Periocular delivery of AAV vectors for treatment of ophthalmic pathologies

Abstract

Provided herein is a method of delivering a pharmaceutical composition comprising an AAV carrier to an ocular or periorbital tissue, comprising periorbital injection of the AAV carrier (e.g., periorbital injection, optionally periorbital injection). In some aspects, the methods are used to treat ophthalmic pathologies (e.g., thyroid eye disease (TED)).

Description

[0001] Cross-referencing of related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 512,546, filed July 7, 2023, and U.S. Provisional Application No. 63 / 616,045, filed December 29, 2023; each application is incorporated herein by reference in its entirety.

[0003] [Reference to the electronically submitted sequence list]

[0004] The contents of the sequence list submitted electronically (name: 4525_119PC02_SequenceListing_ST26.xml; size: 294,812 bytes; and creation date: July 5, 2024) are incorporated herein by reference in their entirety. [Technical Field]

[0006] This disclosure relates to the medical field and gene delivery, including AAV gene therapy for treating ocular pathologies. [Background Technology]

[0008] Ophthalmic pathology significantly impacts the quality of life for millions of people each year and can lead to severe discomfort, vision impairment, or blindness.

[0009] Delivering therapeutic agents to the ophthalmic region and subsequently delivering the kinetics of these therapies to the appropriate locations within or near the eye is crucial for treating a wide range of ophthalmic pathologies.

[0010] Delivering drugs to target tissues with minimal adverse effects and maximum efficacy is one of the major challenges in drug development. Due to the structural and metabolic characteristics of the ophthalmic region, different administration methods (i.e., local, systemic, ocular, and periorbital injections) can result in vastly different concentrations of therapeutic agents delivered to the site of action, often making it difficult to place them within the therapeutic window. Therefore, effectively engineering and delivering therapeutic agents to ophthalmic tissues is a crucial step in developing successful therapies.

[0011] Topical application is the least invasive route of administration, but it is also one of the least effective routes of administration. For example, the bioavailability of topical therapeutic agents is generally low, and their penetration into the corneal and conjunctival epithelium is inefficient.

[0012] Systemic delivery can be achieved via oral administration in tablet or liquid consumable form or via parenteral routes (e.g., intravenous, intramuscular, subcutaneous, and intradermal injectable forms). These methods are not typical for therapeutic delivery in ophthalmology due to low ocular bioavailability and high systemic exposure. Systemic metabolism is another obstacle to systemic administration and significantly reduces the concentration of therapeutic agents that can reach the ocular region.

[0013] Intraocular administration involves the injection or implantation of sterile solutions or devices into the eye via intravitreal, subretinal, or suprachoroidal delivery routes. Intravitreal and subretinal injections are common routes of administration for carrier-based therapeutic agents used to treat intraocular diseases.

[0014] Periocular delivery is another method for introducing therapeutic agents into the ocular region. The subconjunctival space is the most commonly used in clinical practice, but the bioavailability of therapeutic agents is generally poor. Following subconjunctival injection, the bioavailability of therapeutic agents has been established to be roughly similar to that of topical administration and significantly lower than that of intravitreal administration (Subrizi, A. et al., Design Principles of Ocular Drug Delivery Systems: Importance of Drug Payload, Release Rate, and Material Properties. Drug Discov. Today 2019, 24, 1446-1457).

[0015] There is a need to develop delivery strategies for ophthalmic regions that allow for the efficient delivery of carrier-based therapeutics while minimizing iatrogenic complications. [Summary of the Invention]

[0017] Certain aspects of this disclosure relate to a method of treating an ophthalmic pathology in a subject with this need, comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, wherein the administration is a periorbital injection. In some aspects, the periorbital injection is selected from periorbital injection or retroseptal injection. In some aspects, the periorbital injection is selected from the group consisting of subcapsular injection, retrobulbar injection, subconjunctival injection, or periorbital injection. In some aspects, the periorbital injection is a retrobulbar injection. In some aspects, the periorbital injection is a periorbital injection.

[0018] In some respects, the AAV carrier contacts the orbital tissue or extraocular compartments of the group consisting of extraocular adipose tissue, levator lateralis muscle, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, optic nerve, blood vessels, sclera, dura mater sheath, cribriform plate, eyelids, lacrimal gland, lacrimal sac, tear film, or any combination thereof.

[0019] In some respects, the AAV carrier contacts the orbital tissue or extraocular cavity of the group consisting of extraocular adipose tissue, extraocular lateral rectus muscle, extraocular medial rectus muscle, extraocular superior rectus muscle, extraocular inferior rectus muscle, extraocular inferior oblique muscle, extraocular superior oblique muscle, or any combination thereof.

[0020] Certain aspects of this disclosure relate to a method of delivering a pharmaceutical composition comprising an AAV carrier to orbital tissue or an extraocular cavity, comprising periocular injection of the AAV carrier, wherein the orbital tissue or extraocular cavity is selected from the group consisting of extraocular adipose tissue, levator lateralis muscle, lateral rectus muscle, medial lateral rectus muscle, superior lateral rectus muscle, inferior lateral rectus muscle, inferior lateral oblique muscle, superior lateral oblique muscle, optic nerve, blood vessel, sclera, dura mater sheath, cribriform plate, eyelid, lacrimal gland, lacrimal sac, tear film, or any combination thereof.

[0021] In some respects, the orbital tissue or extraocular cavity is selected from the group consisting of extraocular adipose tissue, the lateral rectus muscle, the medial rectus muscle, the superior rectus muscle, the inferior rectus muscle, the inferior oblique muscle, the superior oblique muscle, or any combination thereof.

[0022] In some respects, the volume of the periorbital application composition (e.g., a pharmaceutical composition) is about 0.1 mL to about 10 mL, about 1 mL to about 10 mL, about 0.5 mL to about 10 mL, about 5 mL to about 10 mL, about 0.1 mL to about 5 mL, about 1 mL to about 5 mL, about 0.5 mL to about 5 mL, about 5 mL to about 5 mL, about 0.1 mL to about 1 mL, about 0.25 mL to about 1 mL, about 2.5 mL to about 1 mL, or about 0.5 mL to about 1 mL.

[0023] In some respects, the volume of the drug composition administered includes 0.1 mL to 1 mL injected per eye periorbitally.

[0024] In some respects, the volume of the drug composition administered includes 0.1 mL to 0.5 mL injected per eye periorbitally.

[0025] In some respects, the volume of the drug composition administered includes 0.3 mL to 0.5 mL injected per eye periorbitally.

[0026] In some respects, the volume of the drug composition administered includes approximately 0.4 mL injected per eye periorbitally.

[0027] In some respects, the transduction efficiency of the AAV vector is at least 5% of ocular or periocular tissue cells (e.g., 5%–95%, 10%–95%, 15%–95%, 20%–95%, 25%–95%, 30%–95%, 40%–95%, or 50%–95%).

[0028] In some respects, AAV vectors transduce cells from a group consisting of adipocytes, orbital fibroblasts, and extraocular muscles.

[0029] In some respects, the AAV vector comprises a capsid of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRh8, AAVrh9, AAV9, AAVrh10, AAV10, AAV11, AAV12 and their modified versions.

[0030] In some respects, the capsid serotype is AAV8 or AAV9. In other respects, the capsid serotype is AAV9.

[0031] In some respects, an AAV vector comprises a capsid, a vector genome, and an expression cassette.

[0032] In some respects, the vector genome contains inverted terminal repeats (ITRs).

[0033] In some respects, the expression cassette contains nucleic acid sequences encoding therapeutic agents.

[0034] In some aspects, the vector genome contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 53. In some aspects, the nucleic acid sequence contains a nucleotide sequence corresponding to SEQ ID NO: 53.

[0035] In some respects, therapeutic agents comprise antibodies or their antigen-binding fragments, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, or single-chain antibodies (e.g., nanobodies).

[0036] In some respects, the antibodies are selected from the group consisting of anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-6R, anti-IL-2, anti-IL-17A, anti-IL-11R, anti-TNF-α, anti-CD28, anti-FcRn or their antigen-binding fragments.

[0037] In some respects, the antibodies are selected from the group consisting of anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-2, anti-IL-17A, anti-TNF-α, anti-CD28, or their antigen-binding fragments.

[0038] In some respects, the antibody is selected from the group consisting of tocilizumab, teprotumumab, bevacizumab, ranibizumab, faricimab, infliximab, daclizumab, secukinumab, or antigen-binding fragments thereof.

[0039] In some respects, anti-IGF-1R antibodies include teprotumumab, VRDN-01100 (SEQ ID NO: 78), VRDN-02700 (SEQ ID NO: 81), ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, and rhuMab. IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H1 5. L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H2 8. 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 fragments, variants or derivatives thereof.

[0040] In some respects, the antibody is teprotumumab.

[0041] In some respects, the antibody is an anti-IL-6 antibody. In other respects, the anti-IL-6 antibody or its antigen-binding fragment is tocilizumab, cetuximab, olokizumab, clazakizumab, sirukumab, or levilimab.

[0042] In some respects, the antibody is an anti-IL-6-R antibody. In other respects, the anti-IL-6-R antibody or its antigen-binding fragment is sarilumab or satralizumab.

[0043] In some respects, the antibody is an anti-IL-11 antibody. In other respects, the anti-IL-11 antibody or its antigen-binding fragment is 9MW3811.

[0044] In some respects, the antibody is an anti-IL-11 receptor (anti-IL-11R) antibody. In other respects, the anti-IL-11R antibody or its antigen fragment is BI 765423 or LASN01.

[0045] In some respects, the antibody is an anti-FcRn antibody. In other respects, the anti-FcRn antibody or its antigen-binding fragment is batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, or IMVT-1402.

[0046] In some respects, the antibody is an anti-TSHR antibody. In other respects, the anti-TSHR antibody or its antigen-binding fragment is K1-70.

[0047] In some aspects, after administration of the AAV carrier to a subject, the expression of the therapeutic agent (e.g., an antibody or its antigen-binding fragment) in the subject's ocular muscles is at least about 18 µg / mL and / or the serum concentration of the therapeutic agent is less than about 6 µg / mL. In some aspects, after administration of the AAV carrier to a subject, the expression of the therapeutic agent (e.g., an antibody or its antigen-binding fragment) in the subject's ocular muscles is at least about 18 µg / mL and / or the serum concentration of the therapeutic agent is less than about 6 µg / mL. In some aspects, the concentration in ocular tissue is about 18 µg / mL to about 180 µg / mL, about 18 µg / mL to about 100 µg / mL, about 18 µg / mL to about 60 µg / mL, or about 18 µg / mL to about 30 µg / mL. In some aspects, after administration of the AAV carrier, the therapeutic agent in serum is less than about 6 µg / mL (e.g., about 0.1 to about 5.9 µg / mL).

[0048] In some respects, ophthalmic pathology is the treatment of eye diseases or diseases around the eyes.

[0049] In some respects, ophthalmic pathology is selected from age-related macular degeneration, Behcet's disease, Bietti's crystalline dystrophy, blepharitis, blepharospasm, iris defects, corneal conditions, diabetic retinopathy, dry eye, glaucoma, idiopathic intracranial hypertension, macular edema, ocular histoplasmosis syndrome, conjunctivitis, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt's disease, Usher syndrome, thyroid eye disease (TED), uveitis, orbital tumors, or any combination thereof.

[0050] In some aspects, ophthalmic pathology is thyroid eye disease (TED).

[0051] In some cases, administration involves a single dose.

[0052] In some cases, the administration involves multiple doses.

[0053] In some respects, each dose in the multiple doses is administered to the subject at a single injection site.

[0054] In some respects, each dose in the multi-dose regimen is administered to the subject via multiple injection sites.

[0055] In some respects, the application is bilateral, that is, applied to both eyes (e.g., injected around the eyeball in each eye).

[0056] In some cases, the treatment involves bilateral periorbital injections.

[0057] Some aspects of this disclosure relate to a method of treating thyroid ophthalmopathy in a subject with this need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, said AAV carrier comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof. In some aspects, the administration is a periorbital injection. In some aspects, the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-IGF-1R antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-11 antibody, anti-IL-11R antibody, anti-FcRn antibody, anti-TSHR antibody, or an antigen-binding fragment thereof.

[0058] Some aspects of this disclosure relate to a method for treating an ophthalmic pathology (e.g., thyroid ophthalmopathy) in a subject with such need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, the AAV carrier comprising a nucleic acid sequence encoding an anti-IGF-1R antibody or an antigen-binding fragment thereof, wherein the administration is a periorbital injection.

[0059] Some aspects of this disclosure relate to a method for treating an ophthalmic pathology (e.g., thyroid ophthalmopathy) in a subject with such need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, the AAV carrier comprising a nucleic acid sequence encoding an anti-IL-6 antibody or an antigen-binding fragment thereof, wherein the administration is a periorbital injection.

[0060] Some aspects of the present invention relate to a method for treating an ophthalmic pathology (e.g., thyroid ophthalmopathy) in a subject with such need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, the AAV carrier comprising a nucleic acid sequence encoding an anti-IL-6R antibody or an antigen-binding fragment thereof, wherein the administration is a periorbital injection.

[0061] Some aspects of the present invention relate to a method for treating an ophthalmic pathology (e.g., thyroid ophthalmopathy) in a subject with such need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, the AAV carrier comprising a nucleic acid sequence encoding an anti-IL-11 antibody or an antigen-binding fragment thereof, wherein the administration is a periorbital injection.

[0062] Some aspects of the present invention relate to a method for treating an ophthalmic pathology (e.g., thyroid ophthalmopathy) in a subject with such need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, the AAV carrier comprising a nucleic acid sequence encoding an anti-IL-11R antibody or an antigen-binding fragment thereof, wherein the administration is a periorbital injection.

[0063] Some aspects of the present invention relate to a method for treating an ophthalmic pathology (e.g., thyroid ophthalmopathy) in a subject with such need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, the AAV carrier comprising a nucleic acid sequence encoding an anti-FcRn antibody or an antigen-binding fragment thereof, wherein the administration is a periorbital injection.

[0064] Some aspects of the present invention relate to a method for treating an ophthalmic pathology (e.g., thyroid ophthalmopathy) in a subject with such need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, the AAV carrier comprising a nucleic acid sequence encoding an anti-TSHR antibody or an antigen-binding fragment thereof, wherein the administration is a periorbital injection.

[0065] In some respects, the thyroid eye disease is selected from active Graves' orbital disease and chronic Graves' orbital disease.

[0066] In some respects, the administration is a single dose.

[0067] In some respects, the single dose is administered at a single injection site.

[0068] In some respects, the single dose is administered at multiple injection sites.

[0069] In some respects, the administration is in multiple doses.

[0070] In some respects, the doses in the multiple doses are administered at a single injection site.

[0071] In some respects, each of the multiple doses is administered at a different injection site.

[0072] In some respects, the application is bilateral, that is, applied to both eyes (e.g., periocular injection into each eye).

[0073] In some respects, the anti-IGF-1R antibody or its antigen-binding fragment is teprotumumab.

[0074] In some respects, the anti-IGF-1R antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0075] In some aspects, the VH comprises a VH complementarity-determining region (CDR) 1, a VH CDR 2, and a VH CDR 3. In some aspects, the VH CDR 1 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 9 or 12. In some aspects, the VH CDR 2 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 10, 13, or 15. In some respects, the VHCDR3 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 11, 14, or 16.

[0076] In some aspects, the VL includes a VL complementarity-determining region (CDR) 1, a VL CDR 2, and a VL CDR 3. In some aspects, the VL CDR 1 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 17. In some aspects, the VL CDR 2 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 18, 20, or 22. In some respects, the VL CDR3 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 19, 21, or 23.

[0077] In some respects, the heavy chain variable region is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 24-26.

[0078] In some respects, the light chain variable region is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 29-31.

[0079] In some respects, the anti-IGF-1R antibody comprises a heavy chain and a light chain.

[0080] In some respects, the heavy chain is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 40-42.

[0081] In some respects, the light chain is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 46-48.

[0082] In some aspects, the AAV vector includes an expression cassette containing a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-IGF-1R antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-11 antibody, anti-IL-11R antibody, anti-FcRn antibody, anti-TSHR antibody, or an antigen-binding fragment thereof.

[0083] In some aspects, the AAV vector includes an expression cassette containing a nucleic acid sequence encoding an anti-IGF-1R antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter.

[0084] In some aspects, the AAV vector includes an expression cassette containing a nucleic acid sequence encoding an anti-IL-6 antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter.

[0085] In some aspects, the AAV vector includes an expression cassette containing a nucleic acid sequence encoding an anti-IL-6R antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter.

[0086] In some aspects, the AAV vector includes an expression cassette containing a nucleic acid sequence encoding an anti-IL-11 antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter.

[0087] In some aspects, the AAV vector includes an expression cassette containing a nucleic acid sequence encoding an anti-IL-11R antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter.

[0088] In some aspects, the AAV vector includes an expression cassette containing a nucleic acid sequence encoding an anti-FcRn antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter.

[0089] In some aspects, the AAV vector includes an expression cassette containing a nucleic acid sequence encoding an anti-TSHR antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter.

[0090] In some respects, the promoter is selected from the group consisting of the early CMV enhancer / chicken β-actin (CAG) promoter, CAG, CBA, smCBA, CMV, human elongation factor 1α subunit (EF1α), EF1α with CMV enhancer, CMV promoter with CMV enhancer (CMVe / p), CMV promoter with SV40 intron, or tissue-specific promoter.

[0091] In some respects, the promoter is the CAG promoter.

[0092] In some respects, the promoter is the smCBA promoter.

[0093] In some respects, the expression box also includes introns.

[0094] In some respects, the introns are selected from the group consisting of SV40 introns, MVM introns, CAG introns, or human β-globulin introns.

[0095] In some respects, the expression cassette also includes a nucleic acid sequence encoding a signal peptide, which is operatively linked to a nucleic acid encoding an antibody or an antigen-binding fragment thereof.

[0096] In some respects, the signal peptide is the IL-2 signal peptide or the IL-10 signal peptide.

[0097] In some respects, the expression box also includes poly(A) sequences.

[0098] In some respects, the polymer (A) is a synthetic polymer (A) or bovine growth hormone (BGH) polymer (A).

[0099] In some respects, the AAV vector includes a capsid selected from the group consisting of serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRh8, AAVrh9, AAV9, AAVrh10, AAV10, AAV11, AAV12 and modified versions thereof.

[0100] In some respects, the capsid serotype is AAV8 or AAV9. In other respects, the capsid serotype is AAV9.

[0101] In some respects, AAV vectors include a capsid, a vector genome, and an expression cassette.

[0102] In some respects, vector genomes include inverted terminal repeats (ITRs).

[0103] In some respects, the AAV vector is a recombinant AAV (rAAV) comprising an AAV9 capsid that encapsulates the vector genome, the vector genome comprising a nucleic acid coding sequence encoding an anti-IGF-1R antibody or an antigen-binding fragment thereof (optionally teprotumumab) flanked by a pair of inverted terminal repeats (ITRs).

[0104] In some cases, subjects received approximately 6E11 to 2E13 vector genomes (vg) per eye. In some cases, subjects received approximately 1E12 to 2E13 vg per eye. In some cases, subjects received approximately 3E12 to 1.8E13 vg per eye. In some cases, subjects received approximately 5E12 to 1E13 vg per eye. In some cases, subjects received approximately 6E12 to 9E12 vg per eye. In some cases, subjects received approximately 6E12 vg per eye. In some cases, subjects received approximately 9E12 vg per eye.

[0105] In some respects, administration (e.g., periocular injection) is a single-dose administration to the eye (e.g., unilateral administration). In some respects, a single dose is administered at a single injection site. In some respects, a single dose is administered at multiple injection sites.

[0106] In some respects, administration (e.g., periocular injection) is a multi-dose administration to the eye (e.g., unilateral administration). In some respects, the doses in the multi-dose administration are administered at a single injection site. In some respects, each dose in the multi-dose administration is administered at a different injection site.

[0107] In some respects, the application is bilateral, that is, applied to both eyes (e.g., periocular injection in each eye).

[0108] In some aspects, the vector genome includes a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 142. In some aspects, the vector genome includes a nucleotide sequence corresponding to SEQ ID NO: 142.

[0109] Some aspects of this disclosure relate to a method for increasing teprotumumab expression in the eyes of a subject, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, the AAV carrier comprising a nucleic acid sequence encoding an antibody (teprotumumab) or an antigen-binding fragment thereof, wherein the administration is a periorbital injection, wherein teprotumumab expression in ocular tissue is at least 18 µg / mL, optionally wherein the ocular tissue is an extraocular muscle. In some aspects, the concentration in ocular tissue is about 18 µg / mL to 180 µg / mL, about 18 µg / mL to about 100 µg / mL, about 18 µg / mL to about 60 µg / mL, or about 18 µg / mL to about 30 µg / mL.

[0110] In some respects, teprotumumab concentrations in serum after administration of an AAV vector containing a nucleic acid sequence encoding an antibody against teprotumumab or an antigen-binding fragment thereof are less than about 6 µg / mL (e.g., about 0.1 to about 5.9 µg / mL). [Attached Image Description]

[0112] Figure 1 A schematic diagram of an exemplary retroocular injection is shown. A needle is inserted into the inferior temporal conjunctiva to deliver the AAV payload into the retroocular space within the lateral extraocular muscle cone.

[0113] Figure 2 A schematic diagram of an exemplary retroseptal injection is shown. The needle passes through the inferior orbital septum to deliver the AAV payload to the orbital fat, which is located outside the muscle cone.

[0114] Figure 3 This diagram illustrates an exemplary periocular injection in the inferior temporal region. The needle is inserted parallel to the orbital floor and tangentially at the angle between the lateral third and medial two-thirds of the lower orbital rim in the sagittal plane, and is not directed toward the apex angle.

[0115] Figure 4Immunohistochemical staining and mCherry staining are shown for cross-sections of extraocular fat collected from the posterior aspect of the left eye of two Göttingen miniature pigs in group 4 after retroocular injection with 1E12 vg / eye AAV9.CAG.mCherry.

[0116] Figure 5 Immunohistochemical (IHC) staining and mCherry staining of extraocular muscles collected from the left eyes of two Göttingen miniature pigs in group 4 after retroocular injection with 1E12 vg / eye AAV9.CAG.mCherry were shown.

[0117] Figure 6 The image shows a cross-section of the left eye of a Göttingen miniature pig after retroocular injection with 1E12 vg / eye AAV9.CAG.mCherry and after IHC staining of the mCherry, including different anatomical regions (e.g., cornea, lens, ciliary body, retina, optic disc, small optic nerve attached to the eyeball and extraocular fat attached to the posterior part of the eyeball with some adhering marginal muscles (top panel) plus the separately embedded residual optic nerve (bottom panel)). Note the presence of abundant perioptic nerve fibrous connective tissue and adipocytes around the optic nerve.

[0118] Figures 7A-7B The IHC scores of AAV9.mCherry-stained tissues from three Göttingen miniature pigs after perioral injection are shown. The presence of ( ) was assessed using a semi-quantitative scoring system. Figure 7A ) or intensity ( Figure 7B The presence matrix reflects the total number of mCherry-positive cells in the entire section, regardless of intensity, ranging from 0 to 5 (score: 0 = absent; 1 = minimal, 1–10% of target tissue positive; 2 = mild, 11–25% positive; 3 = moderate, 26–50% positive; 4 = significant, 51–75% positive; and 5 = severe, >75% positive). The intensity score assesses the overall color shading of positive staining and is divided into four score categories, ranging from 0 to 3 (0 = absent; 1 = weak; 2 = intermediate intensity and / or mixed pattern between weak and strong, with no staining category exceeding 50%; and 3 = strong). Other ocular ventricles, brain regions, CNS, and peripheral tissues with a score of 0 were analyzed.

[0119] Figure 8The vector genome copy numbers in all tissues harvested after post-ocular injection in two Göttingen miniature pigs from group 4 following post-ocular injection with 1E12 vg / eye AAV9.CAG.mCherry are shown, as determined by qPCR assay targeting the bGH polyA region at the 3' end of the transgene found in the test strain vector.

[0120] Figure 9 The vector genome (cps = copies; ds = double strands; EOM = extraocular intramuscular; RB = periocular retroocular; VG = vector genome; samples below the limit of quantitation (BLOQ) are plotted as zero by qPCR in ocular and systemic tissues of Göttingen miniature pigs administered with periocular vectorized mCherry).

[0121] Figure 10 This image shows mCherry staining in fat and extraocular muscles following periorbital AAV9.mCherry injection in Göttingen miniature pigs. Each extraocular muscle from the left eye (OS) was divided into three sections, fixed in formalin, sectioned at 5 µm, stained with anti-mCherry antibody, and developed using DAB (3,3-diaminobenzidine) as the chromophore. Representative images from different animals are shown at 1× or 28× magnification.

[0122] Figures 11A-11B This image shows teprotumumab protein expression in Göttingen miniature pigs that received periorbital AAV9.CAG.teprotumumab administration. The expression was visualized at D8, D15, and D22 (left). Figure 11A Serum from each animal (Group 2) after receiving AAV9.CAG.tetumumab administration and on day 22 (right, 2 per animal) Figure 11B Tissue samples from each eye are presented separately. Serum concentrations are reported as ng / mL of serum, and solid tissue concentrations are reported as the amount of teprotumumab (µg) per volume of isolated tissue (mL). The limit of quantification (LLOQ) values ​​are 8 ng / mL for serum, 144 ng / mg for muscle, and 72 ng / mg for fat. BLOQ values ​​are not reported for samples.

[0123] Figure 12 The vector genome is shown in the extraocular tissue of Göttingen miniature pigs that received periocular AAV9.CAG.tetumumab. Animals received postocular AAV9.CAG.tetumumab injection (Group 2) (cps=copy; ds=double strand; OS=left eye; OD=right eye; VG=vector genome).

[0124] Figure 13 The vector genomes in selected tissues of wild-type mice that received either periocular vectorized mCherry or intramuscular vectorized teprotumumab are shown (samples plotted at zero are BLOQ.cps=copy;IM=intramuscular;PO=periocular;VG=vector genome).

[0125] Figures 14A-14C IHC staining of mCherry in cross-sections of the eyeball, liver, and heart following periocular injection in wild-type mice is shown. Representative images of the entire eyeball, liver, and heart from individual animals are also shown. Each tissue was fixed in formalin, sectioned at 5 µm, and stained with anti-mCherry antibody using DAB (3,3'-diaminobenzidine) as the chromophore. For each tissue, note the magnification of representative images from different animals.

[0126] Figures 15A-15B Serum and quadriceps femoris muscle (muscle) teprotumumab concentrations in mice treated with intramuscularly packaged teprotumumab are shown. Samples plotted at zero represent BLOQ values. smCBA = intramuscular AAV9.smCBA.teprotumumab; CAG = intramuscular AAV9.CAG.teprotumumab; IV = intravenous; ns = not significant.

[0127] Figures 16A-16B Serum concentrations of teprotumumab following a single IV Tepezza dose are shown in non-human primates (NHP). Serum teprotumumab concentrations in NHPs were at 20 mg / kg 9 days after IV Tepezza. Figure 16A ) and 50mg / kg ( Figure 16B Teprotumumab was quantified against the Fc region of human IgG using ELISA and plotted as µg of antibody per mL of serum. The LLOQ per well was 50 ng / mL. The only sample below the quantification level was zero at time and plotted as zero on the graph.

[0128] Figures 17A-17B The concentrations of teprotumumab in muscle and fat were shown 9 days after IV Tepezza injection in NHP patients. Ocular muscle concentrations were also shown in NHP patients treated with 20 mg / kg or 50 mg / kg IV Tepezza. Figure 17A ) and fat slices ( Figure 17BThe concentration of teprotumumab in the tissue was determined by ELISA targeting the Fc region of human IgG and plotted as µg of antibody per milliliter of isolated tissue. Intrawell LLOQ was 25 ng / mL (muscle) and 4 ng / mL (fat). IO = inferior oblique muscle; IR = inferior rectus muscle; LR = lateral rectus muscle; MR = medial rectus muscle; SO = superior oblique muscle; SR = superior rectus muscle; PB = periorbital; RB = retroorbital.

[0129] Figures 18A-18B The concentrations of teprotumumab in the ocular muscles or fat were shown separately at 9 days after IV Tepezza treatment in NHP. Teprotumumab concentrations were pooled in all ocular muscles at 9 days after treatment with 20 mg / kg or 50 mg / kg IV Tepezza. Figure 18A ) and fat slices ( Figure 18B In this study, teprotumumab was quantified by ELISA against the Fc region of human IgG and plotted as µg of antibody per mL of isolated tissue. Intrawell LLOQ was 25 ng / mL (muscle) and 4 ng / mL (fat). No samples were below the quantification level, and the mean is indicated at the top of each bar.

[0130] Figures 19A-19B This shows the serum teprotumumab concentration following a single periorbital injection of AAV9.smCBA.teprotumumab in NHP. AAV9.smCBA.teprotumumab was administered at a dose of 1E12 vg / eye (…). Figure 19A ) or 4.65E12 vg / eye ( Figure 19B ) Application. Teprotumumab was quantified against the Fc region of human IgG by ELISA and plotted as µg of antibody per mL of isolated tissue. The LLOQ in the wells was 50 ng / mL, and samples below the quantification level were estimated as LLOQ / 2 for plotting on the graph.

[0131] Figures 20A-20B This study shows the teprotumumab concentrations in each muscle and fat slice in the NHP at 35 days following high-dose AAV9.smCBA.teprotumumab periocular administration. The concentrations in each ocular muscle were determined in the 4.65E 12 vg / ocular dose group. Figure 20A ) and fat slices ( Figure 20BThe concentration of teprotumumab in the tissue was plotted. Teprotumumab was quantified by ELISA against the Fc region of human IgG and plotted as µg of antibody per mL of isolated tissue. Intrawell LLOQ was 25 ng / mL (muscle) and 4 ng / mL (fat). Samples below the quantification level were not included in the figure. IO = inferior oblique muscle; IR = inferior rectus muscle; LR = lateral rectus muscle; MR = medial rectus muscle; SO = superior oblique muscle; SR = superior rectus muscle; PB = periorbital; RB = retroorbital.

[0132] Figures 21A-21B This shows the concentration of teprotumumab in the ocular muscles or fat of NHP 35 days after periocular administration of AAV9.smCBA. The concentrations of teprotumumab were pooled across all ocular muscles ( Figure 21A ) and fat samples ( Figure 21B In the study, teprotumumab was quantified by ELISA against the Fc region of human IgG and plotted as µg of antibody per mL of isolated tissue. The intrawell LLOQ was 25 ng / mL (muscle) and 4 ng / mL (fat). Samples below the quantification level were not included in the plot or used to generate the average, which is indicated at the top of each bar.

[0133] Figures 22A-22B The levels of teprotumumab protein in tumors and serum after intratumoral injection of AAV9.smCBA.teprotumumab or AAV9.CAG.teprotumumab or intraperitoneal injection of teprotumumab antibody in Colo205 tumors of xenografted nude mice are shown. The protein levels of teprotumumab (recombinant or AAV-vectored) on days 15 (n=3), 22 (N=3), and 31 (N=8) were determined by IgG MSD ELISA. ELISA = Enzyme-linked immunosorbent assay; MSD = Meso Scale Discovery; SD = Standard deviation.

[0134] Figure 23The vector genome levels, determined by qPCR, are shown in Colo205 tumors of xenografted nude mice following administration of AAV9.smCBA.tetumumab, AAV9.CAG.tetumumab, AAV9.smCBA.mCherry, or AAV9.CAG.mCherry. The vector genome copy number per microgram of input nucleic acid was determined by primers that identified the bGH poly(A) region in the qPCR assay. Vector genomes were detected in three Tepezza-treated (control) animals, which may be due to sample contamination during sampling. Samples below the quantification level are represented as zero for plotting purposes.

[0135] Figure 24 This study shows the IGF1R protein expression levels in tumors treated with AAV9.smCBA.tetumumab or AAV9.CAG.tetumumab compared to Tepezza treatment. IGF1R protein levels in tumors from intermediate and terminal necropsy animals were determined by IgG MSD ELISA. ELISA = Enzyme-linked immunosorbent assay; MSD = Meso Scale Discovery; SD = Standard deviation. IGF1R levels in tumors from animals treated with AAV9.smCBA.tetumumab, AAV9.CAG.tetumumab, or Tepezza were not significantly different from each other at any time point, but were all lower than those in control animals treated with a medium or untreated animals.

[0136] Figure 25 The correlation between IgG antibody quantification of Tepezza, AAV9.smCBA.tetumumab, and AAV9.CAG.tetumumab using the Gyrolab human IgG assay and the MSD IGF1R binding assay is shown. IgG = Immunoglobulin G; MSD = Meso Scale Discovery.

[0137] Figures 26A-26B The levels of total IGF1R and phosphotyrosine IGF1R in HT-1080 cells after treatment with Tepezza or AAV9.smCBA.tetumumab are shown separately. HT1080 cells were treated with Tepezza, AAV9.smCBA.tetumumab, or AAV9.CAG.tetumumab and stimulated with 500 ng / mL human IGF1. IGF1R and phosphorylated IGF1R were measured by MSD ELISA. For comparison of significant differences in IC50 values ​​for total IGF1R: P = 0.9611 (not significant) and for phosphorylated IGF1R: P = 0.0950 (not significant).

[0138] Figure 27This study demonstrates the basal IGF1R protein levels in primary fibroblasts from Graves' disease and healthy donors after treatment with vector-derived teprotumumab. The effect of AAV9.CAG.teprotumumab transduction supernatant on total IGF1R protein expression in fibroblasts isolated from peripheral blood of healthy (donor 003N) or Graves' disease (donor 002G) patients was shown. Fibroblasts were treated with either a mock supernatant or 500 ng / mL vector-derived teprotumumab (from the construct AAV9.CAG.teprotumumab) for 24–48 hours prior to harvesting and preparation of cell lysates. Total IGF1R protein was determined using an insulin signaling kit (Meso Scale Discovery). Relative basal IGF1R protein levels were calculated relative to the mock-treated samples. Samples were run in two biological replicates for donor 003N and in one biological replicate for donor 002G.

[0139] Figures 28A-28C The percentage (%) of IGF-1R expression in orbital fibroblasts decreased 24 hours after treatment with various concentrations of teprotumumab derived from cells transduced with AAV9.smCBA.teprotumumab (vTepro). Figure 28A The response of orbital fibroblasts derived from TED patients (Dn 002) treated with Tepezza for more than 90 days is shown. Figure 28B The study showed the response of orbital fibroblasts derived from Tepezza naive TED patients (Dn 004). Figure 28C The response of orbital fibroblasts derived from control donors without TED is shown. The percentage reduction in IGF-1R expression was calculated by subtracting the total positive cell intensity value for each positive cell at each concentration level from the mean total positive cell intensity value of the wells treated with IGF-1 alone as a baseline. The reduction in IGF-1R was mainly observed at higher vTepro concentrations, especially for donors 002 and 004.

[0140] Figures 29A-29C The orbital fibroblast donor Dn 002 was shown. Figure 29A ), Dn 004 ( Figure 29B ) and Dn 006 ( Figure 29CThe percentage (%) of decreased IGF-1R expression was measured by treatment with various concentrations of teprotumumab derived from cells transduced with AAV9.smCBA.tepro and IGF-1 at 100 ng / mL for 24 hours. The percentage decrease in IGF-1R expression was calculated by subtracting the total positive cell intensity value for each positive cell at each concentration level from the mean total positive cell intensity value of the wells treated with IGF-1 alone as the baseline.

[0141] Figures 30A-30B The relative changes in auditory brainstem response (ABR) threshold levels at three frequencies relative to baseline were shown. At baseline and 4 weeks post-tetrumb administration of AAV9.smCBA.tetrumb... Figure 30A ) and 12 weeks ( Figure 30B ABRs were measured for each animal. Brain response thresholds were recorded in decibels across frequencies of 5 kHz, 10 kHz, and 20 kHz. The decibel thresholds of each 4-week and 12-week reading were subtracted from the baseline thresholds to determine the amount of change from before AAV treatment.

[0142] Figure 30C Serum teprotumumab concentrations following a single periocular injection of AAV9.smCBA.teprotumumab are shown. AAV9.smCBA.teprotumumab was administered at doses of 4.5E11, 1.5E12, and 4.35E12 vg / eye, and serum samples were collected throughout the 12-week study period. The figure shows the mean concentration at each time point for each dose group, along with standard deviation error bars. Teprotumumab was quantified by ELISA against the Fc region of human IgG and plotted as µg antibody per mL of isolated tissue. The LLOQ per well was 50 ng / mL, and samples below the quantification level were estimated as LLOQ / 2 for plotting on the graph.

[0143] Figures 30D-30E This shows the effect 4 weeks after periorbital administration of AAV9.smCBA.tetumumab. Figure 30D ) or 12 weeks ( Figure 30E The concentration of teprotumumab in all muscle tissue collected from each animal was determined. The mean teprotumumab concentration per animal was determined across the ocular muscle at 4 and 12 weeks. Teprotumumab was quantified by ELISA against the Fc region of human IgG and plotted as µg antibody per mL of isolated tissue. The LLOQ per well was 25 ng / mL (muscle). Samples below the quantification level were not included in the figure. The horizontal dashed line represents the predicted minimum effective protein level (4.5 µg / mL) and the effective protein level (18.5 µg / mL).

[0144] Figures 30F-30G This shows the effect 4 weeks after periorbital administration of AAV9.smCBA.tetumumab. Figure 30F ) or 12 weeks ( Figure 30G The concentration of teprotumumab in each fat fraction was determined. The mean teprotumumab concentration per animal was determined across the fat of the eye at 4 and 12 weeks. Teprotumumab was quantified by ELISA against the Fc region of human IgG and plotted as µg antibody per mL of isolated tissue. The LLOQ per well was 4 ng / mL (fat). Samples below the quantification level were not included in the figure.

[0145] Figure 30H The relative teprotumumab concentrations in each muscle following periocular administration of AAV9.smCBA.teprotumumab are shown. The teprotumumab concentration was determined for each ocular muscle and then normalized to its corresponding dose group mean teprotumumab concentration in the lateral rectus muscle. 100% of the samples contained the same concentration of teprotumumab as the mean lateral rectus muscle within their respective dose groups. Samples below the quantification level are not included in the figure.

[0146] Figure 30I The vector genome in the ocular muscles of NHPs receiving periocular AAV9.smCBA.tetumumab is shown. Vector genome copy number per microgram of input nucleic acid was determined by primers that identified the poly(A) sequence in a qPCR assay. All samples from each animal were averaged and plotted as a single data point within each group, with bars representing the mean for the entire group. Error bars are SEM (cps = copies; OS = left eye; OD = right eye; VG = vector genome).

[0147] Figure 30J The image shows teprotumumab mRNA in the ocular muscles of NHPs that received AAV9.smCBA.teprotumumab perocular region. The mRNA copy number per microgram of input nucleic acid was determined by primers that identified the poly(A) sequence in an RT-qPCR assay. All samples from each animal were averaged and plotted as a single data point within each group, with bars representing the mean for the entire group. Error bars are SEM (cps = copies; OS = left eye; OD = right eye). Note: Data from early necropsy animals have been removed from the figure.

[0148] Figure 30KThe vector genome from the orbital fat of NHPs receiving periocular AAV9.smCBA.tetumumab is displayed. Vector genome copy number per µg of input nucleic acid was determined by primers that identified the poly(A) gene sequence in a qPCR assay. All samples from each animal were averaged and plotted as a single data point within each group; bars represent the average across the entire group. Error bars are shown in the SEM (cps = copies; OS = left eye; OD = right eye; VG = vector genome).

[0149] Figure 30L The image shows mRNA from the orbital fat of NHPs that received periocular AAV9.smCBA.tetumumab. The mRNA copy number per µg of input nucleic acid was determined by primers that identified the poly(A) gene sequence in the RT-qPCR assay. All samples from each animal were averaged and plotted as a single data point for each group; bars represent the mean for the entire group. Error bars are SEM (cps = copies; OS = left eye; OD = right eye). Note: Early necropsy animal data were removed from the figure.

Detailed Implementation Methods

[0151] Some aspects of the present invention relate to a method of treating an ophthalmic pathology in a subject with such a need, comprising administering to the subject a pharmaceutical composition comprising an AAV carrier. In some aspects, the administration is a periorbital injection. In some aspects, the periorbital injection is a periorbital injection or a retroseptal injection. In some aspects, the periorbital injection is a subcapsular injection, a retrobulbar injection, a subconjunctival injection, or a periorbital injection. In some aspects, the periorbital injection is a retrobulbar injection. In some aspects, the periorbital injection is a periorbital injection.

[0152] Some aspects of the present invention relate to a method of delivering a pharmaceutical composition comprising an AAV carrier to orbital tissue or an extraocular cavity, comprising periocular injection of the AAV carrier, wherein the orbital tissue or extraocular cavity is selected from the group consisting of: extraocular adipose tissue, levator lateralis muscle, lateral rectus muscle, medial lateral rectus muscle, superior lateral rectus muscle, inferior lateral rectus muscle, inferior lateral oblique muscle, superior lateral oblique muscle, optic nerve, blood vessels, sclera, dura mater sheath, lamina cribriformis, eyelid, lacrimal gland, lacrimal sac, tear film, or any combination thereof.

[0153] Some aspects of the present invention relate to a method of treating thyroid ophthalmopathy in a subject with this need, comprising administering to the subject a pharmaceutical composition comprising an AAV vector, said AAV vector comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof. In some aspects, the administration is a periorbital injection. In some aspects, said antibody or antigen-binding fragment thereof is selected from the group consisting of: anti-IGF-1R antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-11 antibody, anti-IL-11R antibody, anti-FcRn antibody, anti-TSHR antibody, or an antigen-binding fragment thereof. In some aspects, said AAV vector is recombinant AAV (rAAV) comprising an AAV9 capsid encapsulating a vector genome, said vector genome comprising a nucleic acid coding sequence encoding an anti-IGF-1R antibody or an antigen-binding fragment thereof (optionally teprotumumab), and flanked by a pair of inverted terminal repeats (ITRs).

[0154]

I. Definition

[0155] To make this invention easier to understand, some terms are first defined. Other definitions are set forth throughout the detailed specification.

[0156] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "nucleic acid sequence" is understood to represent one or more nucleic acid sequences unless otherwise stated. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this document.

[0157] Furthermore, the term “and / or” as used herein should be considered as a separate disclosure of each of the two specified features or components, regardless of the presence of the other. Therefore, the term “and / or” in the phrase “A and / or B” as used herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” in the phrase “A, B, and / or C” as used herein is intended to cover each of the following: 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).

[0158] The numerical values ​​presented in this article can be written out, or in scientific notation, i.e., x × 10⁻⁶. y Or expressed in scientific E notation xEy.

[0159] It should be understood that the aspects described herein use the term "comprising," whereas similarly described aspects are provided in the terms "consisting of" and / or "substantially composed of."

[0160] As used herein, the term “about” means approximately, roughly, around, or within a range of. When the term “about” is used with a numerical range, it modifies the range by extending the boundaries to be above and below the stated value. Typically, the term “about” can modify values ​​above and below the stated value by, for example, a 10% variation, upward or downward (higher or lower).

[0161] The quantity or series of numbers preceding the term "at least" is understood to include the number immediately preceding the term "at least," as well as all subsequent numbers or integers that can be reasonably included from the context, up to zero. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in that series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, and 5.18%, regardless of the number of significant figures).

[0162] As used in this article, “not more than” or “less than” is understood to be the value immediately preceding the phrase and the lower value or integer that is reasonable from the context, up to zero. When “not more than” appears before a series of numbers or a range, it should be understood that “not more than” can modify each number in that series or range.

[0163] As used herein, the term "derived from" means a component isolated from or prepared using a particular molecule or organism, or information (e.g., amino acid or nucleic acid sequences) derived from a particular molecule or organism. For example, a nucleic acid sequence (e.g., an AAV vector) derived from a second nucleic acid sequence (e.g., another AAV vector) may include nucleotide sequences that are identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of the polynucleotides disclosed herein, derived strains can be obtained, for example, through natural mutation, artificial directed mutagenesis, or artificial random mutagenesis.

[0164] The term "operably linked" refers to the arrangement of nucleic acid sequences (e.g., coding regions, such as genes of interest) and regulatory sequences (e.g., promoters, enhancers, or silencers) in such a manner that gene expression is permitted when a suitable molecule (e.g., a transcriptional activator protein) binds to the regulatory sequence. In some aspects, the regulatory nucleic acid sequence (e.g., promoter, enhancer, or silencer) is operably linked to the coding region, which is under the "transcriptional control" of the regulatory nucleic acid sequence. The term "operably inserted" refers to the adjacent location of the nucleic acid sequence of interest to a regulatory nucleic acid sequence that guides the transcription and translation of the nucleic acid sequence of interest (i.e., promoting, for example, the production of a polypeptide encoded by the DNA of interest).

[0165] The "sequence identity percentage (%)" or "identity percentage (%)" for a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to those in a reference polynucleotide or polypeptide sequence after alignment of the sequences and, where necessary, the introduction of vacancies to achieve the maximum sequence identity percentage. For the purpose of determining the nucleotide or amino acid sequence identity percentage, alignment can be achieved in various ways 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 algorithm required to achieve maximum alignment of the full length of the sequences being compared. For example, the sequence comparison computer program BLAST can be used to generate sequence identity percentage values.

[0166] As used herein, the term "modified" refers to an altered state or structure of the molecules of the present invention. Molecules can be modified in many ways, including chemically, structurally, and functionally.

[0167] As used herein, the term "synthetic" means prepared, manufactured, and / or produced by hand. The synthesis of polynucleotides or polypeptides or other molecules of the present invention can be chemical or enzymatic.

[0168] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably in this application. These terms refer only to the primary structure of a molecule. Therefore, these terms include double-stranded and single-stranded DNA and double-stranded and single-stranded RNA. As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are defined as molecules comprising two or more covalently linked nucleosides, as generally understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Polynucleotides can be prepared by recombination, enzymatic reaction, or synthesis, for example, by solid-phase chemical synthesis followed by purification. When referring to the sequence of a polynucleotide or nucleic acid, it means the sequence or order of the nucleobase portion of the covalently linked nucleotide or nucleoside, or modifications thereof.

[0169] As used in this article, the term "mRNA" refers to a single-stranded RNA that encodes an amino acid sequence of one or more polypeptide chains.

[0170] A "coding sequence," or sequence that "encodes" a specific molecule (such as 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 in vitro or in vivo when operatively linked to an appropriate regulatory sequence (such as a promoter). The boundaries of a coding sequence are defined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. 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. The transcription termination sequence is typically located at the 3' end of the coding sequence.

[0171] As used herein, the term "promoter" refers to a DNA sequence that is recognized in vivo by cellular machinery, in vitro by synthetic machinery, or in vivo by synthetic machinery introduced into cells for initiating specific transcription of a gene. In some aspects, the term "promoter" is also intended to encompass those nucleic acid elements sufficient to carry out promoter-controlled gene expression, which can be cell-type specific, tissue-specific, or induced by external signals or agents; such elements may be located in the 5' or 3' region of a natural gene. In some aspects, a promoter is a constitutive promoter, a cell-type specific promoter, or an inducible promoter.

[0172] As used herein, the term “tunable promoter” is any promoter whose activity is affected by cis or trans-acting factors (e.g., inducible promoters, such as external signals or agents).

[0173] As used herein, the term “constitutive promoter” is any promoter that directs RNA production in many or all tissue / cell types most of the time, such as the immediate early enhancer / promoter region of the human CMV, which promotes constitutive expression of cloned DNA inserts in mammalian cells.

[0174] As used in this article, the term "ubiquitin promoter" is any promoter that is strongly active in a wide range of cells, tissues, and the cell cycle.

[0175] As used in this article, the term "bidirectional promoter" is an intergenic region located between two divergent genes on the complementary strand of DNA that drives their coordinated transcription in opposite directions.

[0176] As used herein, the term “tissue-specific promoter” refers to a promoter that is active only in certain cell types, i.e., that drives the expression of an operablely linked nucleotide sequence only in certain cell types.

[0177] As used herein, the term "enhancer" is a cis-acting element that stimulates the transcription of nucleic acids (e.g., genes). The term "silencer," as used herein, is a cis-acting element that inhibits the transcription of nucleic acids (e.g., genes). Enhancers can function in either orientation (e.g., they can be associated with coding sequences) and can function at distances of up to several thousand base pairs (kb) from the coding sequence and downstream of the transcribed region.

[0178] As used herein, the term "termination signal sequence" can refer to any genetic element that causes RNA polymerase to terminate transcription, such as the polyadenylation signal sequence. The polyadenylation signal sequence is a recognition region necessary for endonuclease to cleave RNA transcripts, followed by the polyadenylation concordance sequence AATAAA. The polyadenylation signal sequence provides a "poly-A site," that is, a site on the RNA transcript where adenine residues will be added during post-transcriptional polyadenylation.

[0179] As used herein, the term "internal ribosome entry site" or "IRES" refers to an element that facilitates direct ribosome entry into the start codon (such as ATG) of a cistron (protein-coding region), resulting in cap-independent translation of the gene. See, for example, Jackson RJ et al., Trends Biochem Sci 15(12):477-83 (199); Jackson R Jand Kaminski, A. RNA 1(10):985-1000 (1995). "Under the control of translation by IRES" as used herein means that translation is associated with the IRES and proceeds in a cap-independent manner.

[0180] As used herein, the terms “mutation” or “polymorphism” refer to any alteration in gene structure that results in a genetic variant (also known as a “mutant” or “polymorphic allele”). Genetic variants can occur in somatic cells or germline cells. Genetic variants occurring in the germline can be passed on to offspring. Genetic variants can occur in coding or non-coding regions and can be caused by substitution of a single base in the DNA or by deletion, insertion, or rearrangement of a larger segment of a gene or chromosome.

[0181] The term "antonym," as used herein, refers to a nucleic acid that is fully complementary to all or part of a gene, primary transcript, or processed mRNA, thereby interfering with the expression of an endogenous gene. A "complementary" polynucleotide is a polynucleotide capable of base pairing according to the standard Watson-Crick complementarity rule. Specifically, a purine will pair with a pyrimidine to form combinations of guanine and cytosine (G:C) and adenine and thymine (A:T) (in the case of DNA) or adenine and uracil (A:U) (in the case of RNA). It should be understood that two polynucleotides can hybridize even if they are not perfectly complementary, provided that each polynucleotide has at least one region substantially complementary to the other.

[0182] The terms "antisense strand" and "guide strand" refer to the non-coding DNA strand of a gene. The antisense strand serves as a template for the generation of messenger RNA (mRNA) and is complementary to the transcribed mRNA without any alteration during transcription. Nucleic acid molecules that include regions substantially complementary to the mRNA contain sequences substantially identical to the antisense strand, thus possessing sufficient complementarity with the target mRNA sequence to guide target-specific silencing.

[0183] The terms "sense strand" and "lagging strand," as used herein, refer to the coding DNA strand of a gene. Without any alteration during transcription, the sense strand contains the same sequence as the transcribed mRNA (except for the T in the DNA for the U in the transcribed RNA). The antisense and sense strands hybridize to form a double-stranded structure.

[0184] The terms "expression vector" or "expression construct" refer to any type of genetic construct containing nucleic acids, in which all or part of the nucleic acid coding sequence can be transcribed. In some respects, expression vectors or constructs may contain antibody expression cassettes.

[0185] As used herein, the term "delivery vector" or "vector" refers to any medium used to clone and / or transfer nucleic acids into host cells, such as plasmids, bacteriophages, transposons, kinases, chromosomes, artificial chromosomes, viruses, viral particles, etc. A vector can be a replicon to which another nucleic acid fragment can be ligated to induce replication of the ligated fragment. A replicon is any genetic element that functions as a unit of autonomous replication in vivo, i.e., capable of replicating under its own control. The term "delivery vector" or "vector" includes both viral and nonviral media used to introduce nucleic acids into cells in vitro, ex vivo, or in vivo. A large number of vectors are known and used, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. In some aspects, the insertion of a polynucleotide into a suitable vector can be achieved by ligating a suitable polynucleotide fragment into a selected vector having complementary sticky ends. Vectors can be programmed to encode selection markers or reporter genes, thereby providing selection or identification for cells incorporating the vector. Expression of selection markers or reporter genes allows for the identification and / or selection of host cells that incorporate and express additional coding regions contained on the vector. Examples of selectable marker genes known and used in the art include genes conferring resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, diammonium phosphate herbicide, sulfonamides, etc.; and genes used as phenotypic markers, such as anthocyanin regulatory genes, isopentenyltransferase genes, etc. Examples of reporter genes known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered reporter genes. In some aspects, delivery vectors are selected from the group including viral vectors (e.g., AAV vectors), plasmids, lipids, protein particles, bacterial vectors, and lysosomes.

[0186] Some aspects of this invention relate to biological vectors, which may include viruses, particularly attenuated and / or replication-defective viruses.

[0187] "Viral vector" refers to a virus-derived component that can be used to carry and / or deliver a payload (e.g., a nucleic acid sequence containing one or more polynucleotide regions encoding or containing a molecule of interest (e.g., a protein, peptide, or oligonucleotide, or a plurality thereof). Viral vectors can be used to deliver genetic material into cells. Viral vectors can be modified for specific applications. In some aspects, the delivery vector of the present invention is a viral vector selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, or retroviral vectors.

[0188] As used herein, the terms “adeno-associated virus vector” or “AAV vector” or “adeno-associated viral vector” refer to any vector containing or derived from adeno-associated vector components and suitable for infecting mammalian cells, preferably human cells. In some respects, the term AAV vector may refer to AAV-type viral particles or viral particles containing a payload. AAV vectors may be derived from various serotypes, including combinations of serotypes (i.e., “pseudotyped” AAVs) or from various genomes (e.g., single-stranded or self-complementary). In some respects, pseudotyped AAVs include AAV2 ITRs and capsids derived from other known AAV vectors (e.g., AAV2 / 8 or AAV2 / 9, which are generally referred to herein by their capsid serotypes, e.g., AAV8 or AAV9, respectively). Furthermore, AAV vectors may be replication-defective and / or targeting. The term "adeno-associated virus" (AAV) used in this document includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV 14, AAV 15, AAV 16, AAV VS3, AAVrh8, AAVrh10, AAVrh.74, AAV type 9e, AAVrh.20, AAVrh.39, AAVrh.46, AAVrh.73, AAVrh.74, AAVhu.12, AAVhu.21, AAVhu.26, AAVhu.37, AAVhu.51, AAV.RHM4-1, AAV.Anc80, AAV.7m8, AAV.PHP.B, A AV.PHP.eB, AAV2.5, AAV2tYF, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV. HSC14, AAV.HSC15, AAV.HSC16, snake AAV, avian AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, those AAV serotypes and branches disclosed by Gao et al. (J.Virol.78:6381 (2004)) and Moris et al. (Virol.33:375 (2004)), as well as any other AAV now known or subsequently discovered.See, for example, FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th edition, Lippincott-Raven Publishers). In some respects, “AAV vector” includes derivatives of known AAV vectors. In some respects, “AAV vector” includes modified or artificial AAV vectors. In some respects, AAV vectors include heterozygous vectors (e.g., AAV-DJ, AAV-PHP.B, AAV2-ESGHGYF, AAVM41, AAV-LK03, AAV2-BR1, AAV587MTP, AAV-Anc80L65, AAV2-7m8, AAV2HBKO, AAV2YF, AAV6-RGD, or AAV6.2). In some respects, the terms “AAV genome” and “AAV vector” are used interchangeably. In some respects, AAV vectors are modified relative to wild-type AAV serotype sequences. In some respects, the modified AAV vector is a modified AAV6, such as an AAV6 vector containing the RGD peptide (AAV6-RGD vector) or an AAV6 vector containing surface-exposed tyrosine residue mutations as described in Sayroo et al. Gene Ther. 2016 January; 23(1):18-25. In some respects, the AAV6-RGD vector also contains modified amino acids corresponding to Y705, Y731, T492, and K531 (e.g., Y705, Y731F, T492V, and K531E; also referred to as AAV-RGD-Y705-731F+T492V+K531E).

[0189] The "self-complementary adeno-associated virus" or "scAAV" used in this article is a viral vector engineered from naturally occurring AAV, such that the coding region is designed to form an intramolecular double-stranded DNA template. Therefore, after cells are infected with scAAV, instead of waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV combine to form a single double-stranded DNA (dsDNA) unit ready for replication and transcription. In some respects, the coding capacity present in typical rAAV (e.g., approximately 4.7–6 kb) is reduced by about half in scAAV (e.g., 2.4 kb). In some respects, rAAV is scAAV.

[0190] As used herein, “recombinant AAV particle,” “recombinant AAV vector,” “rAAV particle,” or “rAAV vector” refers to an AAV vector comprising a capsid protein and a vector genome (or AAV genome, such as at least one inverted repeat (ITR) region) containing at least one payload region (e.g., an expression cassette comprising a polynucleotide encoding a therapeutic agent). In some aspects, “AAV vector of the present invention” or “AAV vector” refers to an AAV vector comprising a polynucleotide encoding an antibody (e.g., an anti-IGF-1R antibody, an anti-IL6 antibody, an anti-FcRn antibody, an anti-TSHR antibody, an anti-IL11 antibody, an anti-IL6-R antibody, or an anti-IL11R antibody) and a vector genome containing a pair of AAV ITRs, for example encapsulated in an AAV particle.

[0191] As used in this article, “therapeutic agent” means a compound (e.g., a chemical compound), peptide, protein (e.g., an antibody), lipid, carbohydrate, nucleic acid, or any other molecule or compound that can treat, improve, or alleviate the symptoms of a disease, condition, or syndrome, or otherwise has a beneficial effect when administered to a subject suffering from a disease, condition, or syndrome.

[0192] The term “polypeptide” as used herein is intended to cover both the singular and plural “polypeptide”, and includes any chain or strand of two or more amino acids. Therefore, the terms “peptide,” “peptide subunit,” “protein,” “amino acid chain,” “amino acid sequence,” or any other term used herein to refer to a chain or strand of two or more amino acids are included in the definition of “polypeptide,” although each of these terms may have a more specific meaning. The term “polypeptide” may be used in place of or interchangeably with any of these terms. The term also includes polypeptides that have undergone post-translational or post-synthetic modifications, such as conjugation of a palmitoyl group, glycosylation, acetylation, phosphorylation, amidation, derivatization by a known protecting / blocking group, protease cleavage, or modification by a non-naturally occurring amino acid. The term “peptide” as used herein includes full-length peptides and fragments, variants, or derivatives thereof. A “peptide” disclosed herein may be part of a fusion polypeptide containing additional components, such as an Fc domain or an albumin domain, to increase half-life. The peptides described herein may also be derivatized in a variety of different ways. The peptides described herein may include modifications, including, for example, conjugation of a palmitoyl group.

[0193] The term "subunit" refers to a polypeptide capable of assembling with another polypeptide to form a protein (e.g., immunoglobulin (Ig)) comprising two or more subunits. In some aspects, the delivery vector of the present invention may include a coding region for each subunit. For example, a viral vector may include a coding region for the Ig heavy chain (or a variable region of the Ig heavy chain) and a coding region for the Ig light chain (or a variable region of the Ig light chain).

[0194] In this article, the terms “transcriptional regulatory protein,” “transcriptional regulatory factor,” and “transcription factor,” which are used interchangeably, refer to nuclear proteins that bind to DNA response elements and thereby transcribe and regulate the expression of related genes or genes. Transcriptional regulatory proteins typically bind directly to DNA response elements, but in some cases, they bind indirectly to DNA by binding to another protein, which in turn binds to or is bound to the DNA response element.

[0195] As used herein, the terms “self-processing cleavage site” or “self-processing cleavage sequence” refer to a post-translational or co-translational processing cleavage site or sequence. Such a “self-processing cleavage” site or sequence refers to a DNA or amino acid sequence, illustrated herein by examples of 2A sites, sequences or domains, or 2A-like sites, sequences or domains. Hereinafter, a “self-processing peptide” is defined as a peptide expression product encoding a DNA sequence that encodes a self-processing cleavage site or sequence, which, upon translation, mediates rapid intramolecular (cis) cleavage of a protein or polypeptide containing the self-processing cleavage site to produce discrete, mature protein or polypeptide products.

[0196] As used herein, the term "additional protease cleavage site" refers to a sequence introduced in the expression construct of the present invention adjacent to a self-processing cleavage site (e.g., a 2A or 2A-like sequence) and provides 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 protease cleavage sites" are described herein and include, but are not limited to, furin cleavage sites having a concordant sequence RXKRR or RXKR. Such furin cleavage sites can be cleaved by endogenous subtilisin-like proteases (e.g., furin and other serine proteases) located in the protein secretion pathway. In some aspects, other exemplary "additional protease cleavage sites" may be used, as described in Lie et al., Sci Rep 7, 2193 (2017).

[0197] The terms “antibody” and “antibody” refer to immunoglobulin molecules that recognize and specifically bind to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, within at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecules, provided that the antibody exhibits the desired biological activity. Antibodies can be any of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and µ, respectively, based on the identity of their heavy chain constant regions. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations.

[0198] The term “immunoglobulin” is used herein to include antibodies, their functional fragments, 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 other suitable constructs. An immunoglobulin molecule is a protein containing an immunologically active portion of an immunoglobulin heavy chain and an immunoglobulin light chain covalently coupled together and capable of specifically binding to an antigen. Immunoglobulin molecules are 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” may be used interchangeably herein. Immunoglobulin heavy chains are polypeptides containing at least a portion of the antigen-binding domain of an immunoglobulin and at least a portion of the variable region of the immunoglobulin heavy chain. Therefore, immunoglobulin-derived heavy chains share significant amino acid sequence homology regions with members of the immunoglobulin gene superfamily. For example, the heavy chain in the Fab fragment is an immunoglobulin-derived heavy chain. Immunoglobulin light chains are polypeptides containing at least a portion of the antigen-binding domain of an immunoglobulin and at least a portion of the variable region of the immunoglobulin. Therefore, immunoglobulin-derived light chains share significant amino acid homology regions with members of the immunoglobulin gene superfamily. Immunoadhesins are chimeric, antibody-like molecules that combine the functional domains of binding proteins (typically receptors, ligands, cell adhesion molecules, or 1-2 immunoglobulin variable domains) with constant domains of immunoglobulins (typically including hinges or GS linkers and Fc regions). Antigen-binding (Fab) fragments are the antigen-binding regions of antibodies. They consist of one constant domain and one variable domain from each of the heavy and light chains. Regarding the immunoglobulins or antibodies described herein, each fragment of the immunoglobulin coding sequence may be derived from one or more sources or synthesized. Suitable fragments may include coding regions of one or more of, for example, heavy chains, light chains, and / or fragments thereof (e.g., constant or variable regions (CH1, CH2, and / or CH3) of the heavy chain and / or constant or variable regions of the light chain). Alternatively, variable regions of the heavy or light chains may be utilized. Where appropriate, these sequences may be modified from their derived “natural” sequences as described herein. As used herein, the term “immunoglobulin construct” refers to any of the immunoglobulins or fragments thereof encoded and included by the expression cassettes described herein. Immunoglobulin constructs encoded and included by the expression cassettes described herein may be in the form of combinations including immunoglobulin constructs and viral vectors described herein.

[0199] The term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to the portion of a complete antibody that binds to an antigen. An antigen-binding fragment may contain an antigen recognition site of the complete 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 can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially produced.

[0200] The term "nanobody" or "sdAb" refers to a class of antigen-binding fragments, which are single-chain immunoglobulin molecules composed of monomeric variable antibody domains that recognize and specifically bind to antigens.

[0201] The term "monoclonal" antibody or its antigen-binding fragment refers to a homogeneous group of antibodies or antigen-binding fragments that participate in the recognition and binding of a single antigenic determinant or epitope with high specificity. This contrasts with polyclonal antibodies, which typically include different antibodies targeting different antigenic determinants. The term "monoclonal" antibody or its antigen-binding fragment includes full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal" antibody or its antigen-binding fragment refers to any number of such antibodies and their antigen-binding fragments prepared in any manner, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0202] As used in this article, the term "fully human monoclonal antibody" refers to a monoclonal antibody. Monoclonal antibodies can be, but are not limited to, murine, chimeric, humanized, or fully human, depending on the number of sequences derived from the mouse or human genome. Murine monoclonal antibodies contain only antibody-coding sequences derived from mice. Chimeric monoclonal antibodies contain antibody-coding sequences derived from both mice and humans. Humanized monoclonal antibodies are extensions of chimeras in which all regions of the mouse antibody-coding sequence are replaced by human antibody-coding sequences, except for the CDR-coding sequence. Fully human monoclonal antibodies contain only human antibody-coding sequences and no mouse antibody-coding sequences.

[0203] As used in this article, the term "vectored antibody" refers to a delivery vector (e.g., a viral vector) that contains a nucleotide sequence encoding an antibody or a derivative thereof.

[0204] The term "bispecific" or "bifunctional antibody," or its antigen-binding fragment, refers to an artificial hybrid antibody having two distinct heavy / light chain pairs and two distinct binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0205] The term "multispecific antibody" refers to an antibody that has specificity against two or more different epitopes (usually non-overlapping epitopes) or contains more than two different antigen-binding sites.

[0206] The terms “variable region” or “variable domain” used herein are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, usually a portion of the light or heavy chain, typically about 110–120 or 110–125 amino acids in the mature heavy chain and about 90–115 amino acids in the mature light chain, which varies widely in sequence between antibodies and is responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more highly conserved regions within a variable domain are called scaffold regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is assumed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some respects, the variable region is a human variable region. In some respects, the variable region comprises rodent or mouse CDRs and human scaffold regions (FRs). In some respects, the variable region is a primate (e.g., non-human primate) variable region. In some respects, the variable region includes the rodent or mouse CDR and the primate (e.g., non-human primate) skeletal region (FR).

[0207] The terms “VL” and “VL domain” are used interchangeably to refer to the variable region of the light chain of an antibody.

[0208] The terms “VH” and “VH domain” are used interchangeably to refer to the variable region of the heavy chain of an antibody.

[0209] The VH and VL regions can be further subdivided into highly degenerate regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: 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 regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

[0210] The term "Kabat numbering" and similar terms are recognized in the art and refer to a numbering system for amino acid residues in the variable regions of the heavy and light chains of an antibody or its antigen-binding fragment. In some respects, 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, CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31–35, which may optionally include one or two additional amino acids immediately following 35 (referred to as 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 within the antibody light chain molecule are typically located at amino acid positions 24–34 (CDR1), 50–56 (CDR2), and 89–97 (CDR3). In some respects, the CDRs of the antibodies described herein can be determined according to the Kabat numbering scheme.

[0211] The term "Chothia number" and similar terms are recognized in the art and refer to a numbering system for amino acid residues in the variable regions of the heavy and light chains of an antibody or its antigen-binding fragment. In some respects, the CDR can be determined according to the Chothia numbering system (see, for example, Chothia C & Lesk AM (1987) J Mol Biol 196:901-17 and Chothia C et al., (1989) Nature 342:877-83). The term "IMGT® number" and similar terms are recognized in the art and refer to a numbering system for amino acid residues in the variable regions of the heavy and light chains of an antibody or its antigen-binding fragment. In some respects, the CDR can be determined according to the IMGT® numbering system (see, for example, Lefranc MP, et al., (1997) Immunol Today 18:509 and Lefranc MP et al (2003) Dev Comp Immunol 27:55-77). In some respects, the CDR of the antibody described herein can be determined according to the IMGT® numbering scheme.

[0212] The terms “constant region” or “constant domain” as used herein are used interchangeably and have the meanings commonly found in the art. A constant region is an antibody portion, such as the carboxyl-terminal portion of the light chain and / or heavy chain, that does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors. The constant regions of immunoglobulin molecules generally have a more conserved amino acid sequence relative to the variable domains of immunoglobulins. In some respects, antibody or antigen-binding fragments contain a constant region or a portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity.

[0213] As used herein, the term "heavy chain" when referring to antibodies can refer to any distinct type, such as α, δ, ε, γ, and µ, based on constant-domain amino acid sequences that generate IgA, IgD, IgE, IgG, and IgM antibodies, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some respects, the heavy chain is the human heavy chain.

[0214] As used herein, the term "light chain" when referring to antibodies can refer to any distinctive type, such as κ or λ, based on the amino acid sequence of the constant domain. The amino acid sequence of a light chain is well known in the art. In some respects, the light chain is the human light chain.

[0215] The “Fc region” (crystallization fragment region), “Fc domain”, or “Fc” refers to the C-terminal region of the heavy chain of an antibody, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (such as effector cells) or binding to the first component (C1q) of the classical complement system.

[0216] A “natural sequence Fc region” or “natural sequence Fc” contains an amino acid sequence identical to that of an Fc region found in nature. Natural sequence human Fc regions include the natural sequence human IgG1 Fc region; the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as their natural variants. Natural sequence Fc includes various alleles of Fc (see, for example, Jefferis et al., (2009) mAbs 1:1; Vidarsson G. et al. Front Immunol. 5:520 (published online on 20 October 2014)).

[0217] "Fc receptors," or "FcRs," are receptors that bind to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternative splice 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 Fc receptors and elicits the strongest Fc effector function. It is considered equivalent to mouse IgG2a, in relation to the type of activating Fc receptor it binds to. Conversely, human IgG4 elicits the least Fc effector function (Vidarsson G. et al. FrontImmunol. 5:520 (published online on October 20, 2014)). "Fc effector function" refers to the interaction between the antibody Fc region and the Fc receptor or ligand, or the resulting biochemical events.

[0218] Constant regions can be manipulated, for example, using recombinant DNA technology, to eliminate one or more effector functions. An "effector function" refers to the interaction between the antibody's Fc region and an Fc receptor or ligand, or the resulting biochemical event. 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 downregulation of cell surface receptors (e.g., B cell receptors; BCR). Such effector functions typically require a combination of the Fc region and a binding domain (e.g., the antibody variable region). Therefore, a "constant region without Fc function" includes a constant region with reduced or no Fc-mediated one or more effector functions. Furthermore, constant regions can be manipulated, for example, using recombinant DNA technology, to increase antibody specificity, enhance antibody activity, increase antibody half-life, eliminate or reduce off-target effects, or any combination thereof. As used herein, the term "antibody half-life" refers to the time required for half the amount of antibody deposited in a living organism to be metabolized or eliminated by normal biological processes.

[0219] Effector function of antibodies can be reduced or avoided through various methods. Effector function can be reduced or avoided by using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single-chain Fv (scFv), or sdAb composed of monomeric VH or VL domains). Alternatively, so-called non-glycosylated antibodies can be generated by removing sugars linked to specific residues in the Fc region to reduce effector function while retaining other valuable properties of the Fc region (e.g., extended half-life and heterodimerization). Non-glycosylated antibodies can be generated, for example, by deleting or altering the residues to which the sugar is attached, enzymatically removing the sugar, generating the antibody in cultured cells in the presence of glycosylation inhibitors, or expressing the antibody in cells that cannot glycosylate proteins (e.g., bacterial host cells). See, for example, U.S. Publication No. 20120100140. Another approach is to utilize the Fc region from an IgG subclass with reduced effector function. For example, IgG2 and IgG4 antibodies are characterized by having 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 effector function of the antibody because it contains binding sites that largely overlap with the C1q (complement) and IgG-Fc receptor (FcγR) on effector cells of the innate immune system. (Vidarsson G. et al. Front Immunol. 5:520 (published online on October 20, 2014). Therefore, antibodies with reduced or no Fc effector function can be prepared, for example, by generating chimeric Fc regions containing a CH2 domain from an IgG4 type IgG antibody and a CH3 domain from an IgG1 type IgG antibody, or chimeric Fc regions containing a hinge region from IgG2 and a CH2 region from IgG4 (see, for example, Lau C. et al. J. Immunol. 191:4769-4777 (2013)), or mutated Fc regions that result in altered Fc effector function (e.g., reduced or no Fc function). Such mutated Fc regions are known in the art. See, for example, U.S. Publication No. 20120100140 and its cited U.S. and PCT applications, and An et al., mAbs 1:6, 572-579 (2009); the entire contents of which are incorporated herein by reference.

[0220] In some respects, antibodies (e.g., monoclonal antibodies) or their antigen-binding fragments are modified so that they do not bind to the Fc region. See, for example, Saunders K., Front. Immunol., 10:1296 (2019).

[0221] The terms "hinge," "hinge domain," "hinge region," or "antibody hinge region" are used interchangeably and refer to a domain that connects the CH1 domain to the CH2 domain within the heavy chain constant region, including 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 antibody binding region and the effector region, and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions.

[0222] As used in this article, “isotype” refers to the antibody class encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0223] In this article, the phrases “antibody that recognizes antigen” and “antibody that is specific to antigen” are used interchangeably with the term “antibody that specifically binds to antigen”.

[0224] As used herein, “isolated antibody” refers to an antibody that is substantially free of other antibodies with different antigenicity. However, isolated antibodies that specifically bind to epitopes of proteins can be cross-reactive with corresponding proteins of other species.

[0225] In some respects, antibodies (such as monoclonal antibodies) or their antigen-binding fragments are chimeric antibodies. The term "chimeric" antibody or its antigen-binding fragment refers to an antibody or antigen-binding fragment in which the amino acid sequence is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment derived from a mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and ability, while the constant regions are homologous to sequences in an antibody or antigen-binding fragment derived from another (usually human) species to avoid triggering an immune response in that species.

[0226] In some respects, antibodies (e.g., monoclonal antibodies) or their antigen-binding fragments are humanized antibodies. The term “humanized” antibody or its antigen-binding fragment refers to a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof of a non-human (e.g., mouse) antibody or antigen-binding fragment containing a minimal non-human (e.g., mouse) sequence. Typically, humanized antibodies or their antigen-binding fragments are human immunoglobulins 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) with the desired specificity, affinity, and ability (“CDR transplantation”) (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 aspects, humanized antibodies or their antigen-binding fragments may contain at least a portion of an immunoglobulin constant region or structural domain (Fc), typically the Fc of a human immunoglobulin. Methods for generating humanized antibodies are described in U.S. Patent 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 aspects, “humanized antibody” is a resurfacing antibody.

[0227] In some respects, antibodies (e.g., monoclonal antibodies) or their antigen-binding fragments are human antibodies. The term "human" antibody (HuMAb) or its antigen-binding fragment refers to an antibody or antigen-binding fragment having an amino acid sequence derived from a human immunoglobulin locus, wherein such an antibody or antigen-binding fragment is manufactured using any technique known in the art. The definition of a human antibody or antigen-binding fragment includes complete or full-length antibodies and fragments thereof.

[0228] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived from a polypeptide or two or more discontinuous regions of a polypeptide (conformal, nonlinear, discontinuous, or non-continuous epitopes). The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0229] As used herein, the term “fragment of its function” refers to a segment or portion of a protein (e.g., an immunomodulatory protein) that is still capable of performing one or more functions associated with the full-length protein (e.g., stimulating, regulating, modulating, or modifying an immune response).

[0230] As used herein, the term "inhibitor" refers to any agent that reduces the level and / or activity of a protein. Non-limiting examples of inhibitors include blocking antibodies, inhibitory proteins, dsRNAs (e.g., siRNA or shRNA), and vectors such as AAV containing a polynucleotide encoding a blocking antibody, inhibitory protein, or dsRNA (e.g., siRNA or shRNA). As used herein, the term "reduction" is used interchangeably with "silencing," "downregulation," "inhibition," and other similar terms, and includes any degree of reduction.

[0231] As used in this article, a “blocking antibody” is an antibody that does not react when combined with an antigen but prevents other antibodies from binding to that antigen.

[0232] An antibody that “blocks” or “inhibits” or “blocks” is an antibody that, when it binds to a target protein, reduces or inhibits (partially or completely) the binding of the target protein to one or more ligands and / or, when it binds to a target protein, reduces or inhibits (partially or completely) one or more activities or functions of the target protein.

[0233] As used herein, the term "signal peptide" refers to a polypeptide sequence or combination of sequences sufficient to mediate the transport of a polypeptide to the cell surface. Without being bound by any particular theory, the transport of a polypeptide to the cell surface can be mediated by a secretory pathway, including the transport of the polypeptide from the cytoplasm to the endoplasmic reticulum and subsequently to the cell membrane via the Golgi apparatus, wherein the protein may 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 "plaques," etc. Examples of signal peptides include, but are not limited to, endogenous signal peptides of HGH and its variants; endogenous signal peptides of interferons and their variants, including signal peptides of type I, II, and III interferons and their variants; and endogenous signal peptides of known cytokines and their variants, such as signal peptides of erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β and their variants. In some aspects, the signal peptide is the IL-2 signal peptide. In some aspects, the signal peptide is the IL-10 signal peptide. In some aspects, the signal peptide is a modified signal peptide.

[0234] As used herein, the phrase “contacting cells” (e.g., contacting cells with a construct, rAAV carrier, or pharmaceutical composition of this disclosure) includes contacting cells directly or indirectly. In some aspects, contacting cells with a construct, rAAV carrier, or pharmaceutical composition of this disclosure includes contacting cells with the construct, rAAV carrier, or pharmaceutical composition in vitro or in vivo. Thus, for example, the construct, rAAV carrier, or pharmaceutical composition may be physically contacted with cells by an individual performing the method, or alternatively, the construct, rAAV carrier, or pharmaceutical composition may be placed in a situation that would subsequently contact the cells.

[0235] In some respects, cell contact can be achieved in vitro, for example by culturing cells together with the construct or AAV vector.

[0236] In some respects, cells can be contacted in vivo, for example by injecting the construct, rAAV carrier, or pharmaceutical composition of this disclosure into or near the tissue in which the cells reside (e.g., the periorbital region), or by injecting the construct, rAAV carrier, or pharmaceutical composition into another region, such as blood or subcutaneous space, such that the agent subsequently reaches the tissue in which the cells to be contacted reside. For example, the AAV carrier may be encapsulated and / or conjugated with a ligand that guides the AAV carrier to the site of interest. Combinations of in vitro and in vivo contact methods are also possible. For example, cells can be contacted in vitro with the construct, rAAV carrier, or pharmaceutical composition and then transplanted into a subject.

[0237] In some aspects, contacting cells with the construct, rAAV carrier, or pharmaceutical composition of this disclosure includes “introducing” or “delivering” (directly or indirectly) the construct, rAAV carrier, or pharmaceutical composition into cells by promoting or achieving uptake or absorption into cells. In some aspects, introduction into cells includes methods known in the art, such as electroporation and lipid transfection.

[0238] As used herein, the term “in vitro” refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, in a petri dish, etc., rather than within an organism (such as an animal, plant, or microorganism).

[0239] As used in this article, the term "in vivo" refers to events that occur within an organism, such as an animal, plant, or microorganism or its cells or tissues.

[0240] As used herein, the term "transfection" refers to a method of introducing exogenous nucleic acids into cells via non-viral means. Transfection methods include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures. As used herein, the term "transduction" refers to a method of introducing exogenous nucleic acids into cells via viral means (i.e., via viral vectors). The list of reagents that can be transfected or transduced into cells is extensive and includes, for example, siRNA, shRNA, sense and / or antisense sequences, DNA encoding one or more proteins (e.g., therapeutic antibodies) and organized into expression plasmids, such as vectors.

[0241] As used in this article, "off-target" refers to any unintended effect on any one or more targets, genes, RNA transcripts, proteins, or signaling mechanisms.

[0242] As used herein, the term "subject" means any organism to which the compositions of this disclosure (e.g., the rAAV vectors of this disclosure) may be administered, for example, for experimental, diagnostic, preventative, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). Subjects may be persons or animals seeking or requiring treatment, receiving treatment, or in the future receiving treatment, or who are cared for by trained professionals for a particular disease, symptom, syndrome, or condition.

[0243] As used herein, the term "administration" means administering a therapeutic agent (such as the constructs, rAAV carriers, or pharmaceutical compositions disclosed herein) to a subject or system. Administering to animal subjects (e.g., humans) can be via any suitable route (e.g., periorbital injection).

[0244] As used herein, the terms “reapplication,” “repeated application,” and “re-application” refer to the administration of one or more additional doses of a therapeutic agent, such as the constructs, rAAV carriers, or pharmaceutical compositions disclosed herein, following an initial application.

[0245] As used herein, the terms “effective amount,” “therapeutic effective amount,” and “sufficient amount,” such as the constructs, rAAV carriers, or pharmaceutical compositions disclosed herein, refer to an amount sufficient to produce a beneficial or desired outcome (including clinical outcomes) when administered to subjects (including humans). Therefore, “effective amount” or its synonyms depend on the context in which they are used. In some respects, a therapeutically effective amount of an agent (such as the constructs, rAAV carriers, or pharmaceutical compositions disclosed herein) is an amount that produces a beneficial or desired outcome in subjects compared to a control.

[0246] The amount of a given reagent (e.g., the construct, rAAV vector, or pharmaceutical composition disclosed herein) will correspond to such an amount that will vary depending on various factors, such as the given reagent, pharmaceutical formulation, route of administration, type of disease or condition, subject (e.g., age, sex, and / or weight) or the identity of the host to be treated.

[0247] As used herein, the term "preventive effective amount" includes an amount of agent (e.g., the construct, rAAV carrier, or pharmaceutical composition disclosed herein) sufficient to prevent, reduce, or improve symptoms of or one or more symptoms of a disease, condition, syndrome, or condition when administered to a subject who has or is susceptible to such a disease, condition, syndrome, or condition (e.g., an ophthalmic disease, condition, syndrome, or condition). Improvement of a disease, condition, syndrome, or condition includes slowing the course of the disease, condition, syndrome, or condition or reducing the severity of a subsequently developed disease, condition, syndrome, or condition. "Preventive effective amount" can vary depending on the characteristics of the agent, such as the construct, rAAV carrier, or pharmaceutical composition disclosed herein, how the agent is administered, the degree of risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics.

[0248] As used herein, the terms “treat,” “treated,” and “treating” refer to both therapeutic and preventative measures aimed at preventing or reducing (alleviating) an undesirable physical condition, disease, symptom, syndrome, or condition, or at achieving a beneficial or desired clinical outcome. In some respects, treatment reduces or alleviates symptoms associated with a disease, symptom, syndrome, or condition. In other respects, treatment leads to a beneficial or desired clinical outcome. For example, treating an ophthalmic disease, symptom, syndrome, or condition may reduce or alleviate symptoms associated with the ophthalmic disease, symptom, syndrome, or condition, or reduce or alleviate the severity of at least one indicator of the ophthalmic disease, symptom, syndrome, or condition.

[0249] Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms; reduction in the severity of the disease, condition, syndrome, or condition; stabilization of the disease, condition, syndrome, or condition (i.e., no worsening); delay in the onset or slowing of the progression of the disease, condition, syndrome, or condition; improvement or relief of the state of the disease, condition, syndrome, or condition (whether partial or complete), whether detectable or not; improvement in at least one measurable physical parameter, not necessarily perceptible to the patient; or enhancement or improvement of the disease, condition, syndrome, or condition. In some respects, treatment includes eliciting a clinically significant response without producing excessive levels of side effects. In some respects, treatment includes prolonging survival compared to expected survival without treatment.

[0250] As used herein, the terms “amelioration” or “ameliorating” refer to a reduction in the severity of at least one indicator of a disease, symptom, syndrome, or condition.

[0251] As used in this article, the term “preventing” or “prevention” means delaying or postponing the onset, development, or progression of a disease, symptom, syndrome, or condition for a period of time, including weeks, months, or years.

[0252] As used herein, the term "pharmaceutical composition" means a composition comprising the compounds or molecules described herein (such as the rAAV carriers disclosed herein) and formulated with pharmaceutically acceptable excipients. In some respects, pharmaceutical compositions may be approved by government regulatory agencies as part of a treatment regimen for the treatment of diseases, symptoms, syndromes, or conditions in mammals.

[0253] As used herein, the term “pharmaceuticalally acceptable excipient” means any component other than the compounds described herein (e.g., a medium capable of suspending or dissolving the active compound) and having substantially non-toxic and non-inflammatory properties in patients.

[0254] The terms "therapeutic agent" or "therapeutic molecule" include compounds or molecules that, when present in an effective amount, produce a desired therapeutic, pharmacological, and / or physiological effect on a subject in need of them. It includes any compound, such as a small molecule drug or a biological product (e.g., a peptide drug or a nucleic acid drug), that, when administered to a subject, has a measurable or communicable effect on the subject, for example, it alleviates or reduces symptoms of a disease, symptom, syndrome, or condition. In some aspects, a therapeutic molecule is an rAAV carrier containing an expression cassette encoding a therapeutic antibody or a functional fragment thereof.

[0255] As used herein, the term "pathology" refers to any deviation from a state of health. As used herein, the term "pathology" should be understood to include "disease," "symptom," "syndrome," "condition," or any combination thereof.

[0256] As used in this article, the term "ocular" should be understood to mean the eye. Therefore, for example, "ocular pathology" refers to pathologies affecting the eye.

[0257] As used herein, the term “periocular” should be understood to mean the area surrounding the eye (e.g., within the orbit). Thus, for example, “periocular pathology” is a pathology affecting the area around the eye (e.g., within the orbit).

[0258] As used herein, the term “ophthalmology” should be understood to mean the eye, related to the eye, or located near the eye, including surrounding the eye (e.g., within the orbit). Therefore, the term ophthalmology should be understood to include both the ocular and periorbital regions. Thus, for example, “ophthalmic pathology” is a pathology affecting the eye and / or the periorbital region (e.g., within the orbit). Non-limiting examples of ophthalmic pathology include, but are not limited to, age-related macular degeneration, Behçet's disease, Bietti's crystalline dystrophy, blepharitis, blepharospasm, iris defects, corneal conditions, diabetic retinopathy, dry eye, glaucoma, idiopathic intracranial hypertension, macular edema, ocular histoplasmosis syndrome, conjunctivitis, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt's disease, thyroid eye disease (TED), Usher syndrome, and uveitis (e.g., non-infectious uveitis).

[0259] As used in this article, the term "intraocular" refers to a location located inside or through the eye.

[0260] As used in this article, the term "intraorbital" refers to a location within or through the orbit. The term "orbit" refers to the cavity in the skull that houses the eyes, muscles, glands, blood vessels, nerves, etc., all of which are related to the eyes.

[0261] As used in this article, the term "periorbital" refers to the location located around or surrounding the orbit (e.g., the eye tissues surrounding or lining the orbit).

[0262] As used in this article, the term "posterior orbit" refers to a location located behind or occurring behind the orbit.

[0263] As used in this article, the term "posterior to the eyeball" refers to a location located behind the eyeball or in the area or space behind the eyeball.

[0264] As used in this article, the term "posterior septum" refers to a location in the region between the inferior orbital septum and the inferior orbital rim, or in the region occurring between the inferior orbital septum and the inferior orbital rim.

[0265] As used herein, the term "immune response" refers to a biological response within an organism against a foreign agent or abnormal cell, wherein the response protects the organism from such agents / cells and the diseases they cause. An immune response is mediated by the action of immune system cells (such as T lymphocytes (T cells), B lymphocytes (B cells), natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the organism of invasive pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. In some aspects, an immune response includes, for example, the activation or inhibition of T cells (such as effector T cells or Th cells, such as CD4+ or CD8+ T cells), or the inhibition of regulatory T cells (Treg cells).

[0266] As used herein, the terms “immunomodulatory protein,” “immune modulator,” “immunoregulatory protein,” or “immunoregulator” refer to agents, such as components of signaling pathways, that can participate in stimulating, regulating, modulating, or modifying an immune response. “Modulating,” “regulating,” or “modifying” an immune response refers to any alteration in the activity of immune system cells or such cells (e.g., effector T cells). This regulation includes stimulation or suppression of the immune system, which can be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which can have an enhancing function in the tumor microenvironment. In some respects, immunomodulators are located on the surface of T cells. The term “immunomodulatory target” or “immunoregulatory target” refers to an immunomodulatory agent that is targeted and bound by a substance, agent, group, compound, or molecule, and whose activity is altered by that binding. Immunomodulatory targets can include, for example, receptors on the cell surface (“immunomodulatory receptors”) and receptor ligands (“immunomodulatory ligands”). In some respects, immunomodulatory proteins are cytokines. In other respects, immunomodulatory proteins are soluble natural killer (NK) cell, B lymphocyte, T lymphocyte, neutrophil, or macrophage ligands.

[0267] As used in this article, the term "cytokine" refers to one or more factors that exert an effect on cells, such as affecting growth or proliferation.

[0268] As used herein, the term "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., a polynucleotide containing a promoter operatively linked to a nucleic acid encoding a therapeutic molecule) into the cells and / or tissues of an individual to treat, reduce symptoms of, or reduce the likelihood of a pathology or disease. Gene therapy also includes the insertion of repressive transgenes, i.e., transgenes that suppress, reduce, or decrease the expression, activity, or function of endogenous genes or proteins (e.g., undesirable or abnormal (e.g., pathogenic) genes or proteins). Such transgenes can be exogenous. Exogenous molecules or sequences are understood to be molecules or sequences that are not normally present in the cells, tissues, and / or individuals to be treated. Acquired and congenital diseases are both suitable candidates for gene therapy.

[0269]

II. Application around the eyes

[0270] Some aspects of the present invention relate to the periocular delivery of therapeutic agents (e.g., rAAV disclosed herein).

[0271] In some aspects, periocular delivery (e.g., injection) is periocular delivery or retroseptal delivery. In some aspects, periocular delivery is subcapsular injection, retroocular injection, subconjunctival injection, or periocular injection. In some aspects, periocular delivery is retroocular delivery (e.g., injecting an AAV construct into a location located posterior to the eye). In some aspects, periocular delivery is periocular delivery. In some aspects, periocular delivery is retroseptal delivery (e.g., injecting an AAV construct into a location located between the inferior orbital septum and the inferior orbital rim).

[0272] In some respects, retroocular delivery is retroocular injection (see, for example...) Figure 1 In some respects, septal delivery is septal injection (see, for example...). Figure 2 ).

[0273] In some aspects, periorbital application includes injecting a volume of 0.1 mL to 5 mL of the composition (e.g., a pharmaceutical composition) per eye. In other aspects, periorbital application includes injecting a volume of 0.1 mL to 1 mL of the composition (e.g., a pharmaceutical composition) per eye.

[0274] In some aspects, administration includes a volume of 0.1 mL to 1.0 mL of the composition (e.g., a pharmaceutical composition) injected per eye periorbitally. In other aspects, administration includes a volume of 0.1 mL to 0.5 mL of the composition (e.g., a pharmaceutical composition) injected per eye periorbitally.

[0275] In some aspects, the administration includes a volume of about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, 0.9 mL, or about 1.0 mL per eye. In some aspects, the administration includes a volume of about 0.1 mL to 0.25 mL per eye. In some aspects, the administration includes a volume of 0.4 mL per eye.

[0276] In some respects, periocular application allows pharmaceutical compositions containing AAV carriers to contact orbital tissues or extraocular compartments, such as extraocular adipose tissue, levator lateralis muscle, lateral rectus muscle, medial lateral rectus muscle, superior lateral rectus muscle, inferior lateral rectus muscle, inferior lateral oblique muscle, superior lateral oblique muscle, optic nerve, blood vessels, sclera, dura mater sheath, lamina cribriformis, eyelids, lacrimal gland, lacrimal sac, tear film, or any combination thereof.

[0277] In some respects, the AAV carrier contact is selected from the following orbital tissues or extraocular chambers: extraocular adipose tissue, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, or any combination thereof.

[0278] Some aspects of the present invention relate to a method of delivering a pharmaceutical composition comprising an AAV carrier into orbital tissue or an extraocular cavity, the method comprising periocular injection of the AAV carrier, wherein the periocular tissue is selected from the following: extraocular adipose tissue, levator lateralis muscle, lateral rectus muscle, medial lateral rectus muscle, superior lateral rectus muscle, inferior lateral rectus muscle, inferior lateral oblique muscle, superior lateral oblique muscle, optic nerve, blood vessels, sclera, dura mater sheath, cribriform plate, eyelid, lacrimal gland, lacrimal sac, tear film, or any combination thereof.

[0279] In some respects, the orbital tissue or extraocular cavity is selected from the following: extraocular adipose tissue, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, or any combination thereof.

[0280] In some aspects, the method of the present invention provides an AAV vector transduction efficiency of at least 5% (e.g., 5%–95%, 10%–95%, 15%–95%, 20%–95%, 25%–95%, 30%–95%, 40%–95%, or 50%–95%) in target ocular or periocular cells. In some aspects, the AAV vector transduction efficiency is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% in target ocular or periocular cells. In some aspects, the target ocular or periocular cells are fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, epithelial cells, neuroepithelial cells, glial cells, or any combination thereof. In some aspects, the target cells are retroocular cells.

[0281] In some respects, AAV vector transduction is selected from the following cells: adipocytes, orbital fibroblasts, and extraocular muscles.

[0282] In some cases, administration involves periocular injection into the extraocular muscles. In some cases, the extraocular muscles are levator muscles, rectus muscles (e.g., lateral rectus, medial rectus, superior rectus, or inferior rectus), or oblique muscles (e.g., inferior oblique or superior oblique).

[0283] In some respects, administration includes periocular injection into the ocular or periocular cells. In some respects, the ocular or periocular cells are fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, epithelial cells, neuroepithelial cells, glial cells, or any combination thereof. In some respects, the periocular cells are retrobulbar cells.

[0284] In some aspects, after administration of the AAV carrier to a subject, the therapeutic agent (e.g., an antibody or its antigen-binding fragment) expressed in the subject's ocular muscles is at least about 18 µg / mL and / or the serum concentration of the therapeutic agent is less than about 6 µg / mL. In some aspects, the concentration in ocular tissue is about 18 µg / mL to about 180 µg / mL, about 18 µg / mL to about 100 µg / mL, about 18 µg / mL to about 60 µg / mL, or about 18 µg / mL to about 30 µg / mL. In some aspects, after administration of the AAV carrier, the serum concentration of the therapeutic agent is less than about 6 µg / mL (e.g., about 0.1 to about 5.9 µg / mL).

[0285] In some respects, periocular injection allows the expression of nucleic acids encoding therapeutic antibodies (e.g., anti-IGF-1R antibody, anti-IL6 antibody, anti-FcRn antibody, anti-TSHR antibody, anti-IL11 antibody, anti-IL6-R antibody, or anti-IL11R antibody) in cells. In some respects, the cells are fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, epithelial cells, neuroepithelial cells, glial cells, or any combination thereof. In some respects, the periocular cells are retrobulbar cells.

[0286] In some aspects, the AAV vector comprises a capsid of a serotype 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. In some aspects, the capsid serotype is AAV8 or AAV9. In some aspects, the capsid serotype is AAV9.

[0287] In some respects, an AAV vector comprises a capsid, a vector genome, and an expression cassette.

[0288] In some respects, the vector genome contains inverted terminal repeats (ITRs).

[0289] In some respects, the expression cassette contains nucleic acid sequences encoding therapeutic agents for treating ophthalmic pathologies.

[0290] In some respects, ophthalmic pathology is the study of diseases within or around the eye.

[0291] In some respects, ophthalmic pathology is selected from the group consisting of age-related macular degeneration, Behçet's disease, Bietti's crystalline dystrophy, blepharitis, blepharospasm, iris defects, corneal conditions, diabetic retinopathy, dry eye, glaucoma, idiopathic intracranial hypertension, macular edema, ocular histoplasmosis syndrome, conjunctivitis, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt's disease, thyroid ophthalmopathy (TED), Usher syndrome, uveitis, or any combination thereof.

[0292] In some respects, ophthalmic pathology is thyroid eye disease (TED).

[0293] In some cases, periorbital application includes injecting a single dose.

[0294] In some cases, periorbital application involves injecting multiple doses.

[0295] In some respects, each dose in the multi-dose regimen is administered to the subject at a single injection site.

[0296] In some respects, each dose in the multi-dose regimen is administered to the subject via multiple injection sites.

[0297] In some respects, administration (e.g., periocular injection) is a single dose to the eye (e.g., unilateral administration). In some respects, a single dose is administered at a single injection site. In some respects, a single dose is administered at multiple injection sites.

[0298] In some respects, administration (e.g., periocular injection) is multiple-dose administration to the eye (e.g., unilateral administration). In some respects, the doses in multiple doses are administered at a single injection site. In some respects, each dose in multiple doses is administered at a different injection site.

[0299] In some respects, the application is bilateral, that is, applied to both eyes (e.g., periocular injection in each eye).

[0300] In some respects, the subjects were human.

[0301] In some cases, the vector genome (vg) was administered to the subjects at a rate of approximately 6E11 to approximately 2E13 per eye. In some cases, it was administered to the subjects at a rate of approximately 1E12 to approximately 2E13 per eye. In some cases, it was administered to the subjects at a rate of approximately 3E12 to approximately 1.8E13 per eye. In some cases, it was administered to the subjects at a rate of approximately 5E12 to approximately 1E13 per eye. In some cases, it was administered to the subjects at a rate of approximately 6E12 to approximately 9E12 per eye.

[0302] In some respects, the genome of any vector described herein is administered to human subjects in approximately 1E11, 2E11, 3E11, 4E11, 5E11, 6E11, 7E11, 8E11, 9E11, 1E12, 2E12, 3E12, 4E12, 5E12, 6E12, 7E12, 8E12, 9E12, 1E13, 1.1E13, 1.2E13, 1.3E13, 1.4E13, or 1.5E13 doses per eye (i.e., vg / eye).

[0303] In some respects, human subjects were administered approximately 6E12 to approximately 9E12 of any of the vector genomes described herein per eye.

[0304] In some respects, approximately 6E12 of any vector genomes described herein were administered to human subjects per eye.

[0305] In some respects, approximately 9E12 of any vector genomes described herein were administered to human subjects per eye.

[0306] In some aspects, the vector genome contains a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof. In some aspects, the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-IGF-1R antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-11 antibody, anti-IL-11R antibody, anti-FcRn antibody, anti-TSHR antibody, or an antigen-binding fragment thereof. In some aspects, the antibody or antigen-binding fragment thereof is an anti-IGF-1R antibody. In some aspects, the anti-IGF-1R antibody is a teprotumumab antibody. In some aspects, the vector genome contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 53. In some aspects, the vector genome contains a nucleotide sequence corresponding to SEQ ID NO: 53. In some aspects, the vector genome contains a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 142. In some aspects, the vector genome contains a nucleotide sequence corresponding to SEQ ID NO: 142.

[0307] In some cases, periorbital injections are subcapsular, retrobulbar, subconjunctival, or periorbital injections.

[0308] In some respects, periorbital injection is peri-ocular injection.

[0309] [Apply behind the eyeball]

[0310] Some aspects of the present invention relate to retroocular delivery of therapeutic agents.

[0311] In some respects, retroocular delivery is retroocular injection (see, for example...) Figure 1 ).

[0312] In some respects, retroocular injection involves (i) advancing the needle along the inferior temporal wall of the orbit (e.g., with the needle roughly parallel to the orbital floor at an angle of 10–15 degrees), penetrating the orbital septum about 1 cm, usually feeling a “pop”, then reorienting the needle 30–45 degrees upwards towards the nose and advancing it 2.5–3.5 cm until reaching the intraorbital muscle cone space (e.g., aao.org / education / basic-skills / orbital-block-technique).

[0313] In some aspects, retroocular injection involves (i) advancing the needle along the inferior temporal wall of the orbit (e.g., with the needle generally parallel to the orbital floor and angled downwards), (ii) redirecting the needle upwards and nasally toward the orbital apex, and (iii) advancing the needle until it penetrates the muscle cone. In some aspects, advancing the needle along the inferior temporal wall of the orbit involves passing the needle through the junction of the middle and outer thirds of the inferior orbital rim and under the eye.

[0314] In some cases, a straight or curved needle is used for retroocular injection. In others, the injection is advanced to a depth of approximately 30 mm to 35 mm, or to the area behind the eyeball.

[0315] In some cases, retroocular injection delivers a volume of approximately 5 mL to approximately 10 mL.

[0316] In some cases, the injection is administered retroocularly to the lateral aspect of the extraocular muscle cone.

[0317] [Subcapsular application]

[0318] Some aspects of the present invention relate to the subcapsular delivery of therapeutic agents to the ocular fascia (e.g., the application of an AAV construct to a location between the capsule and the sclera, also known as the extrascleral space).

[0319] In some respects, subcapsular delivery is subcapsular injection.

[0320] In some cases, a small conjunctival and ocular fascia tent is raised approximately 5–10 mm from the inferior nasal limbus using a pair of blunt, non-toothed forceps, and can extend as far as the fornix, depending on the patient's anatomy. A small incision is made in the tissue using a pair of ophthalmic scissors to expose the underlying sclera. A blunt-tipped sub-Tenonanaesthesia catheter, attached to an AAV construct syringe, is then inserted through the newly created defect and passed posteriorly, following the curvature of the eyeball, until its tip is considered to have crossed the equator—ensuring the catheter is inserted into the sub-Tenonanaesthesia space (only outside the bare sclera) rather than into the subconjunctival space; the AAV construct is then injected. See, for example, Amin S, et al., A new technique for delivering sub-Tenonanaesthesia in ophthalmic surgery. Br J Ophthalmol; 2001; 86:114-123.

[0321] Subconjunctival administration

[0322] Some aspects of this disclosure relate to subconjunctival delivery of therapeutic agents (e.g., application of an AAV construct to a location between the conjunctiva and sclera). See, for example, Sue Stevens, Community Eye Health. 2009 March; 22(69):15.

[0323] In some respects, subconjunctival delivery is subconjunctival injection.

[0324] [Apply around the eyes]

[0325] Some aspects of this disclosure relate to the periorbital delivery of therapeutic agents (e.g., administration of an AAV construct to a location around the orbital equator). See, for example, Alhassan MB, Kyari F, Ejere HOD. Peribulbar versus retrobulbar anaesthesia for cataract surgery. Cochrane Database of Systematic Reviews 2015, Issue 7. Art. No.: CD004083.

[0326] In some respects, periorbital delivery is septal injection (see, for example...) Figure 2 In some cases, periocular injections involve advancing the needle until it penetrates the orbital septum.

[0327] In some cases, periocular injections use a 25-gauge needle advanced to a depth of approximately 20 mm to approximately 25 mm, or close to the ocular equator. In other cases, the needle is advanced more than 25 mm, passing through the ocular equator. In some cases, periocular injections deliver a volume of approximately 5 mL to approximately 10 mL.

[0328] During periocular injections, the needle is inserted into the extraconical space of the extraocular muscles to avoid the risk of damage to major structures in the intraconical space. Periocular injections are typically performed in the lower space, with additional upper injections administered if necessary.

[0329] In 1986, Bloomberg et al. first introduced periorbital injection as a safer and more effective alternative to retroocular administration. This technique is often used to deliver lidocaine or benzodiazepines before ophthalmic surgery (especially cataract surgery) to provide ocular motility and anesthesia (Bloomberg, LB, J Cataract Refract Surg 12(6):677-679 (1986), Alhassan, MB, et al., Cochrane Database Syst Rev 2015(7):CD004083 (2015), Pucchio, A., et al., Int Ophthalmol 43(5): 1761-1769 (2023)). For deeper extraocular drug penetration, periocular injection has been used to treat ocular diseases such as uveitis, cystoid macular edema and age-related macular degeneration (Chew, EY, et al., Retina 31(2): 284-289(2011), Sodhi, G., et al., Ophthalmology Management 22(August 2018): 30-38(2018)).

[0330] Periorbital injection of corticosteroids has been explored in TED patients and improvements in exophthalmos, inflammation, diplopia and eyelid retraction have been observed without the systemic side effects of oral corticosteroids, such as weight gain, hyperglycemia and hypertension (Bartalena, L., et al., Baillieres Clin Endocrinol Metab11(3):521-536(1997), Ebner, R., et al., BrJ Ophthalmol 88(11):1380-1386(2004), Bordaberry, M., et al., Acta Ophthalmol 87(1):58-64(2009), Alkawas, AA, et al., Clin ExpOphthalmol38(7):692-697(2010), Wang, Y., et al., ExpTher Med 20(3):2031-2038(2020)).

[0331] Periorbital procedure-related events are rare. This is mainly due to the needle being placed in the extra-cone space of the extraocular muscles rather than the deeper intra-cone space (i.e., retroocular injection). The major complication rate of periorbital block in patients undergoing ophthalmic surgery has been reported to be 0.006% (Kazancioglu, L., et al., Turk J Anaesthesiol Reanim45(4): 231-233 (2017)). The risk of bleeding due to vascular puncture is extremely low (0.74% in 16,224 procedures (Davis, DB, et al., J Cataract Refract Surg 20(3): 327-337 (1994)) and no cases in 2,684 procedures (Arnold, PN, et al., J Cataract Refract Surg 18(2): 157-161 (1992)). In a case study of 16,224 periorbital surgeries, other potential risks (such as ocular perforation, drainage hemorrhage, and grand mal seizures) were rare (0.006%, 0.013%, and 0.006%, respectively); no patients experienced cardiac or respiratory depression (Davis 1994).

[0332] In some respects, administration of the disclosed AAV constructs (e.g., AAV9.smCBA.tetumumab or AAV9.CAG.tetumumab) via the periorbital route concentrates exposure at the disease site, namely the extraocular muscles and / or enlarged adipose tissue. In some respects, local delivery restricts IGF1R inhibition to relevant disease cells in the orbit and minimizes systemic exposure, thereby mitigating the off-target toxicities observed with intravenous administration of Tepezza. In some respects, local transduction of adipose tissue in the periorbital space can serve as an additional safety margin, as the tissue can be removed should overexpression or tolerance issues arise, since fat reduction surgery is a routine procedure performed in TED patients.

[0333] In some respects, AAV constructs (e.g., AAV9.smCBA.tetumumab or AAV9.CAG.tetumumab) will be administered as bilateral injections (both eyes). Although bilateral TED can present asymmetrically, true unilateral patients are rare, and patients initially diagnosed with clinically unilateral disease often develop bilateral disease (Strianese, D., et al., BMC Ophthalmol 13: 21 (2013)). In addition, clinical evaluation using computed tomography has shown that 90% of unilateral patients have subclinically bilateral disease (Strianese 2013). For patients with bilateral TED, unilateral intervention may increase ocular discomfort and diplopia due to normalization of the treated eye, while the contralateral eye remains protruding, similar to unilateral orbital myopathy, requiring acute surgical intervention (Kang, MS, et al., J clin Med 9(4) (2020)). In some cases, AAV constructs (such as AAV9.smCBA.tetumumab or AAV9.CAG.tetumumab) are administered as bilateral periorbital injections to TED patients to balance orbital exposure.

[0334] The safety of intraocular gene therapy can be assessed unilaterally to protect vision because multiple barriers within the intraocular space minimize contralateral and systemic exposure; however, the AAV immune response precludes subsequent treatment of the contralateral eye. Since periocular injection occurs outside the typical barriers of intraocular drug delivery (i.e., the sclera, orbital septum, and blood-eye barrier), serum exposure is higher for this route than for intravitreal injection, but lower than for oral administration (Weijtens, O., et al., Am J Ophthalmol 123(3): 358-363 (1997)). In some respects, AAV constructs (e.g., AAV9.smCBA.tetumumab or AAV9.CAG.tetumumab) are administered as unilateral periocular injections to TED patients (e.g., Ted patients with unilateral disease).

[0335] [Apply after interval]

[0336] Some aspects of this disclosure relate to the septal delivery of therapeutic agents (e.g., the application of an AAV construct to a location located or occurring between the inferior orbital septum and the inferior orbital rim).

[0337] In some respects, post-septal delivery is post-septal injection (see, for example) Figure 2 In some respects, post-septal injection involves advancing the needle until it penetrates the orbital septum.

[0338] In some respects, retroseptal injection is a periocular injection limited to the area anterior to the equator of the eye. The septum is a tissue pad found above and below the eye, and the retroseptal injection passes just through the septum. In some respects, the retroseptal injection penetrates the septum by approximately 10 mm to approximately 15 mm.

[0339] III. Ophthalmic Pathology

[0340] Some aspects of this disclosure relate to periocular administration to subjects with ophthalmic pathology.

[0341] Some aspects of the present invention relate to treating subjects suffering from ophthalmic pathology, said ophthalmic pathology including periocular administration of a therapeutic agent for treating the ophthalmic pathology.

[0342] In some respects, ophthalmic pathology is selected from the group consisting of age-related macular degeneration, Behçet's disease, Bietti's crystalline dystrophy, blepharitis, blepharospasm, iris defects, corneal conditions, diabetic retinopathy, dry eye, glaucoma, idiopathic intracranial hypertension, macular edema, ocular histoplasmosis syndrome, conjunctivitis, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt's disease, Usher's syndrome, thyroid eye disease (TED), uveitis, or any combination thereof.

[0343] In some respects, ophthalmic pathology is thyroid eye disease (abbreviated as TED), also known as Graves' eye disease.

[0344] Thyroid eye disease (TED) is an autoimmune disease that affects the muscles and fat behind the eye. TED is sometimes referred to as Graves' eye disease, Graves' orbital disease, Graves' eye, thyroid-associated orbital disease (TAO), and thyroid eye disease.

[0345] Graves' orbital disease (GO) refers to the active or chronic phase of an autoimmune orbital inflammatory thyroid-related disease. "Active" or "dynamic" GO can last from about 6 to 24 months and may be followed by "inactive" or "chronic" GO.

[0346] Some aspects of the present invention relate to a method of treating ophthalmic pathology (e.g., thyroid ophthalmopathy) in a subject with such need, the method comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, wherein the administration is a periorbital injection as described herein.

[0347] IV. Therapeutic Antibodies

[0348] This invention provides polynucleotides (e.g., antibody expression cassettes), vectors, and rAAV particles for delivering and expressing therapeutic agents in cells or subjects. In some aspects, the therapeutic agent is a therapeutic protein. In other aspects, the therapeutic agent is a therapeutic antibody.

[0349] In some respects, antibodies are selected from the group consisting of monoclonal antibodies, bispecific antibodies, nanobodies, and multispecific antibodies.

[0350] In some respects, antibodies are monoclonal antibodies.

[0351] In some respects, therapeutic proteins are antibody fragments selected from Fab, Fab', F(ab')2, Fv fragments, linear antibodies, or single-chain antibodies (e.g., nanobodies).

[0352] In some aspects, the antibody expression cassettes disclosed herein contain nucleic acid sequences encoding heavy chains (HC) and / or light chains (LC). In other aspects, the antibody expression cassettes disclosed herein contain nucleic acid sequences encoding variable heavy chains (VH) and / or variable light chains (VL).

[0353] In some respects, antibodies (e.g., monoclonal antibodies) or their antigen-binding fragments are chimeric antibodies.

[0354] In some respects, antibodies (e.g., monoclonal antibodies) or their antigen-binding fragments are humanized antibodies.

[0355] In some aspects, an antibody (e.g., a monoclonal antibody) or its antigen-binding fragment is a human antibody. In some aspects, a human antibody is a fully human antibody containing an amino acid sequence derived from a human immunoglobulin locus and not containing an amino acid sequence derived from a non-human immunoglobulin locus. In some aspects, a human antibody is a partially human antibody containing both an amino acid sequence derived from a human immunoglobulin locus and an amino acid sequence derived from a non-human immunoglobulin locus. 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 having approximately equal amounts of an amino acid sequence derived from a human immunoglobulin locus and an amino acid sequence derived from a non-human immunoglobulin locus.

[0356] In some respects, therapeutic antibodies are selected from the group consisting of anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-6R, anti-IL-2, anti-IL-17A, anti-TNF-α, anti-CD28 antibody, anti-IL-11, anti-IL-11R, anti-FcRn, anti-thyroid-stimulating hormone receptor (anti-TSHR) or their antigen-binding fragments.

[0357] In some respects, therapeutic antibodies are selected from the group consisting of anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-2, anti-IL-17A, anti-TNF-α, anti-CD28 antibodies, or their antigen-binding fragments. In other respects, therapeutic antibodies are selected from the group consisting of tocilizumab, teprotumumab, bevacizumab, ranibizumab, faricimab, infliximab, daclizumab, secukinumab, or their antigen-binding fragments.

[0358] In some respects, therapeutic antibodies are anti-IGF-1R antibodies, or their antigen-binding fragments. Some examples of anti-IGF-1R antibodies include teprotumumab, VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, and rhuMab. IGFR, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H1 5. L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H2 8. 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 fragments, variants or derivatives thereof.

[0359] In some aspects, the anti-IGF-1R antibody is VRDN-01100, VRDN-02700, or teprotumumab, or a fragment, variant, or derivative thereof. In some aspects, the anti-IGFR antibody comprises SEQ ID NO: 78 (corresponding to VRDN-01100). In some aspects, the anti-IGFR antibody comprises SEQ ID NO: 81 (corresponding to VRDN-002700).

[0360] In some respects, therapeutic antibodies are anti-IL-6 antibodies, or their antigen-binding fragments.

[0361] In some respects, anti-IL-6 antibodies are selected from the group consisting of tocilizumab, cetuximab, olokizumab, clazakizumab, sirukumab, levilimab, TOUR006, or fragments, variants, or derivatives thereof.

[0362] In some respects, anti-IL-6R antibodies are selected from the group consisting of sarilumab, satralizumab, or fragments, variants, or derivatives thereof.

[0363] In some respects, therapeutic antibodies are anti-IL-11 antibodies or their antigen-binding fragments.

[0364] In some respects, anti-IL-11 antibodies are selected from the group consisting of 9MW3811 or fragments, variants or derivatives thereof.

[0365] In some respects, anti-IL-11R antibodies are selected from the group consisting of BI 765423, LASN01, or fragments, variants, or derivatives thereof.

[0366] In some respects, therapeutic antibodies are anti-FcRn antibodies, or their antigen-binding fragments.

[0367] In some respects, anti-FcRn antibodies are selected from the group consisting of batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, IMVT-1402, or fragments, variants, or derivatives thereof.

[0368] In some respects, therapeutic antibodies are anti-TSHR antibodies or their antigen-binding fragments.

[0369] In some respects, the anti-TSHR antibody is K1-70.

[0370] In some aspects, the antibody expression cassette comprises a nucleic acid encoding a signal peptide operatively linked to a nucleic acid encoding an antibody or an antigen-binding fragment thereof. In some aspects, the signal peptide is an endogenous signal peptide of HGH and its variants; an endogenous signal peptide of interferon and its variants, including signal peptides of type I, type II, and type III interferon and their variants; or an endogenous signal peptide of known cytokines and their variants, such as signal peptides of erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β and their variants. In some aspects, the signal peptide is a modified signal peptide. In some aspects, the signal peptide is an IL-2 signal peptide or 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 with SEQ ID NO: 45 or 51.

[0371] In some aspects, compositions comprising a delivery vector (e.g., a viral vector) containing a nucleic acid encoding an immunoglobulin disclosed herein (e.g., an anti-IGF-1R antibody) are suitable for delivery to a subject in need via periocular injection.

[0372] In some aspects, antibody expression cassettes containing nucleic acid sequences encoding therapeutic antibodies (e.g., anti-IGF-1R antibodies) can be packaged in viral vectors (e.g., AAV vectors) disclosed herein, wherein the nucleic acid sequence encoding a therapeutic antibody (e.g., anti-IGF-1R antibody) is operatively linked to a promoter. In some aspects, the promoter can drive the expression of therapeutic antibodies (e.g., anti-IGF-1R antibodies) in host cells (e.g., epithelial cells, neuroepithelial cells, fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, or any combination thereof). In some aspects, polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions containing nucleic acids encoding therapeutic antibodies (e.g., anti-IGF-1R antibodies), can be administered via periocular injection.

[0373] In some respects, polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions containing nucleic acids encoding therapeutic antibodies (e.g., anti-IGF-1R antibodies), can be administered to connective tissue via periocular injection.

[0374] In some respects, polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions containing nucleic acids encoding therapeutic antibodies (e.g., anti-IGF-1R antibodies), can be administered to adipose tissue via periocular injection.

[0375] In some aspects, a polynucleotide (e.g., antibody expression cassette), vector, rAAV particles, or composition containing a nucleic acid encoding a therapeutic antibody (e.g., an anti-IGF-1R antibody), can be administered to the orbital tissue or extraocular cavity via periocular injection. In some aspects, the orbital tissue or extraocular cavity is selected from the group consisting of: extraocular adipose tissue, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, optic nerve, blood vessels, sclera, dura mater sheath, lamina cribriformis, eyelids, lacrimal gland, lacrimal sac, tear film, or any combination thereof. In some aspects, the periocular tissue is retrobulbar tissue.

[0376] In some respects, the orbital tissue or extraocular cavity is selected from the group consisting of: extraocular adipose tissue, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, or any combination thereof.

[0377] In some respects, polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions containing nucleic acids encoding therapeutic antibodies (e.g., anti-IGF-1R antibodies) can be administered via periocular injection into the extraocular muscles and / or fat in the orbital space. In some respects, the extraocular muscles are levator muscles, rectus muscles (e.g., lateral, medial, superior, or inferior rectus muscles), or oblique muscles (e.g., inferior or superior oblique muscles).

[0378] In some respects, polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising nucleic acids encoding therapeutic antibodies (e.g., anti-IGF-1R antibodies) can be administered to the eye or periocular cells via periocular injection. In some respects, the eye or periocular cells are fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, epithelial cells, neuroepithelial cells, glial cells, lymphocytes, or any combination thereof. In some respects, the periocular cells are retrobulbar cells.

[0379] In some respects, polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions containing nucleic acids encoding therapeutic antibodies (e.g., anti-IGF-1R antibodies) are expressed in cells. In some respects, the cells are fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, epithelial cells, neuroepithelial cells, glial cells, or any combination thereof. In some respects, the periocular cells are retrobulbar cells.

[0380] In some respects, subjects are administered via periocular injection a polynucleotide (e.g., antibody expression cassette), vector, rAAV particle, or composition containing a nucleic acid encoding a therapeutic antibody (e.g., an anti-IGF-1R antibody).

[0381] [IV.A.1. Anti-IGF-1R antibody]

[0382] Some aspects of the present invention relate to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising an antibody (e.g., a monoclonal antibody) encoding a nucleic acid that specifically binds to the insulin-like growth factor 1 receptor (IGF-1R) (e.g., human IGF-1R) and its antigen-binding fragment. In some aspects, the encoded anti-IGF-1R antibody is an anti-insulin-like growth factor-1 receptor (anti-IGF-1R) antibody. In some respects, the encoded anti-IGF-1R antibodies include teprotumumab, VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, 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, L16H 16. L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30 The amino acid sequence of 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 lonigutamab or a fragment, variant or derivative thereof.

[0383] In some respects, the encoded anti-IGF-1R antibody contains the amino acid sequence of VRDN-01100, or the amino acid sequence of VRDN-02700, or fragments, variants or derivatives thereof.

[0384] In some aspects, the encoded anti-IGF-1R antibody comprises the amino acid sequence of SEQ ID NO: 78 (corresponding to VRDN-01100). In some aspects, the anti-IGFR antibody comprises SEQ ID NO: 81 (corresponding to VRDN-002700).

[0385] In some respects, the encoded anti-IGF-1R antibody contains the amino acid sequence of teprotumumab, or fragments, variants or derivatives thereof.

[0386] In some respects, the encoded anti-IGF-1R antibody contains the amino acid sequence of lonigutamab, or fragments, variants or derivatives thereof.

[0387] In some aspects, the present invention relates to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions thereof, said polynucleotides, vectors, rAAV particles, or compositions comprising a promoter operatively linked to a nucleic acid encoding an immunoglobulin (e.g., an antibody that binds to IGF-1R or an antigen-binding fragment thereof).

[0388] In some aspects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions of the present invention used in the methods disclosed herein encode antibodies (e.g., monoclonal antibodies or antigen-binding fragments thereof) having a CDR and / or variable region sequence of teprotumumab or having at least 80% identity with its variable region or CDR sequence (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). In some aspects, the gene therapy construct encoding an anti-IGF-1R antibody (e.g., teprotumumab) is a polycistronic (e.g., bicistronic) construct (e.g., comprising a heavy chain and a light chain). In some aspects, the polycistronic (e.g., bicistronic) construct also comprises an F2A or IRES element.

[0389] In some aspects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions disclosed herein comprise nucleic acids encoding antibodies comprising the heavy and light chains of teprotumumab or an antigen-binding fragment thereof. In some aspects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions disclosed herein comprise nucleic acid sequences modified relative to the coding sequence of wild-type (unmodified) teprotumumab.

[0390] In some aspects, the anti-IGF-1R antibody comprises a heavy chain and a light chain. In some aspects, the nucleic acid sequence encoding the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 40–42 (or the HC amino acid sequences disclosed in Table 11). In some aspects, the encoded HC comprises SEQ ID NO: 43 or 85 (or the HC amino acid sequences in Table 12). In some aspects, the nucleic acid sequence encoding the light chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 46–48 (or the LC coding sequences disclosed in Table 13). In some respects, the encoded LC contains SEQ ID NO: 49 or 90 (or the LC amino acid sequence in Table 14).

[0391] In some aspects, the nucleic acid sequence encoding the heavy chain variable region comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 24–26 (or the VH coding sequences disclosed in Table 5). In some aspects, the encoded VH comprises any one of SEQ ID NO: 27, 28, or 86 (or any VH amino acid sequence in Table 6). In some aspects, the nucleic acid sequence encoding the light chain variable region comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 29–31 (or the VL coding sequences disclosed in Table 7). In some respects, the encoded VL contains any one of SEQ ID NO: 32, 33 or 91 (or any one of the VL amino acid sequences in Table 8).

[0392] In some aspects, the anti-IGF-1R antibody comprises a heavy chain and a light chain. In some aspects, the nucleic acid sequence encoding the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 40–42 (or the HC amino acid sequence disclosed in Table 11). In some aspects, the encoded HC comprises SEQ ID NO: 43 (or the HC amino acid sequence in Table 12). In some aspects, the nucleic acid sequence encoding the light chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 46–48 (or the LC coding sequences disclosed in Table 13). In some respects, the encoded LC contains SEQ ID NO: 49 (or the LC amino acid sequence in Table 14).

[0393] In some aspects, the nucleic acid sequence encoding the heavy chain variable region comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 24–26 (or the VH coding sequences disclosed in Table 5). In some aspects, the encoded VH comprises SEQ ID NO: 27 or SEQ ID NO: 28 (or any one of the VH amino acid sequences in Table 6). In some aspects, the nucleic acid sequence encoding the light chain variable region comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 29–31 (or the VL coding sequences disclosed in Table 7). In some respects, the encoded VL contains SEQ ID NO: 32 or SEQ ID NO: 33 (or any VL amino acid sequence in Table 8).

[0394] In some aspects, the heavy chain includes a heavy chain variable region (VH), which includes complementarity-determining regions (CDRs) 1, VH CDR 2, and VH CDR 3. In some aspects, VH CDRs 1–3 correspond to the CDRs of teprotumumab. In some respects, the nucleic acid sequence encoding VH CDR1 contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to SEQ ID NO: 9 or 12 (or the VH CDR1 coding sequences disclosed in Table 3); the nucleic acid sequence encoding VH CDR2 contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to SEQ ID NO: 10, 13, or 15 (or the VHCDR2 coding sequences disclosed in Table 3); and the nucleic acid sequence encoding VH CDR3 contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to SEQ ID NO: 11, 14, or 16 (or the VH CDR2 coding sequences disclosed in Table 3). The CDR3 coding sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the original nucleotide sequence.

[0395] In some aspects, the light chain includes a light chain variable region (VL), which includes complementarity-determining regions (CDR) 1, VL CDR 2, and VL CDR 3. In some aspects, VL CDR 1–3 correspond to the CDRs of teprotumumab. In some respects, the nucleic acid sequence encoding 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 with SEQ ID NO: 17 (or the VL CDR1 coding sequence disclosed in Table 4); the nucleic acid sequence encoding 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 with SEQ ID NO: 18, 20, or 22 (or the VL CDR2 coding sequence disclosed in Table 4); and the nucleic acid sequence encoding VL 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 with SEQ ID NO: 19, 21, or 23 (or the VL CDR1 coding sequence disclosed in Table 4). The CDR3 coding sequence has a nucleotide sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0396] In some aspects, the polynucleotides disclosed herein encode single-domain antibodies (e.g., nanobodies) comprising (i) a heavy chain variable region (VH) including complementarity-determining regions (CDRs) 1, VH CDR2, and / or VH CDR3, or (ii) a light chain variable region (VL) including CDR1, VL CDR2, and / or VL CDR3. In some aspects, the encoded VH CDRs and / or VL CDRs are selected from the corresponding CDRs of teprotumumab.

[0397] In some respects, this article discloses polynucleotides encoding teprotumumab, VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, 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, L16H 16. L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30 The amino acid sequence of H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, L52H52, or a fragment, variant, or derivative thereof.

[0398] In some respects, the polynucleotides disclosed herein encode amino acid sequences of VRDN-01100 or VRDN-02700 or fragments, variants or derivatives thereof.

[0399] In some respects, the polynucleotide disclosed herein encodes the amino acid sequence of SEQ ID NO: 78 (corresponding to VRDN-01100). In some respects, the anti-IGFR antibody comprises SEQ ID NO: 81 (corresponding to VRDN-002700).

[0400] In some respects, the polynucleotides disclosed herein encode the amino acid sequences of teprotumumab or fragments, variants or derivatives thereof.

[0401] In some aspects, the polynucleotides disclosed herein encode antibodies or antigen-binding fragments thereof comprising 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 VLCDRs 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 three VH CDRs as listed in Table 3 and nucleic acids encoding three VL CDRs as listed in Table 4).

[0402] [Table 1. Amino acid sequence of the variable heavy chain CDR (VH CDR) of the anti-IGF-1R antibody]

[0403]

[0404] [Table 2. Amino acid sequence of the variable light chain CDR (VL CDR) of the anti-IGF-1R antibody]

[0405]

[0406] Table 3. Nucleic acid sequence of anti-IGF-1R antibody variable heavy chain CDR (VH CDR)

[0407]

[0408] [Table 4. Nucleic acid sequence of anti-IGF-1R antibody variable light chain CDR (VL CDR)]

[0409]

[0410] In some respects, the polynucleotides disclosed herein comprise the nucleic acid sequences listed in Table 5. In some respects, the polynucleotides disclosed herein encode the antibody variable heavy chain (VH) sequences or antigen-binding fragments thereof listed in Table 6.

[0411] Table 5. Variable Heavy Chain (VH) Nucleic Acid Sequence of Anti-IGF-1R Antibody

[0412]

[0413] Table 6. Amino acid sequence of the variable heavy chain (VH) of the anti-IGF-1R antibody.

[0414]

[0415] In some respects, the polynucleotides disclosed herein comprise the nucleic acid sequences listed in Table 7. In some respects, the polynucleotides disclosed herein encode antibody variable light chains (VLs) comprising the sequences listed in Table 8 or antigen-binding fragments thereof.

[0416] Table 7. Variable Light Chain (VL) Nucleic Acid Sequence of Anti-IGF-1R Antibody

[0417]

[0418] Table 8. Amino acid sequence of the variable light chain (VL) of the anti-IGF-1R antibody.

[0419]

[0420] In some respects, the polynucleotides disclosed herein comprise nucleic acids selected from Table 5 (e.g., nucleic acids encoding VH in Table 6) and nucleic acids from Table 7 (e.g., nucleic acids encoding VL in Table 8).

[0421] In some respects, the polynucleotides disclosed herein comprise the nucleic acid sequences shown in Table 9.

[0422] Table 9. Nucleic acid sequence of the light chain constant region of anti-IGF-1R antibody

[0423]

[0424] In some respects, the polynucleotides disclosed herein comprise the nucleic acid sequences shown in Table 10.

[0425] Table 10. Nucleic acid sequence of the heavy chain constant region of anti-IGF-1R antibody

[0426]

[0427] In some aspects, the polynucleotides disclosed herein comprise the nucleic acid sequences listed in Table 11. In some aspects, the polynucleotides disclosed herein encode antibodies comprising the heavy chain (HC) of antibodies listed in Table 12 or an antigen-binding fragment thereof. In some aspects, the polynucleotides disclosed herein encode signal peptides listed in Table 12.

[0428] Table 11. Anti-IGF-1R antibody heavy chain (HC) nucleic acid sequence

[0429]

[0430]

[0431] [Table 12. Amino acid sequence of the heavy chain (HC) of the anti-IGF-1R antibody]

[0432]

[0433] In some aspects, the polynucleotides disclosed herein comprise the nucleic acid sequences listed in Table 13. In some aspects, the polynucleotides disclosed herein encode antibodies comprising the light chain (LC) of antibodies listed in Table 14 or an antigen-binding fragment thereof. In some aspects, the polynucleotides disclosed herein encode signal peptides listed in Table 14.

[0434] Table 13. Nucleic acid sequence of the light chain (LC) of the anti-IGF-1R antibody.

[0435]

[0436] [Table 14. Amino acid sequence of the light chain (LC) of the anti-IGF-1R antibody]

[0437]

[0438] In some aspects, the polynucleotides disclosed herein comprise the nucleic acids listed in Tables 11 and 13. In some aspects, the polynucleotides disclosed herein encode antibodies comprising the HC and LC of the antibodies listed in Tables 12 and 14 (i.e., the HC of the antibody listed in Table 12 and the LC of the same antibody listed in Table 14).

[0439] In some aspects, the therapeutic protein used in the methods disclosed herein is an antibody (e.g., a monoclonal antibody or its antigen-binding fragment), said antibody having the VH, VL, HC and / or LC sequences of teprotumumab and antibodies having at least 80%, 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 the corresponding VH, VL, HC and / or LC sequences.

[0440] In some aspects, the polynucleotide also includes an adapter sequence operatively linked to a nucleic acid sequence encoding a heavy chain and / or a nucleic acid sequence encoding a light chain. In some aspects, the polynucleotide also includes a pausing element sequence operatively linked to a nucleic acid sequence encoding a heavy chain and / or a nucleic acid sequence encoding a light chain.

[0441] In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an anti-IGF-1R antibody or an antigen-binding fragment thereof (e.g., teprotumumab), said antibody comprising: (i) VH CDR 1–3 (e.g., (1) SEQ ID NO: 9–11; (2) SEQ ID NO: 12–14, or (3) SEQ ID NO: 12, 15, and 16) and VL CDR 1–3 (e.g., (1) SEQ ID NO: 17–19; (2) SEQ ID NO: 17, 20, and 21; or (3) SEQ ID NO: 17, 22, and 23); (ii) VH (e.g., SEQ ID NO: 24–26) and VL (e.g., SEQ ID NO: 29–31); (iii) HC (e.g., SEQ ID NO: 40–42) and LC (e.g., SEQ ID NO: 46–48); or (iv) comprising SEQ ID NO: 12, 15, and 16. The construct of any one of NO: 67 to 75, wherein the construct further comprises one or more of IRES, furin cleavage site, 2A site or dual promoter (e.g. promoter-VH-IRES-VL, etc.).

[0442] In some respects, the polynucleotide includes an open reading frame (ORF) containing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 52-66 or the ORF sequences disclosed in Table 15.

[0443] In some aspects, the antibody expression cassette includes an open reading frame (ORF) containing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 52–66 or the ORF sequences disclosed in Table 15.

[0444] Table 15. Modified ORF nucleic acid sequences encoding anti-IGF-1R antibodies

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452] In some respects, the antibody expression cassette contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of SEQ ID NO: 67–75 or the antibody expression cassette sequences disclosed in Table 16.

[0453] Table 16. Expression cassettes encoding anti-IGF-1R antibodies

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469] [IV.A.2. Anti-IL-6 and anti-IL-6 receptor antibodies]

[0470] Some aspects of the present invention relate to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising nucleic acids encoding an antibody (e.g., a monoclonal antibody) that specifically binds to interleukin-6 (IL-6) or the interleukin-6 receptor (IL-6R) and its antigen-binding fragment.

[0471] In some respects, the encoded antibody is an anti-IL-6 antibody. In some respects, the encoded anti-IL-6 antibody contains the amino acid sequence of tocilizumab, cetuximab, olokizumab, clazakizumab, sirukumab, levilimab, TOUR006, or fragments, variants, or derivatives thereof.

[0472] In some respects, the encoded antibody is an anti-IL-6-R (anti-IL-6R) antibody. In some respects, the encoded anti-IL-6-R antibody contains the amino acid sequence of sarilumab, satralizumab, or fragments, variants, or derivatives thereof.

[0473] In some aspects, the present invention relates to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising a promoter operatively linked to a nucleic acid encoding an immunoglobulin, such as an antibody or antigen-binding fragment thereof that binds to IL-6 or IL-6R.

[0474] In some respects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions used in the methods disclosed herein encode antibodies (e.g., monoclonal antibodies or antigen-binding fragments thereof) 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 their variable region or CDR sequences of Tocilizumab, Siltuximab, Sarilumab, Satralizumab, Olokizumab, Clazakizumab, Sirukumab, Levilimab, TOUR006. In some respects, gene therapy constructs encoding anti-IL-6 antibodies or anti-IL-6R antibodies are polycistronic (e.g., bicistronic) constructs (e.g., containing both heavy and light chains). In some respects, polycistronic (e.g., bicistronic) constructs also contain F2A or IRES elements.

[0475] In some respects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions disclosed herein contain nucleic acids encoding antibodies, said antibodies comprising the heavy and light chains of anti-IL-6 antibodies, anti-IL-6R antibodies, or antigen-binding fragments thereof.

[0476] In some respects, the anti-IL-6 antibody or anti-IL-6R antibody comprises a heavy chain and a light chain. In some respects, the nucleic acid sequence encodes a heavy chain amino acid sequence corresponding to any one of SEQ ID NO: 94, 103, or 104 (or the HC amino acid sequences in Table 17). In some respects, the nucleic acid sequence encodes a light chain amino acid sequence corresponding to any one of SEQ ID NO: 99, 109, or 110 (or the LC amino acid sequences in Table 17).

[0477] In some aspects, the anti-IL-6 antibody or anti-IL-6R antibody comprises a heavy chain variable region and a light chain variable region. In some aspects, the nucleic acid sequence encodes the heavy chain variable region amino acid sequence corresponding to SEQ ID NO: 95 or 105 (or the VH amino acid sequence in Table 17). In some aspects, the nucleic acid sequence encodes the light chain variable region amino acid sequence corresponding to SEQ ID NO: 100 or 111 (or the VL amino acid sequence in Table 17).

[0478] In some aspects, the heavy chain includes a heavy chain variable region (VH), which includes a complementarity-determining region (CDR) 1, VH CDR 2, and VH CDR 3. In some aspects, VH CDRs 1–3 correspond to the CDRs of tocilizumab, cetuximab, sarilumab, satralizumab, olokizumab, clazakizumab, sirukumab, or levilimab. In some respects, the nucleic acid sequence encodes the VH CDR1 amino acid sequence corresponding to SEQ ID NO: 96 (or the VH CDR1 coding sequence disclosed in Table 17); the nucleic acid sequence encodes the VH CDR2 amino acid sequence corresponding to SEQ ID NO: 97 (or the VHCDR2 coding sequence disclosed in Table 17); and the nucleic acid sequence encodes the VH CDR3 amino acid sequence corresponding to SEQ ID NO: 98 (or the VH CDR3 coding sequence disclosed in Table 17).

[0479] In some respects, the nucleic acid sequence encodes the VH CDR1 amino acid sequence corresponding to SEQ ID NO: 106 (or the VH CDR1 coding sequence disclosed in Table 17); the nucleic acid sequence encodes the VH CDR2 amino acid sequence corresponding to SEQ ID NO: 107 (or the VH CDR2 coding sequence disclosed in Table 17); and the nucleic acid sequence encodes the VH CDR3 amino acid sequence corresponding to SEQ ID NO: 108 (or the VHCDR3 coding sequence disclosed in Table 17).

[0480] In some aspects, the light chain includes a light chain variable region (VL), which includes a complementarity-determining region (CDR) 1, VL CDR 2, and VL CDR 3. In some aspects, VL CDR 1–3 correspond to the CDRs of tocilizumab, cetuximab, sarilumab, satralizumab, olokizumab, clazakizumab, sirukumab, or levilimab. In some respects, the nucleic acid sequence encodes the VL CDR1 amino acid sequence corresponding to SEQ ID NO: 101 (or the VL CDR1 coding sequence disclosed in Table 17); the nucleic acid sequence encodes the VL CDR2 amino acid sequence corresponding to amino acid YTS (or the VL CDR2 coding sequence disclosed in Table 17); and the nucleic acid sequence encodes the VLCDR3 amino acid sequence corresponding to SEQ ID NO: 102 (or the VL CDR3 coding sequence disclosed in Table 17).

[0481] In some respects, the nucleic acid sequence encodes the VL CDR1 amino acid sequence corresponding to SEQ ID NO: 112 (or the VL CDR1 coding sequence disclosed in Table 17); the nucleic acid sequence encodes the VL CDR2 amino acid sequence corresponding to SEQ ID NO: 113 (or the VL CDR2 coding sequence disclosed in Table 17); and the nucleic acid sequence encodes the VL CDR3 amino acid sequence corresponding to SEQ ID NO: 114 (or the VL CDR3 coding sequence disclosed in Table 17).

[0482] In some aspects, the polynucleotides disclosed herein encode single-domain antibodies (e.g., nanobodies) comprising (i) a heavy chain variable region (VH) including complementarity-determining regions (CDRs) 1, VH CDR2, and / or VH CDR3, or (ii) a light chain variable region (VL) including CDR1, VL CDR2, and / or VL CDR3. In some aspects, the encoded VH CDRs and / or VL CDRs are selected from the corresponding CDRs of tocilizumab, cetuximab, sarilumab, satalizumab, olokizumab, clazakizumab, sirukumab, or levilimab.

[0483] In some respects, the polynucleotides disclosed herein encode amino acid sequences of tocilizumab, siltuximab, sarilumab, satralizumab, olokizumab, clazakizumab, sirukumab, levilimab, or fragments, variants, or derivatives thereof.

[0484] In some respects, the polynucleotides disclosed herein encode antibodies or antigen-binding fragments of the six CDRs listed in Table 17.

[0485] Table 17. Sequences of anti-IL-6 and anti-IL-6 receptor antibodies

[0486]

[0487]

[0488] [IV.A.3. Anti-IL-11 and anti-IL-11 receptor antibodies]

[0489] Some aspects of the present invention relate to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising nucleic acids encoding an antibody (e.g., a monoclonal antibody) and an antigen-binding fragment thereof, the antibody and its antigen-binding fragment specifically binding to interleukin-11 (IL-11) or an interleukin-11 receptor (IL-11R).

[0490] In some respects, the encoded antibody is an anti-IL-11 (anti-IL-11) antibody. In some respects, the encoded anti-IL-11 antibody contains the amino acid sequence of 9MW3811 or a fragment, variant, or derivative thereof.

[0491] In some respects, the encoded antibody is an anti-IL-11 receptor (anti-IL-11R) antibody. In some respects, the encoded anti-IL-11R antibody contains the amino acid sequence of BI 765423, LASN01 or fragments thereof, variants or derivatives thereof.

[0492] In some aspects, the present invention relates to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising a promoter operatively linked to a nucleic acid encoding an immunoglobulin (e.g., an antibody or antigen-binding fragment thereof that binds to IL-11 or IL-11R).

[0493] In some aspects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions used in the methods disclosed herein encode antibodies (e.g., monoclonal antibodies or antigen-binding fragments thereof) having a CDR and / or variable region sequence of teprotumumab or 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 the variable region or CDR sequence of BI765423, 9MW3811, or LASN01. In some aspects, the gene therapy construct encoding an anti-IL-11 antibody is a polycistronic (e.g., bicistronic) construct (e.g., containing a heavy chain and a light chain). In some aspects, the polycistronic (e.g., bicistronic) construct also contains an F2A or IRES element.

[0494] In some respects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions disclosed herein contain nucleic acids encoding antibodies, said antibodies comprising the heavy and light chains of anti-IL-11 antibodies, anti-IL-11R antibodies, or antigen-binding fragments thereof.

[0495] In some respects, the anti-IL-11 antibody or anti-IL-11R antibody comprises a heavy chain variable region and a light chain variable region. In some respects, the nucleic acid sequence encodes the heavy chain variable region amino acid sequence corresponding to SEQ ID NO: 115 (or the VH amino acid sequence in Table 18). In some respects, the nucleic acid sequence encodes the light chain variable region amino acid sequence corresponding to SEQ ID NO: 119 (or the VL amino acid sequence in Table 18).

[0496] In some aspects, the heavy chain includes a heavy chain variable region (VH), which includes a complementarity-determining region (CDR) 1, VH CDR2, and VH CDR3. In some aspects, VH CDR1–3 correspond to the CDRs of BI 765423, 9MW3811, or LASN01. In some aspects, the nucleic acid sequence encodes the amino acid sequence of VH CDR1 corresponding to SEQ ID NO: 116 (or the VH CDR1 coding sequence disclosed in Table 18); the nucleic acid sequence encodes the amino acid sequence of VH CDR2 corresponding to SEQ ID NO: 117 (or the VH CDR2 coding sequence disclosed in Table 18); and the nucleic acid sequence encodes the amino acid sequence of VH CDR3 corresponding to SEQ ID NO: 118 (or the VH CDR3 coding sequence disclosed in Table 18).

[0497] In some aspects, the light chain includes a light chain variable region (VL), which includes a complementarity-determining region (CDR) 1, VL CDR 2, and VL CDR 3. In some aspects, VL CDR 1–3 correspond to the CDRs of BI 765423, 9MW3811, or LASN01. In some aspects, the nucleic acid sequence encodes the amino acid sequence of VL CDR 1 corresponding to SEQ ID NO: 120 (or the VLCDR 1 coding sequence disclosed in Table 18); the nucleic acid sequence encodes the amino acid sequence of VL CDR 2 corresponding to SEQ ID NO: 121 (or the VL CDR 2 coding sequence disclosed in Table 18); and the nucleic acid sequence encodes the amino acid sequence of VL CDR 3 corresponding to SEQ ID NO: 122 (or the VL CDR 3 coding sequence disclosed in Table 18).

[0498] In some aspects, the polynucleotides disclosed herein encode single-domain antibodies (e.g., nanobodies) comprising (i) a heavy chain variable region (VH) comprising complementarity-determining regions (CDR) 1, VH CDR 2, and / or VH CDR 3, or (ii) a light chain variable region (VL) comprising CDR 1, VL CDR 2, and / or VL CDR 3. In some aspects, the encoded VH CDRs and / or VL CDRs are selected from the corresponding CDRs of BI 765423, 9MW3811, or LASN01.

[0499] In some respects, the polynucleotides disclosed herein encode amino acid sequences of BI 765423, 9MW3811, LASN01 or fragments, variants or derivatives thereof.

[0500] In some respects, the polynucleotides disclosed herein encode antibodies or antigen-binding fragments thereof, said antibodies or antigen-binding fragments comprising the six CDRs listed in Table 18.

[0501] Table 18. Sequences of anti-IL-11 and anti-IL-11 receptor antibodies

[0502]

[0503] [IV.A.4. Anti-FcRn antibody]

[0504] Certain aspects of the present invention relate to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising nucleic acids encoding antibodies (e.g., monoclonal antibodies) and their antigen-binding fragments, said antibodies specifically binding to neonatal Fc receptors (FcRn). In some aspects, the encoded anti-FcRn antibody comprises an amino acid sequence of batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, IMVT-1402, or fragments, variants, or derivatives thereof.

[0505] In some aspects, the present invention relates to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions of the present invention comprising a promoter operatively linked to a nucleic acid encoding an immunoglobulin, such as an antibody or antigen-binding fragment thereof that binds to FcRn.

[0506] In some aspects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions of the present invention used in the methods disclosed herein encode an antibody (e.g., a monoclonal antibody or its antigen-binding fragment) 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 the variable region or CDR sequence of batoclimab, edemamod, rozanolixizumab, nipocalimab, orilanolimab, or IMVT-1402. In some aspects, the gene therapy construct encoding an anti-FcRn antibody is a polycistronic (e.g., bicistronic) construct (e.g., comprising a heavy chain and a light chain). In some respects, polycistronic (e.g., bicistronic) constructs also include F2A or IRES elements.

[0507] In some respects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions disclosed herein contain nucleic acids encoding antibodies, said antibodies containing the heavy and light chains of an anti-FcRn antibody or an antigen-binding fragment thereof.

[0508] In some respects, the anti-FcRn antibody comprises a heavy chain and a light chain. In some respects, the nucleic acid sequence encodes the heavy chain amino acid sequence corresponding to SEQ ID NO: 123 or 132 (or the HC amino acid sequence in Table 19). In some respects, the nucleic acid sequence encodes the light chain amino acid sequence corresponding to SEQ ID NO: 128 or 137 (or the LC amino acid sequence in Table 19).

[0509] In some respects, the anti-FcRn antibody comprises a heavy chain variable region and a light chain variable region. In some respects, the nucleic acid sequence encodes the heavy chain variable region amino acid sequence corresponding to SEQ ID NO: 124 or 133 (or the VH amino acid sequence in Table 19). In some respects, the nucleic acid sequence encodes the light chain variable region amino acid sequence corresponding to SEQ ID NO: 129 or 138 (or the VL amino acid sequence in Table 19).

[0510] In some aspects, the heavy chain includes a heavy chain variable region (VH), which includes complementarity-determining regions (CDRs) 1, VH CDR 2, and VH CDR 3. In some aspects, VH CDRs 1–3 correspond to the CDRs of batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, or IMVT-1402. In some respects, the nucleic acid sequence encodes the VH CDR1 amino acid sequence corresponding to SEQ ID NO: 125 (or the VH CDR1 coding sequence disclosed in Table 19); the nucleic acid sequence encodes the VH CDR2 amino acid sequence corresponding to SEQ ID NO: 126 (or the VH CDR2 coding sequence disclosed in Table 19); and the nucleic acid sequence encodes the VH CDR3 amino acid sequence corresponding to SEQ ID NO: 127 (or the VH CDR3 coding sequence disclosed in Table 19).

[0511] In some respects, the nucleic acid sequence encodes the VH CDR1 amino acid sequence corresponding to SEQ ID NO: 134 (or the VH CDR1 coding sequence disclosed in Table 19); the nucleic acid sequence encodes the VH CDR2 amino acid sequence corresponding to SEQ ID NO: 135 (or the VH CDR2 coding sequence disclosed in Table 19); and the nucleic acid sequence encodes the VH CDR3 amino acid sequence corresponding to SEQ ID NO: 136 (or the VHCDR3 coding sequence disclosed in Table 19).

[0512] In some aspects, the light chain includes a light chain variable region (VL), which includes a complementarity-determining region (CDR) 1, VL CDR2, and VL CDR3. In some aspects, VL CDR1–3 correspond to the CDRs of batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, or IMVT-1402. In some aspects, the nucleic acid sequence encodes the VL CDR1 amino acid sequence corresponding to SEQ ID NO: 130 (or the VL CDR1 coding sequence disclosed in Table 19); the nucleic acid sequence encodes the VL CDR2 amino acid sequence corresponding to the amino acid sequence DDS (or the VL CDR2 coding sequence disclosed in Table 19); and the nucleic acid sequence encodes the VL CDR3 amino acid sequence corresponding to SEQ ID NO: 131 (or the VL CDR3 coding sequence disclosed in Table 19).

[0513] In some respects, the nucleic acid sequence encodes the VL CDR1 amino acid sequence corresponding to SEQ ID NO: 139 (or the VL CDR1 coding sequence disclosed in Table 19); the nucleic acid sequence encodes the VL CDR2 amino acid sequence corresponding to SEQ ID NO: 140 (or the VL CDR2 coding sequence disclosed in Table 19); and the nucleic acid sequence encodes the VL CDR3 amino acid sequence corresponding to SEQ ID NO: 141 (or the VLCDR3 coding sequence disclosed in Table 19).

[0514] In some aspects, the polynucleotides disclosed herein encode single-domain antibodies (e.g., nanobodies) comprising (i) a heavy chain variable region (VH) comprising a complementarity-determining region (CDR) 1, VH CDR2, and / or VH CDR3, or (ii) a light chain variable region (VL) comprising CDR1, VL CDR2, and / or VL CDR3. In some aspects, the encoded VH CDR and / or VL CDR are selected from the corresponding CDRs of batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, or IMVT-1402.

[0515] In some respects, the polynucleotides disclosed herein encode amino acid sequences of batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, IMVT-1402, or fragments, variants, or derivatives thereof.

[0516] In some respects, the polynucleotides disclosed herein encode antibodies or antigen-binding fragments thereof, said antibodies or antigen-binding fragments comprising the six CDRs listed in Table 19.

[0517] Table 19. Anti-FcRn antibody sequences

[0518]

[0519]

[0520] [IV.A.5. Anti-TSHR antibody]

[0521] Some aspects of the present invention relate to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions comprising nucleic acids encoding antibodies (e.g., monoclonal antibodies) and their antigen-binding fragments, said antibodies specifically binding to the thyroid-stimulating hormone receptor (TSHR), also known as the thyroid-stimulating hormone receptor. In some aspects, the encoded anti-TSHR antibody comprises an amino acid sequence of K1-70 or a fragment, variant, or derivative thereof.

[0522] In some aspects, the present invention relates to polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions of the present invention comprising a promoter operatively linked to a nucleic acid encoding an immunoglobulin, such as an antibody or antigen-binding fragment thereof that binds to TSHR.

[0523] In some aspects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions of the present invention used in the methods disclosed herein encode antibodies (e.g., monoclonal antibodies or antigen-binding fragments thereof) having a CDR and / or variable region sequence of teprotumumab or having at least 80% identity with the variable region or CDR sequence of K1-70 (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). In some aspects, the gene therapy construct encoding an anti-TSHR antibody is a polycistronic (e.g., bicistronic) construct (e.g., comprising a heavy chain and a light chain). In some aspects, the polycistronic (e.g., bicistronic) construct also comprises an F2A or IRES element.

[0524] In some respects, the polynucleotides (e.g., antibody expression cassettes), vectors, rAAV particles, or compositions disclosed herein contain nucleic acids encoding antibodies, said antibodies comprising the heavy and light chains of anti-TSHR antibodies or antigen-binding fragments thereof.

[0525] In some respects, the anti-TSHR antibody comprises a heavy chain and a light chain. In some respects, the nucleic acid sequence encodes the heavy chain amino acid sequence corresponding to SEQ ID NO: 146 (or the HC amino acid sequence in Table 20). In some respects, the nucleic acid sequence encodes the light chain amino acid sequence corresponding to SEQ ID NO: 147 (or the LC amino acid sequence in Table 20).

[0526] In some respects, the anti-TSHR antibody includes a heavy chain variable region and a light chain variable region. In some respects, the nucleic acid sequence encodes the heavy chain variable region amino acid sequence corresponding to SEQ ID NO: 148 (or the VH amino acid sequence in Table 20). In some respects, the nucleic acid sequence encodes the light chain variable region amino acid sequence corresponding to SEQ ID NO: 149 (or the VL amino acid sequence in Table 20).

[0527] In some aspects, the heavy chain includes a heavy chain variable region (VH), which includes complementarity-determining regions (CDR) 1, VH CDR2, and VH CDR3. In some aspects, VH CDR1–3 correspond to the CDRs of K1-70. In some aspects, the nucleic acid sequence encodes the VH CDR1 amino acid sequence corresponding to SEQ ID NO: 150 (or the VH CDR1 coding sequence disclosed in Table 20); the nucleic acid sequence encodes the VH CDR2 amino acid sequence corresponding to SEQ ID NO: 151 (or the VH CDR2 coding sequence disclosed in Table 20); and the nucleic acid sequence encodes the VHCDR3 amino acid sequence corresponding to SEQ ID NO: 152 (or the VH CDR3 coding sequence disclosed in Table 20).

[0528] In some aspects, the light chain includes a light chain variable region (VL), which includes a complementarity-determining region (CDR) 1, VL CDR2, and VL CDR3. In some aspects, VL CDR1–3 correspond to the CDRs of K1-70. In some aspects, the nucleic acid sequence encodes the VL CDR1 amino acid sequence corresponding to SEQ ID NO: 153 (or the VL CDR1 coding sequence disclosed in Table 20); the nucleic acid sequence encodes the VL CDR2 amino acid sequence corresponding to the amino acid sequence DNN (or the VL CDR2 coding sequence disclosed in Table 20); and the nucleic acid sequence encodes the VLCDR3 amino acid sequence corresponding to SEQ ID NO: 154 (or the VL CDR3 coding sequence disclosed in Table 20).

[0529] In some aspects, the polynucleotides disclosed herein encode single-domain antibodies (e.g., nanobodies), said single-domain antibodies comprising (i) a heavy chain variable region (VH) including complementarity-determining region (CDR) 1, VH CDR2, and / or VH CDR3, or (ii) a light chain variable region (VL) including CDR1, VL CDR2, and / or VL CDR3. In some aspects, the encoded VH CDRs and / or VL CDRs are selected from the corresponding CDRs of K1-70.

[0530] In some respects, the polynucleotides disclosed herein encode amino acid sequences of K1-70 or fragments, variants or derivatives thereof.

[0531] In some respects, the polynucleotides disclosed herein encode antibodies or antigen-binding fragments thereof, including the six CDRs listed in Table 20.

[0532] Table 20. Anti-TSHR antibody sequences

[0533]

[0534] [IV.A.6. Antigen-binding fragment]

[0535] In some aspects, the antigen-binding fragments of the antibodies described herein are encoded by the polynucleotides disclosed herein. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and scFv, wherein Fab, Fab', F(ab')2, or scFv comprises the heavy chain variable region sequence and the light chain variable region sequence of teprotumumab as described herein. Fab, Fab', F(ab')2, or scFv can be generated by any technique known to those skilled in the art. In some aspects, the antigen-binding fragment (such as Fab, Fab', F(ab')2, or scFv) further includes a group for extending the in vivo half-life of the antibody. This group is also referred to as a "half-life extension group". Any group known to those skilled in the art for extending the in vivo half-life of an antigen-binding fragment (such as Fab, Fab', F(ab')2, or scFv) may be used. For example, the half-life extension group may include an Fc region, a polymer, albumin, or an albumin-binding protein or compound. The polymer may include natural or synthetic, optionally substituted, straight-chain or branched polyalkylene, polyolefin, polyoxyalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxy polyethylene glycol, lactose, starch, dextran, glycogen, or derivatives thereof. Substituents may include one or more hydroxyl, methyl, or methoxy groups. In some aspects, the antigen-binding fragment (such as Fab, Fab', F(ab')2, or scFv) may be modified by adding one or more C-terminal amino acids for attaching a half-life extension group. In some aspects, the half-life extension group is polyethylene glycol or human serum albumin. In some aspects, the antigen-binding fragment (such as Fab, Fab', F(ab')2, or scFv) is fused to the Fc region.

[0536] In some respects, the antibody or its antigen-binding fragment is selected from the group consisting of anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-2, anti-IL-17A, anti-TNF-α, or anti-CD28. In other respects, the antibody or its antigen-binding fragment is selected from the group consisting of tocilizumab, teprotumumab, bevacizumab, ranibizumab, faricimab, infliximab, daclizumab, and secukinumab.

[0537] In some respects, antibodies or their antigen-binding fragments specifically bind to insulin-like growth factor-1 receptor (IGF-1R) (such as human IGF-1R), interleukin-6 (IL-6), interleukin-6 receptor (IL-6-R), interleukin-11 (IL-11), interleukin-11 receptor (IL-11R), or neonatal Fc receptor (FcRn).

[0538] In some respects, the encoded anti-IGF-1R antibodies include teprotumumab, VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, 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, L16H 16. L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L3 The amino acid sequence of 0H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, L52H52 or Lonigutamab, or its antigen-binding fragment.

[0539] In some respects, the encoded anti-IGF-1R antibody includes the amino acid sequence of teprotumumab, or its antigen-binding fragment.

[0540] In some respects, therapeutic antibodies are anti-IGF-1R antibodies, or their antigen-binding fragments. Some examples of anti-IGF-1R antibodies include teprotumumab, VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, 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, L16 H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, L52H52 or Lonigutamab, or fragments, variants or derivatives thereof.

[0541] In some aspects, the anti-IGF-1R antibody is VRDN-01100, VRDN-02700, or teprotumumab, or a fragment, variant, or derivative thereof. In some aspects, the anti-IGFR antibody comprises SEQ ID NO: 78 (corresponding to VRDN-01100). In some aspects, the anti-IGFR antibody comprises SEQ ID NO: 81 (corresponding to VRDN-002700).

[0542] In some respects, the encoded anti-IL-6 antibody contains the amino acid sequence of tocilizumab, cetuximab, olokizumab, clazakizumab, sirukumab, levilimab, TOUR006, or fragments, variants, or derivatives thereof.

[0543] In some respects, the encoded anti-IL-6R antibody contains the amino acid sequence of sarilumab, satralizumab, or fragments, variants, or derivatives thereof.

[0544] In some respects, the encoded anti-IL-11 antibody contains the amino acid sequence of 9MW3811 or a fragment, variant, or derivative thereof.

[0545] In some respects, the encoded anti-IL-11R antibody contains the amino acid sequence of BI 765423, LASN01, or fragments, variants, or derivatives thereof.

[0546] In some respects, the encoded anti-FcRn antibody contains the amino acid sequence of batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, IMVT-1402, or fragments, variants, or derivatives thereof.

[0547] In some respects, the encoded anti-TSHR antibody contains the amino acid sequence of K1-70.

[0548] In some respects, the antibodies (e.g., monoclonal antibodies) or their antigen-binding fragments disclosed herein are modified to give them an enhanced half-life and / or reduced toxicity.

[0549] In some respects, the encoded antibody or its antigen-binding fragment is a human antibody, a humanized antibody, or a chimeric antibody. In some respects, the antibody or its antigen-binding fragment 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 respects, the antibody or its antigen-binding fragment is bispecific or multispecific.

[0550]

IV.A.7. Antibody Expression Kit

[0551] In some respects, this document provides antibody expression cassettes comprising nucleotide sequences encoding a therapeutic antibody or an antigen-binding fragment thereof or a domain thereof (e.g., light chain, heavy chain, variable light chain region and / or variable heavy chain region), and a vector, for example, a vector comprising such an antibody expression cassette for expression in cells.

[0552] In some respects, this document provides antibody expression cassettes comprising nucleotide sequences encoding antibody or antigen-binding fragments as described herein, and antibody or antigen-binding fragments that compete with or bind to the same epitopes as these antibody or antigen-binding fragments.

[0553] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof selected from the group consisting of: anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-6R, anti-IL-11, anti-IL11R, anti-FcRn, anti-IL-2, anti-IL-17A, anti-TNF-α, anti-CD28, or an antigen-binding fragment thereof.

[0554] In some respects, antibody expression cassettes contain nucleic acid sequences encoding antibodies or antigen-binding fragments thereof selected from the group consisting of: anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-2, anti-IL-17A, anti-TNF-α, anti-CD28, or antigen-binding fragments thereof.

[0555] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof selected from the group consisting of tocilizumab, teprotumumab, bevacizumab, ranibizumab, faricimab, infliximab, daclizumab, secukinumab, or an antigen-binding fragment thereof.

[0556] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof selected from the group consisting of: tocilizumab, cetuximab, olokizumab, clazakizumab, sirukumab, levilimab, TOUR006, or fragments, variants or derivatives thereof.

[0557] In some respects, antibody expression cassettes contain nucleic acid sequences encoding antibodies or antigen-binding fragments thereof selected from the group consisting of Sarilumab, Satralizumab, or fragments, variants, or derivatives thereof.

[0558] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof selected from the group consisting of 9MW3811 or a fragment, variant, or derivative thereof.

[0559] In some respects, antibody expression cassettes contain nucleic acid sequences encoding antibodies or antigen-binding fragments thereof selected from the group consisting of BI 765423, LASN01, or fragments, variants, or derivatives thereof.

[0560] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof selected from the group consisting of: batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, IMVT-1402, or fragments, variants or derivatives thereof.

[0561] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody that competes with teprotumumab, bevacizumab, ranibizumab, faricimab, infliximab, daclizumab, secukinumab, or their antigen-binding fragments for binding to the same epitope.

[0562] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody that competes with Tocilizumab, Siltuximab, Olokizumab, Clazakizumab, Sirukumab, Levilimab, TOUR006 or fragments, variants or derivatives thereof to bind to the same epitope.

[0563] In some respects, antibody expression cassettes contain nucleic acid sequences encoding antibodies that compete with Sarilumab, Satralizumab, or fragments, variants, or derivatives thereof for binding to the same epitope.

[0564] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody that competes with 9MW3811 or a fragment, variant, or derivative thereof for binding to the same epitope.

[0565] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody that competes with BI 765423, LASN01, or fragments, variants, or derivatives thereof for binding to the same epitope.

[0566] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody that competes with batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, IMVT-1402 or fragments, variants or derivatives thereof to bind to the same epitope.

[0567] In some respects, antibody expression cassettes contain nucleic acid sequences encoding anti-IGF-1R antibodies or their antigen-binding fragments.

[0568] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding an antibody selected from the following group: teprotumumab, VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, 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, L52H52 or lonigutamab, or its antigen-binding fragment.

[0569] In some respects, the antibody expression cassette contains codes for teprotumumab, VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, 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, L16H16 , 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, L52H52, or lonigutamab competitively bind to the nucleic acid sequences of antibodies that have the same epitope, or their antigen-binding fragments.

[0570] In some respects, the antibody expression cassette contains a nucleic acid sequence encoding VRDN-01100, VRDN-02700, or an antigen-binding fragment thereof.

[0571] In some respects, the antibody expression cassette contains a nucleic acid sequence, or an antigen-binding fragment thereof, encoding an antibody that competes with VRDN-01100 or VRDN-02700 for binding to the same epitope.

[0572] In some aspects, the antibody expression cassette comprises a nucleic acid sequence encoding teprotumumab or an antigen-binding fragment thereof. In some aspects, the antibody expression cassette comprises an open reading frame containing a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 53. In some aspects, the nucleic acid sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 53. In some aspects, the antibody expression cassette further comprises an smCBA promoter (e.g., SEQ ID NO: 145).

[0573] In some respects, antibody expression cassettes contain a nucleic acid sequence encoding an antibody that competes with teprotumumab for binding to the same epitope, or an antigen-binding fragment thereof.

[0574] This document also provides an antibody expression cassette comprising a nucleotide sequence encoding a polypeptide comprising any one of SEQ ID NO: 9–26, 29–31, 34–42, 44, 46–48, 50, 52–75, or a combination thereof.

[0575] This document also provides kits, vectors, or host cells comprising (i) the first antibody expression cassette described herein; and (ii) a delivery vector.

[0576] This document also provides a kit, vector, or host cell comprising (i) a first antibody expression cassette containing a nucleotide sequence encoding any one or a combination of SEQ ID NO: 9–26, 29–31, 34–42, 44, 46–48, 50, 52–75; and (ii) a delivery vector.

[0577] In some aspects, this document provides antibody expression cassettes comprising nucleotide sequences containing three VH domain CDRs, such as the nucleotide sequences of VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein (e.g., see Table 3), wherein the three VH domain CDRs are situated in a VH background. In some aspects, this document provides polynucleotides comprising nucleotide sequences containing three VL domain CDRs, such as the nucleotide sequences of VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (e.g., see Table 4), wherein the three VL domain CDRs are situated in a VL background. In some respects, this document provides antibody expression cassettes (or combinations of polynucleotides) comprising nucleotide sequences containing an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising (i) three VH domain CDRs, for example, nucleotide sequences of VH CDR1, VH CDR2, and VH CDR3 containing any of the antibodies described herein (e.g., see Table 3), for example, wherein the three VH domain CDRs are in a VH background and (ii) three VL domain CDRs, for example, nucleotide sequences of VLCDR1, VL CDR2, and VL CDR3 containing any of the antibodies described herein (e.g., see Table 4), for example, wherein the three VL domain CDRs are in a VL background.

[0578] In some aspects, this document provides antibody expression cassettes comprising nucleotide sequences encoding three VH domain CDRs, for example, peptides comprising VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein (e.g., see Table 3), wherein the three VH domain CDRs are situated in a VH background. In other aspects, this document provides antibody expression cassettes comprising nucleotide sequences encoding three VL domain CDRs, for example, peptides comprising VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (e.g., see Table 4), wherein the three VL domain CDRs are situated in a VL background. In some respects, this document provides antibody expression cassettes (or combinations of polynucleotides) comprising nucleotide sequences encoding an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising (i) three VH domain CDRs, for example, peptides comprising VHCDR1, VH CDR2, and VH CDR3 of any antibody described herein (e.g., see Table 3), for example, wherein the three VH domain CDRs are in a VH background and (ii) three VL domain CDRs, for example, peptides comprising VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (e.g., see Table 4), for example, wherein the three VL domain CDRs are in a VL background.

[0579] In some aspects, the heavy chain includes a heavy chain variable region (VH), which includes complementarity-determining regions (CDRs) 1, VH CDR 2, and VH CDR 3. In some aspects, the modified VH CDRs 1–3 correspond to the CDRs of teprotumumab. In some respects, 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 with SEQ ID NO: 9 or 12; 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 with SEQ ID NO: 10, 13, or 15; and the nucleic acid sequence encoding VHCDR3 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 with SEQ ID NO: 11, 14, or 16.

[0580] In some aspects, the light chain includes a light chain variable region (VL), which includes complementarity-determining regions (CDR) 1, VL CDR 2, and VL CDR 3. In some aspects, VL CDRs 1–3 correspond to the CDRs of teprotumumab. In some respects, the nucleic acid sequence encoding 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 with SEQ ID NO: 17; the nucleic acid sequence encoding 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 with SEQ ID NO: 18, 20, or 22; and the nucleic acid sequence encoding VL 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 with SEQ ID NO: 19, 21, or 23.

[0581] This document also provides antibody expression cassettes encoding antibodies or antigen-binding fragments or domains thereof described herein, and which are optimized through, for example, codon / RNA optimization, substitution with heterologous signal sequences, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding antibodies or antigen-binding fragments or domains thereof (e.g., heavy chain, light chain, VH domain, or VL domain) described herein can be implemented using, for example, the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, for recombinant expression by introducing codon changes (e.g., codon changes encoding the same amino acid due to the degeneracy of the genetic code) and / or eliminating repressive regions in the mRNA, each of which is incorporated herein by reference in its entirety.

[0582] The antibody expression cassettes described herein can be generated from nucleic acids of suitable sources using methods well known in the art, such as PCR and other molecular cloning methods. For example, PCR amplification can be performed using synthetic primers that hybridize to the 3' and 5' ends of a known sequence, using genomic DNA obtained from hybridoma cells that produce antibodies of interest. In some aspects, hybridoma cell lines have accession number DSM ACC 2587 (deposited on October 4, 2003). In other aspects, hybridoma cell lines have accession number DSM ACC 2594 (deposited on September 5, 2003). Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding light and / or heavy chains that encode antibodies or antigen-binding fragments thereof. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding variable light chain regions and / or variable heavy chain regions that encode antibodies or antigen-binding fragments thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and further cloning, for example, for the production of chimeric antibodies and humanized antibodies or antigen-binding fragments thereof.

[0583] The antibody expression cassettes described herein may exist, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be a coding strand or a non-coding (antisense) strand. In some aspects, the antibody expression cassette is cDNA or DNA lacking one or more endogenous introns. In some aspects, the antibody expression cassette is not naturally occurring. In some aspects, the antibody expression cassette is recombinantly generated. In some aspects, the antibody expression cassette is isolated. In some aspects, the antibody expression cassette is substantially pure. In some aspects, the antibody expression cassette is purified from natural components.

[0584] In some respects, the viral vectors disclosed herein comprise antibody expression cassettes containing coding regions for two or more polypeptides, such as heavy and light chains.

[0585] When it is desired that the antibody expression cassette include coding regions for two or more separate polypeptide chains, each additional coding region besides the first is preferably linked to an element that promotes co-expression of the protein in the host cell, such as an internal ribosome entry sequence (IRES) element (see, for example, U.S. Patent No. 4,937,190), a furin cleavage site, a 2A element, or a promoter. In some aspects, when a single vector contains the sequence encoding each subunit of a multi-subunit protein, an IRES, a furin cleavage site, or a 2A element may be used. In cases where the protein of interest is an immunoglobulin with desired specificity, for example, the first coding region (encoding the heavy or light chain of the immunoglobulin) is located downstream of the promoter. The second coding region (encoding the remaining chain of the immunoglobulin) may be located downstream of the first coding region, and the IRES, furin cleavage site, or 2A element may be positioned between the two coding regions, for example, immediately preceding the second coding region. In some aspects, inserting an IRES, furin cleavage site, or 2A element between the sequences of the first gene and the second gene (encoding the heavy and light chains, respectively) can allow both chains to be expressed in the cell at substantially the same level from the same promoter.

[0586] In some aspects, the protein of interest comprises two or more subunits, such as immunoglobulin (Ig). In some aspects, the delivery vector of the present invention may include a coding region for each subunit. For example, a viral vector may include a coding region for the Ig heavy chain (or a variable region of the Ig heavy chain) and a coding region for the Ig light chain (or a variable region of the Ig light chain). In some aspects, the vector includes a first coding region for the variable region of the antibody heavy chain and a second coding region for the variable region of the antibody light chain. In some aspects, the two coding regions may be separated, for example, by means of a 2A self-processing sequence to allow polycistronic transcription of both coding regions.

[0587] A viral vector may include coding regions of two or more proteins of interest. For example, a viral vector may include coding regions of a first protein of interest and a second protein of interest. The first and second proteins of interest may be the same or different.

[0588] The Kozak concordance sequence, Kozak consensus, or Kozak sequence refers to a sequence appearing on eukaryotic mRNA and having a concordance sequence (gcc) gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". In some aspects, the vector contains a nucleotide sequence having at least about 85%, at least about 90%, or at least about 95% sequence identity with the Kozak concordance sequence. In some aspects, the vector contains the Kozak concordance sequence. In some aspects, the vector includes the Kozak concordance sequence after a polynucleotide encoding one or more proteins of interest is inserted into the vector (e.g., at a restriction site downstream of the promoter). For example, the vector may include the nucleotide sequence GCCGCCATG, where ATG is the start codon of the protein of interest. In some aspects, the vector contains the nucleotide sequence GCGGCCGCCATG (SEQ ID NO: 84), where ATG is the start codon of the protein of interest.

[0589] In some respects, this document provides compositions comprising a delivery vector (e.g., a viral vector) containing a nucleic acid encoding a therapeutic agent of interest (e.g., an antibody or an antigen-binding fragment thereof, such as an anti-IGF-1R antibody or an antigen-binding fragment thereof).

[0590]

V. Carrier Construction

[0591] Some aspects of this disclosure relate to vector constructs or expression constructs (e.g., expression cassettes) having a eukaryotic promoter operatively linked to DNA of interest encoding a therapeutic agent of interest (e.g., an antibody or antigen-binding fragment thereof, such as an anti-IGF-1R antibody or its antigen-binding fragment). In some aspects, vector constructs or expression constructs comprising DNA sequences (or corresponding RNA sequences) usable according to this disclosure can be any eukaryotic expression construct containing the DNA or RNA sequence of interest. For example, plasmids or viral constructs (e.g., AAV vectors) can be cleaved to provide linear DNA with linkable ends. These ends bind to exogenous DNA with complementary, similar linkable ends to provide a biologically functional recombinant DNA molecule with intact replicons and desired phenotypic properties. In some aspects, vector constructs or expression constructs are capable of replicating in both eukaryotic and prokaryotic hosts, as is known in the art and is commercially available.

[0592] In some aspects, the vector constructs or expression constructs encoding therapeutic agents of interest (e.g., antibodies or antigen-binding fragments thereof, such as anti-IGF-1R antibodies or antigen-binding fragments thereof) of this disclosure are polycistronic (e.g., bicistronic) constructs (e.g., containing heavy and light chains). In some aspects, the polycistronic (e.g., bicistronic) constructs also contain F2A or IRES elements.

[0593] In some respects, therapeutic agents of interest (such as antibodies or their antigen-binding fragments, such as anti-IGF-1R antibodies or their antigen-binding fragments) are antibodies.

[0594] In some respects, the antibodies are selected from the group consisting of anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-6R, anti-IL-11, anti-IL11R, anti-FcRn, anti-IL-2, anti-IL-17A, anti-TNF-α, anti-CD28, or their antigen-binding fragments.

[0595] In some respects, the antibodies are selected from the group consisting of anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-2, anti-IL-17A, anti-TNF-α, anti-CD28, or their antigen-binding fragments.

[0596] In some respects, the antibody is selected from the group consisting of tocilizumab, teprotumumab, bevacizumab, ranibizumab, faricimab, infliximab, daclizumab, secukinumab, or antigen-binding fragments thereof.

[0597] In some respects, antibodies are anti-IGF-1R antibodies or their antigen-binding fragments.

[0598] In some respects, anti-IGF-1R antibodies include teprotumumab, VRDN-01100, VRDN-02700, ganitumab (AMG 479), figitumumab, CP-751,871, cixutumumab (AMG 655), IMC-A12, dalotuzumab, MK0646, RG1507, robatumumab, 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, 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, L52H52 or Lonigutamab, or its antigen-binding fragment.

[0599] In some respects, the anti-IGF-1R antibody is VRDN-01100 or VRDN-02700, or its antigen-binding fragment.

[0600] In some respects, the anti-IGF-1R antibody is teprotumumab, or its antigen-binding fragment. In some respects, teprotumumab is encoded by 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: 53. In some respects, the nucleic acid sequence comprises the nucleotide sequence corresponding to SEQ ID NO: 53.

[0601] In some aspects, the anti-IGFR antibody comprises SEQ ID NO: 78 (corresponding to VRDN-01100). In some aspects, the anti-IGFR antibody comprises SEQ ID NO: 81 (corresponding to VRDN-002700).

[0602] The exogenous (i.e., donor) DNA used in this 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; both incorporated herein by reference, regarding methods and compositions for eukaryotic expression of DNA of interest).

[0603] In some respects, the vector construct or expression construct contains a promoter to facilitate the expression of DNA of interest within secretory cells. In some respects, 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, promoters derived from the immediate early gene of human CMV (Boshart et al., Cell 41:521-530 (1985)) and promoters derived from RSV long terminal repeats (LTRs) (Gorman et al., Proc. Natl. Acad. Sci.USA 79:6777-6781 (1982)). In some respects, the promoter is the CMV early enhancer / chicken β-actin (CBA) promoter, CAG promoter, CMV, EF1α, EF1α with a CMV enhancer, CMV promoter with a CMV enhancer (CMVe / p), CBA promoter with a CMV enhancer, CMV promoter with an SV40 intron, CBA promoter with both a CMV enhancer and a CAG intron, EF1α promoter with a truncated 5'LTR and chimeric HBG and IgHC introns, or a tissue-specific promoter. In some respects, tissue-specific promoters are muscle-specific promoters. In some respects, muscle-specific promoters are the DES promoter, HSA promoter, MCK promoter, HMCK7 promoter, dMCK promoter, tMCK promoter, CK8e promoter, SPc5-12 promoter, SP-301 promoter, MH promoter, Sk-CRM promoter, or Sk-CRM4 promoter.

[0604] In some respects, the nucleic acid sequence containing the promoter may contain introns. In some respects, the introns are selected from the group consisting of SV40 introns, MVM introns, CAG introns, or human β-globulin introns. In some respects, the CMVp promoter is fused with the SV40 intron.

[0605] Alternatively, the promoter used can be a tissue-specific promoter. For example, if the cell is a fibroblast, the tissue-specific promoter could be the insulin-like growth factor binding protein 2 (IGFBP2) promoter, the fibroblast activating protein (FAP) promoter, or the fibroblast-specific protein 1 (FSP1) promoter. If the cell is a muscle cell, the tissue-specific promoter could be the creatine kinase (MCK) promoter, the tropomyosin I (TNNI2) promoter, the skeletal muscle α-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 cell is an adipocyte, the tissue-specific promoter could be the adiponectin promoter or the adipocyte fatty acid binding protein (AP2) promoter.

[0606] In some aspects, the vector construct or expression construct includes a first promoter and a second promoter. In some aspects, the first and second promoters are different. In some aspects, the first and second promoters are the same. In some aspects, the first and second promoters initiate transcription in the same direction. In some aspects, the first and second promoters initiate transcription in different directions. In some aspects, the first or second promoter is a CMV promoter. In some aspects, the first or second promoter is an EF-1α promoter.

[0607] In some aspects, the nucleic acid sequence encoding the first promoter and the nucleic acid sequence encoding the second promoter are operatively linked. In some aspects, the nucleic acid sequence encoding the first promoter and the nucleic acid sequence encoding the second promoter are operatively linked by a pausing element.

[0608] In some aspects, the polycistronic vectors disclosed herein comprise an internal ribosome entry site (IRES) sequence or a 2A peptide. In some aspects, the constructs of the present invention may also include a protease cleavage site. In some aspects, the protease cleavage site is a furin cleavage site and / or a 2A cleavage site.

[0609] In some aspects, the vector constructs or expression constructs of the present invention may further include other components, such as markers (e.g., antibiotic resistance genes (such as ampicillin resistance genes) or β-galactosidase), to facilitate the selection of cells containing and / or expressing the construct; origins of replication (preferably high copy number origins of replication) for stable replication of the construct in bacterial cells; nuclear localization signals; or other elements that promote the production of the DNA construct, its encoded protein, or both. In some aspects, the vector constructs of the present invention may contain antibiotic resistance genes, including but not limited to neomycin, kanamycin, puromycin, and / or hygromycin. In some aspects, the vector constructs of the present invention may contain ColE1, f1, pUC, p15A, or pMB1 origins of replication.

[0610] In some aspects, the vector construct of the present invention comprises a backbone containing a ColE1 origin of replication and / or a kanamycin resistance gene.

[0611] For eukaryotic expression, the vector construct or expression construct may at least contain a eukaryotic promoter operatively linked to the DNA of interest, which in turn is operatively linked to a polyadenylation sequence. The polyadenylation signal sequence may be selected from any of the various polyadenylation signal sequences known in the art. In some aspects, the polyadenylation signal sequence is the SV40 early polyadenylation signal sequence. In some aspects, the polyadenylation signal sequence is the bovine growth hormone polyadenylation signal sequence (bGHpA). In some aspects, the polyadenylation signal sequence is the human growth hormone polyadenylation signal sequence (hGHpA). In some aspects, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence (SV40pA). The construct may also include one or more introns, which can increase the expression level of the DNA of interest, especially when the DNA of interest is cDNA (e.g., introns that do not contain naturally occurring sequences). Any of the various introns known in the art can be used (e.g., the human β-globin intron, inserted at the 5' position of the DNA of interest in the vector construct or expression construct). In some aspects, the intron is the SV40 intron. In some aspects, the intron is derived from the immunoglobulin heavy chain. In some aspects, the intron is a chimera between the human β-globin and immunoglobulin heavy chain genes.

[0612] In some respects, the polynucleotide contains a polymer (A). In some respects, the polymer (A) is a synthetic polymer (A) or a bovine growth hormone (BGH) polymer (A).

[0613] When it is desired to include coding regions of two or more separate polypeptide chains or subunits of two or more proteins of interest in a viral vector, each additional coding region other than the first is preferably linked to an element that promotes co-expression of these proteins in the host cell, such as an internal ribosome entry sequence (IRES) element (see, for example, U.S. Patent No. 4,937,190), or a 2A element. In some aspects, when a single vector contains the sequence encoding each subunit of a multi-subunit protein, an IRES, a furin cleavage site, or a 2A element may be used. For example, in the case where the protein of interest is an immunoglobulin with desired specificity, a first coding region encoding the heavy or light chain of the immunoglobulin is located downstream of the promoter. A second coding region encoding the remaining chain of the immunoglobulin may be located downstream of the first coding region, and an IRES, furin cleavage site, or 2A element may be positioned between the two coding regions, for example, immediately preceding the second coding region. In some aspects, introducing an IRES, furin cleavage site, or 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 in the cell at substantially the same level from the same promoter.

[0614] In some aspects, the nucleic acid sequence of the vector construct or expression construct includes a promoter, heavy chain, IRES, and light chain sequences in a 5'-3' orientation. In other aspects, the nucleic acid sequence of the construct includes a promoter, light variant, IRES, and heavy chain sequences in a 5'-3' orientation.

[0615] In some aspects, the nucleic acid sequence vector construct or expression construct includes a protease cleavage site. For example, the nucleic acid sequence may include a sequence incorporated into the vector construct or expression construct of the present invention adjacent to a self-processing cleavage site (such as 2A or 2A-like sequences), and provide a means of removing additional amino acids remaining after cleavage of the self-processing cleavage sequence. Exemplary protease cleavage sites are described herein and include, but are not limited to, furin protease cleavage sites having a concordant sequence RXK(R)R. Such furin protease cleavage sites can be cleaved by endogenous subtilisin-like proteases (such as furin and other serine proteases) present in the protein secretion pathway. In some aspects, as described in Lie et al., Sci Rep 7, 2193 (2017), other exemplary “additional protease cleavage sites” may be used.

[0616] In some aspects, the nucleic acid sequence vector construct or expression construct comprises a nucleic acid encoding a signal peptide operatively linked to a nucleic acid encoding an antibody or its antigen-binding fragment that binds to the insulin-like growth factor-1 receptor. In some aspects, the signal peptide is an endogenous signal peptide of HGH and its variants; an endogenous signal peptide of interferon and its variants, including signal peptides of type I, type II, and type III interferon and their variants; or an endogenous signal peptide of known cytokines and their variants, such as signal peptides of erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β and their variants. In some aspects, 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 with SEQ ID NO: 45 or 51. In some aspects, the nucleic acid sequence encoding the signal peptide comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 44 or 50.

[0617] In some respects, the promoter is selected from the group consisting of CAG promoter, CBA promoter, CMV promoter, EF1α promoter, EF1α promoter with CMV enhancer, CMV promoter with CMV enhancer (CMVe / p), CMV promoter with SV40 intron, or tissue-specific promoter. In some respects, the tissue-specific promoter is selected from DES promoter, HSA promoter, MCK promoter, HMCK7 promoter, dMCK promoter, tMCK promoter, CK8e promoter, SPc5-12 promoter, SP-301 promoter, MH promoter, Sk-CRM promoter, and Sk-CRM4 promoter.

[0618] In some respects, the nucleic acid sequence containing the promoter may contain an intron. In some respects, the intron is selected from the group consisting of CAG introns, SV40 introns, MVM introns, or human β-globin introns. In some respects, CMVp is fused with the SV40 intron.

[0619] In some respects, the first and second promoters differ. In some respects, the first and second promoters are the same. In some respects, the first and second promoters initiate transcription in the same direction. In some respects, the first and second promoters initiate transcription in different directions.

[0620] In some aspects, the nucleic acid sequences encoding the first promoter and the second promoter are operatively linked. In other aspects, the nucleic acid sequences encoding the first promoter and the second promoter are operatively linked via pausing elements.

[0621] Vectors used to deliver DNA of interest 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 on its own but acts to enhance or otherwise improve the effect of the active compound.

[0622]

VA1. Delivery carrier

[0623] In some respects, the delivery vector containing nucleic acids encoding or containing a therapeutic agent of interest (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment) is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome.

[0624] In some aspects, this disclosure provides 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) that encodes or contains a nucleic acid encoding or containing a therapeutic agent of interest (e.g., an antibody or an antigen-binding fragment thereof, such as an anti-IGF-1R antibody or an antigen-binding fragment thereof).

[0625] In some respects, the therapeutic effects of the agents of interest (such as antibodies or their antigen-binding fragments, such as anti-IGF-1R antibodies or their antigen-binding fragments) are local, systemic, or both.

[0626] In some aspects, the delivery carriers or compositions disclosed herein are delivered or administered by injection (e.g., via periocular injection). In some aspects, delivery or administration is by infusion. In some aspects, delivery or administration is as a single dose via injection and / or infusion. In some aspects, single-dose administration includes multiple injections or infusions. In some aspects, delivery or administration is as multiple doses via injection and / or infusion. In some aspects, each of the multiple-dose administrations includes multiple injections or infusions.

[0627] In some respects, the delivery carriers or compositions disclosed herein are suitable for delivery to epithelial tissues, neuroepithelial tissues, connective tissues, muscle tissues and / or adipose tissues (e.g., via periocular injection).

[0628] In some respects, the delivery carriers or compositions disclosed herein are suitable for delivery to ophthalmic areas (i.e., the ocular or periocular area, to or near the eye (e.g., one or both eyes)), for example, delivery carriers or compositions are suitable for periocular application.

[0629] In some respects, delivery or application is to the orbital tissue or extraocular cavity. In some respects, the orbital tissue or extraocular cavity is selected from the group consisting of: extraocular adipose tissue, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, optic nerve, blood vessels, sclera, dura mater sheath, cribriform plate, eyelids, lacrimal gland, lacrimal sac, or tear film.

[0630] In some respects, the orbital tissue or extraocular cavity is selected from the group consisting of: extraocular adipose tissue, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, or any combination thereof.

[0631] In some respects, the periorbital tissue is the tissue behind the eyeball.

[0632] In some respects, delivery or application (e.g., via periocular injection) is to the extraocular muscles. In some respects, the extraocular muscles are levator muscles, rectus muscles (e.g., lateral, medial, superior, or inferior rectus muscles), or oblique muscles (e.g., inferior or superior oblique muscles). In some respects, delivery or application is to connective tissue. In some respects, delivery or application is to adipose tissue.

[0633] In some respects, delivery or administration (e.g., via periocular injection) is to the cells of the eye or periocular region. In some respects, the cells of the eye or periocular region are fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, epithelial cells, neuroepithelial cells, glial cells, or any combination thereof. In some respects, the cells of the periocular region are the cells behind the eyeball.

[0634] In some aspects, the therapeutic agent of interest (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment, contained in 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) or composition is produced in a cell. In some aspects, the cell is a fibroblast, adipocyte, myofibroblast, myocyte, muscle cell, epithelial cell, neuroepithelial cell, glial cell, or any combination thereof. In some aspects, the periocular cells are retroocular cells.

[0635] In some cases, application is to the periorbital and / or retroorbital tissues (e.g., via periorbital injection). In other cases, application is to the periorbital and / or retroorbital muscles via periorbital injection.

[0636] In some cases, administration via retroocular injection is to the posterior ocular muscles (e.g., the retroocular space within the extraocular muscle cone). In some cases, administration is to the levator palpebrae superioris muscle. In some cases, administration is to the rectus muscles (e.g., the lateral, medial, superior, or inferior rectus muscles). In some cases, administration is to the oblique muscles (e.g., the inferior or superior oblique muscles).

[0637] In some cases, retroocular injection is administered to the periorbital or retroorbital connective tissue. In some cases, it is administered to fibroblasts in the periorbital or retroorbital connective tissue. In some cases, it is administered to myofibroblasts in the periorbital or retroorbital connective tissue. In some cases, it is administered to adipocytes in the periorbital or retroorbital connective tissue. In some cases, it is administered to myocytes in the periorbital or retroorbital connective tissue.

[0638] In some respects, the delivery vector contains a nucleic acid sequence encoding a therapeutic agent. In some respects, the therapeutic agent is a therapeutic protein. In some respects, the therapeutic protein is a therapeutic antibody. In some respects, the therapeutic antibody is selected from the group consisting of: anti-IGF-1R, anti-VEGF, anti-IL-6, anti-IL-2, anti-IL-17A, anti-TNF-α, anti-CD28, or antigen-binding fragments thereof. In some respects, the therapeutic antibody is selected from the group consisting of: tocilizumab, teprotumumab, bevacizumab, ranibizumab, faricimab, infliximab, daclizumab, secukinumab, or antigen-binding fragments thereof. In some respects, the therapeutic antibody is an anti-IGF-1R antibody or its antigen-binding fragment. In some respects, the therapeutic antibody is teprotumumab. In some respects, therapeutic antibodies include: (i) VH CDR 1–3 (e.g., (1) SEQ ID NO: 9–11; (2) SEQ ID NO: 12–14, or (3) SEQ ID NO: 12, 15, and 16) and VL CDR 1–3 (e.g., (1) SEQ ID NO: 17–19; (2) SEQ ID NO: 17, 20, and 21; or (3) SEQ ID NO: 17, 22, and 23); (ii) VH (e.g., SEQ ID NO: 24–26) and VL (e.g., SEQ ID NO: 29–31); (iii) HC (e.g., SEQ ID NO: 40–42) and LC (e.g., SEQ ID NO: 46–48); or (iv) containing SEQ ID NO: 12, 15, and 16. NO: Any of 67 to 75, wherein the vector construct or expression construct (e.g., antibody expression cassette) further comprises one or more of IRES, furin cleavage site, 2A site, dual promoter (e.g., promoter-VH-IRES-VL, etc.) or signal peptide (e.g., IL-2 or IL-10 signal peptide).

[0639]

VA2. Viral Vector

[0640] In some aspects, the delivery vector containing DNA of interest (e.g., nucleic acids encoding a therapeutic agent of interest (e.g., an antibody or an antigen-binding fragment thereof, such as an anti-IGF-1R antibody or an antigen-binding fragment thereof)) is a viral vector. In some aspects, the viral vector used according to this disclosure consists of viral particles derived from naturally occurring viruses, which have been genetically modified to be viral replication-deficient and express recombinant genes according to this disclosure. Once the virus delivers its genetic material into a cell, it does not produce additional infectious virus, but rather introduces exogenous recombinant genes into the cell, preferably into the cell's genome.

[0641] In some respects, the viral vector is a retrovirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia virus, and poliovirus vector. In some respects, retroviral vectors are less preferred because retroviruses require replicating cells, and target organs may include slowly replicating and / or terminally differentiated cells. In some respects, adenovirus and AAV are preferred viral vectors because these viruses effectively infect slowly replicating and / or terminally differentiated cells. In some respects, the delivery vector (e.g., a viral vector) is selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, or retroviral vectors.

[0642] Using a replication-defective virus as a viral vector, infectious viral particles containing DNA or RNA corresponding to the DNA of interest can be generated by introducing a viral construct into a recombinant cell line, which provides the missing component essential for viral replication. In some aspects, transformation of a recombinant cell line with a recombinant viral vector does not result in the generation of replicating viruses, for example, through homologous recombination of the viral sequence of the recombinant cell line with the introduced viral vector. Methods for generating replication-defective viral particles containing the nucleic acid of interest are known in the art and 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).

[0643] [VI. Adeno-associated virus (AAV)-mediated gene therapy]

[0644] In some aspects, this disclosure relates to periocular delivery of a vector comprising DNA of interest (e.g., nucleic acid encoding a therapeutic agent of interest (e.g., an antibody or an antigen-binding fragment thereof, such as an anti-IGF-1R antibody or an antigen-binding fragment thereof)), wherein said vector is an AAV vector. AAV belongs to the genus Dependent Virus (DVV). Dependovirus AAV is a small parvovirus with several characteristics 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. AAV has not been associated with any human or animal diseases and does not appear to alter the physiological characteristics of host cells after integration. Finally, AAV is stable under a wide range of physical and chemical conditions, which is beneficial for production, storage, and transportation requirements.

[0645] The AAV genome, a linear single-stranded DNA molecule, contains approximately 4,700 nucleotides (the AAV-2 genome contains 4,681 nucleotides) and typically includes an internal non-repetitive region flanked by inverted terminal repeats (ITRs). The ITR is approximately 145 nucleotides long (AAV-1 has a 143-nucleotide ITR) and has multiple functions, including serving as an origin of replication and as a packaging signal for the viral genome.

[0646] The non-repetitive portion of the genome comprises two large open reading frames (ORFs) called the AAV replication (rep) and capsid (cap) regions. These ORFs encode replication and capsid gene products, respectively: these products (i.e., proteins) allow for the replication, assembly, and packaging of the complete AAV virion. More specifically, at least four viral protein families are expressed from the AAV rep region: Rep 78, Rep 68, Rep 52, and Rep 40, all named for their readily apparent molecular weights. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.

[0647] AAV is a helper-dependent virus, requiring co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) to form a fully functional AAV virion. Without 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 a free form, but does not produce infectious virions. Subsequent helper virus infection "rescues" the integrated genome, allowing it to replicate and be packaged into the viral capsid, thus reconstructing an infectious virion. Although AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Therefore, for example, human AAV will replicate in canine cells that have already been infected with canine adenovirus.

[0648] In some respects, to generate recombinant AAV (rAAV) virions containing DNA of interest, 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, the wt AAV gene (called the cofactor functional gene, which includes rep and cap) and the helper virus functional gene (called the helper function gene) can be provided in one or more plasmids, thus eliminating the need for wt AAV and helper viruses in rAAV virion production. The cofactor and helper function gene products are expressed in the host cell, where they act on the rAAV vector containing the heterologous gene. The heterologous gene is then replicated and packaged as if it were the wt AAV genome, forming recombinant AAV virions. When patient cells are transduced with the resulting rAAV virions, the DNA enters and is expressed in the patient cells. Because the patient cells lack the rep and cap genes and the helper function genes, the rAAV virions cannot further replicate and package their genome. Furthermore, without the source of the rep and cap genes, wt AAV virions cannot form in the patient's cells. See, for example, U.S. Patent Application Publication No. 2003 / 0147853.

[0649] In some aspects, the AAV vector of this disclosure may comprise or be derived from any natural or recombinant AAV serotype. According to this disclosure, the AAV serotype may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, and AAV12. In some aspects, the AAV vector is the AAV8 serotype. In some aspects, the AAV vector is the AAV9 serotype. In some respects, AAV carriers are AAV13, AAV14, AAV15, AAV16, AAVS3, AAVrh.74, AAV type 9e, AAVrh.20, AAVrh.39, AAVrh.46, AAVrh.73, AAVrh.74, AAVhu.12, AAVhu.21, AAVhu.26, AAVhu.37, AAVhu.51, AAV.RHM4-1, AAV.Anc80, AAV.7m8, AAV.PHP.B, The serotypes are AAV.PHP.eB, AAV2.5, AAV2tYF, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16. In some aspects, the AAV vector is modified relative to the wild-type AAV serotype sequence. In some aspects, the modified AAV vector is a modified AAV6 vector. In some aspects, the modified AAV6 is an AAV6-RGD vector. In some respects, the AAV6-RGD vector also contains modified amino acids corresponding to Y705, Y731, T492, and K531 (e.g., Y705, Y731F, T492V, and K531E; also referred to as AAV-RGD-Y705-731F+T492V+K531E). In some respects, the AAV serotype is a synthetic serotype (e.g., AAV-DJ, AAV-PHP.B, AAV2-ESGHGYF, AAVM41, AAV-LK03, AAV2-BR1, AAV587MTP, AAV-Anc80L65, AAV2-7m8, AAV2HBKO, AAV2YF, AAV6-RGD, or AAV6.2 capsid). In some respects, the AAV serotype is AAV8. In some respects, the AAV serotype is AAV9. In some respects, the AAV serotype is AAV6.In some respects, the AAV serotype is modified AAV6, such as AAV6 containing the RGD peptide (AAV6-RGD) or AAV6 containing a mutation of surface-exposed tyrosine residues as described, for example, in Sayroo et al. Gene Ther. 2016 January; 23(1):18-25. In some respects, the AAV serotype is AAV-DJ.

[0650] Some aspects of the present invention relate to an AAV delivery vector comprising a polynucleotide and an antibody expression cassette, the antibody expression cassette containing a nucleic acid encoding a therapeutic agent. In some aspects, the therapeutic agent is suitable for the treatment of ophthalmic pathology. In some aspects, the therapeutic agent is an antibody. In some aspects, the therapeutic agent is an anti-IGF-1R antibody.

[0651] In some aspects, the AAV carriers disclosed herein are suitable for delivery to ophthalmic regions (i.e., intraocular and / or periocular regions). In some aspects, the AAV carriers are suitable for delivery to ophthalmic regions (i.e., intraocular and / or periocular regions) by injection (e.g., periocular injection).

[0652] In some respects, the AAV vector disclosed herein can be administered in combination with one or more other therapeutic agents.

[0653] In some aspects, the AAV carriers described herein can be delivered in combination with one or more other therapeutic agents (e.g., simultaneously or sequentially). In some aspects, the one or more other therapeutic agents can be administered before, simultaneously with, or after the administration of the AAV carriers described herein.

[0654]

VI.A.1. AAV carrier components

[0655] In some aspects, the AAV vector, construct, or nucleic acid of the present invention comprises a flanking ITR, a promoter, a nucleic acid sequence encoding a therapeutic agent, and a poly(A) sequence. In some aspects, the AAV vector, construct, or nucleic acid of the present invention further comprises an adapter. In some aspects, the AAV vector, construct, or nucleic acid of the present invention comprises two or more promoters.

[0656] In some aspects, the expression cassette contains nucleic acids having a 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: 67-75.

[0657] In some aspects, the vectors, constructs, nucleic acids or expression cassettes of the present invention contain nucleic acid sequences corresponding to SEQ ID NO: 52-75.

[0658] In some aspects, the vector or construct of the present invention comprises a backbone, for example, including an origin of replication (oriR) and / or an antibiotic resistance gene. In some aspects, the backbone comprises an origin of replication of the coliform E1 gene (ColE1) and / or a kanamycin resistance gene (KanR). In some aspects, the backbone is adapted for use in an AAV payload vector (e.g., an expression cassette comprising 5' and 3' ITRs). In some aspects, the backbone may be a puc57 backbone or a modified version thereof. In some aspects, the backbone comprises a filler sequence.

[0659]

VI.A.2. Inverted Terminal Repeat (ITR)

[0660] In some aspects, the AAV vector of the present invention (e.g., an rAAV vector) comprises a viral genome having at least one ITR region and a payload region, such as a polynucleotide encoding a therapeutic protein, an antibody or an antigen-binding fragment thereof, such as an anti-IGF-1R antibody or an antigen-binding fragment thereof. In some aspects, the AAV vector comprises an antibody expression cassette disclosed herein. In some aspects, the AAV vector has two ITRs (i.e., a pair of ITRs). The two ITRs are flanked at the 5' and 3' ends of the payload region (e.g., the antibody expression cassette). The ITR functions as an origin of replication, which includes a replication recognition site. The ITR contains complementary and symmetrically arranged sequence regions. The ITRs incorporated into the AAV vector of the present invention may consist of naturally occurring polynucleotide sequences or recombinant-derived polynucleotide sequences.

[0661] ITRs may originate from the same serotype as the capsid, selected from any serotype listed herein, or derivatives thereof. ITRs may be serotypes different from the capsid. In some aspects, the AAV vector has more than one ITR. In a non-limiting example, the AAV vector has a viral genome comprising two ITRs. In some aspects, the ITRs are of the same serotype as each other. In some aspects, the ITRs are of different serotypes. Non-limiting examples include zero, one, or two ITRs having the same serotype as the capsid. In some aspects, both ITRs of the AAV vector are AAV2 ITRs.

[0662] Independently, each ITR can be approximately 75 to approximately 175 nucleotides in length. ITRs can be approximately 100 to 105 nucleotides in length, approximately 106 to 110 nucleotides in length, approximately 111 to 115 nucleotides in length, approximately 116 to 120 nucleotides in length, approximately 121 to 125 nucleotides in length, approximately 126 to 130 nucleotides in length, approximately 131 to 135 nucleotides in length, approximately 136 to 140 nucleotides in length, approximately 141 to 145 nucleotides in length, or approximately 146 to 150 nucleotides in length. In some aspects, the ITR is approximately 140 to 142 nucleotides in length. Non-limiting examples of ITR lengths are approximately 102, approximately 140, approximately 141, approximately 142, approximately 145 nucleotides in length, and those having at least 95% identity with such lengths.

[0663] In some aspects, the AAV vector contains a nucleic acid sequence encoding a therapeutic agent, such as a therapeutic antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment, which may be located near the 5' end of the inverted ITR in the vector. In some aspects, the AAV vector contains a nucleic acid sequence encoding a therapeutic agent, such as a therapeutic antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment, which may be located near the 3' end of the inverted ITR in the vector. In some aspects, the AAV vector contains a nucleic acid sequence encoding a therapeutic agent, such as a therapeutic antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment, which may be located near the 5' end of the inverted ITR in the vector. In some aspects, the AAV vector contains a nucleic acid sequence encoding a therapeutic agent, such as a therapeutic antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment, which may be located near the 3' end of the inverted ITR in the vector. In some aspects, the AAV vector comprises a nucleic acid sequence encoding a therapeutic agent, such as a therapeutic antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment, which may be located between the 5' end of the inverted ITR and the 3' end of the inverted ITR in the vector. In other aspects, the AAV vector comprises a nucleic acid sequence encoding a therapeutic agent, such as a therapeutic antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment, which may be located between the 3' end of the inverted ITR and the 5' end of the inverted ITR in the vector (e.g., halfway between the 5' end of the inverted ITR and the 3' end of the inverted ITR or halfway between the 3' end of the inverted ITR and the 5' end of the inverted ITR).

[0664] In some aspects, the AAV vector comprises a nucleic acid sequence encoding a therapeutic agent, such as a therapeutic antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment, which may be located downstream or upstream of the 5' or 3' end of the ITR (e.g., an inverted or flipped ITR) for 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 nucleotides.

[0665] As another non-limiting example, the AAV vector comprises a nucleic acid sequence encoding a therapeutic agent, such as a therapeutic antibody, like an anti-IGF-1R antibody or its antigen-binding fragment, which may be located downstream or upstream of the 5' or 3' end of the ITR (e.g., an inverted or flipped ITR) for 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 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.

[0666] In some aspects, the AAV vector contains a nucleic acid sequence encoding a therapeutic agent (e.g., a therapeutic antibody, such as an anti-IGF-1R antibody or its antigen-binding fragment), said therapeutic agent being located at the first 1%, first 2%, first 3%, first 4%, first 5%, first 6%, first 7%, first 8%, first 9%, first 10%, first 15%, first 20%, first 25% or more of the nucleotides upstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the vector.

[0667] As another non-limiting example, the AAV vector contains a nucleic acid sequence encoding a therapeutic agent (e.g., a therapeutic antibody, such as an anti-IGF-1R antibody or its antigen-binding fragment), said therapeutic agent being located at the first 1%, first 2%, first 3%, first 4%, first 5%, first 6%, first 7%, first 8%, first 9%, first 10%, first 15%, first 20%, first 25%, or more of the first 1% to first 25%, first 20%, or first 25%, of the first 1% to first 25%, first 20%, or first 25%, of the first 1% to first 25%, first 25%, or first 25%, of the first 1% to first 25%, first 25%, or first 25%, of the first 1% to first 25%, first 25%, first 25%, or first 25%, of the first 1% to first 25%, first 25%, or first 25%, of the first 1% to first 25%, first 25%, or first 25%, of the first 10% to first 25%, first 25%, or first 25%, of the first 15% to first 25%, or first 25%, of the first 15% to first 25%, or first 25%, of the first 15% to first 25%, or first 25%, of the first 2 ...%, or first 25%, of the first

[0668] In some respects, the 5' and 3' ITRs comprise the 5' ITRs and 3' ITRs disclosed in Table 16. In some respects, the 5' and 3' ITRs comprise SEQ ID NO: 143 and 144, respectively.

[0669] [VI.A.3. Promoters and Enhancers]

[0670] In some respects, the payload region of an AAV vector contains at least one element to enhance nucleic acid specificity and / or expression. Non-limiting examples of elements that enhance nucleic acid specificity and expression include, for example, promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers, and introns. In some respects, the enhancer is a CMV enhancer.

[0671] The expression of the nucleic acids of the present invention may require specific promoters after delivery or integration into the genomic DNA of target cells, including but not limited to species-specific, inducible, tissue-specific or cell cycle-specific promoters (Parr et al., Nat. Med. 3:1145-9 (1997); the entire contents of which are incorporated herein by reference).

[0672] In some respects, a promoter is considered effective when it drives the expression of a nucleic acid encoding a protein (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or antigen-binding fragment), the protein being carried by the payload region of an AAV vector. In other respects, a promoter is considered effective when it drives the expression of a protein (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or antigen-binding fragment), where the targeted cells are ophthalmic (i.e., ocular or periorbital) cells.

[0673] Promoters can be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters and mammalian promoters. In some aspects, promoters can be human promoters. In some aspects, promoters can be truncated. Promoters that drive or promote expression in most tissues include, but are not limited to, cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC). In some aspects, promoters are CMV early enhancer / chicken β-actin (CAG) promoters, CAG, CBA, smCBA, CMV, human elongation factor 1α subunit (EF1α), EF1α with a CMV enhancer, CMV promoters with a CMV enhancer (CMVe / p), CMV promoters with SV40 introns, or tissue-specific promoters.

[0674] In some respects, the promoter is the CAG, CBA, or smCBA promoter. In some respects, the promoter is the CAG promoter (e.g., SEQ ID NO: 155). In some respects, the promoter is the smCBA promoter (e.g., SEQ ID NO: 145).

[0675] In some respects, tissue-specific promoters are eye tissue-specific promoters. In other respects, eye tissue-specific promoters are the human rhodopsin kinase (GRK1) promoter, the mouse pyramidal inhibition (CAR) promoter, or the human red opsin (RedO) promoter.

[0676] In some respects, tissue-specific expression elements can be used to restrict expression to certain cell types.

[0677] Non-limiting examples of muscle-specific promoters include the mammalian creatine kinase (MCK) promoter, the mammalian desmin (DES) promoter, the HSA 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, the mammalian tropomyosin I (TNNI2) promoter, and the mammalian skeletal muscle α-actin (ASKA) promoter (see, for example, U.S. Patent Publication US 20110212529, the entire contents of which are incorporated herein by reference). Non-limiting examples of tissue-specific expression elements in neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synaptophysin (Syn), methyl-CpG-binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabolite glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin small gene ηβ2, proenkephalin (PPE), enkephalin (Enk), and the excitatory amino acid transporter 2 (EAAT2) promoter. Non-limiting examples of tissue-specific expression elements in astrocytes include glial fibrillary acidic protein (GFAP) and the EAAT2 promoter.

[0678] Non-limiting examples of tissue-specific expression elements in oligodendrocytes include the myelin basic protein (MBP) promoter.

[0679] 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.

[0680] Non-limiting examples of tissue-specific expression elements in adipocytes include the adiponectin promoter and the adipocyte fatty acid-binding protein (AP2) promoter.

[0681] In some respects, the promoter is an inducible promoter. In other respects, the promoter is a bidirectional promoter.

[0682] In some respects, a promoter can be a combination of two or more components of the same or different initiating or parental promoters, including but not limited to CMV, CAG, EF1a, and CBA. In some respects, the promoter is the CMV early enhancer / chicken β-actin (CAG) promoter, CAG, CBA, CMV, EF1α, EF1α with a CMV enhancer, CMV promoter with a CMV enhancer (CMVe / p), or CMV promoter with an SV40 intron.

[0683] In some respects, the promoter composition comprises a CMV enhancer, a CBA promoter, and a CAG intron.

[0684] In some respects, AAV vectors contain ubiquitous promoters. Non-limiting examples of ubiquitous promoters include, for example, CMV, CBA (including derivatives CAG, CBh, etc.), EF-la, PGK, UBC, GUSB (hGBp), and UCOE (HNRPA2B1-CBX3 promoter).

[0685] In some respects, the promoter is not cell-specific. In some respects, the promoter is the ubiquitin c (UBC) promoter. The UBC promoter can be 300–350 nucleotides in size. In some respects, the promoter is the β-glucuronidase (GUSB) promoter. In some respects, the promoter is the neurofilament light chain (NFL) promoter. In some respects, the construct can be AAV-promoter-CMV / globin intron-regulatory polynucleotide-RBG, where AAV can be self-complementary and AAV can be DJ serotype.

[0686] In some aspects, the AAV vector contains the Pol III promoter. In some aspects, the AAV vector contains the PI promoter. In some aspects, the AAV vector contains the FXN promoter. In some aspects, the promoter is the phosphoglycerate kinase 1 (PGK) promoter. In some aspects, the promoter is the chicken β-actin (CBA) promoter. In some aspects, the promoter is the CAG promoter, which is a construct containing a cytomegalovirus (CMV) enhancer fused to the chicken β-actin (CBA) promoter, and with chimeric introns. In some aspects, the promoter is the cytomegalovirus (CMV) promoter. In some aspects, the promoter is the EF1α promoter. In some aspects, the promoter is the EF1α promoter fused to the CMV enhancer. In some aspects, the promoter is the CMV promoter fused to the CMV enhancer. In some aspects, the promoter is the CMV promoter fused to the SV40 intron. In some aspects, the AAV vector contains the H1 promoter.

[0687] In some respects, the AAV vector contains an enhancer element, a promoter, and / or a 5'UTR intron. The enhancer element (also referred to herein as an "enhancer") may be, but is not limited to, a CMV enhancer, the promoter may be, but is not limited to, EF1α, CMV, CBA, UBC, GUSB, NSE, Synapsin, MeCP2, and GFAP promoters, and the 5'UTR / intron may be, but is not limited to, a chimera between SV40, CAG, CBA-MVM (mouse parvovirus), human β-globin, immunoglobulin heavy chain, human β-globin, and immunoglobulin heavy chain genes. In some respects, the enhancer is a CMV enhancer. In some respects, the enhancer, promoter, and / or intron 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. 5'UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5'UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) Synapsin promoter; (8) MeCP2 promoter; (9) GFAP promoter; (10) H1 promoter; (11) U6 promoter; (12) CMV promoter, CMV enhancer; (13) EF1α promoter, CMV enhancer; or (14) CMV promoter, SV40 intron; (15) Human β-globin intron and immunoglobulin heavy chain intron chimera, EF1α promoter, CMV enhancer, CMV promoter, SV40 intron. In some respects, the promoter is the cytomegalovirus (CMV) promoter. In some respects, the intron is the SV40 intron, MVM intron, or human β-globin intron in the vector. In some respects, the promoter is the CBA promoter. In some respects, the promoter is the EF1α promoter. In some respects, the promoter is a CMV promoter fused with a CMV enhancer. In some respects, the promoter is a CMV enhancer fused with an EF1α promoter. In some respects, the promoter is a CMV promoter fused with an SV40 intron. In some respects, the AAV vector contains an engineered promoter. In some respects, the AAV vector contains a CMV early enhancer / chicken β-actin (CAG) promoter. In some respects, the AAV vector contains a promoter derived from a naturally expressed protein.

[0688]

VI.A.4. Untranslated Region (UTR)

[0689] The wild-type untranslated region (UTR) of a gene is transcribed but not translated. Typically, the 5' UTR begins at the transcription start site and ends at the start codon, while the 3' UTR begins immediately after the stop codon and continues until the transcription is terminated.

[0690] Features commonly found in genes that are highly expressed in specific target organs can be engineered into the UTR to enhance the stability and production of transcripts.

[0691] The wild-type 5' untranslated region (UTR) includes features that play a role in translation initiation. The Kozak sequence, known to be involved in the ribosomal initiation of translation of many genes, is typically included in the 5' UTR. The Kozak sequence has a concordant sequence CCRRCCAUGG, where R is a purine (adenine or guanine) located three bases upstream of the start codon (ATG), followed by another 'G'. In some aspects, the 5' UTR of the AAV vectors of this disclosure includes the Kozak sequence. In other aspects, the 5' UTR of the AAV vectors of this disclosure does not include the Kozak sequence.

[0692] Wild-type 3'UTRs are known to contain extensions of adenosine and uridine. These AU-enriched features are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-enriched elements (AREs) can be classified into three classes (Chen et al., 1995, the entire contents of which are incorporated herein by reference). Class I AREs, such as but not limited to c-Myc and MyoD, contain several scattered AUUUA motifs in their U-enriched regions. Class II AREs, such as but not limited to GM-CSF and TNF-α, have two or more overlapping UUAUUUA(U / A) nonamers. Class III AREs, such as but not limited to c-Jun and myogenin, are less well-defined. These U-enriched regions do not contain AUUUA motifs. Most proteins that bind to AREs are known to destabilize messengers, while members of the ELAV family, especially HuR, have been documented to increase mRNA stability. HuR binds AREs of all three classes. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding in vivo, thereby stabilizing the information.

[0693] The introduction, removal, or modification of 3'UTR AU enrichment elements (AREs) can be used to modulate the stability of polynucleotides. When engineering specific polynucleotides, such as payload regions of a viral genome, one or more copies of AREs can be introduced to make the polynucleotide less stable, thereby limiting translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability and thus increase translation and the production of the resulting protein.

[0694] In some aspects, the 3'UTR of the AAV vector of this disclosure may include an oligo (dT) sequence for adding a poly-A tail. In some aspects, the AAV vector of this disclosure may include at least one miRNA seed, binding site, or full-length sequence. A microRNA (or miRNA or miR) is a 19-25 nucleotide non-coding RNA that binds to a nucleic acid target site and downregulates gene expression, either by reducing the stability of the nucleic acid molecule or by inhibiting translation. The microRNA sequence contains a "seed" region, i.e., a sequence in the 2-8 region of the mature microRNA, which has perfect Watson-Crick complementarity with the miRNA target sequence of the nucleic acid.

[0695] In some respects, the AAV vectors disclosed herein can be engineered to include, alter, or remove at least one miRNA binding site, sequence, or seed region.

[0696] Any UTR of any gene known in the art may be incorporated into the AAV vector of this disclosure. These UTRs, or portions thereof, may be positioned in the same orientation as the gene from which they are selected, or may be altered in orientation or position. In some aspects, the UTRs used in the AAV vectors of this disclosure may be reversed, shortened, lengthened, or made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term "alteration" means, in relation to a UTR, that the UTR is altered in some way relative to a reference sequence. For example, a 3' or 5' UTR may be altered relative to a wild-type or native UTR by changing its orientation or position as described above, or by including additional nucleotides, deletions, substitutions, or translocations of nucleotides. In some aspects, the AAV vectors of this disclosure contain at least one artificial UTR that is not a variant of a wild-type UTR. In some aspects, the AAV vectors of this disclosure contain UTRs selected from transcript families whose proteins share common functions, structures, characteristics, or properties.

[0697]

VI.A.5. Polyadenylation sequence

[0698] In some aspects, the AAV vectors or expression cassettes of this disclosure contain at least one polyadenylated sequence. The AAV vectors of this disclosure may contain a polyadenylated sequence between the 3' end of the payload coding sequence and the 5' end of the 3' ITR.

[0699] In some respects, the length of the polyadenylated sequence, or "poly(A) sequence," can range from non-existent to approximately 500 nucleotides.

[0700] In some respects, the lengths of the polyadenylated sequences are approximately 10–100, 10–90, 10–80, 10–70, 10–60, 10–55, 10–50, 20–100, 20–90, 20–80, 20–70, 20–60, 20–55, 20–50, 30–100, 30–90, and 30. ~80, about 30~70, about 30~60, about 30~55, about 30~50, about 40~100, about 40~90, about 40~80, about 40~70, about 40~60, about 40~55, about 40~50, about 45~100, about 45~90, about 45~80 or about 45~70, about 45~60, about 45~55, about 45~50 nucleotides.

[0701] In some aspects, the AAV vector or expression cassette contains nucleic acid encoding a protein of interest (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment), which may be located upstream of a polyadenylated sequence in the vector. In other aspects, the AAV vector contains nucleic acid encoding a protein of interest (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment), which may be located downstream of a promoter (such as, but not limited to, EF1α, CMV, U6, CAG, CBA, EF1α with a CMV enhancer, a CMV promoter with an SV40 intron, a CMV promoter with a CMV enhancer, or a CBA promoter with an SV40 intron, an MVM intron, a human β-globulin intron, an immunoglobulin heavy chain intron, or a chimera of a human β-globulin intron and an immunoglobulin heavy chain intron).

[0702] In some respects, the AAV vector or expression cassette contains nucleic acid encoding a protein of interest (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment), which may be located downstream of the promoter in the vector for 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 and / or polyadenylated sequences.

[0703] In some aspects, the AAV vector or expression cassette contains a rabbit globulin polyadenylation (poly A) signal sequence. In some aspects, the AAV vector or expression cassette contains a human growth hormone polyadenylation (poly A) signal sequence. In some aspects, the AAV vector or expression cassette contains a human growth hormone polyadenylation (poly A) (hGHpA) signal sequence. In some aspects, the AAV vector contains a bovine growth hormone polyadenylation (poly A) (bGHpA) signal sequence. In some aspects, the AAV vector contains a synthetic (SYN) polyadenylation (poly A) (SYNpA) signal sequence.

[0704]

VI.A.6. Introns

[0705] In some aspects, the payload region of the AAV vector disclosed herein includes at least one element for enhanced expression, such as one or more introns or portions thereof. Non-limiting examples of introns include MVM (67–97 bps), F.IX truncated intron 1 (300 bps), β-globulin SD / immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor / immunoglobulin splice acceptor (500 bps), SV40 late splice donor / splicing acceptor (19S / 16S) (180 bps), and hybrid adenovirus splice donor / IgG splice acceptor (230 bps). In some aspects, non-limiting examples of introns include, but are not limited to, SV40 introns, CAG introns, or chimeric introns that may be a chimera of human β-globulin introns and human immunoglobulin heavy chain introns.

[0706] In some respects, the length of an intron or intronic portion can be between approximately 100 and approximately 500 nucleotides. In other respects, an intron can have a length between approximately 80–100, approximately 80–120, approximately 80–140, approximately 80–160, approximately 80–180, approximately 80–200, approximately 80–250, approximately 80–300, approximately 80–350, approximately 80–400, approximately 80–450, approximately 80–500, approximately 200–300, approximately 200–400, approximately 200–500, approximately 300–400, approximately 300–500, or approximately 400–500 nucleotides.

[0707] In some aspects, the AAV vector or expression cassette may contain chimeric introns. In some aspects, the AAV vector or expression cassette may contain SV40 introns. In some aspects, the AAV vector or expression cassette may contain immunoglobulin heavy chain introns. In some aspects, the AAV vector or expression cassette may contain human β-globulin introns. In some aspects, the AAV vector or expression cassette may contain a chimera of human β-globulin introns and immunoglobulin heavy chain introns.

[0708] In some aspects, the encoded therapeutic agent of interest (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment) may be located downstream of a promoter in an expression vector (such as, but not limited to, CMV, U6, HI, CBA, CAG, or a CBA promoter having introns (such as SV40, MVM introns, human β-globulin introns, human immunoglobulin heavy chain introns, chimeras of human β-globulin introns and human immunoglobulin heavy chain introns, or other introns known in the art). In some aspects, the introns are selected from the group consisting of SV40 introns, CAG introns, MVM introns, human β-globulin introns, human immunoglobulin heavy chain introns, or chimeras of human β-globulin introns and human immunoglobulin heavy chain introns.

[0709] In addition, the encoded therapeutic agent of interest (e.g., an antibody or its antigen-binding fragment, such as an anti-IGF-1R antibody or its antigen-binding fragment) can also be located upstream of the polyadenylated sequence in the expression vector. In some respects, the encoded therapeutic protein, such as an antibody (e.g., a monoclonal antibody) or its antigen-binding fragment or fusion protein (e.g., an Fc fusion protein), or therapeutic peptide, may be located downstream of the promoter in the vector for 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 and / or polyadenylated sequences.

[0710]

VI.A.7. Filling Sequences

[0711] In some aspects, the AAV vector includes one or more filler sequences (also referred to as "filler sequences"). In some aspects, the AAV vector includes one or more filler sequences to make the length of the AAV vector the optimal size for the package. In some aspects, the AAV vector includes at least one filler sequence to make the length of the AAV vector about 2.0 to 2.5 kb, for example about 2.3 kb. In some aspects, the vector skeleton includes filler sequences.

[0712] In some respects, AAV vectors contain one or more filler sequences to reduce the likelihood that hairpin structures of the vector genome (such as regulatory polynucleotides described herein) will be read as inverted terminal repeats (ITRs) during expression and / or packaging.

[0713] In some respects, the AAV vector is a single-stranded (ss) AAV vector and contains one or more filler sequences with a length between approximately 0.1 kb and approximately 3.8 kb.

[0714] In some respects, the AAV vector is a self-complementary (sc) AAV vector and contains one or more filler sequences having a length between about 0.1 kb and about 1.5 kb.

[0715] In some respects, the AAV vector contains any portion of the filler sequence. The vector may contain, 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.

[0716] In some aspects, the AAV vector is a single-stranded (ss) AAV vector and contains one or more filler sequences to make the length of the AAV vector approximately 4.6 kb. In some aspects, the AAV vector contains at least one filler sequence located at the 3' of the 5' ITR sequence. In some aspects, the AAV vector contains at least one filler sequence located at the 5' of the promoter sequence. In some aspects, the AAV vector contains at least one filler sequence located at the 3' of the polyadenylation signal sequence. In some aspects, the AAV vector contains at least one filler sequence located at the 5' of the 3' ITR sequence. In some aspects, the AAV vector contains at least one filler sequence located between two intron sequences. In some aspects, the AAV vector contains at least one filler sequence located within an intron sequence. In some aspects, the AAV vector contains two filler sequences, with the first filler sequence located at the 3' of the 5' ITR sequence and the second filler sequence located at the 3' of the polyadenylation signal sequence. In some aspects, the AAV vector contains two filler sequences, with the first filler sequence located at the 5' of the promoter sequence and the second filler sequence located at the 3' of the polyadenylation signal sequence. In other aspects, the AAV vector contains two filler sequences, with the first filler sequence located at the 3' of the 5' ITR sequence and the second filler sequence located at the 5' of the 5' ITR sequence.

[0717] In some aspects, the AAV vector is a self-complementary (sc) AAV vector and contains one or more filler sequences to make the length of the AAV vector about 2.3 kb. In some aspects, the AAV vector contains at least one filler sequence and the filler sequence is located at the 3' of the 5' ITR sequence. In some aspects, the AAV vector contains at least one filler sequence and the filler sequence is located at the 5' of the promoter sequence. In some aspects, the AAV vector contains at least one filler sequence and the filler sequence is located at the 3' of the polyadenylation signal sequence. In some aspects, the AAV vector contains at least one filler sequence and the filler sequence is located at the 5' of the 3' ITR sequence.

[0718] In some aspects, the AAV vector contains at least one filler sequence located between two intron sequences. In some aspects, the AAV vector contains at least one filler sequence located within an intron sequence. In some aspects, the AAV vector contains two filler sequences, with a first filler sequence located at the 3' of the 5' ITR sequence and a second filler sequence located at the 3' of the polyadenylation signal sequence. In some aspects, the AAV vector contains two filler sequences, with a first filler sequence located at the 5' of the promoter sequence and a second filler sequence located at the 3' of the polyadenylation signal sequence. In some aspects, the AAV vector contains two filler sequences, with a first filler sequence located at the 3' of the 5' ITR sequence and a second filler sequence located at the 5' of the 5' ITR sequence.

[0719] In some aspects, the AAV vector may include one or more filler sequences between one or more regions of the AAV vector. In some aspects, the filler region may be located before the region, such as, but not limited to, the payload region, ITR, promoter region, intron region, enhancer region, and / or polyadenylation signal sequence region. In some aspects, the filler region may be located after the region, such as, but not limited to, the payload region, ITR, promoter region, intron region, enhancer region, and / or polyadenylation signal sequence region. In some aspects, the filler region may be located both before and after the region, such as, but not limited to, the payload region, ITR, promoter region, intron region, enhancer region, and / or polyadenylation signal sequence region.

[0720] In some aspects, the AAV vector may include one or more filling sequences that bifurcate at least one region of the AAV vector. The bifurcation region of the AAV vector may contain 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 in the filling sequence region at 5'.

[0721] In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 10% of the region is located at the 5' end of the padding sequence and approximately 90% of the region is located at the 3' end of the padding sequence. In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 20% of the region is located at the 5' end of the padding sequence and approximately 80% of the region is located at the 3' end of the padding sequence. In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 30% of the region is located at the 5' end of the padding sequence and approximately 70% of the region is located at the 3' end of the padding sequence. In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 40% of the region is located at the 5' end of the padding sequence and approximately 60% of the region is located at the 3' end of the padding sequence. In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 50% of the region is located at the 5' end of the padding sequence and approximately 50% of the region is located at the 3' end of the padding sequence. In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 60% of the region is located at the 5' end of the padding sequence and approximately 40% of the region is located at the 3' end of the padding sequence. In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 70% of the region lies at the 5' end of the padding sequence and approximately 30% of the region lies at the 3' end of the padding sequence. In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 80% of the region lies at the 5' end of the padding sequence and approximately 20% of the region lies at the 3' end of the padding sequence. In some aspects, the padding sequence may cause at least one region to bifurcate, such that approximately 90% of the region lies at the 5' end of the padding sequence and approximately 10% of the region lies at the 3' end of the padding sequence.

[0722] In some aspects, the AAV vector includes a padding sequence after the 5' ITR. In some aspects, the AAV vector includes a padding sequence after the promoter region. In some aspects, the AAV vector includes a padding sequence after the payload region. In some aspects, the AAV vector includes a padding sequence after the intron region. In some aspects, the AAV vector includes a padding sequence after the enhancer region. In some aspects, the AAV vector includes a padding sequence after the polyadenylation signal sequence region. In some aspects, the AAV vector includes a padding sequence before the promoter region. In some aspects, the AAV vector includes a padding sequence before the payload region. In some aspects, the AAV vector includes a padding sequence before the intron region.

[0723] In some aspects, the AAV vector includes a padding sequence preceding the enhancer region. In some aspects, the AAV vector includes a padding sequence preceding the polyadenylation signaling sequence region. In some aspects, the AAV vector includes a padding sequence preceding the 3' ITR. In some aspects, the padding sequence may be located between two regions, such as, but not limited to, the 5' ITR and the promoter region. In some aspects, the padding sequence may be located between two regions, such as, but not limited to, the 5' ITR and the payload region.

[0724] In some aspec...

Claims

1. A method for treating an ophthalmic pathology in a subject who requires treatment, comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, wherein the administration is a periocular injection.

2. The method according to claim 1, wherein the periorbital injection is a periorbital injection or a retro-septal injection.

3. The method according to claim 2, wherein the periorbital injection is a subcapsular injection, retroocular injection, subconjunctival injection, or periorbital injection.

4. The method according to claim 2 or 3, wherein the periorbital injection is a retroocular injection.

5. The method according to claim 2 or 3, wherein the periorbital injection is a periocular injection.

6. The method according to any one of claims 1 to 5, wherein the AAV carrier contact is selected from the orbital tissue or extraocular cavity of the following: extraocular adipose tissue, levator lateralis muscle, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, optic nerve, blood vessel, sclera, dura mater sheath, cribriform plate, eyelid, lacrimal gland, lacrimal sac, tear film, or any combination thereof.

7. The method according to any one of claims 1 to 6, wherein the AAV carrier contact is selected from the orbital tissue or extraocular cavity of the following: extraocular adipose tissue, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, or any combination thereof.

8. A method of delivering a pharmaceutical composition comprising an AAV carrier to orbital tissue or an extraocular cavity, comprising periocular injection of the AAV carrier, wherein the orbital tissue or extraocular cavity is selected from the following: extraocular adipose tissue, levator lateralis muscle, lateral rectus muscle, medial lateral rectus muscle, superior lateral rectus muscle, inferior lateral rectus muscle, inferior lateral oblique muscle, superior lateral oblique muscle, optic nerve, blood vessel, sclera, dura mater sheath, cribriform plate, eyelid, lacrimal gland, lacrimal sac, tear film, or any combination thereof.

9. The method of claim 8, wherein the orbital tissue or extraocular cavity is selected from the following: extraocular adipose tissue, lateral rectus muscle, medial rectus muscle, superior rectus muscle, inferior rectus muscle, inferior oblique muscle, superior oblique muscle, or any combination thereof.

10. The method according to any one of claims 1 to 9, wherein the administration or delivery comprises injecting a volume of about 0.1 mL to about 10 mL of the pharmaceutical composition per eye periorbital area.

11. The method according to any one of claims 1 to 9, wherein the administration or delivery comprises injecting a volume of 0.1 mL to 1 mL of the pharmaceutical composition per eye periorbital area.

12. The method according to any one of claims 1 to 11, wherein the administration comprises injecting a volume of 0.1 mL to 0.5 mL of the pharmaceutical composition per eye periorbital area.

13. The method according to any one of claims 1 to 12, wherein the administration comprises injecting a volume of 0.3 mL to 0.5 mL of the pharmaceutical composition per eye periorbital area.

14. The method according to any one of claims 2 to 13, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AAV carrier contacts the eye or periocular tissue.

15. The method according to any one of claims 1 to 14, wherein the AAV vector transduces target ocular or periocular cells.

16. The method according to any one of claims 1 to 15, wherein the AAV vector transduction efficiency is at least 5% (e.g., 5% to 95%, 10% to 95%, or 25% to 95%) in one or more target eye or periocular cells.

17. The method according to claim 15 or 16, wherein the target eye or periorbital cells are selected from: fibroblasts, adipocytes, myofibroblasts, myocytes, muscle cells, epithelial cells, neuroepithelial cells, glial cells, or any combination thereof.

18. The method according to any one of claims 1 to 16, wherein the AAV vector comprises a capsid selected from the serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVVRh8, AAVrh9, AAV9, AAVrh10, AAV10, AAV11, AAV12 and modified versions thereof.

19. The method of claim 18, wherein the capsid serotype is AAV8 or AAV9.

20. The method according to any one of claims 1 to 19, wherein the AAV vector comprises a capsid, a vector genome, and an expression cassette.

21. The method of claim 20, wherein the vector genome comprises inverted terminal repeats (ITRs).

22. The method of claim 20 or 21, wherein the expression cassette comprises a nucleic acid sequence encoding a therapeutic agent.

23. The method of claim 22, wherein the therapeutic agent comprises an antibody.

24. The method of claim 23, wherein the antibody comprises an anti-IGF-1R antibody or an antigen-binding fragment thereof.

25. The method according to claim 23 or 24, wherein the antibody is selected from the group consisting of tocilizumab, teprotumumab, bevacizumab, ranibizumab, faricimab, infliximab, daclizumab, secukinumab, or antigen-binding fragments thereof.

26. The method according to any one of claims 23 to 25, wherein the antibody is teprotumumab.

27. The method according to any one of claims 1 to 7 or 10 to 26, wherein the ophthalmic pathology is an ocular disease or a periorbital disease.

28. The method of claim 27, wherein the ophthalmic pathology is selected from the following: age-related macular degeneration, Behçet's disease, Bietti's crystalline dystrophy, blepharitis, blepharospasm, iris defects, corneal conditions, diabetic retinopathy, dry eye, glaucoma, idiopathic intracranial hypertension, macular edema, ocular histoplasmosis syndrome, conjunctivitis, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt's disease, thyroid eye disease, Usher syndrome, uveitis, orbital carcinoma, or any combination thereof.

29. The method according to any one of claims 1 to 7 or 10 to 28, wherein the ophthalmic pathology is thyroid ophthalmopathy.

30. The method according to any one of claims 1 to 29, wherein the administration is a single dose.

31. The method of claim 30, wherein a single dose is administered at a single injection site.

32. The method of claim 30, wherein a single dose is administered at multiple injection sites.

33. The method according to any one of claims 1 to 32, wherein the administration is in multiple doses.

34. The method of claim 33, wherein each of the multiple doses is administered at a single injection site.

35. The method of claim 33, wherein each of the multiple doses is administered at a different injection site.

36. The method according to any one of claims 1 to 35, wherein the application is bilateral or unilateral.

37. The method of claim 36, wherein the administration includes bilateral periorbital injection.

38. A method of treating thyroid ophthalmopathy in a subject with this need, comprising administering to the subject a pharmaceutical composition comprising an AAV carrier, said AAV carrier comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof. It is administered via periorbital injection. The antibody or its antigen-binding fragment is selected from the group consisting of anti-IGF-1R antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-11 antibody, anti-IL-11R antibody, anti-FcRn antibody, anti-TSHR antibody or its antigen-binding fragment.

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

40. The method of claim 38 or 39, wherein the administration is a single dose.

41. The method of claim 40, wherein a single dose is administered at a single injection site.

42. The method of claim 40, wherein a single dose is administered at multiple injection sites.

43. The method according to claim 38 or 39, wherein the administration is in multiple doses.

44. The method of claim 43, wherein each of the multiple doses is administered at a single injection site.

45. The method of claim 43, wherein each of the multiple doses is administered at a different injection site.

46. ​​The method according to any one of claims 38 to 45, wherein the application is bilateral or unilateral.

47. The method according to any one of claims 38 to 46, The antibody is an anti-IGF-1R antibody. Optionally, the anti-IGF-1R antibody or its antigen-binding fragment is teprotumumab.

48. The method according to any one of claims 38 to 47, The anti-IGF-1R antibody or its antigen-binding fragment contains a heavy chain variable region (VH) and a light chain variable region (VL). VH includes VH complementarity-determining regions (CDRs) 1, VH CDR 2, and VH CDR 3. VH CDR1 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 9 or 12; VH CDR2 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 10, 13, or 15; and VH CDR3 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 11, 14, or 16; VL includes VL complementarity determination regions (CDR1), VL CDR2, and VL CDR3. VL CDR1 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 17; VL CDR2 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 18, 20, or 22; and VL CDR3 is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 19, 21, or 23.

49. The method of claim 48, wherein the heavy chain variable region is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 24-26.

50. The method according to claim 48 or 49, wherein the light chain variable region is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 29-31.

51. The method according to any one of claims 48 to 50, wherein the anti-IGF-1R antibody comprises a heavy chain and a light chain, wherein the heavy chain is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 40 to 42.

52. The method according to any one of claims 48 to 51, wherein the light chain is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 46 to 48.

53. The method according to any one of claims 38 to 46, wherein the antibody is an anti-IL-6 antibody, optionally wherein the anti-IL-6 antibody or its antigen-binding fragment is tocilizumab, cetuximab, olokizumab, clazakizumab, sirukumab, or levilimab.

54. The method according to any one of claims 38 to 46, wherein the antibody is an anti-IL-6R antibody, optionally wherein the anti-IL-6 antibody or its antigen-binding fragment is sarilumab or satralizumab.

55. The method according to any one of claims 38 to 46, wherein the antibody is an anti-IL-11 antibody, optionally wherein the anti-IL-11 antibody or its antigen-binding fragment is 9MW3811.

56. The method according to any one of claims 38 to 46, wherein the antibody is an anti-IL-11-R antibody, optionally wherein the anti-IL-11R antibody or its antigen-binding fragment is BI 765423 or LASN01.

57. The method according to any one of claims 38 to 46, wherein the antibody is an anti-FcRn antibody, optionally wherein the anti-FcRn antibody or its antigen-binding fragment is batoclimab, evgartigimod, rozanolixizumab, nipocalimab, orilanolimab, or IMVT-1402.

58. The method according to any one of claims 38 to 46, wherein the antibody is an anti-TSHR antibody, and optionally, wherein the anti-TSHR antibody or its antigen-binding fragment is K1-70.

59. The method according to any one of claims 1 to 52, wherein the AAV vector comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

53.

60. The method according to any one of claims 38 to 59, wherein the AAV vector comprises an expression cassette, the expression cassette comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-IGF-1R antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-IL-11 antibody, anti-IL-11R antibody, anti-FcRn antibody, anti-TSHR antibody, or an antigen-binding fragment thereof.

61. The method of claim 60, wherein the promoter is selected from the group consisting of CMV early enhancer / chicken β-actin (CAG) promoter, CAG, CBA, smCBA, CMV, human elongation factor 1α subunit (EF1α), EF1α with CMV enhancer, CMV promoter with CMV enhancer (CMVe / p), CMV promoter with SV40 intron, or tissue-specific promoter.

62. The method of claim 60 or 61, wherein the promoter is a CAG promoter.

63. The method of claim 60 or 61, wherein the promoter is the smCBA promoter.

64. The method according to any one of claims 60 to 63, wherein the expression cassette further comprises introns.

65. The method of claim 62, wherein the intron is selected from the group consisting of SV40 introns, MVM introns, CAG introns, or human β-globulin introns.

66. The method according to any one of claims 38 to 63, wherein the nucleic acid sequence encoding an anti-IGF-1R antibody or an antigen-binding fragment thereof further comprises a nucleic acid encoding a signal peptide.

67. The method of claim 66, wherein the signal peptide is an IL-2 signal peptide or an IL-10 signal peptide.

68. The method according to any one of claims 60 to 67, wherein the expression cassette further comprises a poly(A) sequence.

69. The method of claim 68, wherein the polymer (A) is a synthetic polymer (A) or a bovine growth hormone (BGH) polymer (A).

70. The method according to any one of claims 38 to 69, wherein the AAV vector comprises a capsid of a serotype 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.

71. The method of claim 70, wherein the capsid serotype is AAV8 or AAV9.

72. The method according to any one of claims 38 to 71, wherein the AAV vector comprises a capsid, a vector genome, and an expression cassette.

73. The method of claim 72, wherein the vector genome comprises inverted terminal repeats (ITRs).

74. The method according to any one of claims 1 to 73, wherein the AAV vector is a recombinant AAV (rAAV) comprising an AAV9 capsid encapsulating a vector genome comprising a nucleic acid coding sequence of an anti-IGF-1R antibody or an antigen-binding fragment thereof (optionally teprotumumab) and side-attached with a pair of inverted terminal repeats (ITRs).

75. The method according to any one of claims 1 to 74, wherein each eye of the subject receives about 6E12 to about 2E13 vector genomes (vg), optionally administered unilaterally or bilaterally.

76. The method according to any one of claims 1 to 52 or 70 to 75, wherein the AAV vector 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 with SEQ ID NO:

142.

77. The method according to any one of claims 22 to 76, wherein the concentration of the therapeutic agent in the ocular muscles of the subject is at least about 18 µg / mL and / or the serum concentration of the therapeutic agent is less than about 6 µg / mL.

78. The method according to claim 77, wherein the concentration of the therapeutic agent in the ocular tissue is about 18 µg / mL to 180 µg / mL, about 18 µg / mL to 100 µg / mL, about 18 µg / mL to 60 µg / mL, or about 18 µg / mL to 30 µg / mL.

79. A method for expressing teprotumumab in the eye of a subject, comprising administering an rAAV vector to the subject, the rAAV vector comprising an AAV9 capsid encapsulating a vector genome comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof for teprotumumab, and having a pair of inverted terminal repeats (ITRs) attached to the side. The procedure involves periocular injection. The concentration of teprotumumab in ocular tissue must be at least 18 µg / mL. Optionally, the ocular tissue is the eye muscle.

80. The method of claim 79, wherein teprotumumab is present in the serum of the subject at a concentration of less than 6 µg / mL.

81. The method according to claim 79 or 80, wherein the concentration in the ocular tissue is about 18 µg / mL to 180 µg / mL, about 18 µg / mL to 100 µg / mL, about 18 µg / mL to 60 µg / mL, or about 18 µg / mL to 30 µg / mL.

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