vector comprises at least one filler sequence to provide an AAV vector length of approximately 4.6 kb. In some embodiments, the vector backbone comprises a filler sequence.

[0468] In some embodiments, the AAV vector comprises one or more filler sequences to reduce the likelihood that a hairpin structure in the vector genome (e.g., a modulating polynucleotide described herein) may be read as an inverted terminal repeat (ITR) during expression and / or packaging. In some embodiments, the AAV vector comprises at least one filler sequence to have an AAV vector length of about 2.0-2.5 kb, e.g., about 2.3 kb. In some embodiments, the AAV vector comprises at least one filler sequence to have an AAV vector length of about 4.6 kb.

[0469] In some embodiments, the AAV vector is a single-stranded (ss) AAV vector and has an average length of between about 0.1 kb and about 3.8 kb, for example, but not limited to, about 0.1 kb, about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 0.6 kb, about 0.7 kb, about 0.8 kb, about 0.9 kb, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, about 1.5 kb, or about 1.6 kb. , about 1.7 kb, about 1.8 kb, about 1.9 kb, about 2 kb, about 2.1 kb, about 2.2 kb, about 2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb, about 2.9 kb, about 3 kb, about 3.1 kb, about 3.2 kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, or about 3.8 kb in length.

[0470] In some embodiments, the AAV vector is a self-complementary (sc) AAV vector and includes one or more filler sequences having a length between about 0.1 kb and about 1.5 kb, for example, but not limited to, about 0.1 kb, about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 0.6 kb, about 0.7 kb, about 0.8 kb, about 0.9 kb, about 1 kb, about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, or about 1.5 kb.

[0471] In some embodiments, the AAV vector comprises any portion of a filler sequence, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the filler sequence.

[0472] In some embodiments, the AAV vector is a single-stranded (ss) AAV vector and contains one or more filler sequences, so that the length of the AAV vector is about 4.6 kb. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located 3' to the 5' ITR sequence. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located 5' to the promoter sequence. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located 3' to the polyadenylation signal sequence. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located 5' to the 3' ITR sequence. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located between two intron sequences. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located within an intron sequence. In some embodiments, the AAV vector comprises two filler sequences, the first filler sequence being located 3' to the 5' ITR sequence and the second filler sequence being located 3' to the polyadenylation signal sequence. In some embodiments, the AAV vector comprises two filler sequences, the first filler sequence being located 5' to the promoter sequence and the second filler sequence being located 3' to the polyadenylation signal sequence. In some embodiments, the AAV vector comprises two filler sequences, the first filler sequence being located 3' to the 5' ITR sequence and the second filler sequence being located 5' to the 5' ITR sequence.

[0473] In some embodiments, the AAV vector is a self-complementary (sc) AAV vector and contains one or more filler sequences, so that the length of the AAV vector is about 2.3 kb. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located 3' to the 5' ITR sequence. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located 5' to the promoter sequence. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located 3' to the polyadenylation signal sequence. In some embodiments, the AAV vector contains at least one filler sequence, and the filler sequence is located 5' to the 3' ITR sequence.

[0474] In some embodiments, the AAV vector comprises at least one filler sequence, the filler sequence being located between two intron sequences. In some embodiments, the AAV vector comprises at least one filler sequence, the filler sequence being located within the intron sequence. In some embodiments, the AAV vector comprises two filler sequences, the first filler sequence being located 3' to the 5' ITR sequence, and the second filler sequence being located 3' to the polyadenylation signal sequence. In some embodiments, the AAV vector comprises two filler sequences, the first filler sequence being located 5' to the promoter sequence, and the second filler sequence being located 3' to the polyadenylation signal sequence. In some embodiments, the AAV vector comprises two filler sequences, the first filler sequence being located 3' to the 5' ITR sequence, and the second filler sequence being located 5' to the 5' ITR sequence.

[0475] In some embodiments, the AAV vector can include one or more filler sequences between one or more regions of the AAV vector. In some embodiments, the filler region can be located before a region, for example, but not limited to, the payload region, ITR, promoter region, intron region, enhancer region, and / or polyadenylation signal sequence region. In some embodiments, the filler region can be located after a region, for example, but not limited to, the payload region, ITR, promoter region, intron region, enhancer region, and / or polyadenylation signal sequence region. In some embodiments, the filler region can be located before and after a region, for example, but not limited to, the payload region, ITR, promoter region, intron region, enhancer region, and / or polyadenylation signal sequence region.

[0476] In some embodiments, the AAV vector can comprise one or more filler sequences that bifurcate at least one region of the AAV vector. The bifurcate region of the AAV vector can comprise about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the region 5' of the filler sequence region.

[0477] In some embodiments, the filler sequence can be bifurcated into at least one region such that about 10% of the region is located 5' to the filler sequence and about 90% of the region is located 3' to the filler sequence. In some embodiments, the filler sequence can be bifurcated into at least one region such that about 20% of the region is located 5' to the filler sequence and about 80% of the region is located 3' to the filler sequence. In some embodiments, the filler sequence can be bifurcated into at least one region such that about 30% of the region is located 5' to the filler sequence and about 70% of the region is located 3' to the filler sequence. In some embodiments, the filler sequence can be bifurcated into at least one region such that about 40% of the region is located 5' to the filler sequence and about 60% of the region is located 3' to the filler sequence. In some embodiments, the filler sequence can be bifurcated into at least one region such that about 50% of the region is located 5' to the filler sequence and about 50% of the region is located 3' to the filler sequence. In some embodiments, the filler sequence can be bifurcated into at least one region such that about 60% of the region is located 5' to the filler sequence and about 40% of the region is located 3' to the filler sequence. In some embodiments, the filler sequence can be bifurcated into at least one region such that about 70% of the region is located 5' to the filler sequence and about 30% of the region is located 3' to the filler sequence. In some embodiments, the filler sequence can be bifurcated into at least one region such that about 80% of the region is located 5' to the filler sequence and about 20% of the region is located 3' to the filler sequence. In some embodiments, the filler sequence can be bifurcated into at least one region such that about 90% of the region is located 5' to the filler sequence and about 10% of the region is located 3' to the filler sequence.

[0478] In some embodiments, the AAV vector comprises a filler sequence after the 5'ITR. In some embodiments, the AAV vector comprises a filler sequence after the promoter region. In some embodiments, the AAV vector comprises a filler sequence after the payload region. In some embodiments, the AAV vector comprises a filler sequence after the intron region. In some embodiments, the AAV vector comprises a filler sequence after the enhancer region. In some embodiments, the AAV vector comprises a filler sequence after the polyadenylation signal sequence region. In some embodiments, the AAV vector comprises a filler sequence before the promoter region. In some embodiments, the AAV vector comprises a filler sequence before the payload region. In some embodiments, the AAV vector comprises a filler sequence before the intron region.

[0479] In some embodiments, the AAV vector comprises a filler sequence before the enhancer region.In some embodiments, the AAV vector comprises a filler sequence before the polyadenylation signal sequence region.In some embodiments, the AAV vector comprises a filler sequence before the 3'ITR.In some embodiments, the filler sequence can be located between two regions, for example, but not limited to, the 5'ITR and the promoter region.In some embodiments, the filler sequence can be located between two regions, for example, but not limited to, the 5'ITR and the payload region.

[0480] In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the intron region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the enhancer region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the polyadenylation signal sequence region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the promoter region and the payload region.

[0481] In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, a promoter region and an intron region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, a promoter region and an enhancer region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, a promoter region and a polyadenylation signal sequence region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, a promoter region and a 3'ITR.

[0482] In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the payload region and the intron region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the payload region and the enhancer region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the payload region and the polyadenylation signal sequence region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the payload region and the 3'ITR.

[0483] In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, an intron region and an enhancer region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, an intron region and a polyadenylation signal sequence region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, an intron region and a 3' ITR. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, an enhancer region and a polyadenylation signal sequence region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, an enhancer region and a 3' ITR. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, a polyadenylation signal sequence region and a 3' ITR.

[0484] In some embodiments, the AAV vector can include two filler sequences, which can be located between two of the regions described herein. IV.A.7 Methods for Producing Recombinant AAV

[0485] The present disclosure also provides a method for producing AAV particles by viral genome replication in a viral-replicating cell, the method comprising contacting a viral-replicating cell with an AAV polynucleotide or AAV genome (e.g., an AAV vector of the present disclosure). In the context of the present disclosure, an AAV vector disclosed herein, for example, an AAV vector comprising at least one polynucleotide encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, is considered an AAV payload construct vector.

[0486] In some aspects, the AAV particles are produced by a method comprising the steps of: (1) co-transfecting competent bacterial cells with a bacmid vector and either a viral construct vector and / or an AAV payload construct vector; (2) isolating the resulting viral construct expression vector and AAV payload construct expression vector and separately transfecting them into viral-replicating cells; (3) isolating and purifying the resulting payload and viral construct particles comprising the viral construct expression vector or the AAV payload construct expression vector; (4) co-infecting viral-replicating cells with both the AAV payload and viral construct particles comprising the viral construct expression vector or the AAV payload construct expression vector; and (5) harvesting and purifying the viral particles comprising the parvovirus genome.

[0487] In one aspect, the present disclosure provides a method for producing AAV particles, the method comprising: (1) simultaneously co-transfecting mammalian cells, such as, but not limited to, HEK293 cells, with constructs expressing a payload region (e.g., a polynucleotide encoding a therapeutic protein or peptide of the disclosure), the rep and cap genes, and a helper construct; and (2) harvesting and purifying AAV particles comprising the viral genome.

[0488] In some embodiments, AAV particles can be produced in virus-replicating cells, including insect cells. Growth conditions for insect cells in culture and the production of heterologous products in insect cells in culture are well known in the art, see, for example, U.S. Patent No. 6,204,059.

[0489] Viral replicating cells can be selected from any organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Viral replicating cells can include mammalian cells, such as A549, WEH1, 3T3, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO.W138, HeLa, HEK293, Saos, C2C12, L cells, HT1080, HepG2, and primary mammalian fibroblasts, hepatocytes, and myoblasts. Viral replicating cells include, but are not limited to, cells derived from mammalian species, including humans, monkeys, mice, rats, rabbits, and hamsters, or cell types including, but not limited to, fibroblasts, hepatocytes, tumor cells, cell line transformed cells, etc.

[0490] The viral production disclosed herein describes processes and methods for producing AAV particles that contact target cells to deliver a payload, e.g., a recombinant viral construct, comprising a polynucleotide sequence encoding a payload, such as an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof, as disclosed herein.

[0491] In some embodiments, AAV particles can be produced in viral replication cells, including mammalian cells.Viral replication cells commonly used for the production of recombinant AAV particles include, but are not limited to, 293 cells, COS cells, HeLa cells, and KB cells.

[0492] In some embodiments, AAV particles are produced in mammalian cells in which all three VP proteins are expressed in a stoichiometry approaching 1:1:10 (VP1:VP2:VP3). The regulatory mechanism that allows for this controlled level of expression involves the production of two mRNAs, one for VP1 and the other for VP2 and VP3, produced by differential splicing.

[0493] In some embodiments, AAV particles are produced in mammalian cells using a triple transfection method in which the payload construct, parvoviral Rep and Cap, and helper construct are contained within three different constructs. The triple transfection method of the three components of AAV particle production can be utilized to produce small lots of virus for assays, including transduction efficiency, target tissue (tropism) assessment, and stability.

[0494] In some embodiments, the viral construct vector and the AAV payload construct vector can be respectively integrated into bacmid, also known as baculovirus plasmid, by transposon donor / acceptor system, by standard molecular biology techniques known and performed by those skilled in the art.Transfection of separate viral replicating cell populations produces two baculoviruses, one containing the viral construct expression vector and the other containing the AAV payload construct expression vector.The two baculoviruses can be used to infect a single viral replicating cell population for the production of AAV particles.

[0495] Baculovirus expression vectors for producing viral particles in insect cells, including, but not limited to, Spodoptera frugiperda (Sf9) cells, provide high-titer viral particle production. Recombinant baculoviruses encoding viral construct expression vectors and AAV payload construct expression vectors initiate productive infection of virus-replicating cells. Infectious baculovirus particles released from the primary infection secondarily infect additional cells in culture, exponentially infecting the entire cell culture population over several infection cycles that are a function of the initial multiplicity of infection; see, e.g., Urabe, M. et al., J. Virol. 2006 Feb; 80 (4): 1874-85, the contents of which are incorporated herein by reference in their entirety.

[0496] The production of AAV particles using baculovirus in insect cell systems can address the known genetic and physical instability of baculovirus. Baculovirus-infected virus-producing cells are harvested in aliquots that can be frozen and stored in liquid nitrogen, and the aliquots retain viability and infectivity for infection of large-scale virus-producing cell cultures (Wasilko DJ et al., Protein Expr Purif. 2009 Jun;65(2):122-32).

[0497] In some embodiments, stable viral replication cells permissive to baculovirus infection are engineered with at least one stable integrated copy of any of the elements required for AAV replication and viral particle production, including, but not limited to, at least one of the entire AAV genome, the Rep and Cap genes, the Rep gene, the Cap gene, each Rep protein as a separate transcription cassette, each VP protein as a separate transcription cassette, AAP (assembly activating protein), or a baculovirus helper gene with a native or non-native promoter.

[0498] In some embodiments, AAV particle production can be modified to increase the scale of production. Transfection of replicating cells in large-scale culture formats can be performed according to any method known in the art.

[0499] In some embodiments, cell culture bioreactors can be used for large-scale virus production. In some cases, the bioreactor comprises a stirred tank reactor. IV.A.8 Cell lysis

[0500] Cells of the present disclosure, including but not limited to virus-producing cells, can be subjected to cell lysis according to any method known in the art. Cell lysis can be performed to obtain one or more agents (e.g., virus particles) present within any cell of the present disclosure.

[0501] Cell lysis methods can be chemical or mechanical. Chemical cell lysis typically involves contacting one or more cells with one or more lysing agents. Mechanical lysis typically involves subjecting one or more cells to one or more lysis conditions and / or one or more lytic forces. In some embodiments, chemical lysis can be used to lyse cells. As used herein, the term "lysing agent" refers to any agent that can assist in the disruption of cells. In some cases, the lysing agent is introduced into a solution referred to as a lysis solution or lysis buffer. As used herein, the term "lysis solution" refers to a solution (typically an aqueous solution) that contains one or more lysing agents. In addition to a lysing agent, the lysis solution can include one or more buffers, solubilizing agents, surfactants, preservatives, cryoprotectants, enzymes, enzyme inhibitors, and / or chelating agents.

[0502] The salt concentration can be increased or decreased to obtain a concentration effective for rupturing cell membranes. Lysing agents, including detergents, can include ionic detergents or non-ionic detergents. Detergents can function to disaggregate or lyse cellular structures, including but not limited to cell membranes, cell walls, lipids, carbohydrates, lipoproteins, and glycoproteins.

[0503] In some embodiments, mechanical cell lysis is performed. Mechanical cell lysis methods may include the use of one or more lysis conditions and / or one or more lytic powers. As used herein, the term "lysis conditions" refers to a state or situation that promotes cell destruction. Lysis conditions may include a certain temperature, pressure, osmotic purity, salt concentration, etc. In some embodiments, the lysis conditions include an increase or decrease in temperature. In some embodiments, the lysis conditions include a change in temperature to promote cell destruction. Cell lysis performed according to such embodiments may include freeze-thaw lysis.

[0504] As used herein, the term "lytic force" refers to physical activity used to disrupt cells. Lytic forces may include, but are not limited to, mechanical force, sonic force, gravity, optical force, electrical force, etc. Cell lysis performed by mechanical force is referred to herein as "mechanical lysis." Mechanical forces that can be used in accordance with mechanical lysis may include high shear fluid force.

[0505] In some embodiments, methods for harvesting AAV particles without lysis can be used for efficient and scalable AAV particle production. In a non-limiting example, AAV particles can be produced by culturing AAV particles lacking heparin-binding sites in cell culture, thereby allowing the AAV particles to migrate into the supernatant, collecting the supernatant from the culture, and isolating the AAV particles from the supernatant, as described in US Patent Application Publication No. 20090275107. IV.A.9 AAV purification

[0506] The cell lysate containing the virus particles can be subjected to clarification. Clarification refers to the first step taken in the purification of virus particles from the cell lysate. Clarification serves to prepare the lysate for further purification by removing larger insoluble debris. Clarification steps can include, but are not limited to, centrifugation and filtration.

[0507] In some embodiments, AAV particles can be purified from clarified cell lysates by one or more methods of chromatography. Chromatography refers to any number of methods known in the art for separating one or more elements from a mixture. Such methods can include, but are not limited to, ion exchange chromatography (e.g., cation exchange chromatography and anion exchange chromatography), immunoaffinity chromatography, and size exclusion chromatography. V. Methods of Treatment and Use

[0508] Certain embodiments of the present disclosure are directed to the use of polynucleotides (e.g., antibody expression cassettes), vectors, and rAAVs to treat a subject in need thereof. Some embodiments of the present disclosure are directed to methods for delivering a gene therapy encoding an anti-IGF-1R antibody or antigen-binding fragment thereof to a subject in need thereof. In some embodiments, the method includes administering a delivery vector (e.g., a viral vector, a non-viral vector, a plasmid, a lipid, a protein particle, a bacterial vector, or a lysosome) to a subject. In certain embodiments, the methods disclosed herein include delivery or administration of a polynucleotide (e.g., an antibody expression cassette), a vector, a rAAV, or a composition disclosed herein to or near the eye (e.g., one or both eyes), for example, intraocularly, retroorbitally or periorbitally, retrobulbarly, intramuscularly near the eye (e.g., the levator and / or glabellar muscles), connective tissue near the eye, or any combination thereof. In some embodiments, the periorbital or retroorbital tissue is selected from muscle, connective tissue, and / or adipose tissue. In some embodiments, administration is to an extraocular muscle. In some embodiments, the extraocular muscle is the levator or glabellar muscle. In some embodiments, administration is to connective tissue. In some embodiments, administration is transconjunctival to the periorbital space. In some embodiments, administration is intralymphatic to the preauricular or submandibular lymph nodes. In some embodiments, delivery or administration is to retroorbital or periorbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by injection. In some embodiments, delivery or administration is by injection and / or infusion as a single dose. In some embodiments, single dose administration comprises multiple injections or infusions.

[0509] In some embodiments, the anti-IGF-1R antibody or antigen-binding fragment thereof is expressed in muscle, connective tissue, or adipose tissue. In some embodiments, the anti-IGF-1R antibody or antigen-binding fragment thereof is produced in muscle, connective tissue, and adipose tissue.

[0510] In some embodiments, the methods include administering a gene therapy encoding an anti-IGF-1R antibody or antigen-binding fragment thereof, comprising administering (i) a gene encoding an anti-IGF-1R antibody or antigen-binding fragment thereof, the antibody comprising: (i) a VH CDR1-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) and a VL CDR2-3 (e.g., SEQ ID NOs: 7-9, 10-12, or 13-15) (ii) CDR1-3 (e.g., SEQ ID NO: 16-18, 19-21, or 22-24); (ii) VH (e.g., SEQ ID NO: 26 or 27) and VL (e.g., SEQ ID NO: 30 or 31); (iii) HC (e.g., SEQ ID NO: 36 or 37) and LC (e.g., SEQ ID NO: 40 or 41); or (iv) any one of SEQ ID NOs: 68-76, further comprising one or more of an IRES, a furin cleavage site, a 2a site, a dual promoter (e.g., promoter-VH-IRES-VL, etc.), or a signal peptide (e.g., an IL-2 or IL-10 signal peptide), and the vector construct or expression construct (e.g., antibody expression cassette) comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof (e.g., teprotumumab).

[0511] In some embodiments, the method includes administering a gene therapy construct encoding an anti-IGF-1R antibody (e.g., teprotumumab) that is a multicistronic (e.g., bicistronic) construct (e.g., comprising a heavy chain and a light chain). In some embodiments, the multicistronic (e.g., bicistronic) construct further comprises an F2A or IRES element.

[0512] In some embodiments, the anti-IGF-1R antibody is selected from the group consisting of teprotumumab, VRDN-01100 (SEQ ID NO: 113), VRDN-02700 (SEQ ID NO: 116), ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L2 9H29, L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52, or an antigen-binding fragment thereof.

[0513] In some embodiments, the anti-IGF-1R antibody is VRDN-01100 or VRDN-02700, or an antigen-binding fragment thereof. In some embodiments, the anti-IGFR antibody comprises SEQ ID NO: 113 (corresponding to VRDN-01100). In some embodiments, the anti-IGFR antibody comprises SEQ ID NO: 116 (corresponding to VRDN-002700).

[0514] In some embodiments, the anti-IGF-1R antibody is teprotumumab or an antigen-binding fragment thereof.

[0515] In some embodiments, the present disclosure is directed to a method of delivering gene therapy to connective tissue (e.g., periorbital, retroorbital). In some embodiments, the gene therapy is administered (e.g., by injection) to the periorbital or retrobulbar space, and then the antibody or antigen-binding fragment thereof is produced in fibroblasts, myocytes, or adipocytes. In some embodiments, the therapeutic effect of the anti-IGF-1R antibody or antigen-binding fragment thereof is local, systemic, or both.

[0516] In some aspects, the present disclosure is directed to a method of delivering a gene therapy to a subject in need thereof, comprising administering via injection to a muscle, intralymphatic, periorbital or retrobulbar tissue, or other delivery site disclosed herein. In some aspects, administration is to an extraocular muscle. In some aspects, the extraocular muscle is the levator or glabellar muscle. In some aspects, administration is to connective tissue. In some aspects, administration is transconjunctival to the periorbital space. In some aspects, administration is intralymphatic to a preauricular or submandibular lymph node.

[0517] Some aspects of the present disclosure are directed to methods of delivering a nucleic acid to a cell of a subject, the method comprising administering to a fibroblast, muscle cell, or adipocyte of the subject an adeno-associated virus (AAV) capsid comprising a nucleic acid comprising a promoter operably linked to a polynucleotide encoding an antibody (e.g., a monoclonal antibody) or antigen-binding fragment thereof disclosed herein, thereby delivering the nucleic acid to the fibroblast, muscle cell, or adipocyte of the subject.

[0518] In some aspects, the methods disclosed herein can be carried out by administering a gene therapy composition comprising a polynucleotide (e.g., an antibody expression cassette), vector, rAAV particle, or composition of the present disclosure, a cell comprising a polynucleotide (e.g., an antibody expression cassette), vector, or rAAV particle of the present disclosure, a cell comprising a nucleic acid encoding an anti-IGF-1R alpha antibody or antigen-binding fragment thereof integrated into its genomic DNA, or a pharmaceutical composition comprising any of the above. Thus, the methods disclosed herein that describe administering a polynucleotide (e.g., an antibody expression cassette), vector, or rAAV particle of the present disclosure can also be carried out by administering any of these compositions.

[0519] In some aspects, the methods disclosed herein can be carried out by administering a gene therapy composition comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to insulin-like growth factor 1 receptor (IGF-1R) or an antigen-binding fragment thereof.

[0520] In some embodiments, the methods disclosed herein can be carried out by administering a gene therapy composition comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising (i) a heavy chain variable region (VH) comprising complementarity-determining region (CDR) 1, VH CDR2, and VH CDR3, and (ii) a light chain variable region (VL) comprising CDR1, VL CDR2, and VL CDR3. In some embodiments, VH CDR1-3 and VL CDR1-3 are derived from the corresponding CDRs of teprotumumab.

[0521] In some aspects, the methods disclosed herein can be used with any of the following antibodies: teprotumumab, VRDN-01100 (SEQ ID NO: 113), VRDN-02700 (SEQ ID NO: 116), ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, ...

Claims

1. 1. A recombinant adeno-associated virus (rAAV) particle comprising a capsid and a vector genome, wherein the vector genome comprises inverted terminal repeats (ITRs) and an antibody expression cassette, the antibody expression cassette comprising: (a) a promoter; (b) (i) a nucleotide sequence encoding a VH complementarity-determining region (CDR) 1 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7 or 10, (ii) a nucleotide sequence encoding a VH CDR2 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, 11, or 14, and (iii) a VH a nucleic acid sequence encoding the heavy chain variable region (VH) of an anti-insulin-like growth factor 1 receptor (anti-IGF-1R) antibody or antigen-binding fragment thereof, comprising a nucleotide sequence encoding a CDR3 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9, 12, or 15; and (c) (i) a nucleotide sequence encoding a VL CDR1 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16, (ii) a nucleotide sequence encoding a VL CDR2 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17, 20, or 23, and (iii) a VL a nucleic acid sequence encoding the light chain variable region (VL) of an anti-IGF-1R antibody or antigen-binding fragment thereof, comprising a nucleotide sequence encoding CDR3 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18, 21, or 24; 1. An rAAV particle comprising:

2. 2. The rAAV particle of claim 1, wherein the nucleic acid sequence encoding the VH of the anti-IGF-1R antibody or antigen-binding fragment thereof comprises (i) the nucleotide sequence having 100% identity to SEQ ID NO: 7 or 10, (ii) the nucleotide sequence having 100% identity to SEQ ID NO: 8, 11 or 14, and (iii) the nucleotide sequence having 100% identity to SEQ ID NO: 9, 12 or 15; and the nucleic acid sequence encoding the VL of the anti-IGF-1R antibody or antigen-binding fragment thereof comprises (i) the nucleotide sequence having 100% identity to SEQ ID NO: 16, (ii) the nucleotide sequence having 100% identity to SEQ ID NO: 17, 20 or 23, and (iii) the nucleotide sequence having 100% identity to SEQ ID NO: 18, 21 or 24.

3. (a) a promoter; (b) (i) a nucleotide sequence encoding a VH complementarity-determining region (CDR) 1 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7 or 10, (ii) a nucleotide sequence encoding a VH CDR2 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, 11, or 14, and (iii) a VH a nucleic acid sequence encoding a heavy chain variable region (VH) of an anti-insulin-like growth factor 1 receptor (anti-IGF-1R) antibody or antigen-binding fragment thereof, comprising a nucleotide sequence encoding a CDR3 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9, 12, or 15; and (c) (i) a nucleotide sequence encoding a VL CDR1 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16, (ii) a nucleotide sequence encoding a VL CDR2 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17, 20, or 23, and (iii) a VL a nucleic acid sequence encoding the light chain variable region (VL) of an anti-IGF-1R antibody or antigen-binding fragment thereof, comprising a nucleotide sequence encoding CDR3 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18, 21, or 24; An antibody expression cassette comprising:

4. A recombinant adeno-associated virus (rAAV) particle comprising a capsid and a vector genome, wherein the vector genome comprises an inverted terminal repeat (ITR) and an antibody expression cassette, and the antibody expression cassette comprises: (a) a promoter; and (b) A nucleic acid sequence encoding an anti-IGF-1R antibody or antigen-binding fragment thereof, wherein the anti-IGF-1R antibody or antigen-binding fragment thereof is selected from the group consisting of VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cizutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, lobatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, and rhuMab. IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H1 6, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H3 0, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52, or a fragment, variant, or derivative thereof.

1. An rAAV particle comprising:

5. An rAAV particle described in any one of claims 1, 2 and 4, or an antibody expression cassette described in claim 3, comprising a sequence encoding a signal peptide, wherein the signal peptide is an IL-2 or IL-10 signal peptide.

6. 5. The rAAV particle of any one of claims 1, 2 and 4, or the antibody expression cassette of claim 3, wherein the antibody expression cassette comprises a linker sequence selected from an internal ribosome entry site (IRES) sequence, a proteolytic cleavage site, or a combination thereof.

7. 7. The rAAV particle or antibody expression cassette of claim 6, wherein the proteolytic cleavage site comprises a furin cleavage site, a 2A cleavage site, or a combination thereof.

8. 6. The rAAV particle or antibody expression cassette of claim 5, wherein the signal peptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 119 or 120.

9. 5. The rAAV particle of claim 1, 2, or 4, or the antibody expression cassette of claim 3, wherein the promoter is a CBA promoter, a CMV promoter, an EF1α promoter, a CAG promoter, or a tissue-specific promoter.

10. 10. The rAAV particle or antibody expression cassette of claim 9, wherein the promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 47-51, 83, or 93.

11. 5. The rAAV particle of any one of claims 1, 2, and 4, or the antibody expression cassette of claim 3, wherein the promoter is a muscle-specific promoter selected from the group consisting of a desmin (DES) promoter, a human skeletal muscle alpha-actin (HSA) promoter, a myosin creatine kinase (MCK) promoter, an alpha myosin heavy chain myosin creatine kinase 7 (HMCK7) promoter, a double MCK enhancer MCK (dMCK) promoter, a triple MCK enhancer MCK (tMCK) promoter, a double MCK enhancer muscle-type creatine kinase 8e (CK8e) promoter, an SPc5-12 promoter, an SP-301 promoter, an alpha myosin heavy chain (MHC) promoter, an Sk-CRM promoter, or an Sk-CRM4 promoter.

12. 5. The rAAV particle of any one of claims 1, 2, and 4, or the antibody expression cassette of claim 3, wherein the antibody expression cassette comprises an intron selected from a CAG intron, an SV40 intron, an MVM intron, or a human beta-globin intron, or any combination thereof.

13. 13. The rAAV particle or antibody expression cassette of claim 12, wherein the intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46 or 82.

14. 10. The rAAV particle of claim 1, 2, or 4, or the antibody expression cassette of claim 3, wherein the promoter comprises a first promoter and a second promoter that are different.

15. An rAAV particle or antibody expression cassette as described in claim 14, wherein the first and second promoters initiate transcription in the same direction.

16. An rAAV particle or antibody expression cassette as described in claim 14, wherein the first and second promoters initiate transcription in different directions.

17. 15. The rAAV particle or antibody expression cassette of claim 14, wherein the antibody expression cassette comprises a pause element between the first and second promoters.

18. 18. The rAAV particle or antibody expression cassette of claim 17, wherein the pause element comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

54.

19. The rAAV particle of any one of claims 1, 2 and 4, or the antibody expression cassette of claim 3, wherein the anti-IGF-1R antibody is teprotumumab.

20. 5. The rAAV particle of any one of claims 1, 2 and 4, or the antibody expression cassette of claim 3, wherein the nucleic acid sequence encoding the VH comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs:25-27.

21. 5. The rAAV particle of any one of claims 1, 2 and 4, or the antibody expression cassette of claim 3, wherein the nucleic acid sequence encoding the VL comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 29-31.

22. 5. The rAAV particle of any one of claims 1, 2 and 4, or the antibody expression cassette of claim 3, wherein the nucleic acid sequence encoding the heavy chain (HC) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 35-37.

23. 5. The rAAV particle of any one of claims 1, 2 and 4, or the antibody expression cassette of claim 3, wherein the nucleic acid sequence encoding the light chain (LC) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 39-41.

24. 5. The rAAV particle of claim 1, 2, or 4, or the antibody expression cassette of claim 3, wherein the antibody expression cassette further comprises a poly(A) sequence selected from bGHpA, hGHpA, SV40pA, or synthetic pA.

25. 25. The rAAV particle or antibody expression cassette of claim 24, wherein the poly(A) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52 or 53.

26. 10. The rAAV particle of any one of claims 1, 2, and 4, or the antibody expression cassette of claim 3, wherein the antibody expression cassette comprises an open reading frame (ORF) comprising a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 57-67 and 94-97.

27. 5. The rAAV particle of any one of claims 1, 2 and 4, or the antibody expression cassette of claim 3, wherein the antibody expression cassette comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 68-76.

28. The rAAV particle of any one of claims 1, 2 and 4, wherein the AAV ITRs comprise a pair of ITRs flanking the antibody expression cassette.

29. An rAAV particle according to any one of claims 1, 2 and 4, or an antibody expression cassette according to claim 3, wherein the antibody expression cassette is packaged in an AAV capsid having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRh8, AAVrh9, AAV9, AAVrhlO, AAV10, AAV11, AAV12, and modified versions thereof.

30. A host cell comprising an rAAV particle described in any one of claims 1, 2 and 4, or an antibody expression cassette described in claim 3.

31. A composition comprising an rAAV particle described in any one of claims 1, 2 and 4, or an antibody expression cassette described in claim 3, and a carrier.

32. 1. A composition for use in a method for expressing an anti-IGF-1R antibody or antigen-binding fragment thereof in a cell, the composition comprising the rAAV particle of any one of claims 1, 2 and 4, or the antibody expression cassette of claim 3, the method comprising administering the rAAV particle or the antibody expression cassette to the cell, thereby expressing the anti-IGF-1R antibody or antigen-binding fragment thereof in the cell.

33. 33. The composition of claim 32, wherein the cells are fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, or any combination thereof.

34. 33. The composition of claim 32, wherein the administration is in vitro.

35. 33. The composition of claim 32, wherein the administration is in vivo.

36. A composition for expressing an anti-IGF-1R antibody or an antigen-binding fragment thereof in a subject requiring expression of the anti-IGF-1R antibody or an antigen-binding fragment thereof, the composition comprising an rAAV particle described in any one of claims 1, 2 and 4, or an antibody expression cassette described in claim 3.

37. 37. The composition of claim 36, wherein the subject has a thyroid eye disease selected from active Graves' orbital disease and chronic Graves' orbital disease.

38. A composition for use in a method for treating thyroid eye disease in a patient in need thereof, comprising an rAAV particle described in any one of claims 1, 2 and 4, or an antibody expression cassette described in claim 3, said method comprising administering said rAAV particle or said antibody expression cassette to said subject, thereby expressing said anti-IGF-1R antibody or its antigen-binding fragment in said subject and treating said thyroid eye disease.

39. 39. The composition of claim 38, wherein the thyroid eye disease is selected from active Graves' orbital disease and chronic Graves' orbital disease.

40. 39. The composition of claim 38, wherein the administration is suitable for delivery of the rAAV particles or the antibody expression cassette to an ocular delivery site, a retro-orbital or periorbital delivery site, a retrobulbar delivery site, an extraocular muscle delivery site, a connective tissue delivery site, or any combination of these delivery sites.

41. 39. The composition of claim 38, wherein the administration is by injection or infusion.

42. 39. The composition of claim 38, wherein the administration is intramuscular (IM), intravenous (IV), intralymphatic, intraocular, retroorbital, periorbital, retrobulbar, or any combination thereof.

43. 39. The composition of claim 38, wherein the administration is suitable for delivery to retroorbital or periorbital fibroblasts, adipocytes, myofibroblasts, muscle cells, or any combination thereof.

44. 39. The composition of claim 38, wherein the administration is to an extraocular muscle.

45. 39. The composition of claim 38, wherein the administration is (i) to connective tissue, (ii) transconjunctivally to the periorbital space, or (iii) intralymphatic, including injection or infusion into preauricular or submandibular lymph nodes.

46. 39. The composition of claim 38, wherein the administration is a single dose.

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