Treatment of ocular diseases using recombinant viral vectors encoding anti-VEGF FABs

Abstract

Provided herein are methods for treating neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of a steroid.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 412,087, filed September 30, 2022, and U.S. Provisional Application No. 63 / 421,741, filed November 2, 2022, the disclosures of each of which are incorporated by reference herein in their entirety and for which priority is claimed.

[0002] Sequence Listing This application contains a computer readable sequence listing that has been submitted herewith in XML file format, the disclosure of which is incorporated herein by reference in its entirety. The sequence listing XML file submitted herewith is entitled "12656-177-228_SEQ_LISTING.xml", was created on September 20, 2023, and is 80,588 bytes in size.

[0003] 1. Field Provided herein are methods for treating neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR) in a subject in need thereof, the methods comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of a steroid. [Background technology]

[0004] 2.Background The human eye is a highly complex, well-developed sensory organ that is prone to a host of diseases and disorders. Approximately 285 million people worldwide are visually impaired, of which 39 million are blind and 246 million have moderate to severe vision impairment (World Health Organization, 2012, World Data on Visual Impairment 2010, Geneva: World Health Organization). Some of the leading causes of blindness include cataracts (47%), glaucoma (12%), age-related macular degeneration (AMD) (9%), and diabetic retinopathy (5%) (World Health Organization, 2007, Global Initiative to End Avoidable Blindness: Plan of Action 2006-2011, Geneva: World Health Organization).

[0005] Many ocular diseases and disorders with ocular pathological symptoms can be traced to genetic alterations or protein dysregulation (Stone et al., 2017, Ophthalmology 124(9):1314-1331). Recent advances in genomics and proteomics have had a profound impact on understanding the disease mechanisms and / or genetic basis underlying such ocular diseases or symptoms. Gene therapy has been used in the treatment of certain ocular diseases (see, e.g., International Patent Application No. PCT / US2017 / 027650 (WO 2017 / 181021)). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Patent Application No. PCT / US2017 / 027650 (International Publication No. 2017 / 181021) [Non-patent literature]

[0007] [Non-Patent Document 1] Stone et al., 2017, Ophthalmology 124(9):1314–1331 Summary of the Invention [Problem to be solved by the invention]

[0008] There is a significant unmet medical need for therapies that specifically address the underlying genetic abnormalities to treat ocular pathologies. [Means for solving the problem]

[0009] (3. Overview) Provided herein are methods for treating neovascular age-related macular degeneration (nAMD) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of a steroid.

[0010] Also provided herein is a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye, and the steroid treatment comprises administering a therapeutically effective amount of a steroid to the subject's eye.

[0011] Also provided herein are methods of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of triamcinolone acetonide.

[0012] Also provided herein is a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of difluprednate.

[0013] In certain embodiments of the methods provided herein, the method is a method for treating neovascular age-related macular degeneration (nAMD). In certain embodiments, the method is a method for treating diabetic retinopathy (DR).

[0014] In certain embodiments of the methods provided herein, a recombinant viral vector is administered to the suprachoroidal space of the subject's eye. In certain embodiments, the recombinant viral vector is administered by injection into the suprachoroidal space of the eye using a suprachoroidal drug delivery device. In certain embodiments, the suprachoroidal drug delivery device is a microinjector. In other embodiments, the recombinant viral vector is administered to the subretinal space of the subject's eye. In certain embodiments, the method does not include performing a vitrectomy on the subject's eye. In certain embodiments, the subretinal administration includes performing a vitrectomy on the subject's eye. In certain embodiments, the vitrectomy is a partial vitrectomy. In other embodiments, the recombinant viral vector is administered to the subretinal space via the suprachoroidal space of the subject's eye. In certain embodiments, the recombinant viral vector is administered using a subretinal drug delivery device that includes a catheter that can be inserted and tunneled through the suprachoroidal space toward the posterior pole, where a thin needle injects into the subretinal space. In certain embodiments, the anti-hVEGF treatment involves catheterizing and tunneling a subretinal drug delivery device through the suprachoroidal space to administer a recombinant viral vector.

[0015] In certain embodiments, the steroid treatment comprises administering a therapeutically effective amount of a corticosteroid. In certain embodiments of the methods provided herein, the corticosteroid is triamcinolone acetonide. In other embodiments, the corticosteroid is difluprednate. In certain embodiments of the methods provided herein, the steroid is triamcinolone acetonide. In other embodiments, the steroid is difluprednate.

[0016] In certain embodiments of the methods provided herein, the anti-hVEGF treatment comprises administering a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye, and the steroid treatment comprises administering triamcinolone acetonide to the subject's eye. In certain embodiments, triamcinolone acetonide is administered after the recombinant viral vector is administered. In other embodiments, triamcinolone acetonide is administered before the recombinant viral vector is administered. In certain embodiments, triamcinolone acetonide is administered to the subject's eye within about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute after the administration of the recombinant viral vector. In certain embodiments, triamcinolone acetonide is administered to the subject's eye by injection. In certain embodiments, triamcinolone acetonide is administered to the subject's eye by a single injection. In certain embodiments, steroid treatment consists of a single injection of triamcinolone acetonide to the subject's eye. In certain embodiments, triamcinolone acetonide is administered to a different quadrant of the eye than the recombinant viral vector. In certain embodiments, triamcinolone acetonide is administered to the sub-Tenon's capsule of the eye. In certain embodiments, triamcinolone acetonide is administered at a dose of about 40 mg. In certain embodiments, triamcinolone acetonide is administered in a volume of about 1 mL.

[0017] In certain embodiments of the methods provided herein, the anti-hVEGF treatment comprises administering a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye, and the steroid treatment comprises administering difluprednate to the subject's eye. In certain embodiments, difluprednate is administered daily to the subject's eye. In certain embodiments, the steroid treatment comprises administering difluprednate four times daily. In certain embodiments, difluprednate is administered four times daily for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. In certain embodiments, difluprednate is administered four times daily for about 4 weeks. In certain embodiments, the steroid treatment comprises administering difluprednate three times daily. In certain embodiments, difluprednate is administered three times daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week. In certain embodiments, difluprednate is administered three times daily for about one week. In certain embodiments, the steroid treatment comprises administering difluprednate twice daily. In certain embodiments, difluprednate is administered twice daily for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least one week. In certain embodiments, difluprednate is administered twice daily for about one week. In certain embodiments, the steroid treatment comprises administering difluprednate once daily. In certain embodiments, difluprednate is administered once daily for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least one week. In certain embodiments, difluprednate is administered once daily for about one week. In certain embodiments, difluprednate is administered to the subject's eye for a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or at least 7 weeks. In certain embodiments, difluprednate is administered to the subject's eye for a period of about 7 weeks.In certain embodiments, the steroid treatment comprises administering difluprednate once on the first day of steroid treatment, followed by four times a day for about four weeks, followed by three times a day for about one week, followed by twice a day for about one week, followed by once a day for about one week. In certain embodiments, the steroid treatment comprises administering difluprednate once on the first day of steroid treatment, followed by four times a day for about four weeks, followed by three times a day for about one week, followed by twice a day for about one week, followed by once a day for about one week. In certain embodiments, the difluprednate is administered in the form of an ophthalmic emulsion. In certain embodiments, the ophthalmic emulsion comprises 0.5 mg / mL (0.05%) difluprednate. In certain embodiments, each administration of difluprednate comprises instilling one drop of the ophthalmic emulsion into the subject's eye. In certain embodiments, each administration of difluprednate consists of instilling one drop of ophthalmic emulsion into the subject's eye. In certain embodiments, difluprednate is first administered to the subject's eye within about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day after administration of the recombinant viral vector. In certain embodiments, difluprednate is first administered to the subject's eye on the same day that the recombinant viral vector is administered. In certain embodiments, the first administration of difluprednate occurs after the first administration of the recombinant viral vector.

[0018] In certain embodiments of the methods provided herein, the anti-hVEGF antigen-binding fragment is a Fab, F(ab')2, or a single-chain variable fragment (scFv).

[0019] In certain embodiments, the anti-hVEGF antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 and a light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3. In certain embodiments, the anti-hVEGF antigen-binding fragment comprises (a) a heavy chain comprising heavy chain CDR1-3 of the amino acid sequence of SEQ ID NO:2 and (b) a light chain comprising light chain CDR1-3 of the amino acid sequence of SEQ ID NO:1. In certain embodiments, the anti-hVEGF antigen-binding fragment comprises (a) a heavy chain comprising heavy chain CDR1-3 of the amino acid sequence of SEQ ID NO:4 and (b) a light chain comprising light chain CDR1-3 of the amino acid sequence of SEQ ID NO:3. In certain embodiments, the anti-hVEGF antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NO:14-16 or SEQ ID NOs:14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NO:17-19 or SEQ ID NOs:20, 18, and 21. In certain embodiments, administration of a recombinant viral vector delivers a therapeutically effective amount of the anti-hVEGF antigen-binding fragment to the retina of the human subject. In certain embodiments, the therapeutically effective amount of the anti-hVEGF antigen-binding fragment is produced by the subject's retinal cells. In certain embodiments, the recombinant viral vector is an rAAV vector. In certain embodiments, the recombinant viral vector is an rAAV8 vector.

[0020] In certain embodiments, the recombinant viral vector comprises an expression cassette encoding an anti-hVEGF antigen-binding fragment, the expression cassette being flanked by AAV2 inverted terminal repeats (ITRs), the expression cassette comprising: CB7 promoter consisting of chicken β-actin promoter and CMV enhancer, chicken β-actin intron, IL-2 signal peptide, a heavy chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2; a self-cleaving furin (F) / F2A linker, a second IL-2 signal peptide, and The light chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding Rabbit β-globin poly(A) signal Includes.

[0021] In certain embodiments, the recombinant viral vector comprises the nucleotide sequence of SEQ ID NO:56.

[0022] In certain embodiments, the recombinant viral vector is administered to about 2.5 x 10 eyes. 11 In certain embodiments, the recombinant viral vector is administered at a dose of about 5.0 x 10 genome copies per eye. 11 In certain embodiments, the recombinant viral vector is administered at a dose of about 1.0 x 10 genome copies per eye. 12 It is administered in a dose of genome copies.

[0023] Also provided herein is a kit for use in the methods of treating neovascular age-related macular degeneration (nAMD) provided herein, the kit comprising a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment and a steroid.

[0024] Also provided herein is a kit for use in the methods of treating diabetic retinopathy (DR) provided herein, the kit comprising a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment and a steroid.

[0025] In certain embodiments of the methods provided herein, the recombinant viral vector is formulated to be suitable for administration to the suprachoroidal space of the eye of a subject.In certain embodiments, the recombinant viral vector is formulated to be suitable for administration to the subretinal space of the eye of a subject.In certain embodiments, the steroid is triamcinolone acetonide.In certain embodiments, the steroid is difluprednate.

[0026] Also provided herein is the use of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment and a steroid in the manufacture of a medicament for the treatment of neovascular age-related macular degeneration (nAMD) as provided herein.Also provided herein is the use of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment and a steroid in the manufacture of a medicament for the treatment of diabetic retinopathy (DR) as provided herein.

[0027] In certain embodiments of the methods provided herein, the recombinant viral vector is formulated to be suitable for administration to the suprachoroidal space of the eye of a subject.In certain embodiments, the recombinant viral vector is formulated to be suitable for administration to the subretinal space of the eye of a subject.In certain embodiments, the steroid is triamcinolone acetonide.In certain embodiments, the steroid is difluprednate.

[0028] 3.1 Exemplary Embodiments 1. A method of treating neovascular age-related macular degeneration (nAMD) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; a. the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; b. the steroid treatment comprises administering a therapeutically effective amount of a steroid to the eye of the subject; method. 2. A method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; a. The anti-hVEGF treatment comprises administering a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye; b. the steroid treatment comprises administering a therapeutically effective amount of a steroid to the eye of the subject; method. 3. A method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; a. the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; b. the steroid treatment comprises administering a therapeutically effective amount of triamcinolone acetonide to the subject's eye; method. 4. A method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; a. the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; b. the steroid treatment comprises administering a therapeutically effective amount of difluprednate to the eye of the subject; method. 5. The method of any one of embodiments 2-4, which is a method of treating neovascular age-related macular degeneration (nAMD). 6. The method of any one of embodiments 2-4, which is a method of treating diabetic retinopathy (DR). 7. The method of any one of embodiments 1 or 3-6, wherein the recombinant viral vector is administered to the suprachoroidal space of the subject's eye. 8. The method of any one of embodiments 1 to 7, wherein the recombinant viral vector is administered by injection into the suprachoroidal space of the eye using a suprachoroidal drug delivery device. 9. The method of embodiment 8, wherein the suprachoroidal drug delivery device is a microinjector. 10. The method of any one of embodiments 1 or 3-6, wherein the recombinant viral vector is administered to the subretinal space of the subject's eye. 11. The method of embodiment 10, which does not include performing a vitrectomy on the subject's eye. 12. The method of embodiment 10, wherein the subretinal administration comprises performing a vitrectomy on the subject's eye. 13. The method of embodiment 12, wherein the vitrectomy is a partial vitrectomy. 14. The method of embodiment 10 or embodiment 11, wherein the recombinant viral vector is administered to the subretinal space via the suprachoroidal space of the subject's eye. 15. The method of embodiment 14, wherein the recombinant viral vector is administered using a subretinal drug delivery device comprising a catheter that can be inserted and tunneled through the suprachoroidal space toward the posterior pole, where a thin needle injects into the subretinal space. 16. The method of embodiment 15, wherein the anti-hVEGF treatment comprises catheterizing and tunneling a subretinal drug delivery device through the suprachoroidal space to administer a recombinant viral vector. 17. The method of any one of embodiments 1-16, wherein the steroid treatment ameliorates or prevents intraocular inflammation. 18. The method of any one of embodiments 1 to 17, wherein the steroid treatment ameliorates or prevents intraocular inflammation associated with the dose of the recombinant viral vector, the number of suprachoroidal injections, and / or the location of the suprachoroidal injections. 19. The method of any one of embodiments 1, 2, and 4-18, wherein the steroid is administered topically. 20. The method of any one of embodiments 1, 2, and 5-19, wherein the steroid is a corticosteroid. 21. The method of any one of embodiments 1, 2, and 5-20, wherein the steroid is triamcinolone acetonide. 22. The method of any one of embodiments 1, 2, and 5-20, wherein the steroid is difluprednate. 23.a. The anti-hVEGF treatment comprises administering to the suprachoroidal space of the subject's eye a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; b. The method of any one of embodiments 1-3, 5-9, and 17-21, wherein the steroid treatment comprises administering triamcinolone acetonide to the eye of the subject. 24. The method of any one of embodiments 3, 5-21, and 23, wherein triamcinolone acetonide is administered after administering the recombinant viral vector. 25. The method of any one of embodiments 3, 5-21, and 23, wherein triamcinolone acetonide is administered prior to administering the recombinant viral vector. 26. The method of any one of embodiments 3, 5-21, and 23-25, wherein triamcinolone acetonide is administered to the eye of the subject within about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute of administration of the recombinant viral vector. 27. The method of any one of embodiments 3, 5-18, 20, 21, and 23-26, wherein triamcinolone acetonide is administered by injection into the eye of the subject. 28. The method of embodiment 27, wherein triamcinolone acetonide is administered by single injection into the subject's eye. 29. The method of any one of embodiments 3, 5-18, 20, 21, and 23-28, wherein the steroid treatment consists of a single injection of triamcinolone acetonide into the subject's eye. 30. The method of any one of embodiments 3, 5-18, 20, 21, and 23-29, wherein triamcinolone acetonide is administered in a different quadrant of the eye than the recombinant viral vector. 31. The method of any one of embodiments 3, 5-18, 20, 21, and 23-30, wherein triamcinolone acetonide is administered sub-Tenon's capsule of the eye. 32. The method of any one of embodiments 3, 5-18, 20, 21, and 23-31, wherein triamcinolone acetonide is administered at a dose of about 40 mg. 33. The method of any one of embodiments 3, 5-18, 20, 21, and 23-32, wherein triamcinolone acetonide is administered in a volume of about 1 mL. 34.a. The anti-hVEGF treatment comprises administering to the suprachoroidal space of the subject's eye a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; b. the steroid treatment comprises administering difluprednate to the subject's eye; The method of any one of embodiments 1, 2, 4-9, 17-20 and 22. 35. The method of any one of embodiments 4-20, 22, and 34, wherein difluprednate is administered daily to the subject's eye. 36. The method of embodiment 35, wherein the steroid treatment comprises administering difluprednate four times daily. 37. The method of embodiment 36, wherein difluprednate is administered four times daily for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. 38. The method of embodiment 37, wherein difluprednate is administered four times daily for about four weeks. 39. The method of any one of embodiments 35-38, wherein the steroid treatment comprises administering difluprednate three times daily. 40. The method of embodiment 39, wherein difluprednate is administered three times daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week. 41. The method of embodiment 40, wherein difluprednate is administered three times a day for about one week. 42. The method of any one of embodiments 35-41, wherein the steroid treatment comprises administering difluprednate twice daily. 43. The method of embodiment 42, wherein difluprednate is administered twice daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week. 44. The method of embodiment 44, wherein difluprednate is administered twice daily for about 1 week. 45. The method of any one of embodiments 35-44, wherein the steroid treatment comprises administering difluprednate once daily. 46. ​​The method of embodiment 45, wherein difluprednate is administered once daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week. 47. The method of embodiment 46, wherein difluprednate is administered once daily for about 1 week. 48. The method of any one of embodiments 4-20, 22, and 34-47, wherein difluprednate is administered to the eye of the subject for a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or at least 7 weeks. 49. The method of embodiment 48, wherein difluprednate is administered to the eye of the subject for a period of about 7 weeks. 50. The method of embodiment 49, wherein the steroid treatment comprises administering difluprednate once on the first day of steroid treatment, followed by four times a day for about four weeks, followed by three times a day for about one week, followed by twice a day for about one week, followed by once a day for about one week. 51. The method of embodiment 49, wherein the steroid treatment consists of administering difluprednate once on the first day of steroid treatment, followed by four times a day for about four weeks, followed by three times a day for about one week, followed by twice a day for about one week, followed by once a day for about one week. 52. The method of any one of embodiments 4-20, 22, and 34-51, wherein difluprednate is administered in the form of an ophthalmic emulsion. 53. The method of embodiment 52, wherein the ophthalmic emulsion comprises 0.5 mg / mL (0.05%) difluprednate. 54. The method of embodiment 52 or embodiment 53, wherein each administration of difluprednate comprises instilling one drop of ophthalmic emulsion into the subject's eye. 55. The method of embodiment 52 or embodiment 53, wherein each administration of difluprednate consists of instilling one drop of ophthalmic emulsion into the subject's eye. 56. The method of any one of embodiments 4-20, 22, and 34-55, wherein difluprednate is first administered to the subject's eye within about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day of administration of the recombinant viral vector. 57. The method of embodiment 56, wherein difluprednate is first administered to the subject's eye on the same day that the recombinant viral vector is administered. 58. The method of embodiment 56 or embodiment 57, wherein the first administration of difluprednate occurs after the first administration of the recombinant viral vector. 59. The method of any one of embodiments 1-58, wherein the anti-hVEGF antigen-binding fragment is a Fab, F(ab')2, or a single-chain variable fragment (scFv). 60. The method of any one of embodiments 1-59, wherein the anti-hVEGF antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 and a light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3. 61. The method of any one of embodiments 1 to 60, wherein the anti-hVEGF antigen-binding fragment comprises (a) a heavy chain comprising heavy chain CDRs 1 to 3 of the amino acid sequence of SEQ ID NO: 2 and (b) a light chain comprising light chain CDRs 1 to 3 of the amino acid sequence of SEQ ID NO: 1. 62. The method of any one of embodiments 1 to 60, wherein the anti-hVEGF antigen-binding fragment comprises (a) a heavy chain comprising heavy chain CDRs 1 to 3 of the amino acid sequence of SEQ ID NO: 4 and (b) a light chain comprising light chain CDRs 1 to 3 of the amino acid sequence of SEQ ID NO: 3. 63. The method of any one of embodiments 1-62, wherein the anti-hVEGF antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 or SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 17-19 or SEQ ID NOs: 20, 18, and 21. 64. The method of any one of embodiments 1-63, wherein administering the recombinant viral vector delivers a therapeutically effective amount of the anti-hVEGF antigen-binding fragment to the retina of said human subject. 65. The method of embodiment 65, wherein the therapeutically effective amount of the anti-hVEGF antigen-binding fragment is produced by retinal cells of the subject. 66. The method of any one of embodiments 1-65, wherein the recombinant viral vector is an rAAV vector. 67. The method of any one of embodiments 1-66, wherein the recombinant viral vector is an rAAV8 vector. 68. The recombinant viral vector comprises an expression cassette encoding an anti-hVEGF antigen-binding fragment, wherein the expression cassette is flanked by AAV2 inverted terminal repeats (ITRs), and the expression cassette is a. CB7 promoter consisting of chicken β-actin promoter and CMV enhancer; b. chicken β-actin intron, ciIL-2 signal peptide, ii. a heavy chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2; iii. a self-cleaving furin (F) / F2A linker; iv. a second IL-2 signal peptide, and v. The light chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding d. Rabbit β-globin poly(A) signal 68. The method of any one of embodiments 1-67, comprising: 69. The method of any one of embodiments 1-68, wherein the recombinant viral vector comprises the nucleotide sequence of SEQ ID NO: 56. 70. Recombinant viral vectors were administered at a rate of approximately 2.5 × 10 per eye. 11 The method of any one of embodiments 1-69, wherein the dose is administered in a dose of genome copies. 71. Recombinant viral vectors were administered at a rate of approximately 5.0 × 10 per eye. 11 The method of any one of embodiments 1-69, wherein the dose is administered in a dose of genome copies. 72. Recombinant viral vectors are administered at a rate of approximately 1.0 × 10 per eye. 12 The method of any one of embodiments 1-69, wherein the dose is administered in a dose of genome copies. 73. The method of any one of embodiments 1-72, wherein the recombinant viral vector is administered by double suprachoroidal injection. 74. The method of any one of embodiments 1-72, wherein the recombinant viral vector is administered by a single suprachoroidal injection. 75. A kit for use in the method of treating neovascular age-related macular degeneration (nAMD) of any one of embodiments 1-5 and 7-74, comprising: a. a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; and b. steroids Kit including: 76. A kit for use in the method of treating diabetic retinopathy (DR) of any one of embodiments 2-4 and 6-74, comprising: a. a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; and b. steroids Kit including: 77. The kit of embodiment 75 or embodiment 76, wherein the recombinant viral vector is formulated to be suitable for administration to the suprachoroidal space of the subject's eye. 78. The kit of embodiment 75 or embodiment 76, wherein the recombinant viral vector is formulated to be suitable for administration to the subretinal space of the subject's eye. 79. The kit of any one of embodiments 75-78, wherein the steroid is triamcinolone acetonide. 80. The kit of any one of embodiments 75-78, wherein the steroid is difluprednate. 81. Use of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment and a steroid in the manufacture of a medicament for the treatment of neovascular age-related macular degeneration (nAMD) according to any one of embodiments 1 to 5 and 7 to 74. 82. Use of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment and a steroid in the manufacture of a medicament for the treatment of diabetic retinopathy (DR) according to any one of embodiments 2 to 4 and 6 to 74. 83. The use of embodiment 81 or embodiment 82, wherein the recombinant viral vector is formulated to be suitable for administration to the suprachoroidal space of the subject's eye. 84. The use of embodiment 81 or embodiment 82, wherein the recombinant viral vector is formulated to be suitable for administration to the subretinal space of the subject's eye. 85. The use of any one of embodiments 81-84, wherein the steroid is triamcinolone acetonide. 86. The use of any one of embodiments 81-84, wherein the steroid is difluprednate. [Brief explanation of the drawings]

[0029] 4. Brief description of the drawings [Figure 1] Amino acid sequence of ranibizumab (top; SEQ ID NOS: 2 and 1) showing five different residues in bevacizumab Fab (bottom; SEQ ID NOS: 4 and 3). The start of the variable and constant heavy (VH and CH) and light (VL and VC) chains is indicated by an arrow (→), and the CDRs are underlined. Non-consensus glycosylation sites ("G-sites") and tyrosine-O-sulfation sites ("Y-sites") are indicated. [Figure 2] Glycans that can be attached to HuGlyFabVEGFi (Source: Bondt et al., 2014, Mol & Cell Proteomics 13.1:3029-3039). [Figure 3] Amino acid sequences of hyperglycosylated variants of ranibizumab (top; SEQ ID NOs: 62 and 61) and bevacizumab Fab (bottom; SEQ ID NOs: 4 and 3). The start of the variable and constant heavy chains (VH and CH) and light chains (VL and VC) are indicated by arrows (→), and CDRs are underlined. Non-consensus glycosylation sites ("G sites") and tyrosine-O-sulfation sites ("Y sites") are indicated. Four hyperglycoslated variants are indicated with an asterisk (*). [Figure 4] Schematic diagram of the AAV8-anti-VEGFfab genome. [Figure 5]A subretinal drug delivery device manufactured by Janssen Pharmaceuticals, Inc., which includes a catheter that can be inserted and tunneled through the suprachoroidal space toward the posterior pole, where a thin needle injects into the subretinal space. [Figure 6A] Illustration of the posterior juxtascleral depot procedure. Figure 6A shows a small incision made to expose the sclera, followed by insertion of the cannula tip. Figures 6B, 6C, and 6D show the curved portion of the cannula shaft being inserted while maintaining the cannula tip in direct apposition to the scleral surface. [Figure 6B] Illustration of the posterior juxtascleral depot procedure. Figure 6A shows a small incision made to expose the sclera, followed by insertion of the cannula tip. Figures 6B, 6C, and 6D show the curved portion of the cannula shaft being inserted while maintaining the cannula tip in direct apposition to the scleral surface. [Figure 6C] Illustration of the posterior juxtascleral depot procedure. Figure 6A shows a small incision made to expose the sclera, followed by insertion of the cannula tip. Figures 6B, 6C, and 6D show the curved portion of the cannula shaft being inserted while maintaining the cannula tip in direct apposition to the scleral surface. [Figure 6D] Illustration of the posterior juxtascleral depot procedure. Figure 6A shows a small incision made to expose the sclera, followed by insertion of the cannula tip. Figures 6B, 6C, and 6D show the curved portion of the cannula shaft being inserted while maintaining the cannula tip in direct apposition to the scleral surface. [Figure 7-1] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-2]Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-3] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-4] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-5] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-6] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-7]Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-8] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-9] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-10] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-11] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-12]Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 7-13] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 41-51). Amino acid substitutions (shown in bold in the bottom line) can be made in AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated by "HVR" = hypervariable region. [Figure 8A] A microvolume injector drug delivery device manufactured by Altaviz. Figure 8A depicts a microvolume injector drug delivery device, and Figure 8B depicts the components of the microvolume injector drug delivery device. [Figure 8B] A microvolume injector drug delivery device manufactured by Altaviz. Figure 8A depicts a microvolume injector drug delivery device, and Figure 8B depicts the components of the microvolume injector drug delivery device. [Figure 9A] A drug delivery device manufactured by Visionisti OY. Specifically, FIG. 9A shows an injection adapter that can convert a 30g short subcutaneous needle into a suprachoroidal / subretinal needle. The device can control the length of the needle tip exposed from the distal tip of the adapter. Adjustments can be made in 10 μL increments. The device has the ability to adjust for suprachoroidal delivery and / or subretinal delivery from outside the eye. FIG. 9B shows a needle adapter guide that can hold the eyelid open and hold the needle at the optimal angle and depth for delivery. The needle adapter is secured in a stabilization device. The needle adapter is an all-in-one tool for standardized and optimized in-clinic suprachoroidal and / or subretinal injections. [Figure 9B]A drug delivery device manufactured by Visionisti OY. Specifically, FIG. 9A shows an injection adapter that can convert a 30g short subcutaneous needle into a suprachoroidal / subretinal needle. The device can control the length of the needle tip exposed from the distal tip of the adapter. Adjustments can be made in 10 μL increments. The device has the ability to adjust for suprachoroidal delivery and / or subretinal delivery from outside the eye. FIG. 9B shows a needle adapter guide that can hold the eyelid open and hold the needle at the optimal angle and depth for delivery. The needle adapter is secured in a stabilization device. The needle adapter is an all-in-one tool for standardized and optimized in-clinic suprachoroidal and / or subretinal injections. DETAILED DESCRIPTION OF THE INVENTION

[0030] 5. Detailed Description 5.1 Overview Provided herein are methods for treating neovascular age-related macular degeneration (nAMD) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of a steroid.

[0031] Also provided herein is a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye, and the steroid treatment comprises administering a therapeutically effective amount of a steroid to the subject's eye.

[0032] Also provided herein is a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of triamcinolone acetonide.

[0033] Also provided herein is a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of difluprednate.

[0034] In certain embodiments of the methods provided herein, the method is a method for treating neovascular age-related macular degeneration (nAMD). In certain embodiments, the method is a method for treating diabetic retinopathy (DR).

[0035] In certain embodiments, nAMD and DR can be treated using the methods disclosed in Sections 5.3 and 5.4.

[0036] In certain embodiments, the anti-hVEGF treatment provided herein comprises administering a recombinant viral vector described in Section 5.2. In certain embodiments, the recombinant viral vector is administered as described in Section 5.3. In certain embodiments, the steroid treatment is administered as described in Section 5.4.

[0037] In certain embodiments, the anti-hVEGF treatments provided herein comprise delivery of a fully human post-translationally modified (HuPTM) antibody against VEGF to the retina / vitreous humor of the eye(s) of a patient (human subject) diagnosed with neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR).

[0038] Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intrabodies, heteroconjugate antibodies, monovalent antibodies, and antigen-binding fragments of full-length antibodies and fusion proteins of the above. Such antigen-binding fragments include, but are not limited to, single domain antibodies (heavy chain antibody variable domains (VHH) or nanobodies), Fab, F(ab')2, and scFv (single chain variable fragments) (collectively referred to herein as "antigen-binding fragments") of full-length anti-VEGF antibodies (preferably full-length anti-VEGF monoclonal antibodies (mAbs)). In a preferred embodiment, the fully human post-translationally modified antibody against VEGF is a fully human post-translationally modified antigen-binding fragment of a monoclonal antibody (mAb) against VEGF ("HuPTMFabVEGFi"). In a further preferred embodiment, the HuPTMFabVEGFi is a fully human glycosylated antigen-binding fragment of an anti-VEGF mAb ("HuGlyFabVEGFi"). For compositions and methods that may be used in accordance with the embodiments described herein, see International Patent Application Publication No. WO / 2017 / 180936 (International Patent Application No. PCT / US2017 / 027529, filed April 14, 2017), International Patent Application Publication No. WO2017 / 181021 (International Patent Application No. PCT / US2017 / 027650, filed April 14, 2017), International Patent Application Publication No. WO2019 / 067540 (International Patent Application No. WO2019 / 067540, filed September 26, 2018), and the like. See also International Patent Application No. PCT / US2018 / 052855, filed on 2018 / 09 / 04, International Patent Application Publication No. WO2020 / 206098 (International Patent Application No. PCT / US2020 / 026356, filed April 20, 2020), and International Patent Application Publication No. WO2021 / 041373 (International Patent Application No. PCT / US2020 / 047733, filed August 25, 2020), each of which is incorporated herein by reference in its entirety. In an alternative embodiment, a full-length mAb can be used.Delivery can be achieved via gene therapy, for example, by administering a viral vector or other DNA expression construct encoding an anti-VEGF antigen-binding fragment or mAb (or hyperglycosylated derivative) to the choroidal space, subretinal space (via a transvitreal approach or using a catheter through the choroidal space), the intraretinal space, the vitreous cavity, and / or the outer surface of the sclera (i.e., juxtascleral administration) in the eye(s) of a patient (human subject) diagnosed with neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR), to create a permanent depot in the eye that continuously supplies human PTMs, e.g., human glycosylated transgene products. See, e.g., the modes of administration described in Section 5.3.2.

[0039] In certain embodiments, the patient is known to be responsive to treatment with an anti-VEGF antigen-binding fragment injected intravitreally prior to treatment with gene therapy. In certain embodiments, the patient has previously been treated with Lucentis® (ranibizumab), EYLEA® (aflibercept), and / or Avastin® (bevacizumab), and is known to be responsive to one or more of said Lucentis® (ranibizumab), EYLEA® (aflibercept), and / or Avastin® (bevacizumab).

[0040] Subjects to which such viral vectors or other DNA expression constructs are delivered must be responsive to the anti-VEGF antigen-binding fragment encoded by the transgene in the viral vector or expression construct. To determine responsiveness, the anti-hVEGF antigen-binding fragment transgene product (e.g., produced in cell culture, bioreactor, etc.) can be administered directly to the subject, such as by intravitreal injection.

[0041] The HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, encoded by the transgene can include, but are not limited to, an antigen-binding fragment of an antibody that binds hVEGF, such as bevacizumab; an anti-hVEGF Fab portion, such as ranibizumab; or such a bevacizumab or ranibizumab Fab portion that has been engineered to contain additional glycosylation sites in the Fab domain (see, e.g., Courtois et al., 2016, mAbs 8:99-112, which is incorporated by reference in its entirety, for a description of a bevacizumab derivative that is hyperglycosylated in the Fab domain of the full-length antibody).

[0042] The recombinant vector used to deliver the transgene must have tropism for human retinal cells or photoreceptor cells. Such vectors can include non-replicating recombinant adeno-associated viral vectors ("rAAV"), particularly those with an AAV8 capsid. However, other viral vectors, including but not limited to lentiviral vectors, vaccinia viral vectors, or non-viral expression vectors referred to as "naked DNA" constructs, may also be used. Preferably, the HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, transgene should be controlled by appropriate expression control elements, such as the CB7 promoter (chicken β-actin promoter and CMV enhancer), RPE65 promoter, or opsin promoter, to name a few, and may also contain other expression control elements (e.g., introns, e.g., chicken β-actin intron, minute virus of mouse (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), β-globin, or β-actin introns) that enhance expression of the transgene driven by the vector. These may include splice donor / immunoglobulin heavy chain splice (spice) acceptor introns, adenovirus splice donor / immunoglobulin splice acceptor introns, SV40 late splice donor / splice acceptor (19S / 16S) introns, and hybrid adenovirus splice donor / IgG splice acceptor introns and polyA signals, such as rabbit β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.

[0043] In a preferred embodiment, the gene therapy construct is designed to express both heavy and light chains. More specifically, the heavy and light chains should be expressed in approximately equal amounts, in other words, the heavy and light chains are expressed in approximately a 1:1 ratio of heavy to light. The coding sequences for the heavy and light chains can be engineered into a single construct in which the heavy and light chains are separated by a cleavable linker or IRES, resulting in the expression of separate heavy and light chain polypeptides. For example, see Section 5.2.4 for specific leader sequences and Section 5.2.5 for specific IRES, 2A, and other linker sequences that can be used with the methods and compositions provided herein.

[0044] In certain embodiments, the gene therapy construct is supplied as a freeze-sterilized, single-use solution of the AAV vector active ingredient in a formulation buffer. In certain embodiments, a pharmaceutical composition suitable for subretinal administration comprises a suspension of a recombinant (e.g., rHuGlyFabVEGFi) vector in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In certain embodiments, the construct is formulated in Dulbecco's phosphate-buffered saline and 0.001% Pluronic F68, pH 7.4.

[0045] A therapeutically effective dose of the recombinant vector must be administered subretinally and / or intraretinally (e.g., by subretinal injection via a transvitreal approach (surgical procedure) or subretinal administration via the suprachoroidal space) in a volume ranging from ≧0.1 mL to ≦0.5 mL, preferably 0.1 to 0.30 mL (100 to 300 μl), and most preferably 0.25 mL (250 μl). A therapeutically effective dose of the recombinant vector must be administered suprachoroidally (e.g., by suprachoroidal injection) in a volume of 100 μl or less, for example, 50 to 100 μl. A therapeutically effective dose of the recombinant vector should be administered to the outer surface of the sclera in a volume of 500 μl or less, e.g., in a volume of 500 μl or less, e.g., 10-20 μl, 20-50 μl, 50-100 μl, 100-200 μl, 200-300 μl, 300-400 μl, or 400-500 μl. Subretinal injection is a surgical procedure performed by a trained retinal surgeon and involves partial vitrectomy with the subject under local anesthesia and injection of gene therapy into the retina (see, e.g., Campochiaro et al., 2017, Hum Gen Ther 28(1):99-111, incorporated herein by reference in its entirety). In certain embodiments, subretinal administration is performed via the suprachoroidal space using a subretinal drug delivery device that includes a catheter that can be inserted and tunneled through the suprachoroidal space toward the posterior pole, where a thin needle injects into the subretinal space (see, for example, Baldassarre et al., 2017, Subretinal Delivery of Cells via the Suprachoroidal Space: Janssen Trial, in Schwartz et al. (eds.) Cellular Therapies for Retinal Disease, Springer, Cham; International Patent Application Publication No. 2016 / 040635, each of which is incorporated herein by reference in its entirety).The suprachoroidal administration procedure involves administering a drug to the suprachoroidal space of the eye and is typically performed using a suprachoroidal drug delivery device, such as a microinjector equipped with a microneedle (see, for example, Hariprasad, 2016, Retinal Physician 13:20-23; Goldstein, 2014, Retina Today 9(5):82-87, each of which is incorporated herein by reference in its entirety). Suprachoroidal drug delivery devices that can be used to place expression vectors in the suprachoroidal space according to the embodiments described herein include, but are not limited to, suprachoroidal drug delivery devices manufactured by Clearside® Biomedical, Inc. (see, for example, Hariprasad, 2016, Retinal Physician 13:20-23). Subretinal drug delivery devices that can be used to place an expression vector into the subretinal space via the suprachoroidal space according to the embodiments described herein include, but are not limited to, subretinal drug delivery devices manufactured by Janssen Pharmaceuticals, Inc. (see, for example, International Patent Application Publication No. 2016 / 040635). In certain embodiments, administration to the outer surface of the sclera is performed by a juxtascleral drug delivery device that includes a cannula, the tip of which can be inserted and maintained in direct apposition to the scleral surface. See Section 5.3.2 for more details on various modes of administration. Suprachoroidal, subretinal, juxtascleral, intravitreal, subconjunctival and / or intraretinal administration should result in delivery of a soluble transgene product to the retina, vitreous humor and / or aqueous humor. Expression of the transgene product (e.g., the encoded anti-VEGF antibody) by retinal cells, e.g., rod cells, cone cells, retinal pigment epithelial cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, and / or Müller cells, results in delivery and maintenance of the transgene product in the retina, vitreous humor, and / or aqueous humor. In certain embodiments, the concentration of the transgene product is increased to a C of at least 0.330 μg / mL in the vitreous humor or 0.110 μg / mL in the aqueous humor (anterior chamber of the eye). minA maintenance dose of 1.70-6.60 μg / mL of transgene product is desired for 3 months, followed by a vitreous C min Concentration and / or chamber C in the range of 0.567 to 2.20 μg / mL min Concentrations must be maintained. However, because the transgene product is continuously produced, maintaining lower concentrations may be effective. In certain embodiments, the concentration of the transgene product can be measured in patient samples of vitreous and / or aqueous humor obtained from the anterior chamber of the treated eye. Alternatively, the vitreous concentration can be estimated and / or monitored by measuring the patient's serum concentration of the transgene product—the ratio of systemic to vitreous exposure of the transgene product is approximately 1:90,000. (See, e.g., the vitreous and serum concentrations of ranibizumab reported in Table 5 of Xu L et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616-1624, 1621, and 1623, which is incorporated herein by reference in its entirety.)

[0046] Vector transgenes have the potential to spread to unintended recipients through shedding (release of the vector without infecting target cells and being cleared from the body via feces or bodily fluids), shedding (transgene replication and transfer from target cells), or germline transmission (gene transmission to offspring via semen). Vector shedding can be determined, for example, by measuring vector DNA in biological fluids such as tears, serum, or urine using quantitative polymerase chain reaction. In some embodiments, no vector gene copies are detectable in biological fluids (e.g., tears, serum, or urine) at any time after vector administration. In some embodiments, fewer than 1000, fewer than 500, fewer than 100, fewer than 50, or fewer than 10 vector gene copies / 5 μL are detectable in biological fluids (e.g., tears, serum, or urine) at any time after administration by quantitative polymerase chain reaction. In certain embodiments, no more than 210 vector gene copies / 5 μL are detectable in serum. In some embodiments, fewer than 1000, fewer than 500, fewer than 100, fewer than 50, or fewer than 10 vector gene copies / 5 μL are detectable by quantitative polymerase chain reaction in biological fluids (e.g., tears, serum, or urine) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks after administration. In certain embodiments, no vector gene copies are detectable in serum by 14 weeks after administration of the vector.

[0047] The embodiments described herein have several advantages over standard therapeutic treatments, including repeated ocular injections of high-dose boluses of VEGF inhibitors, resulting in peak and trough levels that dissipate over time. Sustained expression of the transgene product antibody, as opposed to repeated antibody injections, allows for more consistent levels of antibody to be present at the site of action, requiring fewer injections and resulting in fewer visits, which is less risky and more convenient for the patient. Consistent protein production may lead to better clinical outcomes, as rebound edema in the retina is less likely to occur. Furthermore, antibodies expressed from transgenes are post-translationally modified differently from those injected directly due to the different microenvironments present during and after translation. Without being bound by any particular theory, this results in antibodies with different diffusion, bioactivity, distribution, affinity, pharmacokinetics, and immunogenicity characteristics, resulting in "bio-better" antibodies delivered to the site of action compared to directly injected antibodies.

[0048] Furthermore, antibodies expressed from transgenes in vivo are less likely to contain degradation products associated with recombinantly produced antibodies, such as protein aggregation and oxidation. Aggregation is a problem associated with protein production and storage due to high protein concentrations, surface interactions with manufacturing equipment and containers, and purification using certain buffer systems. These conditions that promote aggregation are not present with transgene expression in gene therapy. Oxidation, such as methionine, tryptophan, and histidine oxidation, also occurs during protein production and storage due to stressful cell culture conditions, metal and air contact, and impurities in buffers and excipients. Proteins expressed from transgenes in vivo may also oxidize under stressful conditions. However, humans and many other organisms are equipped with antioxidant defense systems that not only reduce oxidative stress but also sometimes repair and / or reverse oxidation. Therefore, proteins produced in vivo are less likely to be in an oxidized form. Both aggregation and oxidation can affect efficacy, pharmacokinetics (clearance), and immunogenicity.

[0049] The production of HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, should provide a "bio-better" molecule for the treatment of neovascular age-related macular degeneration (nAMD) and / or diabetic retinopathy (DR), and can be achieved via gene therapy, for example, by administering a viral vector or other DNA expression construct encoding a HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, to the choroidal space, subretinal space, or outer surface of the sclera of the eye(s) of a patient (human subject) diagnosed with neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) (e.g., by choroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the choroidal space, or a posterior juxtascleral depot procedure), thereby creating a permanent depot in the eye that continuously delivers fully human, post-translationally modified, e.g., human glycosylated, sulfated, transgene products produced by the transduced retinal cells. The cDNA construct for FabVEGFi must contain a signal peptide that ensures proper co-translational and post-translational processing (glycosylation and protein sulfation) by the transduced retinal cells. Such signal sequences used by retinal cells include, but are not limited to:

[0050] [ka] Examples include:

[0051] For signal peptides that can be used, see, e.g., Stern et al., 2007, Trends Cell. Mol. Biol., 2:1-17 and Daltom & Barton, 2014, Protein Sci, 23:517-525, each of which is incorporated by reference in its entirety.

[0052] As an alternative or additional treatment to gene therapy, HuP™FabVEGFi products, e.g., HuGlyFabVEGFi glycoproteins, can be produced in human cell lines by recombinant DNA technology and administered via intravitreal injection to patients diagnosed with neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR). HuP™FabVEGFi products, e.g., glycoproteins, can also be administered to patients with neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR).

[0053] Human cell lines that can be used for recombinant glycoprotein production include, but are not limited to, human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080, HKB-11, CAP, HuH-7, and retinal cell lines, PER.C6 or RPE, to name a few. (For a review of human cell lines that can be used for the recombinant production of HuP™FabVEGFi products, e.g., HuGlyFabVEGFi glycoproteins, see, e.g., Dumont et al., 2015, Crit. Rev. Biotechnol. (Early Online, published online September 18, 2015, pp. 1-13), "Human cell lines for biopharmaceutical manufacturing: history, status, and future," which is incorporated herein by reference in its entirety.) To ensure complete glycosylation, particularly sialylation and tyrosine sulfation, cell lines used for production can be enhanced by engineering the host cells to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferase) and / or the TPST-1 and TPST-2 enzymes responsible for tyrosine O-sulfation in retinal cells.

[0054] The combination of delivering HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, to the eye / retina simultaneously with the delivery of other available treatments is encompassed by the methods provided herein. The additional treatments can be administered before, simultaneously with, or after the gene therapy treatment. Available treatments for neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) that can be combined with the gene therapy provided herein include, but are not limited to, laser photocoagulation, photodynamic therapy using verteporfin, and intravitreal (IVT) injections using anti-VEGF agents, including, but not limited to, pegaptanib, ranibizumab, aflibercept, or bevacizumab. Additional treatment using anti-VEGF agents, e.g., biologics, is sometimes referred to as "rescue" therapy.

[0055] Unlike small molecule drugs, biologics typically consist of a mixture of numerous variants with different modifications or morphologies that have different potencies, pharmacokinetics, and safety profiles. It is not essential that any molecule produced in either gene therapy or protein therapy approaches be fully glycosylated and sulfated. Rather, the population of glycoproteins produced must have sufficient glycosylation (approximately 1% to approximately 10% of the population), including 2,6-sialylation and sulfation, to demonstrate efficacy. The goal of the gene therapy treatments provided herein is to slow or halt the progression of retinal degeneration and slow or prevent vision loss with minimal intervention / invasive procedures. Efficacy can be monitored by measuring best-corrected visual acuity (BCVA), intraocular pressure, slit-lamp biomicroscopy, indirect ophthalmoscopy, SD-OCT (SD-optical coherence tomography), and electroretinography (ERG). Signs of other safety events, including vision loss, infection, inflammation, and retinal detachment, can also be monitored. Retinal thickening can be monitored to determine the effectiveness of the treatments provided herein. Without being bound by any particular theory, retinal thickening can be used as a clinical readout, and the greater the reduction in retinal thickening or the longer the time to retinal thickening, the more effective the treatment. Retinal thickening can be determined, for example, by SD-OCT, a three-dimensional imaging technique that uses low-coherence interferometry to determine the echo time delay and amplitude of backscattered light reflected from an object of interest. OCT can be used to scan layers of a tissue sample (e.g., the retina) with an axial resolution of 3-15 μm, and SD-OCT improves axial resolution and scanning speed over previous forms of technology (Schuman, 2008, Trans. Am. Opthamol. Soc. 106:426-458). Retinal function can be determined, for example, by ERG. ERG is a non-invasive electrophysiological test of retinal function approved by the FDA for use in humans that examines the light-sensitive cells (rods and cones) of the eye and their connected ganglion cells, specifically their response to flashing light stimuli.

[0056] 5.2 Constructs and Formulations For use in the methods provided herein, viral vectors or other DNA expression constructs encoding anti-VEGF antigen-binding fragments or hyperglycosylated derivatives of anti-VEGF antigen-binding fragments are included. The viral vectors and other DNA expression constructs provided herein include any suitable method for delivering a transgene to target cells (e.g., retinal pigment epithelial cells). Means of transgene delivery include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetic modified mRNA, unmodified mRNA, small molecules, biologically inactive molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a targeted vector, e.g., a vector targeted to retinal pigment epithelial cells.

[0057] In some aspects, the disclosure provides a nucleic acid for use, wherein the nucleic acid encodes a HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, operably linked to a promoter selected from the group consisting of a CB7 promoter (chicken beta-actin promoter and CMV enhancer), a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, an MMT promoter, an EF-1 alpha promoter, a UB6 promoter, a chicken beta-actin promoter, a CAG promoter, an RPE65 promoter, and an opsin promoter. In certain embodiments, the HuPTMFabVEGFi is operably linked to the CB7 promoter.

[0058] In certain embodiments, provided herein are recombinant vectors comprising one or more nucleic acids (e.g., polynucleotides). The nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more sequences selected from the group consisting of a promoter sequence, a sequence of a gene of interest (transgene, e.g., an anti-VEGF antigen-binding fragment), an untranslated region, and a termination sequence. In certain embodiments, the viral vectors provided herein comprise a promoter operably linked to a gene of interest.

[0059] In certain embodiments, the nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein can be codon optimized, for example, by any codon optimization technique known to those of skill in the art (see, e.g., the review by Quax et al., 2015, Mol Cell 59:149-161).

[0060] In a specific embodiment, the construct described herein is Construct I, which comprises the following components: (1) AAV8 inverted terminal repeats flanking an expression cassette, (2) control elements including a) a CB7 promoter containing a CMV enhancer / chicken β-actin promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal, and (3) nucleic acid sequences encoding the heavy and light chains of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, ensuring equal expression of heavy and light chain polypeptides.

[0061] In another specific embodiment, the construct described herein is Construct II, which comprises the following components: (1) AAV2 inverted terminal repeats flanking the expression cassette, (2) control elements including a) a CB7 promoter containing a CMV enhancer / chicken β-actin promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal, and (3) nucleic acid sequences encoding the heavy and light chains of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, ensuring equal expression of heavy and light chain polypeptides.

[0062] In certain embodiments, the construct comprises an expression cassette encoding an anti-hVEGF antigen-binding fragment, the expression cassette being flanked by AAV2 inverted terminal repeats (ITRs), the expression cassette comprising: CB7 promoter consisting of chicken β-actin promoter and CMV enhancer, chicken β-actin intron, IL-2 signal peptide, a heavy chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2; a self-cleaving furin (F) / F2A linker, a second IL-2 signal peptide, and The light chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding Rabbit β-globin poly(A) signal Includes.

[0063] In certain embodiments, the constructs described herein are illustrated in FIG.

[0064] 5.2.1 mRNA In certain embodiments, the vectors provided herein are modified mRNAs encoding a gene of interest (e.g., a transgene, e.g., an anti-VEGF antigen-binding fragment portion). Synthesis of modified and unmodified mRNAs for transgene delivery to retinal pigment epithelial cells is taught, for example, in Hansson et al., J. Biol. Chem., 2015, 290(9):5661-5672, which is incorporated herein by reference in its entirety. In certain embodiments, modified mRNAs encoding an anti-VEGF antigen-binding fragment portion are provided herein.

[0065] 5.2.2 Viral vectors Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV8), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus, vaccinia virus, and retroviral vectors. Retroviral vectors include murine leukemia virus (MLV)- and human immunodeficiency virus (HIV)-based vectors. Alphavirus vectors include Semliki Forest virus (SFV) and Sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors provided herein are modified to be replication-deficient in humans. In certain embodiments, the viral vector is a hybrid vector, e.g., an AAV vector packaged in a "disabled" adenoviral vector. In certain embodiments, provided herein are viral vectors comprising a viral capsid from a first virus and a viral envelope protein from a second virus. In a particular embodiment, the second virus is vesicular stomatitus virus (VSV). In a more particular embodiment, the envelope protein is VSV-G protein.

[0066] In certain embodiments, the viral vectors provided herein are HIV-based viral vectors. In certain embodiments, the HIV-based vectors provided herein comprise at least two polynucleotides, the gag and pol genes being derived from the HIV genome and the env gene being derived from another virus.

[0067] In certain embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In certain embodiments, the herpes simplex virus-based vectors provided herein are modified so that they do not contain one or more immediate early (IE) genes, making them non-cytotoxic.

[0068] In certain embodiments, the viral vector provided herein is an MLV-based viral vector. In certain embodiments, the MLV-based vector provided herein comprises up to 8 kb of heterologous DNA instead of viral genes.

[0069] In certain embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In certain embodiments, the lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are derived from non-human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are packaged in lentiviral capsids. In certain embodiments, the lentiviral vectors provided herein comprise one or more of the following elements: a long terminal repeat sequence, a primer binding site, a polypurine tract, an att site, and an encapsidation site.

[0070] In certain embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In certain embodiments, the alphavirus vectors provided herein are recombinant replication-defective alphaviruses. In certain embodiments, the alphavirus replicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying functional heterologous ligands on their virion surface.

[0071] In certain embodiments, the viral vectors provided herein are AAV-based viral vectors. In a preferred embodiment, the viral vectors provided herein are AAV8-based viral vectors. In certain embodiments, the AAV8-based viral vectors provided herein retain tropism for retinal cells. In certain embodiments, the AAV-based vectors provided herein encode the AAV rep gene (required for replication) and / or the AAV cap gene (required for capsid protein synthesis). Multiple AAV serotypes have been identified. In certain embodiments, the AAV-based vectors provided herein comprise components derived from one or more serotypes of AAV. In certain embodiments, the AAV-based vectors provided herein comprise capsid components derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAVrhlO. In preferred embodiments, the AAV-based vectors provided herein comprise components derived from one or more of the AAV8, AAV9, AAV10, AAV11, or AAVrhlO serotypes.

[0072] In certain embodiments, an AAV8 vector is provided that includes a viral genome comprising an expression cassette for expression of a transgene flanked by ITRs under the control of regulatory elements and a viral capsid having the amino acid sequence of an AAV8 capsid protein, or a viral capsid that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (SEQ ID NO: 48) while retaining the biological function of the AAV8 capsid. In certain embodiments, the encoded AAV8 capsid has the sequence of SEQ ID NO: 48 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions while retaining the biological function of the AAV8 capsid. Figure 7 provides, in the line labeled SUBS, a comparative alignment of the amino acid sequences of capsid proteins of various AAV serotypes with potential amino acids that may be substituted at certain positions in the aligned sequences based on the comparison. Thus, in certain embodiments, an AAV8 vector comprises an AAV8 capsid variant with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions identified in the SUBS line of Figure 7 that are not present at that position in the native AAV8 sequence.

[0073] In certain embodiments, the AAV used in the methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068, which are incorporated by reference in their entireties. In certain embodiments, the AAV used in the methods described herein includes one of the following amino acid insertions: LGETTRP (SEQ ID NO:59) or LALGETTRP (SEQ ID NO:60), as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated by reference in its entirety. In certain embodiments, the AAV used in the methods described herein is AAV.7m8, which is described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282 and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated by reference in its entirety. In certain embodiments, the AAV used in the methods described herein is any AAV disclosed in U.S. Patent No. 9,585,971, such as AAV-PHP.B. In certain embodiments, the AAV used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: U.S. Pat. Nos. 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, 9,284,357, and 9,409,955. Nos. 3, 9,169,299, 9,193,956, 9,458517, and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, and 2017 / 0051257, and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0074] AAV8-based viral vectors are used in certain of the methods described herein. The nucleic acid sequences of AAV-based viral vectors and methods for producing recombinant AAV and AAV capsids are taught, for example, in U.S. Patent No. 7,282,199, U.S. Patent No. 7,790,449, U.S. Patent No. 8,318,480, U.S. Patent No. 8,962,332 and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety. In one aspect, provided herein is an AAV (e.g., AAV8)-based viral vector encoding a transgene (e.g., an anti-VEGF antigen-binding fragment). In certain embodiments, provided herein is an AAV8-based viral vector encoding an anti-VEGF antigen-binding fragment. In more specific embodiments, provided herein is an AAV8-based viral vector encoding ranibizumab.

[0075] In certain embodiments, single-stranded AAV (ssAAV) can be used as described above. In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al., 2001, Gene Therapy, Vol. 8, No. 16, pp. 1248-1254; and U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0076] In certain embodiments, the viral vector used in the methods described herein is an adenovirus-based viral vector. Recombinant adenovirus vectors may be used to deliver anti-VEGF antigen-binding fragments. The recombinant adenovirus may be a first-generation vector with an E1 deletion, with or without an E3 deletion, in which an expression cassette is inserted into either deleted region. The recombinant adenovirus may be a second-generation vector containing a complete or partial deletion of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus inverted terminal repeats and packaging signal (phi). The transgene is inserted between the packaging signal and the 3'ITR, with or without a stuffer sequence, to maintain the genome near the wild-type size of approximately 36 kb. Exemplary protocols for the generation of adenoviral vectors can be found in Alba et al., 2005, "Gutless adenovirus: last generation adenovirus for gene therapy," Gene Therapy 12:S18-S27, which is incorporated herein by reference in its entirety.

[0077] In certain embodiments, the viral vector used in the methods described herein is a lentivirus-based viral vector.Recombinant lentivirus vectors can be used to introduce anti-VEGF antigen-binding fragments.Four plasmids are used to create the construct: a Gag / pol sequence-containing plasmid, a Rev sequence-containing plasmid, an envelope protein-containing plasmid (i.e., VSV-G), and a cis-plasmid carrying packaging elements and the anti-VEGF antigen-binding fragment gene.

[0078] For lentiviral vector production, the four plasmids are co-transfected into cells (i.e., HEK293-based cells), for which polyethyleneimine or calcium phosphate, among others, can be used as transfection agents. The lentivirus in the supernatant is then harvested (lentivirus must bud from the cells to be active, so cell harvesting is not necessary / should not be performed). The supernatant is filtered (0.45 μm), followed by the addition of magnesium chloride and benzonase. Further downstream processes vary, but the most GMP-compliant use is TFF and column chromatography. Others use ultracentrifugation with or without column chromatography. An exemplary protocol for lentiviral vector production is described in Lesch et al., 2011, "Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors." Gene Therapy 18:531-538 and Ausubel et al., 2012, "Production of CGMP-Grade Lentiviral Vectors," Bioprocess Int. 10(2):32-43, both of which are incorporated herein by reference in their entirety.

[0079] In certain embodiments, vectors for use in the methods described herein encode an anti-VEGF antigen-binding fragment (e.g., ranibizumab) such that, upon introduction of the vector into a relevant cell (e.g., a retinal cell in vivo or in vitro), the cell expresses a glycosylated and / or tyrosine-sulfated variant of the anti-VEGF antigen-binding fragment. In certain embodiments, the expressed anti-VEGF antigen-binding fragment comprises a glycosylation and / or tyrosine sulfation pattern.

[0080] 5.2.3 Promoters and Modifiers of Gene Expression In certain embodiments, the vectors provided herein comprise components that modulate gene delivery or gene expression (e.g., "expression control elements"). In certain embodiments, the vectors provided herein comprise components that modulate gene expression. In certain embodiments, the vectors provided herein comprise components that affect cell binding or targeting. In certain embodiments, the vectors provided herein comprise components that affect the localization of a polynucleotide (e.g., a transgene) within a cell after uptake. In certain embodiments, the vectors provided herein comprise components that can be used, for example, as a detectable or selectable marker to detect or select cells that have taken up the polynucleotide.

[0081] In certain embodiments, the viral vectors provided herein comprise one or more promoters. In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is an inducible promoter. Inducible promoters may be preferred so that transgene expression can be turned on and off depending on the desired therapeutic effect. Such promoters include, for example, hypoxia-inducible promoters and drug-inducible promoters, such as promoters induced by rapamycin and related drugs. Hypoxia-inducible promoters include promoters with HIF-binding sites; for teachings of hypoxia-inducible promoters, see, for example, Schodel et al., 2011, Blood 117(23):e207-e217 and Kenneth and Rocha, 2008, Biochem J. 414:19-29, each of which is incorporated by reference. Additionally, hypoxia-inducible promoters that can be used in the constructs include the erythropoietin promoter and the N-WASP promoter (for teachings of hypoxia-inducible promoters, see Tsuchiya, 1993, J. Biochem. 113:395 for a disclosure of the erythropoietin promoter and Salvi, 2017, Biochemistry and Biophysics Reports 9:13-21 for a disclosure of the N-WASP promoter, both of which are incorporated by reference). Alternatively, the construct may contain a drug-inducible promoter, for example, a promoter inducible by administration of rapamycin and related analogs (for disclosure of drug-inducible promoters, see, e.g., International Patent Application Publication Nos. WO 94 / 18317, WO 96 / 20951, WO 96 / 41865, WO 99 / 10508, WO 99 / 10510, WO 99 / 36553, and WO 99 / 41258, and U.S. Patent No. US 7,067,526 (disclosing rapamycin analogs), which are incorporated herein by reference). In certain embodiments, the promoter is a hypoxia-inducible promoter. In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site.In certain embodiments, the promoter comprises a HIF-1α binding site. In certain embodiments, the promoter comprises a HIF-2α binding site. In certain embodiments, the HIF binding site comprises an RCGTG motif. For details regarding the location and sequence of HIF binding sites, see, e.g., Schodel et al., Blood, 2011, 117(23):e207-e217, which is incorporated herein by reference in its entirety. In certain embodiments, the promoter comprises a binding site for a hypoxia-inducible transcription factor other than a HIF transcription factor. In certain embodiments, the viral vectors provided herein comprise one or more IRES sites that are preferentially translated in hypoxia. For teachings regarding hypoxia-inducible gene expression and factors involved therein, see, e.g., Kenneth and Rocha, Biochem J., 2008, 414:19-29, which is incorporated herein by reference in its entirety.

[0082] In certain embodiments, the promoter is the CB7 promoter (see Dinculescu et al., 2005, Hum Gene Ther 16:649-663, incorporated herein by reference in its entirety). In some embodiments, the CB7 promoter comprises other expression control elements that enhance expression of a transgene driven by the vector. In certain embodiments, other expression control elements include a chicken β-actin intron and / or a rabbit β-globin polyA (pol A) signal. In certain embodiments, the promoter comprises a TATA box. In certain embodiments, the promoter comprises one or more elements. In certain embodiments, one or more promoter elements may be inverted or moved relative to each other. In certain embodiments, the promoter elements are positioned to function cooperatively. In certain embodiments, the promoter elements are positioned to function independently. In certain embodiments, the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus (RS) long terminal repeat, and the rat insulin promoter. In certain embodiments, the vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs. In certain embodiments, the vectors provided herein comprise one or more tissue-specific promoters (e.g., retinal pigment epithelial cell-specific promoters). In certain embodiments, the viral vectors provided herein comprise an RPE65 promoter. In certain embodiments, the vectors provided herein comprise a VMD2 promoter.

[0083] In certain embodiments, the viral vectors provided herein comprise one or more regulatory elements other than a promoter. In certain embodiments, the viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein comprise a repressor. In certain embodiments, the viral vectors provided herein comprise an intron or a chimeric intron. In certain embodiments, the viral vectors provided herein comprise a polyadenylation sequence.

[0084] 5.2.4 Signal peptide In certain embodiments, the vectors provided herein comprise a component that modulates protein delivery. In certain embodiments, the viral vectors provided herein comprise one or more signal peptides. Signal peptides may also be referred to herein as "leader sequences" or "leader peptides." In certain embodiments, the signal peptide enables the transgene product (e.g., the anti-VEGF antigen-binding fragment portion) to achieve proper packaging (e.g., glycosylation) in the cell. In certain embodiments, the signal peptide enables the transgene product (e.g., the anti-VEGF antigen-binding fragment portion) to achieve proper localization in the cell. In certain embodiments, the signal peptide enables the transgene product (e.g., the anti-VEGF antigen-binding fragment portion) to achieve secretion from the cell. Examples of signal peptides to be used in connection with the vectors and transgenes provided herein can be found in Table 1.

[0085] [Table 1]

[0086] 5.2.5 Polycistronic Messages - IRES and F2A Linker Internal ribosome entry site. A single construct can be engineered to encode both heavy and light chains separated by a cleavable linker or IRES, resulting in the expression of separate heavy and light chain polypeptides by transduced cells. In certain embodiments, the viral vectors provided herein deliver polycistronic (e.g., bicistronic) messages. For example, the viral construct can encode heavy and light chains separated by an internal ribosome entry site (IRES) element (see, e.g., Gurtu et al., 1996, Biochem. Biophys. Res. Comm. 229(1):295-8, incorporated herein by reference in its entirety, for an example of the use of an IRES element to create a bicistronic vector). The IRES element bypasses the ribosome scanning model and initiates translation at an internal site. The use of IRES in AAV is described, for example, in Furling et al., 2001, Gene Ther 8(11):854-73, which is incorporated herein by reference in its entirety. In certain embodiments, the bicistronic message is contained within a viral vector while limiting the size of the polynucleotide(s) therein. In certain embodiments, the bicistronic message is contained within an AAV virus-based vector (e.g., an AAV8-based vector).

[0087] Furin-F2A linker. In other embodiments, the viral vectors provided herein encode heavy and light chains separated by a cleavable linker, e.g., a self-cleaving furin / F2A (F / F2A) linker (Fang et al., 2005, Nature Biotechnology 23:584-590 and Fang, 2007, Mol Ther 15:1153-9, each incorporated by reference in its entirety).

[0088] For example, a furin-F2A linker can be incorporated into the expression cassette to separate the heavy and light chain coding sequences, resulting in a construct with the following structure: leader-heavy chain-furin site-F2A site-leader-light chain-polyA.

[0089] The F2A site, having the amino acid sequence LLNFDLLKLAGDVESNPGP (SEQ ID NO: 26), self-processes, resulting in a "cleavage" between the last G and P amino acid residues. Additional linkers that can be used include, but are not limited to, the following:

[0090] [ka]

[0091] The peptide bond is skipped when the ribosome encounters the F2A sequence in the open reading frame, resulting in either termination or continued translation of the downstream sequence (light chain). This self-processing sequence results in a series of additional amino acids at the C-terminus of the heavy chain. However, these additional amino acids are then cleaved by host cell furin at a furin site located immediately before the F2A site and after the heavy chain sequence, and further cleaved by carboxypeptidase. The resulting heavy chain may have one, two, three, or more additional amino acids included at its C-terminus, depending on the furin linker used and the sequence of the carboxypeptidase that cleaves the linker in vivo (see, e.g., Fang et al., 2005, Nature Biotechnol. Advance online publication April 17; Fang et al., 2007, Molecular Therapy 15(6):1153-1159; Luke, 2012, Innovations in Biotechnology, Chapter 8, pp. 161-186). Furin linkers that can be used include a stretch of four basic amino acids, such as RKRR (SEQ ID NO: 31), RRRR (SEQ ID NO: 32), RRKR (SEQ ID NO: 33), or RKKR (SEQ ID NO: 34). When the linker is cleaved by a carboxypeptidase, additional amino acids may remain, such that an additional zero, one, two, three, or four amino acids, e.g., R, RR, RK, RKR, RRR, RRK, RKK, RKRR (SEQ ID NO: 31), RRRR (SEQ ID NO: 32), RRKR (SEQ ID NO: 33), or RKKR (SEQ ID NO: 34), remain at the C-terminus of the heavy chain. In certain embodiments, when the linker is cleaved by a carboxypeptidase, no additional amino acids remain. In certain embodiments, no more than 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, or 20% but more than 0% of the antibody, e.g., antigen-binding fragment, population generated by the constructs for use in the methods described herein have 1, 2, 3, or 4 amino acids remaining at the C-terminus of the heavy chain after cleavage.In certain embodiments, between 0.5 and 1%, 0.5% and 2%, 0.5% and 3%, 0.5% and 4%, 0.5% and 5%, 0.5% and 10%, 0.5% and 20%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 1% and 10%, 1% and 20%, 2% and 3%, 2% and 4%, 2% and 5%, 2% and 10%, 2% and 20%, 3% and 4%, 3% and 5%, 3% and 10%, 3% and 20%, 4% and 5%, 4% and 10%, 4% and 20%, 5% and 10%, 5% and 20%, or 10% and 20% of the antibody, e.g., antigen-binding fragment, population produced by the constructs for use in the methods described herein have 1, 2, 3, or 4 amino acids remaining at the C-terminus of the heavy chain after cleavage. In certain embodiments, the furin linker has the sequence RXK / RR (SEQ ID NO: 35), such that additional amino acids at the C-terminus of the heavy chain are R, RX, RXK, RXR, RXKR (SEQ ID NO: 36), or RXRR (SEQ ID NO: 37), where X is any amino acid, e.g., alanine (A). In certain embodiments, no additional amino acids may remain at the C-terminus of the heavy chain.

[0092] In certain embodiments, the expression cassette described herein is contained in a viral vector, while limiting the size of the polynucleotide(s) therein. In certain embodiments, the expression cassette is contained in an AAV virus-based vector (e.g., an AAV8-based vector).

[0093] 5.2.6 Untranslated Regions In certain embodiments, the viral vectors provided herein comprise one or more untranslated regions (UTRs), such as 3' and / or 5' UTRs. In certain embodiments, the UTRs are optimized for a desired level of protein expression. In certain embodiments, the UTRs are optimized for transgene mRNA half-life. In certain embodiments, the UTRs are optimized for transgene mRNA stability. In certain embodiments, the UTRs are optimized for transgene mRNA secondary structure.

[0094] 5.2.7 Inverted terminal repeats In certain embodiments, the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences. The ITR sequences can be used to package a recombinant gene expression cassette into the virion of the viral vector. In certain embodiments, the ITRs are derived from AAV, such as AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(1):364-379; U.S. Patent Nos. 7,282,199, 7,790,449, 8,318,480, 8,962,332, and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety).

[0095] 5.2.8 Transgenes The HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, encoded by the transgene can include, but are not limited to, an antigen-binding fragment of an antibody that binds VEGF, such as bevacizumab; an anti-VEGF Fab portion, such as ranibizumab; or such a bevacizumab or ranibizumab Fab portion that has been engineered to contain additional glycosylation sites in the Fab domain (see, e.g., Courtois et al., 2016, mAbs 8:99-112, which is incorporated by reference in its entirety, for a description of a bevacizumab derivative that is hyperglycosylated in the Fab domain of the full-length antibody).

[0096] In certain embodiments, the vectors provided herein encode an anti-VEGF antigen-binding fragment transgene. In certain embodiments, the anti-VEGF antigen-binding fragment transgene is controlled by expression control elements suitable for expression in retinal cells. In certain embodiments, the anti-VEGF antigen-binding fragment transgene comprises the bevacizumab Fab portion of the light and heavy chain cDNA sequences (SEQ ID NOs: 10 and 11, respectively). In certain embodiments, the anti-VEGF antigen-binding fragment transgene comprises ranibizumab light and heavy chain cDNA sequences (SEQ ID NOs: 12 and 13, respectively). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a bevacizumab Fab comprising the light and heavy chains of SEQ ID NOs: 3 and 4, respectively. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:4.In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 3 and a heavy chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes hyperglycosylated ranibizumab comprising the light and heavy chains of SEQ ID NOs: 1 and 2, respectively. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 2. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 2.In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1 and a heavy chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the C-terminal lysine of SEQ ID NO: 2 is removed after translation of the antigen-binding fragment and before the antigen-binding fragment is secreted.

[0097] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a hyperglycosylated bevacizumab Fab comprising the light and heavy chains of SEQ ID NOs: 3 and 4 with one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S (light chain). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a hyperglycosylated ranibizumab comprising the light and heavy chains of SEQ ID NOs: 1 and 2 with one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S (light chain). The sequence of the antigen-binding fragment transgene cDNA can be found, for example, in Table 2. In certain embodiments, the sequence of the antigen-binding fragment transgene cDNA is obtained by replacing the signal sequence of SEQ ID NOs: 10 and 11 or SEQ ID NOs: 12 and 13 with one or more of the signal sequences listed in Table 1.

[0098] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences of the six bevacizumab CDRs. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences of the six ranibizumab CDRs. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 of ranibizumab (SEQ ID NOs: 20, 18, and 21). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1-3 of ranibizumab (SEQ ID NOs: 14-16). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1-3 of ranibizumab (SEQ ID NOs: 14, 15, and 63). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1 to 3 of bevacizumab (SEQ ID NOs: 17 to 19). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1 to 3 of bevacizumab (SEQ ID NOs: 14 to 16). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1 to 3 of bevacizumab (SEQ ID NOs: 14, 15, and 63). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1 to 3 of ranibizumab (SEQ ID NOs: 20, 18, and 21) and a light chain variable region comprising light chain CDRs 1 to 3 of ranibizumab (SEQ ID NOs: 14 to 16). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1 to 3 of bevacizumab (SEQ ID NOs: 17 to 19) and a light chain variable region comprising light chain CDRs 1 to 3 of bevacizumab (SEQ ID NOs: 14 to 16).In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the heavy chain CDRs 1 to 3 of ranibizumab (SEQ ID NOs: 20, 18, and 21) and a light chain variable region comprising the light chain CDRs 1 to 3 of ranibizumab (SEQ ID NOs: 14, 15, and 63). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the heavy chain CDRs 1 to 3 of bevacizumab (SEQ ID NOs: 17 to 19) and a light chain variable region comprising the light chain CDRs 1 to 3 of bevacizumab (SEQ ID NOs: 14, 15, and 63).

[0099] It will be understood that reference to the heavy chain variable region CDR(s) and / or light chain variable region(s) of a particular antibody encompasses all CDR definitions known to those of skill in the art. Exemplary CDRs according to various numbering systems are shown in the table below.

[0100] [Table 2]

[0101] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the light chain CDR1, light chain CDR2, and light chain CDR3 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the light chain CDR1, light chain CDR2, and light chain CDR3 of the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising (a) a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 2, and (b) a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising (a) a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 4, and (b) a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 of the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising (a) a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 2, and (b) a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 of the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising (a) a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of the amino acid sequence of SEQ ID NO: 4, and (b) a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 of the amino acid sequence of SEQ ID NO: 1.

[0102] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:18, and SEQ ID NO:21. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:63.

[0103] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:18, and SEQ ID NO:21, and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:18, and SEQ ID NO:21, and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:63. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising (a) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and (b) a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 63.

[0104] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1 to 3 of SEQ ID NOs: 14 to 16, and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1 to 3 of SEQ ID NOs: 14 to 16, wherein the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1 to 3 of SEQ ID NOs: 14 to 16, wherein the 8th and 11th amino acid residues of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) do not retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). The second amino acid residue (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) is not acetylated. In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1 to 3 of SEQ ID NOs: 14 to 16, wherein the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) is not acetylated. In a preferred embodiment, the chemical modification(s) or absence of chemical modifications (as the case may be) described herein is determined by mass spectrometry.

[0105] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 to 16, and the second Q in QQYSTVPWT (SEQ ID NO: 16) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 to 16, and the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWT (SEQ ID NO: 16) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14-16, wherein the second Q in QQYSTVPWT (SEQ ID NO: 16) is not acetylated. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14-16, wherein the two Ns in SASQDISNYLN (SEQ ID NO: 14) each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWT (SEQ ID NO: 16) is not acetylated. In preferred embodiments, the chemical modification(s) or the absence (as the case may be) of chemical modifications described herein is determined by mass spectrometry.

[0106] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1-3 of SEQ ID NOs: 14, 15, and 63, in which the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu)), and the 2nd amino acid residue of light chain CDR3 (i.e., QQYSTV The second Q in PWTF (SEQ ID NO: 63) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (PyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1-3 of SEQ ID NOs: 14, 15, and 63, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) is not acetylated.In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1-3 of SEQ ID NOS: 14-16, wherein the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu)), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) is not acetylated. In preferred embodiments, the chemical modification(s) or absence (as the case may be) of chemical modifications described herein is determined by mass spectrometry.

[0107] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 14, 15, and 63, and the second Q in QQYSTVPWTF (SEQ ID NO: 63) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 14, 15, and 63, and the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWTF (SEQ ID NO: 63) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 63, wherein the second Q in QQYSTVPWTF (SEQ ID NO: 63) is not acetylated. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 63, wherein the two Ns in SASQDISNYLN (SEQ ID NO: 14) each bear one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWTF (SEQ ID NO: 63) is not acetylated. In preferred embodiments, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0108] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 of SEQ ID NOs: 20, 18, and 21, and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 of SEQ ID NOs: 20, 18, and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) bears one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) bears one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu). and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated.In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 of SEQ ID NOs: 20, 18, and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) bears one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) bears one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated. In preferred embodiments, the chemical modification(s) or absence of chemical modifications (as the case may be) described herein is determined by mass spectrometry.

[0109] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, 18, and 21, wherein the N in GYDFTHYGMN (SEQ ID NO: 20) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 20, 18, and 21, wherein M in GYDFTHYGMN (SEQ ID NO: 20) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in GYDFTHYGMN (SEQ ID NO: 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 20, 18, and 21, and N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 20, 18, and 21, wherein M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated. In preferred embodiments, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0110] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and a heavy chain variable region comprising heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of the heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1 to 3 of SEQ ID NOs: 14 to 16 and a heavy chain variable region comprising heavy chain CDRs 1 to 3 of SEQ ID NOs: 20, 18, and 21, and wherein (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the heavy chain CDR3 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the heavy chain CDR4 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the heavy chain CDR5 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the heavy chain CDR6 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the heavy chain C (1) the last amino acid residue of R1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); (2) the 8th and 11th amino acid residues of light chain CDR1 (i.e., two Ns in SASQDISNYLN (SEQ ID NO: 14)) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu).In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and a heavy chain variable region comprising heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) is not acetylated, and the last amino acid residue of the heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated. In certain embodiments, the antigen-binding fragment comprises a heavy chain CDR1 of SEQ ID NO: 20, in which (1) the ninth amino acid residue of the heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), (2) the third amino acid residue of the heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and (3) the last amino acid residue of the heavy chain CDR1 (i.e., GYDFTHYGMN (2) the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) is not acetylated. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0111] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 14 to 16 and a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the second Q in QQYSTVPWT (SEQ ID NO: 16) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the N in GYDFTHYGMN (SEQ ID NO: 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 14 to 16 and a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 20, 18, and 21, and (1) M in GYDFTHYGMN (SEQ ID NO: 20) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu). (1) the N in GYDFTHYGMN (SEQ ID NO: 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); (2) the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second Q in QQYSTVPWT (SEQ ID NO: 16) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 14 to 16 and a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the second Q in QQYSTVPWT (SEQ ID NO: 16) is not acetylated, and the N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated.In certain embodiments, the antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 20, wherein (1) M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); and N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated; and (2) two N in SASQDISNYLN (SEQ ID NO: 14) each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second Q in QQYSTVPWT (SEQ ID NO: 16) is not acetylated. In preferred embodiments, the chemical modification(s) or absence of chemical modifications (as the case may be) described herein is determined by mass spectrometry.

[0112] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and a heavy chain variable region comprising heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of the heavy chain CDR1 (i.e., the N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1-3 of SEQ ID NOs: 14, 15, and 63 and a heavy chain variable region comprising heavy chain CDRs 1-3 of SEQ ID NOs: 20, 18, and 21, wherein (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the heavy chain (1) the last amino acid residue of CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); (2) the eighth and eleventh amino acid residues of light chain CDR1 (i.e., two Ns in SASQDISNYLN (SEQ ID NO: 14)) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu).In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1-3 of SEQ ID NOs: 14, 15, and 63, and a heavy chain variable region comprising heavy chain CDRs 1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) is not acetylated, and the last amino acid residue of heavy chain CDR1 (i.e., the N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated. In certain embodiments, the antigen-binding fragment comprises a heavy chain CDR1 of SEQ ID NO: 20, in which (1) the ninth amino acid residue of the heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), (2) the third amino acid residue of the heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and (3) the last amino acid residue of the heavy chain CDR1 (i.e., GYDFTHYGMN (2) the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) is not acetylated. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0113] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the second Q in QQYSTVPWTF (SEQ ID NO: 63) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the N in GYDFTHYGMN (SEQ ID NO: 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein (1) M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu). (1) the N in GYDFTHYGMN (SEQ ID NO: 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); (2) the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second Q in QQYSTVPWTF (SEQ ID NO: 63) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the second Q in QQYSTVPWTF (SEQ ID NO: 63) is not acetylated and the N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated.In certain embodiments, the antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 20, wherein (1) M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); and N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated; and (2) two N in SASQDISNYLN (SEQ ID NO: 14) each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second Q in QQYSTVPWTF (SEQ ID NO: 63) is not acetylated. In preferred embodiments, the chemical modification(s) or absence of chemical modifications (as the case may be) described herein is determined by mass spectrometry.

[0114] Also provided herein in a specific aspect are anti-VEGF antigen-binding fragments comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and transgenes encoding such antigen-VEGF antigen-binding fragments, wherein the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not bear one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises light chain CDR1 to 3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to 3 of SEQ ID NOs: 20, 18, and 21, wherein the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises light chain CDR1 to 3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to 3 of SEQ ID NOs: 20, 18, and 21, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not retain one or more of the following chemical modifications. In certain embodiments, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any method according to the embodiments described herein. In preferred embodiments, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0115] Also provided herein in certain aspects are anti-VEGF antigen-binding fragments, and transgenes encoding such antigen-VEGF antigen-binding fragments, comprising the amino acid sequences of SEQ ID NOs: 14-16 and 20, 18, and 21, wherein the second Q in QQYSTVPWT (SEQ ID NO: 16) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14-16 and 20, 18, and 21, wherein the two Ns in SASQDISNYLN (SEQ ID NO: 14) each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWT (SEQ ID NO: 16) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14-16 and SEQ ID NOs: 20, 18, and 21, wherein the second Q in QQYSTVPWT (SEQ ID NO: 16) is not acetylated. In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14-16 and SEQ ID NOs: 20, 18, and 21, wherein the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWT (SEQ ID NO: 16) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any method according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0116] Also provided herein in certain aspects are anti-VEGF antigen-binding fragments, and transgenes encoding such antigen-VEGF antigen-binding fragments, comprising light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) does not bear one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (PyroGlu). In certain embodiments, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) does not retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NOS: 14, 15, and 63 and heavy chain CDRs 1-3 of SEQ ID NOS: 20, 18, and 21, wherein the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any of the methods according to the embodiments described herein.In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0117] Also provided herein in certain aspects are anti-VEGF antigen-binding fragments, and transgenes encoding such antigen-VEGF antigen-binding fragments, comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the second Q in QQYSTVPWTF (SEQ ID NO: 63) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (PyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the two Ns in SASQDISNYLN (SEQ ID NO: 14) each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWTF (SEQ ID NO: 63) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, and the second Q in QQYSTVPWTF (SEQ ID NO: 63) is not acetylated. In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the two Ns in SASQDISNYLN (SEQ ID NO: 14) each bear one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWTF (SEQ ID NO: 63) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any method according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0118] Also provided herein in a specific aspect are anti-VEGF antigen-binding fragments comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and transgenes encoding such antigen-VEGF antigen-binding fragments, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not bear one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (PyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1 to CDR3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to CDR3 of SEQ ID NOs: 20, 18, and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu). and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated.In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any of the methods according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0119] Also provided herein in certain aspects are anti-VEGF antigen-binding fragments, and transgenes encoding such antigen-VEGF antigen-binding fragments, comprising the amino acid sequences of SEQ ID NOs: 14-16 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein N in GYDFTHYGMN (SEQ ID NO: 20) does not bear one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14-16 and SEQ ID NOs: 20, 18, and 21, wherein M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); and N in GYDFTHYGMN (SEQ ID NO: 20) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14-16 and SEQ ID NOs: 20, 18, and 21, wherein N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated. In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14-16 and SEQ ID NOs: 20, 18, and 21, wherein M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); and N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any method according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or the absence (as the case may be) of the chemical modifications described herein are determined by mass spectrometry.

[0120] Also provided herein in a specific aspect are anti-VEGF antigen-binding fragments comprising light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and transgenes encoding such antigen-VEGF antigen-binding fragments, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not bear one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (PyroGlu). In certain embodiments, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu). and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated.In certain embodiments, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu). The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any of the methods according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0121] Also provided herein in certain aspects are anti-VEGF antigen-binding fragments, and transgenes encoding such antigen-VEGF antigen-binding fragments, comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the N in GYDFTHYGMN (SEQ ID NO: 20) does not bear one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (PyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in GYDFTHYGMN (SEQ ID NO: 20) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 15, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated. In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 15, and 63, and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); and N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any method according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0122] Also provided herein in a specific aspect are anti-VEGF antigen-binding fragments comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and transgenes encoding such antigen-VEGF antigen-binding fragments, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises light chain CDR1 to CDR3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to CDR3 of SEQ ID NOs: 20, 18, and 21, and (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of heavy chain CDR1 (i.e., GY (2) the 8th and 11th amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14)) do not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu).In certain embodiments, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) is not acetylated. In certain embodiments, the antigen-binding fragment comprises light chain CDR1 to CDR3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to CDR3 of SEQ ID NOs: 20, 18, and 21, in which (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and (2) the 8th and 11th amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWT (SEQ ID NO: 16)) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any method according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or the absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0123] Also provided herein in certain aspects are anti-VEGF antigen-binding fragments comprising the amino acid sequences of SEQ ID NOs: 14-16 and SEQ ID NOs: 20, 18, and 21, and transgenes encoding such antigen-VEGF antigen-binding fragments, wherein the N in GYDFTHYGMN (SEQ ID NO: 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWT (SEQ ID NO: 16) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14 to 16 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein (1) M in GYDFTHYGMN (SEQ ID NO: 20) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and (2) GYDFTHYGMN (SEQ ID NO: 18) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and (3) M in GYDFTHYGMN (SEQ ID NO: 20) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in GYDFTHYGMN (SEQ ID NO: 18) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and (4) M in GYDFTHYGMN (SEQ ID NO: 20) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in GYDFTHYGMN (SEQ ID NO: 18) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and (5) M in GYDFTHYGMN (SEQ ID NO: 20) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in GYDFTHYGMN (SEQ ID NO: 18) has one or more of the following chemical modifications: acetylation, deamidation, and pyrog (1) N in sequence number 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); (2) two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second Q in QQYSTVPWT (SEQ ID NO: 16) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14-16 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated, and the second Q in QQYSTVPWT (SEQ ID NO: 16) is not acetylated.In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14 to 16 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, in which (1) M in GYDFTHYGMN (SEQ ID NO: 20) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated, and (2) two N in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWT (SEQ ID NO: 16) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any of the methods according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0124] Also provided herein in certain aspects are anti-VEGF antigen-binding fragments, and transgenes encoding such antigen-VEGF antigen-binding fragments, comprising light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, in which (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of heavy chain CDR1 (i.e., (1) the N in GYDFTHYGMN (SEQ ID NO: 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); (2) the 8th and 11th amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu).In certain embodiments, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) is not acetylated. In certain embodiments, the antigen-binding fragment comprises light chain CDRs 1 to 3 of SEQ ID NOs: 14, 15, and 63 and heavy chain CDRs 1 to 3 of SEQ ID NOs: 20, 18, and 21, in which: (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) retains one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu); (2) the 8th and 11th amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each retain one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 63)) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any method in accordance with the embodiments described herein. In a preferred embodiment, the chemical modification(s) or the absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0125] Also provided herein in certain aspects are anti-VEGF antigen-binding fragments, and transgenes encoding such antigen-VEGF antigen-binding fragments, comprising the amino acid sequences of SEQ ID NOs: 14, 16, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the N in GYDFTHYGMN (SEQ ID NO: 20) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWTF (SEQ ID NO: 63) does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 16, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein (1) M in GYDFTHYGMN (SEQ ID NO: 20) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and (1) the N in (SEQ ID NO: 20) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), (2) the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWTF (SEQ ID NO: 63) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 16, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, wherein the N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated, and the second Q in QQYSTVPWTF (SEQ ID NO: 63) is not acetylated.In certain embodiments, the antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 14, 16, and 63 and the amino acid sequences of SEQ ID NOs: 20, 18, and 21, in which (1) M in GYDFTHYGMN (SEQ ID NO: 20) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), N in WINTYTGEPTYAADFKR (SEQ ID NO: 18) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and N in GYDFTHYGMN (SEQ ID NO: 20) is not acetylated, and (2) two N in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second Q in QQYSTVPWTF (SEQ ID NO: 63) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any of the methods according to the embodiments described herein. In a preferred embodiment, the chemical modification(s) or absence of chemical modification(s) (as the case may be) described herein is determined by mass spectrometry.

[0126] [Table 3] TIFF2025534347000007.tif249169TIFF2025534347000008.tif217169

[0127] 5.2.9 Constructs In certain embodiments, viral vectors provided herein comprise the following elements, in the following order: a) a constitutive or hypoxia-inducible promoter sequence and b) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion). In certain embodiments, the sequence encoding the transgene comprises multiple ORFs separated by an IRES element. In certain embodiments, the ORFs encode heavy and light chain domains of an anti-VEGF antigen-binding fragment. In certain embodiments, the sequence encoding the transgene comprises multiple subunits in a single ORF separated by an F / F2A sequence. In certain embodiments, the sequence comprising the transgene encodes heavy and light chain domains of an anti-VEGF antigen-binding fragment separated by an F / F2A sequence. In certain embodiments, viral vectors provided herein comprise the following elements, in the following order: a) a constitutive or hypoxia-inducible promoter sequence and b) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion), the transgene comprising the signal peptide of VEGF-A (SEQ ID NO: 5), and the transgene encoding light and heavy chain sequences separated by an IRES element. In certain embodiments, the viral vectors provided herein comprise the following elements, in the following order: a) a constitutive or hypoxia-inducible promoter sequence and b) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion), wherein the transgene comprises the signal peptide of VEGF-A (SEQ ID NO: 5), and the transgene encodes light and heavy chain sequences separated by a cleavable F / F2A sequence.

[0128] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or hypoxia-inducible promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion), i) a second UTR sequence, j) a fourth linker sequence, k) a polyA sequence, l) a fifth linker sequence, and m) a second ITR sequence.

[0129] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or hypoxia-inducible promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion), i) a second UTR sequence, j) a fourth linker sequence, k) a polyA sequence, l) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene comprises the signal peptide of VEGF-A (SEQ ID NO: 5), and the transgene encodes light and heavy chain sequences separated by a cleavable F / F2A sequence.

[0130] In some embodiments, the AAV (AAV viral vector) provided herein comprises the following elements, in the following order: a) a constitutive or hypoxia-inducible promoter sequence and b) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion). In some embodiments, the transgene is a fully human post-translationally modified (HuPTM) antibody against VEGF. In some embodiments, the fully human post-translationally modified antibody against VEGF is a fully human post-translationally modified antigen-binding fragment of a monoclonal antibody (mAb) against VEGF ("HuPTMFabVEGFi"). In some embodiments, the HuPTMFabVEGFi is a fully human glycosylated antigen-binding fragment of an anti-VEGF mAb ("HuGlyFabVEGFi"). In alternative embodiments, a full-length mAb can be used. In some embodiments, the AAV used to deliver the transgene must have tropism for human retinal cells or photoreceptor cells. Such AAVs include non-replicating recombinant adeno-associated viral vectors ("rAAV"), particularly those having an AAV8 capsid. In certain embodiments, the viral vector or other DNA expression construct described herein is Construct I, which comprises the following components: (1) AAV8 inverted terminal repeats flanking the expression cassette, (2) a CB7 promoter containing a CMV enhancer / chicken β-actin promoter, (2) a regulatory element comprising a chicken β-actin intron, and (3) a nucleic acid sequence encoding the heavy and light chains of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, ensuring equal expression of the heavy and light chain polypeptides. In some embodiments, the recombinant viral vector comprises the nucleotide sequence of SEQ ID NO: 56. In some embodiments, the viral vector comprises a vector genome comprising SEQ ID NO: 56 (ITR-CB7-CI-aVEGFv3-rBG-ITR). In some embodiments, the vector genome comprises SEQ ID NO: 14 of US Pat. No. 1,197,937 (hereby incorporated by reference).In some embodiments, the vector genome comprises any one of the sequences disclosed in U.S. Pat. No. 1,197,937 (incorporated herein by reference). In some embodiments, the vector genome comprises a 5' AAV-2 inverted terminal repeat, an expression cassette consisting of contiguous nucleotides 198 to 3733 of SEQ ID NO:56 (equivalent to SEQ ID NO:14 of U.S. Pat. No. 1,197,937 (incorporated herein by reference)), and a 3' AAV-2 inverted terminal repeat. In some embodiments, the viral vector comprises a signal peptide. In some embodiments, the signal peptide is MYRMQLLLLIALSLALVTNS (SEQ ID NO:55). In some embodiments, the signal peptide is derived from the IL-2 signal sequence. In some embodiments, the viral vector comprises a signal peptide from any signal peptide disclosed in Table 1, e.g., MNFLLSWVHWSLALLLYLHHAKWSQA (VEGF-A signal peptide) (SEQ ID NO: 5), MERAAPSRRVPLPLLLLGGLALLAAGVDA (fibulin-1 signal peptide) (SEQ ID NO: 6), MAPLRPLLILALLAWVALA (vitronectin signal peptide) (SEQ ID NO: 7), MRLLAKIICLMLWAICVA (complement factor H signal peptide) (SEQ ID NO: 8), MRLLAFLSLALALVLQETGT (opticin signal peptide) (SEQ ID NO: 9), MKWVTFISLLFLFSSAYS (albumin signal peptide) (SEQ ID NO: 22), MAFLWLLSCWALLGTTFG (chymotrypsinogen signal peptide) (SEQ ID NO: 23), MYRMQLLSCIALILALVTNS (interleukin-2 signal peptide) (SEQ ID NO: 24), MNLLLILTFVAAAVA (trypsinogen-2 signal peptide) (SEQ ID NO: 25), or MYRMQLLLLIALSLALVTNS (mutant interleukin-2 signal peptide) (SEQ ID NO: 55). In some embodiments, the recombinant viral vector encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the recombinant viral vector encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 58.In some embodiments, the recombinant viral vector encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58. In certain embodiments, the signal peptide is post-translationally removed from the first and second polypeptides such that the secreted transgene product is composed of a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the C-terminal lysine of SEQ ID NO: 2 is removed from the transgene product after translation and before the transgene product is secreted.

[0131] In a specific embodiment, the construct described herein is Construct I, which comprises the following components: (1) AAV8 inverted terminal repeats flanking an expression cassette, (2) control elements including a) a CB7 promoter containing a CMV enhancer / chicken β-actin promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal, and (3) nucleic acid sequences encoding the heavy and light chains of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, ensuring equal expression of the heavy and light chain polypeptides.

[0132] In another specific embodiment, the construct described herein is Construct II, which comprises the following components: (1) AAV2 inverted terminal repeats flanking the expression cassette, (2) control elements including a) a CB7 promoter comprising a CMV enhancer / chicken β-actin promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal, and (3) nucleic acid sequences encoding the heavy and light chains of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, ensuring equal expression of the heavy and light chain polypeptides. In a specific embodiment, the construct comprises an expression cassette encoding an anti-hVEGF antigen-binding fragment, wherein the expression cassette is flanked by AAV2 inverted terminal repeats (ITRs), and the expression cassette comprises CB7 promoter consisting of chicken β-actin promoter and CMV enhancer, chicken β-actin intron, IL-2 signal peptide, a heavy chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2; a self-cleaving furin (F) / F2A linker, a second IL-2 signal peptide, and The light chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding Rabbit β-globin poly(A) signal Includes.

[0133] 5.2.10 Vector Production and Testing The viral vectors provided herein can be produced using host cells. The viral vectors provided herein can be produced using mammalian host cells, such as A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. The viral vectors provided herein can be produced using host cells derived from humans, monkeys, mice, rats, rabbits, or hamsters.

[0134] Host cells are stably transformed with sequences encoding the transgene and associated elements (i.e., vector genome) and means for producing virus in the host cell, e.g., replication and capsid genes (e.g., AAV rep and cap genes). For methods of generating recombinant AAV vectors having AAV8 capsids, see Section IV of the detailed description of U.S. Pat. No. 7,282,199, incorporated herein by reference in its entirety. The genome copy titer of the vector can be determined, e.g., by TAQMAN® analysis. Virions can be recovered, e.g., by CsCl2 sedimentation.

[0135] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from the vectors described herein and thus, for example, indicate vector efficacy. For example, the PER.C6® cell line (Lonza), cell lines derived from human embryonic retinal cells or retinal pigment epithelial cells, e.g., the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®), can be used to assess transgene expression. Once expressed, the expressed product (i.e., HuGlyFabVEGFi) can be characterized, including determining the glycosylation and tyrosine sulfation patterns associated with HuGlyFabVEGFi. Glycosylation and tyrosine sulfation patterns and methods for determining same are discussed in PCT / US2017 / 027650, which is incorporated herein by reference. Furthermore, benefits resulting from glycosylation / sulfation of HuGlyFabVEGFi expressed by cells can be determined using assays known in the art, e.g., the methods described in PCT / US2017 / 027650.

[0136] 5.2.11 Composition Compositions are described that include a vector encoding the transgene described herein and a suitable carrier. Suitable carriers (e.g., for suprachoroidal, subretinal, juxtascleral, intravitreal, subconjunctival, and / or intraretinal administration) can be readily selected by those skilled in the art.

[0137] In certain embodiments of the methods provided herein, the recombinant viral vector is formulated to be suitable for administration to the suprachoroidal space of the subject's eye. In certain embodiments, the recombinant viral vector is formulated to be suitable for administration to the subretinal space of the subject's eye. In some embodiments, the formulated composition is a formulation and / or pharmaceutical composition described in WO2021 / 071835, WO2022 / 076549, WO2022 / 076591, or WO2022 / 076595, each of which is incorporated herein by reference. In some embodiments, the formulated composition comprises (a) a recombinant viral vector (e.g., rHuGlyFabVEGFi), (b) potassium chloride at a concentration of about 0.2 g / L, (c) monopotassium phosphate at a concentration of about 0.2 g / L, (d) sodium chloride at a concentration of about 5.84 g / L, (e) anhydrous disodium phosphate at a concentration of about 1.15 g / L, (f) sucrose at a concentration of about 4% weight / volume (40 g / L), (g) poloxamer 188, polysorbate 20, or polysorbate 80 at a concentration of about 0.001% weight / volume (0.01 g / L), and (h) water, at a pH of about 7.4. In some embodiments, the formulated composition comprises (a) a recombinant viral vector (e.g., rHuGlyFabVEGFi), (b) potassium chloride at a concentration of about 0.2 g / L, (c) monopotassium phosphate at a concentration of about 0.2 g / L, (d) sodium chloride at a concentration of about 5.84 g / L, (e) anhydrous disodium phosphate at a concentration of about 1.15 g / L, (f) sucrose at a concentration of about 4% w / v (40 g / L), (g) poloxamer 188 at a concentration of about 0.001% w / v (0.01 g / L), and (h) water, at a pH of about 7.4. In some embodiments, the formulated composition comprises (a) a recombinant viral vector (e.g., rHuGlyFabVEGFi), (b) potassium chloride at a concentration of about 2.70 mM, (c) monopotassium phosphate at a concentration of about 1.47 mM, (d) sodium chloride at a concentration of about 100 mM, (e) anhydrous disodium phosphate at a concentration of about 8.10 mM, (f) sucrose at a concentration of about 117 mM (4% w / v), (g) poloxamer 188 at a concentration of about 0.001% w / v (0.01 g / L), and (h) water, at a pH of about 7.4. In one embodiment, the formulated composition is set forth in the following table:

[0138] [Table 4]

[0139] In certain embodiments, the gene therapy construct is supplied as a frozen, sterile, single-use solution of the AAV vector active ingredient in a formulation buffer. In certain embodiments, a pharmaceutical composition suitable for subretinal administration comprises a suspension of a recombinant (e.g., rHuGlyFabVEGFi) vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In certain embodiments, the gene therapy construct is formulated in Dulbecco's phosphate-buffered saline and 0.001% Pluronic F68, pH 7.4.

[0140] 5.3 Gene therapy Methods for administering a therapeutically effective amount of a transgene construct to a human subject with an ocular disease, particularly an ocular disease caused by increased neovascularization, are described. More specifically, methods for administering a therapeutically effective amount of a transgene construct to a patient with neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) are described for suprachoroidal, subretinal, juxtascleral, intravitreal, subconjunctival, and / or intraretinal administration (e.g., suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior juxtascleral depot procedure).

[0141] Methods are described for administering a therapeutically effective amount of a transgene construct to a patient diagnosed with neovascular age-related macular degeneration or diabetic retinopathy (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), or subretinal administration via the suprachoroidal space) in the suprachoroidal, subretinal, juxtascleral, intravitreal, subconjunctival, and / or intraretinal manner.

[0142] Also provided herein are methods for administering a therapeutically effective amount of a transgene construct suprachoroidally, subretinally, juxtasclerally, intravitreally, subconjunctivally, and / or intraretinally (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior juxtascleral depot procedure), and methods for administering a therapeutically effective amount of a transgene construct to the retinal pigment epithelium.

[0143] 5.3.1 Methods for delivering recombinant viral vectors In one aspect, provided herein is a method of subretinal administration for treating neovascular age-related macular degeneration or diabetic retinopathy without vitrectomy, comprising administering a recombinant viral vector provided herein to the subretinal space of the eye of a human subject in need of treatment, thereby expressing a transgene product and resulting in treatment of neovascular age-related macular degeneration or diabetic retinopathy, wherein the method does not involve performing a vitrectomy on the eye of the human patient. In certain embodiments, the administering step comprises administering a recombinant viral vector to the subretinal space of the eye of the human subject via the suprachoroidal space of the eye of the human subject. In certain embodiments, the administering step is by use of a subretinal drug delivery device including a catheter that can be inserted and tunneled through the suprachoroidal space toward the posterior pole, where a thin needle injects into the subretinal space at the posterior pole. In certain embodiments, the administering step comprises inserting and tunneling a catheter of the subretinal drug delivery device through the suprachoroidal space.

[0144] In another aspect, provided herein is a method for treating neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the subretinal space of the eye of a human subject in need of treatment, thereby expressing a transgene product and resulting in treatment of neovascular age-related macular degeneration or diabetic retinopathy, wherein the method does not include performing a vitrectomy on the eye of the human patient. In certain embodiments, the administering step comprises administering a recombinant viral vector to the subretinal space of the eye of the human subject via the suprachoroidal space of the eye of the human subject. In certain embodiments, the administering step is by using a subretinal drug delivery device including a catheter that can be inserted and tunneled through the suprachoroidal space toward the posterior pole, where a thin needle injects into the subretinal space. In certain embodiments, the administering step comprises inserting and tunneling a catheter of the subretinal drug delivery device through the suprachoroidal space.

[0145] In one aspect, provided herein is a method of subretinal administration accompanied by vitrectomy for treating neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the subretinal space of an eye of a human subject in need of treatment, such that a transgene product is expressed, resulting in treatment of neovascular age-related macular degeneration or diabetic retinopathy, and performing a vitrectomy on the eye of the human patient. In certain embodiments, the vitrectomy is a partial vitrectomy.

[0146] In another aspect, provided herein is a method for treating neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the subretinal space of an eye of a human subject in need of treatment, such that a transgene product is expressed, resulting in treatment of the neovascular age-related macular degeneration or diabetic retinopathy, and the method comprises performing a vitrectomy on the eye of the human patient. In certain embodiments, the vitrectomy is a partial vitrectomy.

[0147] In a preferred embodiment, provided herein is a method of suprachoroidal administration for treating neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the suprachoroidal space of the eye of a human subject in need of treatment, such that the transgene product is expressed, resulting in treatment of neovascular age-related macular degeneration or diabetic retinopathy. In certain embodiments, the administration step involves injecting the recombinant viral vector into the suprachoroidal space using a suprachoroidal drug delivery device. In certain embodiments, the suprachoroidal drug delivery device is a microinjector.

[0148] In certain embodiments, delivery to the subretinal or suprachoroidal space can be performed using the methods and / or devices described and disclosed in International Publication Nos. WO2016 / 042162, WO2017 / 046358, WO2017 / 158365 and WO2017 / 158366, each of which is incorporated herein by reference in its entirety.

[0149] There are currently available technologies for suprachoroidal space (SCS) delivery. Preclinically, SC injections have been achieved using scleral flap techniques, catheters, and standard hypodermic needles, as well as microneedles. Hollow, 750 μm-long microneedles (Clearside Biomedical, Inc.) can be inserted into the area and have shown promise in clinical trials. Microneedles designed with force-sensing technology for SC injections are available, as described by Chitnis et al. (Chitnis, GD et al. A resistance-sensing mechanical injector for the precise delivery of liquids to target tissue. Nat Biomed Eng 3, pp. 621-631 (2019). https: / / doi.org / 10.1038 / s41551-019-0350-2). Oxular Limited is developing a delivery system (Oxulumis) that advances an illuminated cannula into the suprachoroidal space. The Orbit device (Gyroscope) is a specially designed system that allows cannulation of the suprachoroidal space using a flexible cannula. A microneedle inside the cannula is advanced into the subretinal space, enabling targeted dose delivery. Ab interno access to the SCS can also be achieved using a microstent that acts as a minimally invasive glaucoma surgery (MIGS) device. Examples include the CyPass® microstent (Alcon, Fort Worth, Texas, USA) and the iStent® (Glaukos), which are surgically implanted to provide a conduit from the anterior chamber to the SCS for aqueous humor to flow without forming a filtering bleb. Other devices intended for suprachoroidal delivery include those described in UK Patent Publication No. GB2531910A and U.S. Patent No. 10,912,883.

[0150] In some embodiments, the suprachoroidal drug delivery device is a syringe with a 1-millimeter 30-gauge needle. In some embodiments, the syringe has a larger diameter (e.g., a 29-gauge needle). During injection using this device, the needle penetrates to the base of the sclera, and the drug-containing liquid enters the suprachoroidal space, leading to the expansion of the suprachoroidal space. As a result, there is tactile and visual feedback during injection. After injection, the fluid flows to the posterior side and is absorbed predominantly in the choroid and retina. This results in the production of the transgene protein from all retinal cell layers and choroidal cells. The use of this type of device and procedure allows for a quick and easy in-clinic procedure with a low risk of complications.

[0151] In certain embodiments, the recombinant viral vector is administered by multiple suprachoroidal injections. In certain embodiments, the recombinant viral vector is administered by triple suprachoroidal injections. In certain embodiments, the recombinant viral vector is administered by double suprachoroidal injections. In certain embodiments, the first injection in the right eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock and 11 o'clock positions), and the second injection in the same eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock and 5 o'clock positions). In certain embodiments, the first injection in the right eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock and 5 o'clock positions), and the second injection in the same eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock and 11 o'clock positions). In certain embodiments, the first injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock and 2 o'clock positions) and the second injection in the same eye is administered in the inferior nasal quadrant (i.e., between the 7 o'clock and 8 o'clock positions). In certain embodiments, the first injection in the left eye is administered in the inferior nasal quadrant (i.e., between the 7 o'clock and 8 o'clock positions) and the second injection in the same eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock and 2 o'clock positions).

[0152] In certain embodiments, the recombinant viral vector is administered by a single suprachoroidal injection. In certain embodiments, the single injection in the right eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock and 11 o'clock positions). In certain embodiments, the single injection in the right eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock and 5 o'clock positions). In certain embodiments, the single injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock and 2 o'clock positions). In certain embodiments, the single injection in the left eye is administered in the inferior nasal quadrant (i.e., between the 7 o'clock and 8 o'clock positions).

[0153] In one aspect, provided herein are methods of administering to the outer surface of the sclera to treat neovascular age-related macular degeneration or diabetic retinopathy, the methods comprising administering a recombinant viral vector provided herein to the outer surface of the sclera in an eye of a human subject in need of treatment, resulting in expression of a transgene product and treatment of neovascular age-related macular degeneration or diabetic retinopathy. In certain embodiments, the administering step is by use of a juxtascleral drug delivery device comprising a cannula, the tip of which can be inserted and maintained in direct apposition to the scleral surface. In certain embodiments, the administering step comprises inserting the tip of the cannula and maintaining it in direct apposition to the scleral surface.

[0154] In another aspect, provided herein are methods for treating neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the outer surface of the sclera in an eye of a human subject in need of treatment, resulting in expression of a transgene product and treatment of the neovascular age-related macular degeneration or diabetic retinopathy. In certain embodiments, the administering step is by use of a juxtascleral drug delivery device that includes a cannula, the tip of which can be inserted and maintained in direct apposition to the scleral surface. In certain embodiments, the administering step includes inserting the tip of the cannula and maintaining it in direct apposition to the scleral surface.

[0155] In one aspect, provided herein is a method of intravitreal administration for neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the vitreous cavity of an eye of a human subject in need of treatment, such that a transgene product is expressed, resulting in treatment of neovascular age-related macular degeneration or diabetic retinopathy. In certain embodiments, the administering step is by injecting the recombinant viral vector into the vitreous cavity using an intravitreal drug delivery device. In certain embodiments, the intravitreal drug delivery device is a microinjector.

[0156] In another aspect, provided herein is a method for treating neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the vitreous cavity of an eye of a human subject in need of treatment, such that a transgene product is expressed, resulting in treatment of the neovascular age-related macular degeneration or diabetic retinopathy. In certain embodiments, the administering step is by injecting the recombinant viral vector into the vitreous cavity using an intravitreal drug delivery device. In certain embodiments, the intravitreal drug delivery device is a microinjector.

[0157] In one aspect, provided herein is a method of subretinal administration accompanied by vitrectomy for treating neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the subretinal space of an eye of a human subject in need of treatment, such that a transgene product is expressed, resulting in treatment of neovascular age-related macular degeneration or diabetic retinopathy, and the method comprises performing a vitrectomy on the eye of the human patient. In certain embodiments, the vitrectomy is a partial vitrectomy.

[0158] In another aspect, provided herein is a method for treating neovascular age-related macular degeneration or diabetic retinopathy, comprising administering a recombinant viral vector provided herein to the subretinal space of an eye of a human subject in need of treatment, such that a transgene product is expressed, resulting in treatment of the neovascular age-related macular degeneration or diabetic retinopathy, and the method comprises performing a vitrectomy on the eye of the human patient. In certain embodiments, the vitrectomy is a partial vitrectomy.

[0159] In one aspect, provided herein is a method of subretinal administration for treating neovascular age-related macular degeneration or diabetic retinopathy, the method comprising administering a recombinant viral vector provided herein to the subretinal space surrounding the optic disc, fovea, and macula located at the back of the eye of a human subject in need of treatment, such that a transgene product is expressed, resulting in treatment of neovascular age-related macular degeneration or diabetic retinopathy, and the method does not comprise performing a vitrectomy on the eye of the human patient. In certain embodiments, the injection step is by intravitreal injection. In certain embodiments, the intravitreal administration method results in uniform expression of the transgene product throughout the eye (e.g., expression levels at the injection site vary by less than 5%, 10%, 20%, 30%, 40%, or 50% compared to expression levels in other areas of the eye). In certain embodiments, transvitreal injection involves inserting a sharp needle into the sclera through the upper or lower eye and passing the sharp needle throughout the vitreous to inject the recombinant viral vector into the subretinal space on the opposite side. In certain embodiments, the needle is inserted at the 2 or 10 o'clock position. In certain embodiments, transvitreal injection involves inserting a trocar into the sclera and inserting a cannula through the trocar and through the vitreous to inject the recombinant viral vector into the subretinal space on the opposite side.

[0160] In certain embodiments, the transgene product is an anti-hVEGF antibody. In certain embodiments, the anti-hVEGF antibody is an anti-hVEGF antigen-binding fragment. In certain embodiments, the anti-hVEGF antigen-binding fragment is a Fab, F(ab')2, or single-chain variable fragment (scFv). In certain embodiments, the anti-hVEGF antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In certain embodiments, the anti-hVEGF antibody comprises light chain CDRs 1-3 of SEQ ID NOs: 14-16 and heavy chain CDRs 1-3 of SEQ ID NOs: 17-19 or SEQ ID NOs: 20, 18, and 21. In certain embodiments, the ocular pathology is associated with nAMD, dry age-related macular degeneration (dry AMD), retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR). In certain embodiments, the ocular pathology is associated with nAMD or DR.

[0161] In certain embodiments of the methods described herein, the administering step delivers a therapeutically effective amount of a recombinant viral vector to the retina of said human subject.

[0162] In certain embodiments of the methods described herein, the therapeutically effective amount of the transgene product is produced by human retinal cells of said human subject.

[0163] In certain embodiments of the methods described herein, a therapeutically effective amount of the transgene product is produced by human photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells and / or retinal pigment epithelial cells in the external limiting membrane of said human subject.

[0164] In certain embodiments of the methods described herein, the human photoreceptor cells are cone cells and / or rod cells.

[0165] In certain embodiments of the methods described herein, the retinal ganglion cells are midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells and / or Müller glia.

[0166] In certain embodiments of the methods described herein, the recombinant viral vector is an rAAV vector (e.g., an rAAV8, rAAV2, rAAV2tYF, or rAAV5 vector).

[0167] In certain embodiments of the methods described herein, the recombinant viral vector is an rAAV8 vector.

[0168] In certain embodiments of the methods described herein, delivering to the eye comprises delivering to the retina, choroid and / or vitreous humor of the eye.

[0169] 5.3.2 Target patient population The subject to be treated according to the method described herein can be any mammal, for example, rodent, domestic animal, for example, dog or cat, or primate, for example, non-human primate.In a preferred embodiment, the subject is human.In certain embodiments, the method provided herein is for administering to patients diagnosed with ocular disease (for example, wet AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME) or diabetic retinopathy (DR) (particularly wet AMD or DR)), particularly ocular disease caused by increased neovascularization.

[0170] In certain embodiments, the methods provided herein are for administration to patients diagnosed with severe AMD. In certain embodiments, the methods provided herein are for administration to patients diagnosed with attenuating AMD.

[0171] In certain embodiments, the methods provided herein are for administration to patients diagnosed with severe wet AMD. In certain embodiments, the methods provided herein are for administration to patients diagnosed with attenuated wet AMD.

[0172] In certain embodiments, the methods provided herein are for administration to a patient diagnosed with AMD who has been identified as responsive to treatment with an anti-VEGF antibody.

[0173] In certain embodiments, the methods provided herein are for administration to a patient diagnosed with AMD who has been identified as responsive to treatment with an anti-VEGF antigen-binding fragment.

[0174] In certain embodiments, the methods provided herein are for administration to a patient diagnosed with AMD who has been identified as responsive to treatment with an intravitreally injected anti-VEGF antigen-binding fragment prior to treatment with gene therapy.

[0175] In certain embodiments, the methods provided herein are for administration to patients diagnosed with AMD who have been identified as responsive to treatment with Lucentis® (ranibizumab), EYLEA® (aflibercept), and / or Avastin® (bevacizumab).

[0176] In certain embodiments, a patient diagnosed with AMD is identified as responsive to treatment with an anti-VEGF antigen-binding fragment (e.g., ranibizumab) if the patient has an improvement in exudate after intravitreal injection of the anti-VEGF antigen-binding fragment before treatment with gene therapy. In certain embodiments, a patient diagnosed with AMD is identified as responsive to treatment with an anti-VEGF antigen-binding fragment (e.g., ranibizumab) if the patient has an improvement in exudate and has a central retinal thickness (CRT) of less than 400 μm after intravitreal injection of the anti-VEGF antigen-binding fragment before treatment with gene therapy. In some embodiments, the anti-VEGF antigen-binding fragment is intravitreally injected into the patient at 0.5 mg per month for two months before treatment with gene therapy. In other embodiments, the anti-VEGF antigen-binding fragment is intravitreally injected into the patient at 0.5 mg per month for three months before treatment with gene therapy. In preferred embodiments, a patient has an improvement in exudate if they have an improvement in intraretinal (juxtafoveal 3 mm) exudate of more than 50 μm or 30% compared to levels before intravitreal injection of the anti-VEGF antigen-binding fragment, or an improvement in central subfield thickness of more than 50 μm or 30% as determined by CRC compared to levels before intravitreal injection of the anti-VEGF antigen-binding fragment.

[0177] In certain embodiments, the methods provided herein are for administration to patients diagnosed with AMD who have a condition other than exudate that contributes to increased CRT (i.e., pigment epithelial detachment (PED) or subretinal hyperreflective material (SHRM)) and who have exudate (intraretinal or subretinal) of <75 μm as determined by CRC.

[0178] In certain embodiments of the methods described herein, the patient has a BCVA in the eye to be treated that is ≦20 / 20 and ≧20 / 400 before treatment. In certain embodiments, the patient has a BCVA in the eye to be treated that is ≦20 / 25 and ≧20 / 125 (≦83 and ≧44 ETDRS letters) before treatment. In certain embodiments, the patient has a BCVA in the eye to be treated that is ≦20 / 63 and ≧20 / 400 before treatment.

[0179] In certain embodiments of the methods described herein, the patient has a negative or low serum titer result (≦300) for a recombinant viral vector neutralizing antibody (NAb). In certain embodiments, the patient has a negative or low serum titer result (≦300) for an AAV8 Nab. In other embodiments, the patient has a serum titer result >300 for a recombinant viral vector Nab. In certain embodiments, the patient has a serum titer result >300 for an AAV8 Nab. In certain embodiments, the methods described herein are effective for treating AMD or DR in patients with a negative or low serum titer result (≦300) for an AAV8 Nab or in patients with a serum titer result >300 for a recombinant viral vector Nab. In certain embodiments, the methods described herein are effective for treating AMD and DR in patients with a negative or low serum titer result (≦300) for an AAV8 Nab, as well as in patients with a serum titer result >300 for a recombinant viral vector Nab.

[0180] In certain embodiments of the methods described herein, the patient is not on concurrent anticoagulant therapy.

[0181] In certain embodiments, the methods provided herein are for administration to patients diagnosed with severe diabetic retinopathy. In certain embodiments, the methods provided herein are for administration to patients diagnosed with attenuated diabetic retinopathy.

[0182] In certain embodiments, the methods provided herein are for administration to patients diagnosed with moderate to severe NPDR. In certain embodiments, the methods provided herein are for administration to patients diagnosed with severe NPDR. In certain embodiments, the methods provided herein are for administration to patients diagnosed with mild PDR. In certain embodiments, the methods provided herein are for administration to patients diagnosed with moderate PDR.

[0183] In certain embodiments of the methods described herein, the patient has an Early Treatment Diabetic Retinopathy Study (ETDRS) BCVA letter score of between ≦78 and ≧44 in the eye to be treated prior to treatment.

[0184] In certain embodiments, the methods provided herein are for administration to a patient with an ETDRS-DRSS level of 47, 53, 61, or 65. In certain embodiments, the methods provided herein are for administration to a patient with an ETDRS-DRSS level of 47. In certain embodiments, the methods provided herein are for administration to a patient with an ETDRS-DRSS level of 53. In certain embodiments, the methods provided herein are for administration to a patient with an ETDRS-DRSS level of 61. In certain embodiments, the methods provided herein are for administration to a patient with an ETDRS-DRSS level of 65.

[0185] In certain embodiments, the subject treated according to the methods described herein is female. In certain embodiments, the subject treated according to the methods described herein is male. In certain embodiments, the subject treated according to the methods described herein can be of any age. In certain embodiments, the subject treated according to the methods described herein is 18 years of age or older. In certain embodiments, the subject treated according to the methods described herein is between 18 and 89 years of age. In certain embodiments, the subject treated according to the methods described herein is between 25 and 89 years of age. In certain embodiments, the subject treated according to the methods described herein has DR secondary to type 1 diabetes. In certain embodiments, the subject treated according to the methods described herein has DR secondary to type 2 diabetes. In certain embodiments, the subject treated according to the methods described herein is 18 years of age or older with DR secondary to type 1 or type 2 diabetes. In certain embodiments, the subject treated according to the methods described herein is between 18 and 89 years of age with DR secondary to type 1 or type 2 diabetes.

[0186] In certain embodiments, the subject treated in accordance with the methods described herein is a female of non-childbearing potential.

[0187] In certain embodiments, the subject treated according to the methods described herein is phakic. In other certain embodiments, the subject treated according to the methods described herein is pseudophakic.

[0188] In certain embodiments, subjects treated according to the methods described herein have a hemoglobin A1c≦12% (as confirmed by laboratory evaluation).

[0189] In certain embodiments, subjects treated according to the methods described herein have a best corrected visual acuity (BCVA) in the eye to be treated of ≧69 ETDRS letters (approximate Snellen equivalent of 20 / 40 or better).

[0190] In certain embodiments, a method for treating a subject with diabetic retinopathy (DR), wherein the subject has at least one eye with DR, comprises the following steps: (1) determining the subject's ETDRS-DR Severity Scale (DRSS) level; and (2) administering an expression vector encoding an anti-human vascular endothelial growth factor (hVEGF) antibody to the subretinal space or suprachoroidal space of the eye of a human subject when the subject's ETDRS-DRSS is at level 47, 53, 61, or 65; A method is provided herein, comprising:

[0191] In some embodiments, the method further comprises obtaining or having obtained a biological sample from the subject and determining that the subject has a serum level of hemoglobin A1c of 10% or less.

[0192] In some embodiments, the method prevents progression of retinopathy to a proliferative stage in a subject.

[0193] In certain embodiments, a method for treating a subject with diabetic retinopathy, wherein the subject has at least one eye with moderate to severe non-proliferative diabetic retinopathy (NPDR), comprises the steps of: (1) determining the subject's ETDRS-DR Severity Scale (DRSS) level; and (2) administering an expression vector encoding an anti-human vascular endothelial growth factor (hVEGF) antibody to the subretinal or suprachoroidal space of the eye of a human subject when the subject's ETDRS-DRSS is at level 47; A method is provided herein, comprising:

[0194] In certain embodiments, a method for treating a subject with diabetic retinopathy, wherein the subject has at least one eye with severe NPDR, comprises the following steps: (1) determining the subject's ETDRS-DR Severity Scale (DRSS) level; and (2) administering an expression vector encoding an anti-human vascular endothelial growth factor (hVEGF) antibody to the subretinal or suprachoroidal space of the eye of a human subject when the subject's ETDRS-DRSS is at level 53; A method is provided herein, comprising:

[0195] In certain embodiments, a method for treating a subject with diabetic retinopathy, wherein the subject has at least one eye with mild proliferative diabetic retinopathy (PDR), comprises the following steps: (1) determining the subject's ETDRS-DR Severity Scale (DRSS) level; and (2) administering an expression vector encoding an anti-human vascular endothelial growth factor (hVEGF) antibody to the subretinal or suprachoroidal space of the eye of a human subject when the subject's ETDRS-DRSS is at level 61; A method is provided herein, comprising:

[0196] In certain embodiments, a method for treating a subject with diabetic retinopathy, wherein the subject has at least one eye with moderate PDR, comprises the following steps: (1) determining the subject's ETDRS-DR Severity Scale (DRSS) level; and (2) administering an expression vector encoding an anti-human vascular endothelial growth factor (hVEGF) antibody to the subretinal or suprachoroidal space of the eye of a human subject when the subject's ETDRS-DRSS is at level 65; A method is provided herein, comprising:

[0197] In certain embodiments of the methods described herein, the patient has a negative or low serum titer result (≦300) for recombinant viral vector neutralizing antibodies (NAbs). In certain embodiments, the patient has a negative or low serum titer result (≦300) for AAV8 Nabs. In other embodiments, the patient has a serum titer result >300 for recombinant viral vector Nabs. In certain embodiments, the patient has a serum titer result >300 for AAV8 Nabs.

[0198] ETDRS-DR Severity Scale (DRSS) levels are determined using standard 4 wide-field digital stereo fundus photographs or equivalent, and they can also be measured by monoscopic or stereo photography according to Li et al., 2010, Retina Invest Ophthalmol Vis Sci. 2010;51:3184-3192 or similar methods.

[0199] 5.3.3 Dosage and Mode of Administration A therapeutically effective dose of the recombinant vector should be administered subretinally and / or intraretinally (e.g., by subretinal injection via a transvitreal approach (surgical procedure) or via the suprachoroidal space) in a volume ranging from ≧0.1 mL to ≦0.5 mL, preferably 0.1 to 0.30 mL (100 to 300 μl), and most preferably 0.25 mL (250 μl). A therapeutically effective dose of the recombinant vector should be administered suprachoroidally (e.g., by suprachoroidal injection) in a volume of 100 μl or less, for example, 50 to 100 μl. A therapeutically effective dose of the recombinant vector should be administered to the outer surface of the sclera in a volume of 500 μl or less, e.g., 10-20 μl, 20-50 μl, 50-100 μl, 100-200 μl, 200-300 μl, 300-400 μl, or 400-500 μl. A therapeutically effective dose of the recombinant vector may also be administered to the outer surface of the sclera in two or more injections in a volume of 500 μl or less, e.g., 10-20 μl, 20-50 μl, 50-100 μl, 100-200 μl, 200-300 μl, 300-400 μl, or 400-500 μl. Two or more injections may be administered during the same visit.

[0200] In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to a subject as a single dosage form. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to a subject as a single injection. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to a subject as a single injection per eye.

[0201] In certain embodiments, the recombinant vector is administered to the suprachoroid (e.g., by suprachoroidal injection). In certain embodiments, suprachoroidal administration (e.g., injection into the suprachoroidal space) is performed using a suprachoroidal drug delivery device. Suprachoroidal drug delivery devices are often used in suprachoroidal administration procedures, including administration of drugs to the suprachoroidal space of the eye (see, for example, Hariprasad, 2016, Retinal Physician 13: 20-23; Goldstein, 2014, Retina Today 9(5): 82-87; Baldassarre et al., 2017, each of which is incorporated herein by reference in its entirety). Choroidal drug delivery devices that can be used to place expression vectors in the subretinal space according to the embodiments described herein include, but are not limited to, the choroidal drug delivery device manufactured by Clearside® Biomedical, Inc. (see, e.g., Hariprasad, 2016, Retinal Physician 13:20-23) and the MedOne choroidal catheter.

[0202] In certain embodiments, the suprachoroidal drug delivery device is a syringe equipped with a 1-millimeter 30-gauge needle. During injection using this device, the needle penetrates to the base of the sclera, and the drug-containing liquid enters the suprachoroidal space, leading to the expansion of the suprachoroidal space. As a result, there is tactile and visual feedback during injection. After injection, the fluid flows to the posterior side and is absorbed predominantly in the choroid and retina. This results in the production of transgene proteins from all retinal cell layers and choroidal cells. The use of this type of device and procedure allows for quick, easy in-clinic procedures with low risk of complications. A maximum volume of 100 μl can be injected into the suprachoroidal space.

[0203] In certain embodiments, the recombinant vector is administered subretinally via the suprachoroidal space using a subretinal drug delivery device. In certain embodiments, the subretinal drug delivery device is a catheter that is inserted and tunneled through the suprachoroidal space around the back of the eye during a surgical procedure to deliver the drug to the subretinal space (see FIG. 5). This procedure allows the vitreous to remain intact, thus reducing the risk of complications (such as gene therapy spillage and retinal detachment and macular holes), does not involve vitrectomy, and the resulting bleb can spread more diffusely, allowing more of the retinal surface area to be transduced in a smaller volume. The risk of cataract induction after this procedure is minimized, which is desirable for younger patients. Furthermore, this procedure allows for safer delivery of the bleb below the fovea than the standard transvitreal approach, which is desirable for patients with inherited retinal diseases affecting central vision, where the target cells for transduction are in the macula. This procedure is also advantageous for patients with neutralizing antibodies (NABs) against AAV present in the systemic circulation, which may affect other delivery routes (e.g., suprachoroidal and intravitreal). Furthermore, this method has been shown to create a bleb with less outflow from the retinotomy site than the standard transvitreal approach. Subretinal drug delivery devices, originally manufactured by Janssen Pharmaceuticals, Inc. and now by Orbit Biomedical Inc. (see, e.g., Subretinal Delivery of Cells via the Suprachoroidal Space: Janssen Trial, in Schwartz et al. (eds.) Cellular Therapies for Retinal Disease, Springer, Cham. International Patent Application Publication No. WO2016 / 040635), can be used for this purpose.

[0204] In certain embodiments, the recombinant vector is administered to the outer surface of the sclera (e.g., by use of a juxtascleral drug delivery device comprising a cannula, the tip of which can be inserted and maintained in direct apposition to the scleral surface). In certain embodiments, administration to the outer surface of the sclera is performed using a posterior juxtascleral depot procedure, in which the drug is aspirated into a blunt-tip curved cannula and then delivered in direct contact with the outer surface of the sclera without puncturing the eye. In particular, after making a small incision to expose the sclera, the cannula tip is inserted (see Figure 6A). The curved portion of the cannula shaft is inserted, maintaining the cannula tip in direct apposition to the scleral surface (see Figures 6B-6D). After the cannula is fully inserted (Figure 6D), the drug is slowly injected while maintaining gentle pressure along the tip and sides of the cannula shaft with a sterile cotton swab. This delivery method avoids the risk of intraocular infection and retinal detachment, side effects commonly associated with injecting therapeutic agents directly into the eye.

[0205] A dose that maintains a transgene product concentration of at least 0.330 μg / mL in the vitreous humor or 0.110 μg / mL in the aqueous humor (anterior chamber of the eye) Cmin for three months is desired, after which a vitreous Cmin concentration of the transgene product in the range of 1.70-6.60 μg / mL and / or an ocular chamber Cmin concentration in the range of 0.567-2.20 μg / mL should be maintained. However, because the transgene product is produced continuously (either under the control of a constitutive promoter or induced by hypoxic conditions when a hypoxia-inducible promoter is used), maintaining lower concentrations may be effective. Vitreous humor concentrations can be measured directly in patient samples of fluid collected from the vitreous humor or anterior chamber, or can be estimated and / or monitored by measuring the patient's serum concentration of the transgene product—the ratio of systemic to vitreous exposure of the transgene product is approximately 1:90,000. (See, e.g., the vitreous and serum concentrations of ranibizumab reported in Table 5 of Xu L et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616-1624, 1621, and 1623, which is incorporated herein by reference in its entirety.)

[0206] In certain embodiments, described herein is a microvolume syringe delivery system (see FIGS. 8A and 8B) manufactured by Altaviz (see, e.g., International Patent Application Publication No. WO 2013 / 177215, U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906), which can be used for any of the administration routes described herein for ocular administration. The microvolume syringe delivery system can include a gas drive module, which provides powerful delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume syringe delivery system can include a hydraulic drive and gas drive module to provide a constant administration rate, which in turn can include a low-force activation lever to control fluid delivery. In certain embodiments, the microvolume injector delivery system can be used for a microvolume injector that is a microvolume injector with dose guidance, for example, with a suprachoroidal needle (e.g., a Clearside® needle), a subretinal needle, an intravitreal needle, a juxtascleral needle, a subconjunctival needle, and / or an intraretinal needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., by having precise injection flow rate control and dose guidance); (b) surgery by one surgeon, one hand, one finger; (c) pneumatic actuation of 10 μL increment doses; (d) decoupling from the vitrectomy machine; (e) 400 μL syringe dose; (f) digitally guided delivery; (g) digitally recorded delivery; and (h) tip independence (e.g., a MedOne 38 g needle and a Dorc 41 g needle can be used for subretinal delivery, while a Clearside® needle and a Visionisti OY adapter can be used for subretinal delivery).

[0207] In certain embodiments of the methods described herein, the recombinant vector is administered to the suprachoroid (e.g., by suprachoroidal injection). In certain embodiments, suprachoroidal administration (e.g., injection into the suprachoroidal space) is performed using a suprachoroidal drug delivery device. Suprachoroidal drug delivery devices are often used in suprachoroidal administration procedures, including administration of drugs to the suprachoroidal space of the eye (see, for example, Hariprasad, 2016, Retinal Physician 13:20-23; Goldstein, 2014, Retina Today 9(5):82-87; Baldassarre et al., 2017, each of which is incorporated herein by reference in its entirety). Choroidal drug delivery devices that can be used to place an expression vector in the choroidal space according to the embodiments described herein include, but are not limited to, the choroidal drug delivery device manufactured by Clearside® Biomedical, Inc. (see, e.g., Hariprasad, 2016, Retinal Physician 13:20-23) and the MedOne choroidal catheter. In another embodiment, a choroidal drug delivery device that can be used according to the methods described herein includes a microvolume injector delivery system manufactured by Altaviz (see Figures 8A and 8B), which can be used for any of the administration routes described herein for ocular administration (see, e.g., International Patent Application Publication No. WO2013 / 177215, U.S. Patent Application Publication No. 2019 / 0175825, and U.S. Patent Application Publication No. 2019 / 0167906). The microvolume injector delivery system may include a gas drive module that provides powerful delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume injector delivery system may include a hydraulic drive and gas drive module to provide a constant dosing rate and a low force actuation lever to in turn control fluid delivery.The microvolume injector is a microvolume injector with dose guidance and can be used, for example, with a suprachoroidal needle (e.g., a Clearside® needle) or a subretinal needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., by having precise injection flow rate control and dose guidance); (b) surgery by one surgeon, one hand, one finger; (c) pneumatic actuation of 10 μL increments; (d) decoupling from the vitrectomy machine; (e) 400 μL syringe dose; (f) digitally guided delivery; (g) digitally recorded delivery; and (h) tip independence (e.g., a MedOne 38 g needle and a Dorc 41 g needle can be used for subretinal delivery, while a Clearside® needle and a Visionisti OY adapter can be used for suprachoroidal delivery). In another embodiment, a suprachoroidal drug delivery device that can be used in accordance with the methods described herein is a tool manufactured by Visionisti OY, comprising a normal-length hypodermic needle with an adapter (and preferably also a needle guide), which converts the normal-length hypodermic needle into a suprachoroidal needle by controlling the length of the needle tip exposed from the adapter (see Figures 9A and 9B) (see, for example, U.S. Design Patent No. D878,575 and International Patent Application Publication No. WO2016 / 083669). In certain embodiments, the suprachoroidal drug delivery device is a syringe with a 1-millimeter 30-gauge needle. During injection using this device, the needle penetrates to the base of the sclera, and the drug-containing liquid enters the suprachoroidal space, leading to the expansion of the suprachoroidal space. As a result, there is tactile and visual feedback during injection. After injection, the fluid flows posteriorly and is predominantly absorbed in the choroid and retina. This results in the production of a therapeutic product from all retinal cell layers and choroidal cells. The use of this type of device and procedure allows for a quick, easy in-office procedure with a low risk of complications. A maximum volume of 100 μl can be injected into the suprachoroidal space.

[0208] In certain embodiments, intravitreal administration is performed using an intravitreal drug delivery device, including a microvolume injector delivery system (see Figures 8A and 8B) manufactured by Altaviz, which can be used for any of the administration routes described herein for ocular administration (see, e.g., International Patent Application Publication No. WO 2013 / 177215, U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906). The microvolume injector delivery system may include a gas-driven module, which provides powerful delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume injector delivery system may include a hydraulic drive and a gas-driven module to provide a constant administration rate, which in turn includes a low-force activation lever to control fluid delivery. The microvolume injector is a microvolume injector with dose guidance and can be used, for example, with an intravitreal needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., by having precise injection flow rate control and dose guidance), (b) surgery with one surgeon, one hand, and one finger, (c) pneumatic actuation of 10 μL increment doses, (d) decoupling from the vitrectomy machine, (e) 400 μL syringe dose, (f) digitally guided delivery, (g) digitally recorded delivery, and (h) tip independence. In certain embodiments, subretinal administration is performed using a subretinal drug delivery device, including a microvolume injector delivery system (see FIGS. 8A and 8B) manufactured by Altaviz, which can be used for any of the administration routes described herein for ocular administration (see, e.g., International Patent Application Publication No. WO 2013 / 177215, U.S. Patent Application Publication No. 2019 / 0175825, and U.S. Patent Application Publication No. 2019 / 0167906). The microvolume injector delivery system may include a gas drive module that provides powerful delivery and improved accuracy, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906.Additionally, the microvolume injector delivery system may include a hydraulic and gas drive module to provide a constant injection rate, and a low-force actuation lever to control fluid delivery in turn. The microvolume injector is a microvolume injector with dose guidance, and can be used, for example, with a subretinal needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., by having precise injection flow rate control and dose guidance), (b) surgery by one surgeon, one hand, one finger, (c) pneumatic actuation of 10 μL increments, (d) decoupling from the vitrectomy machine, (e) 400 μL syringe dose, (f) digitally guided delivery, (g) digitally recorded delivery, and (h) tip independence (e.g., MedOne 38 g needles and Dorc 41 g needles can be used for subretinal delivery, while Clearside® needles and Visionisti OY adapters can be used for suprachoroidal delivery).

[0209] In certain embodiments, the recombinant vector is administered to the outer surface of the sclera (e.g., by use of a juxtascleral drug delivery device comprising a cannula, the tip of which can be inserted and maintained in direct apposition to the scleral surface). In certain embodiments, administration to the outer surface of the sclera is performed using a posterior juxtascleral depot procedure, in which the drug is aspirated into a blunt-tip curved cannula and then delivered in direct contact with the outer surface of the sclera without puncturing the eye. In particular, after making a small incision to expose the sclera, the cannula tip is inserted (see Figure 6A). The curved portion of the cannula shaft is inserted, maintaining the cannula tip in direct apposition to the scleral surface (see Figures 6B-6D). After the cannula is fully inserted (Figure 6D), the drug is slowly injected while maintaining gentle pressure along the tip and sides of the cannula shaft with a sterile cotton swab. This delivery method avoids the risk of intraocular infection and retinal detachment, side effects commonly associated with injecting therapeutic agents directly into the eye. In certain embodiments, juxtascleral administration is performed using a juxtascleral drug delivery device, including a microvolume injector delivery system (see Figures 8A and 8B) manufactured by Altaviz, which can be used for any of the administration routes described herein for ocular administration (see, e.g., International Patent Application Publication No. WO 2013 / 177215, U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906). The microvolume injector delivery system may include a gas-driven module, which provides powerful delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume injector delivery system may include a hydraulic drive and a gas-driven module to provide a constant administration rate, which in turn includes a low-force activation lever to control fluid delivery. The microvolume injector is a microvolume injector with dose guidance and can be used, for example, with a juxtascleral needle.Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., by having precise injection flow rate control and dose guidance), (b) surgery by one surgeon, one hand, one finger, (c) pneumatic actuation of 10 μL increment dose, (d) decoupling from the vitrectomy machine, (e) 400 μL syringe dose, (f) digitally guided delivery, (g) digitally recorded delivery, and (h) tip independent.

[0210] In certain embodiments, the dosage is measured by genome copies or genome copy number per ml administered to the patient's eye (e.g., suprachoroidally, subretinally, intravitreally, juxtascleral, subconjunctivally, and / or intraretinally (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior juxtascleral depot procedure). In certain embodiments, the dosage is measured by genome copies or genome copy number per ml. In certain embodiments, the dosage is measured by 2.4 x 10 per ml. 11 Genome copies ~ 1 x 10 per ml 13 In a specific embodiment, 2.4 x 10 genome copies per ml are administered. 11 Genome copies per ml: 5 x 10 11 In another specific embodiment, 5 x 10 genome copies per ml are administered. 11 Genome copies ~ 1 x 10 per ml 12 In another specific embodiment, 1 x 10 genome copies per ml are administered. 12 Genome copies per ml: 5 x 10 12 In another specific embodiment, 5 x 10 genome copies per ml are administered. 12 Genome copies ~ 1 x 10 per ml 13 In another specific embodiment, about 2.4 x 10 genome copies per ml are administered. 11 In another specific embodiment, about 5 x 10 genome copies per ml are administered. 11 In another specific embodiment, about 1 x 10 genome copies per ml are administered. 12 In another specific embodiment, about 5 x 10 genome copies per ml are administered. 12In another specific embodiment, about 1 x 10 genome copies per ml are administered. 13 A genome copy is administered.

[0211] In one particular embodiment, 1×10 9 ~1×10 12 In certain embodiments, 3 x 10 genome copies are administered. 9 ~2.5×10 11 In certain embodiments, 1 x 10 genome copies are administered. 9 ~2.5×10 11 In certain embodiments, 1 x 10 genome copies are administered. 9 ~1×10 11 In certain embodiments, 1 x 10 genome copies are administered. 9 ~5×10 9 In certain embodiments, 6 x 10 genome copies are administered. 9 ~3×10 10 In certain embodiments, 4 x 10 genome copies are administered. 10 ~1×10 11 In certain embodiments, 2 x 10 genome copies are administered. 11 ~1×10 12 In certain embodiments, about 3 x 10 genome copies are administered. 9 Genome copies are administered (approximately 1.2 x 10 per ml in a volume of 250 μl) 10 In another specific embodiment, about 1 x 10 10 Genome copies are administered (approximately 4 x 10 per ml in a volume of 250 μl) 10 In another specific embodiment, about 6×10 10 Genome copies are administered (approximately 2.4 x 10 per ml in a volume of 250 μl) 11 In another specific embodiment, about 1.6 x 10 11 Genome copies are administered (approximately 6.2 x 10 per ml in a volume of 250 μl) 11 In another specific embodiment, about 1.55 x 10 11 Genome copies are administered (approximately 6.2 x 10 per ml in a volume of 250 μl)11 In another specific embodiment, about 1.6 x 10 11 Genome copies are administered (approximately 6.4 x 10 per ml in a volume of 250 μl) 11 In another specific embodiment, about 2.5 x 10 11 Genome copies (approximately 1.0 × 10 in a volume of 250 μl) 12 (equivalent to ) is administered.

[0212] In one particular embodiment, about 3.0 x 10 per eye 13 In certain embodiments, up to 3.0 x 10 genome copies are administered per eye. 13 A genome copy is administered.

[0213] In one particular embodiment, about 6.0 x 10 per eye 10 In one particular embodiment, about 1.6 x 10 genome copies are administered per eye. 11 In one particular embodiment, about 2.5 x 10 genome copies are administered per eye. 11 In one particular embodiment, about 5.0 x 10 genome copies are administered per eye. 11 In certain embodiments, about 3 x 10 genome copies are administered per eye. 12 In certain embodiments, about 1 x 10 genome copies per ml per eye are administered. 12 In certain embodiments, about 2.5 x 10 genome copies are administered per ml per eye. 12 A genome copy is administered.

[0214] In one particular embodiment, about 6.0 x 10 per eye 10 In certain embodiments, about 1.6 x 10 genome copies are administered by subretinal injection per eye. 11 In certain embodiments, about 2.5 x 10 genome copies are administered per eye by subretinal injection. 11 In certain embodiments, about 3.0 x 10 genome copies are administered by subretinal injection. 13Genome copies are administered by subretinal injection. In certain embodiments, up to 3.0 x 10 genome copies per eye are administered. 13 The genome copies are administered by subretinal injection.

[0215] In one particular embodiment, about 2.5 x 10 per eye 11 In certain embodiments, about 5.0 x 10 genome copies are administered per eye by suprachoroidal injection. 11 Genome copies are administered by suprachoroidal injection. In certain embodiments, about 3 x 10 genome copies are administered per eye. 12 Genome copies are administered by suprachoroidal injection. In certain embodiments, about 2.5 x 10 genome copies are administered per eye. 11 In certain embodiments, about 5.0 x 10 genome copies are administered per eye via a single suprachoroidal injection. 11 In certain embodiments, about 3.0 x 10 genome copies are administered per eye by double suprachoroidal injection. 13 Genome copies are administered by suprachoroidal injection. In certain embodiments, up to 3.0 x 10 genome copies per eye are administered. 13 Genome copies are administered by suprachoroidal injection. In certain embodiments, about 2.5 x 10 genome copies are administered per ml per eye. 12 Genome copies are administered by a single suprachoroidal injection in a volume of 100 μl. In certain embodiments, about 2.5×10 genome copies are administered per ml per eye. 12 genome copies are administered by double suprachoroidal injection, each injection in a volume of 100 μl.

[0216] In certain embodiments, about 1.5 x 10 per administration or per eye 11 Genome copies are administered by suprachoroidal injection. In certain embodiments, about 2.5 x 10 genome copies are administered per administration or per eye. 11 In certain embodiments, about 5.0 x 10 genome copies are administered per administration or per eye by suprachoroidal injection. 11 Genome copies are administered by suprachoroidal injection. In certain embodiments, about 1.0 x 10 genome copies are administered per administration or per eye. 12Genome copies are administered by suprachoroidal injection. In certain embodiments, about 1.5 x 10 genome copies are administered per administration or per eye. 12 Genome copies are administered by suprachoroidal injection. In certain embodiments, about 2.5 x 10 genome copies are administered per eye. 11 In certain embodiments, about 2.5 x 10 genome copies are administered per eye via a single suprachoroidal injection. 11 Genome copies are administered by a single suprachoroidal injection in a volume of about 100 μl. In certain embodiments, about 5×10 genome copies are administered per eye. 11 Genome copies are administered by a single suprachoroidal injection. In certain embodiments, about 5 x 10 genome copies are administered per eye. 11 Genome copies are administered by a single suprachoroidal injection in a volume of about 100 μl. In certain embodiments, about 5×10 genome copies are administered per administration or per eye. 11 Genome copies are administered by double suprachoroidal injection. In certain embodiments, about 5 x 10 genome copies are administered per eye. 11 Genome copies are administered by double suprachoroidal injection, each injection in a volume of 100 μl. In certain embodiments, about 1×10 genome copies are administered per eye. 12 In certain embodiments, about 1 x 10 genome copies are administered per eye via a single suprachoroidal injection. 12 Genome copies are administered by a single suprachoroidal injection in a volume of about 100 μl. In certain embodiments, about 1.5×10 genome copies are administered per eye. 12 In certain embodiments, about 1.5 x 10 genome copies are administered per eye via a single suprachoroidal injection. 12 The genome copies are administered by a single suprachoroidal injection in a volume of approximately 100 μl.

[0217] As used herein, unless otherwise specified, the term "about" means within plus or minus 10% of a given value or range.

[0218] In certain embodiments, the term "about" is inclusive of the exact number recited.

[0219] When used in phrases such as "between A and B" or "between A and B," the term "between" refers to a range that includes both A and B.

[0220] In certain embodiments, an infrared thermal camera can be used to detect changes in the thermal profile of the ocular surface after administration of a solution that is colder than body temperature, potentially allowing visualization of the spread of the solution within the SCS and determining whether administration was successful. This is because, in certain embodiments, the formulation containing the recombinant vector to be administered is first frozen and then thawed at room temperature (68-72°F) for a short period (e.g., at least 30 minutes) before administration. Therefore, the formulation is colder than the human eye (approximately 92°F) at the time of injection (and may even be colder than room temperature). Drug products are typically used within four hours of thawing, with the warmest solutions being at room temperature. In a preferred embodiment, the procedure is videotaped with infrared video.

[0221] Infrared thermal cameras can detect small changes in temperature. They capture infrared energy through a lens and convert the energy into an electronic signal. The infrared light is focused onto an infrared sensor array, which converts the energy into a thermal image. Infrared thermal cameras can be used for any of the administration routes described herein, including suprachoroidal administration, subretinal administration, subconjunctival administration, intravitreal administration, or administration using a slow infusion catheter into the suprachoroidal space. In certain embodiments, the infrared thermal camera is a FLIR T530 infrared thermal camera. The FLIR T530 infrared thermal camera can capture small temperature differences with an accuracy of ±3.6°F. The camera has an infrared resolution of 76,800 pixels. The camera also utilizes a 24° lens that captures a smaller field of view. The smaller field of view, combined with the high infrared resolution, provides a more detailed thermal profile of what the operator is imaging. However, other infrared cameras with different capabilities and accuracy for capturing small temperature changes, different infrared resolution, and / or different lens strengths can be used.

[0222] In certain embodiments, the infrared thermal camera is a FLIR T420 infrared thermal camera. In certain embodiments, the infrared thermal camera is a FLIR T440 infrared thermal camera. In certain embodiments, the infrared thermal camera is a Fluke Ti400 infrared thermal camera. In certain embodiments, the infrared thermal camera is a FLIRE60 infrared thermal camera. In certain embodiments, the infrared thermal camera has an infrared resolution of 75,000 pixels or greater. In certain embodiments, the infrared thermal camera has a thermal sensitivity of 0.05°C or less at 30°C. In certain embodiments, the infrared thermal camera has a field of view (FOV) of 25° x 25° or less.

[0223] In certain embodiments, an iron filter is used in conjunction with an infrared thermal camera to detect changes in the thermal profile of the ocular surface. In a preferred embodiment, the use of an iron filter allows for the generation of a false color image, with the warmest or hottest areas colored white, intermediate temperatures red and yellow, and the coldest or coldest areas black. In certain embodiments, other types of filters may also be used to generate a false color image of the thermal profile.

[0224] The thermal profile of each administration method may be different. For example, in one embodiment, a successful suprachoroidal injection can be characterized by (a) a slow, wide radial spread of dark color, (b) an initial very dark color, and (c) a gradual change of the injectate to a lighter color, i.e., a temperature gradient indicated by the lighter color. In one embodiment, an unsuccessful suprachoroidal injection can be characterized by (a) no spread of dark color and (b) a slight change in color localized at the injection site without any distribution. In certain embodiments, the small localized temperature drop is the result of the cannula (low temperature) contacting the ocular tissue (high temperature). In one embodiment, a successful intravitreal injection can be characterized by (a) no spread of dark color, (b) an initial change to a very dark color localized at the injection site, and (c) a gradual and uniform change to a darker color throughout the eye. In one embodiment, extraocular outflow can be characterized by (a) a rapid flow outside the outer surface of the eye, (b) an initial very dark color, and (c) a rapid change to a lighter color.

[0225] 5.3.4 Sampling and Efficacy Monitoring The effect of the methods of treatment provided herein on visual impairment may be measured by BCVA (best corrected visual acuity), intraocular pressure, slit lamp biomicroscopy, and / or indirect ophthalmoscopy. Extraocular movements may also be assessed. Intraocular pressure measurements may be performed using Tonopen or Goldmann applanation tonometry. Slit lamp examination may include evaluation of the eyelids / lashes, conjunctiva / sclera, cornea, anterior chamber, iris, lens, and / or vitreous.

[0226] In certain embodiments, the effect of the methods provided herein on visual impairment can be measured by whether the eye of a human patient treated by the methods described herein achieves a BCVA of greater than 43 letters after treatment (e.g., 46-50 weeks or 98-102 weeks after treatment). A BCVA of 43 letters corresponds to the approximate Snellen equivalent of 20 / 160. In certain embodiments, the eye of a human patient treated by the methods described herein achieves a BCVA of greater than 43 letters after treatment (e.g., 46-50 weeks or 98-102 weeks after treatment).

[0227] In certain embodiments, the effect of the methods provided herein on visual impairment can be measured by whether the eye of a human patient treated by the methods described herein achieves a BCVA of greater than 84 letters after treatment (e.g., 46-50 weeks or 98-102 weeks after treatment). A BCVA of 84 letters corresponds to the approximate Snellen equivalent of 20 / 20. In certain embodiments, the eye of a human patient treated by the methods described herein achieves a BCVA of greater than 84 letters after treatment (e.g., 46-50 weeks or 98-102 weeks after treatment). BCVA testing can be performed at a distance of 4 meters using an ETDRS chart. For participants with reduced vision (unable to correctly read ≥20 letters at 4 meters), BCVA testing can be performed at a distance of 1 meter.

[0228] The effect of the methods of treatment provided herein on physical changes in the eye / retina can be measured by SD-OCT (SD Optical Coherence Tomography).

[0229] Efficacy can be monitored as measured by electroretinography (ERG).

[0230] The effectiveness of the methods of treatment provided herein can be monitored by measuring signs of vision loss, infection, inflammation, and other safety events, including retinal detachment.

[0231] Retinal thickening can be monitored to determine the effectiveness of the treatments provided herein. Without being bound by any particular theory, retinal thickening can be used as a clinical readout, and the greater the reduction in retinal thickening or the longer the time until retinal thickening, the more effective the treatment. Retinal function can be determined, for example, by ERG. ERG is a non-invasive electrophysiological test of retinal function approved by the FDA for use in humans, which examines the light-sensitive cells (rods and cones) of the eye and their connected ganglion cells, particularly their response to flashing light stimuli. Retinal thickening can be determined, for example, by SD-OCT. SD-OCT is a three-dimensional imaging technique that uses low-coherence interferometry to determine the echo time delay and amplitude of backscattered light reflected from an object of interest. OCT can be used to scan layers of tissue samples (e.g., the retina) with an axial resolution of 3-15 μm, and SD-OCT improves axial resolution and scanning speed over previous forms of the technique (Schuman, 2008, Trans. Am. Opthamol. Soc. 106:426-458).

[0232] The efficacy of the methods provided herein can also be measured by the change from baseline in the National Eye Institute Visual Function Questionnaire, Rasch score version (NEI-VFQ-28-R) (composite score, activity limitation domain score, and socio-emotional functioning domain score). The efficacy of the methods provided herein can also be measured by the change from baseline in the National Eye Institute Visual Function Questionnaire, 25-item version (NEI-VFQ-25) (composite score and mental health subscale score). The efficacy of the methods provided herein can also be measured by the change from baseline in the Macular Treatment Satisfaction Questionnaire (MacTSQ) (composite score; safety, efficacy, and discomfort domain score, and informational and convenience domain score).

[0233] In certain embodiments, the effectiveness of the methods described herein is reflected by improvement in vision at about 4 weeks, 12 weeks, 6 months, 12 months, 24 months, 36 months, or other desired time points. In certain embodiments, improvement in vision is characterized by an increase in BCVA, for example, an increase of 1 letter, 2 letters, 3 letters, 4 letters, 5 letters, 6 letters, 7 letters, 8 letters, 9 letters, 10 letters, 11 letters, or 12 letters or more. In certain embodiments, improvement in vision is characterized by an increase in visual acuity from baseline of 5%, 10%, 15%, 20%, 30%, 40%, 50% or more.

[0234] In certain embodiments, the effectiveness of the methods described herein is reflected by a reduction in central retinal thickening (CRT) at about 4 weeks, 12 weeks, 6 months, 12 months, 24 months, 36 months, or other desired time point, for example, a 5%, 10%, 15%, 20%, 30%, 40%, 50% or more reduction in central retinal thickening from baseline.

[0235] In certain embodiments, there is no inflammation in the eye after treatment, or there is little inflammation in the eye after treatment (e.g., a 10%, 5%, 2%, 1% or less increase in the level of inflammation from baseline). The effect of the methods provided herein on visual impairment can be measured by optokinetic nystagmus (OKN).

[0236] In certain embodiments, the proportion of subjects who develop ocular inflammation (e.g., a 10% or greater, 5% or greater, 2% or greater, or 1% or greater increase in the level of ocular inflammation from baseline) after administration of an anti-VEGF treatment and a steroid treatment described herein is less than three-quarters, less than half, less than one-quarter, or less than one-tenth of all subjects in the population of subjects. In certain embodiments, the proportion of subjects who develop ocular inflammation (e.g., a 10% or greater, 5% or greater, 2% or greater, or 1% or greater increase in the level of ocular inflammation from baseline) after administration of an anti-VEGF treatment and a steroid treatment described herein is reduced by about 5%, 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 100% compared to the proportion in a reference population. In certain embodiments, the proportion of subjects experiencing ocular inflammation (e.g., an increase in the level of ocular inflammation from baseline of 10% or more, 5% or more, 2% or more, or 1% or more) after administration of an anti-VEGF treatment and a steroid treatment described herein is about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, or about 5% to about 15% of subjects in a reference population. , about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. In certain embodiments, the reference population consists of individuals administered an ocular treatment for neovascular age-related mascular degeneration or diabetic retinopathy, wherein the ocular treatment is not one of those described herein, e.g., in Sections 5.2, 5.3, and / or 5.4.In certain embodiments, the reference population consists of individuals who have been treated with ocular treatment for neovascular age-related mascular degeneration or diabetic retinopathy, such as, for example, anti-VEGF treatment as described herein in Section 5.2 and / or Section 5.3, but who have not been treated with steroid treatment as described herein in Section 5.4.

[0237] Without being bound by theory, this vision screening uses the principle of the OKN involuntary reflex to objectively assess whether a patient's eyes can track a moving target. By using the OKN, no verbal communication is required between the examiner and the patient. As such, the OKN can be used to measure visual acuity in preverbal and / or nonverbal patients. In certain embodiments, the OKN is used to measure visual acuity in patients who are 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years old. In certain embodiments, an iPad is used to measure visual acuity by detecting the OKN reflex while the patient is watching an activity on the iPad.

[0238] Without being bound by theory, this vision screening utilizes the principle of the OKN involuntary reflex to objectively assess whether a patient's eyes can track a moving target. By using the OKN, no verbal communication is required between the examiner and the patient. As such, the OKN can be used to measure visual acuity in preverbal and / or nonverbal patients. In certain embodiments, the OKN is used to measure visual acuity in patients who are 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or under 5 years of age. In another specific embodiment, the OKN is used to measure visual acuity in patients aged 1-2 months, 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-7 months, 7-8 months, 8-9 months, 9-10 months, 10-11 months, 11 months to 1 year, 1-1.5 years, 1.5-2 years, 2-2.5 years, 2.5-3 years, 3-3.5 years, 3.5-4 years, 4-4.5 years, or 4.5-5 years. In another specific embodiment, the OKN is used to measure visual acuity in patients aged 6 months to 5 years. In one specific embodiment, an iPad is used to measure visual acuity by detection of the OKN reflex while the patient watches movements on the iPad.

[0239] If the human patient is a child, visual function can be assessed using an optokinetic nystagmus (OKN)-based approach or a modified OKN-based approach.

[0240] Vector shedding can be determined by measuring vector DNA in biological fluids, such as tears, serum, or urine, using, for example, quantitative polymerase chain reaction (PCR). In some embodiments, no vector gene copies are detectable in urine at any time after administration of the vector. In some embodiments, fewer than 1000, 500, 100, 50, or 10 vector gene copies / 5 μL are detectable in biological fluids (e.g., tears, serum, or urine) by quantitative polymerase chain reaction at any time after administration. In certain embodiments, 210 vector gene copies / 5 μL or less are detectable in serum. In some embodiments, fewer than 1000, 500, 100, 50, or 10 vector gene copies / 5 μL are detectable in biological fluids (e.g., tears, serum, or urine) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks after administration by quantitative polymerase chain reaction. In certain embodiments, no vector gene copies are detectable in biological fluids (e.g., tears, serum, or urine) by 14 weeks after administration of the vector. In some embodiments, no vector gene copies are detectable in biological fluids (e.g., tears, serum, or urine) at any time after administration of the vector.

[0241] In some embodiments, patients treated according to the methods provided herein are monitored for the occurrence of foveal-involved diabetic macular edema (CI-DME), cataracts, neovascularization, retinal detachment, diabetic complications, vascular regression, areas of leakage, and / or areas of retinal nonperfusion. The occurrence of CI-DME, cataracts, neovascularization, retinal detachment, diabetic complications, vascular regression, areas of leakage, and areas of retinal nonperfusion can be assessed by any method known in the art or provided herein. Diabetic complications that occur in a subject may require panretinal photocoagulation (PRP), anti-VEGF therapy, and / or surgical intervention. Diabetic complications may be sight-threatening. Cataracts that occur in a subject may require surgery. In some embodiments, vital signs (e.g., heart rate, blood pressure) of patients treated according to the methods provided herein may be monitored.

[0242] The safety of the methods of treatment described herein can be assessed by assays known in the art. In certain embodiments, the safety of the methods of treatment described herein is assessed by serum chemistry measurements, such as glucose, blood urea nitrogen, creatinine, sodium, potassium, chloride, carbon dioxide, calcium, total protein albumin, total bilirubin, direct bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and / or creatine kinase levels. In certain embodiments, the safety of the methods of treatment described herein is assessed by hematological measurements, such as platelets, hematocrit, hemoglobin, red blood cells, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, mean corpuscular volume, mean corpuscular hemoglobin content, and / or mean corpuscular hemoglobin concentration. In certain embodiments, the safety of the methods of treatment described herein is assessed by urinalysis, e.g., dipstick testing for glucose, ketone, protein, and / or blood levels (microscopic evaluation can be completed, if necessary). In certain embodiments, the safety of the methods of treatment described herein is assessed by measurements of blood clotting (e.g., prothrombin time and / or partial thromboplastin time) or by measurements of hemoglobin A1c.

[0243] In certain embodiments, the effectiveness of the methods provided herein is determined by statistical analysis. Statistical inference can be performed at a significance level of two-sided α=0.2. Statistical endpoints can be summarized with corresponding 80% confidence intervals.

[0244] The efficacy of the methods provided herein can be determined by Fisher's exact test, in which a treated population is tested against the historical response rate (eg, 5%) in an untreated population.

[0245] 5.4 Steroid regimens In certain embodiments, a method of treating neovascular age-related macular degeneration (nAMD) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, The anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector provided herein comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; The steroid treatment comprises administering a therapeutically effective amount of a steroid to the subject's eye. A method is provided herein.

[0246] In certain other embodiments, a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; The anti-hVEGF treatment comprises administering a therapeutically effective amount of a recombinant viral vector provided herein comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye; The steroid treatment comprises administering a therapeutically effective amount of a steroid to the subject's eye. A method is provided herein.

[0247] Also provided is a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector provided herein comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of triamcinolone acetonide. A method is provided herein.

[0248] Also provided is a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector provided herein comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment, and the steroid treatment comprises administering to the eye of the subject a therapeutically effective amount of difluprednate. A method is provided herein.

[0249] In certain embodiments, the method is a method for treating neovascular age-related macular degeneration (nAMD). In certain embodiments, the method is a method for treating diabetic retinopathy (DR).

[0250] In certain embodiments, the anti-hVEGF treatment comprises administering a recombinant viral vector described in Section 5.2.

[0251] In certain embodiments, the recombinant viral vector is administered as described in Section 5.3. In certain embodiments, the recombinant viral vector is administered to the suprachoroidal space of the subject's eye. In certain embodiments, the recombinant viral vector is administered by injection into the suprachoroidal space of the eye using a suprachoroidal drug delivery device. In certain embodiments, the suprachoroidal drug delivery device is a microinjector. In another embodiment, the recombinant viral vector is administered to the subretinal space of the subject's eye.

[0252] In certain embodiments, the steroid treatment comprises topically administering a therapeutically effective amount of a steroid. In certain embodiments, the topical administration of the steroid reduces or prevents intraocular inflammation. In certain embodiments, the steroid treatment comprises administering a therapeutically effective amount of a corticosteroid. In certain embodiments, the administration of the corticosteroid reduces or prevents intraocular inflammation. In certain embodiments, the steroid treatment comprises topically administering a therapeutically effective amount of a corticosteroid. In certain embodiments, the corticosteroid is selected from the group consisting of cortisone, hydrocortisone, fludrocortisone acetate, prednisolone, prednisone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, triamcinolone acetonide, difluprednate, and fluorometholone. In certain embodiments, the corticosteroid is triamcinolone acetonide. In certain embodiments, the corticosteroid is difluprednate. In certain embodiments, the steroid treatment comprises administering a therapeutically effective amount of triamcinolone acetonide. In certain embodiments, the steroid treatment comprises administering a therapeutically effective amount of difluprednate.

[0253] In certain embodiments provided herein, the anti-hVEGF treatment comprises administering a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye, and the steroid treatment comprises administering triamcinolone acetonide to the subject's eye. In certain embodiments, triamcinolone acetonide is administered after the recombinant viral vector is administered. In other embodiments, triamcinolone acetonide is administered before the recombinant viral vector is administered. In certain embodiments, triamcinolone acetonide is administered to the subject's eye within about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute after the administration of the recombinant viral vector. In certain embodiments, triamcinolone acetonide is administered to the eye of the subject about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute before administration of the recombinant viral vector. In certain embodiments, triamcinolone acetonide is administered to the eye of the subject about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute after administration of the recombinant viral vector. In certain embodiments, triamcinolone acetonide is administered to the subject's eye about 20 to 24 hours, about 16 to 20 hours, about 12 to 16 hours, about 8 to 12 hours, about 4 to 8 hours, about 3 to 4 hours, about 2 to 3 hours, about 1 to 2 hours, about 50 to 60 minutes, about 40 to 50 minutes, about 30 to 40 minutes, about 20 to 30 minutes, about 10 to 20 minutes, about 9 to 10 minutes, about 8 to 9 minutes, about 7 to 8 minutes, about 6 to 7 minutes, about 5 to 6 minutes, about 4 to 5 minutes, about 3 to 4 minutes, about 2 to 3 minutes, about 1 to 2 minutes, or less than about 1 minute prior to administration of the recombinant viral vector.In certain embodiments, triamcinolone acetonide is administered to the subject's eye about 20-24 hours, about 16-20 hours, about 12-16 hours, about 8-12 hours, about 4-8 hours, about 3-4 hours, about 2-3 hours, about 1-2 hours, about 50-60 minutes, about 40-50 minutes, about 30-40 minutes, about 20-30 minutes, about 10-20 minutes, about 9-10 minutes, about 8-9 minutes, about 7-8 minutes, about 6-7 minutes, about 5-6 minutes, about 4-5 minutes, about 3-4 minutes, about 2-3 minutes, about 1-2 minutes, or less than about 1 minute after administration of the recombinant viral vector. In certain embodiments, triamcinolone acetonide is administered to the subject's eye by injection. In certain embodiments, triamcinolone acetonide is administered to the subject's eye by a single injection. In certain embodiments, the steroid treatment consists of a single injection of triamcinolone acetonide into the subject's eye. In certain embodiments, the triamcinolone acetonide is administered to a different quadrant of the eye than the recombinant viral vector. In certain embodiments, the triamcinolone acetonide is administered sub-Tenon's. In certain embodiments, the triamcinolone acetonide is administered at a dose of about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg. In certain embodiments, the triamcinolone acetonide is administered at a dose of about 40 mg. In certain embodiments, triamcinolone acetonide is administered at a dose of between about 5 mg to about 10 mg, about 10 mg to about 15 mg, about 15 mg to about 20 mg, about 20 mg to about 25 mg, about 25 mg to about 30 mg, about 30 mg to about 35 mg, about 35 mg to about 40 mg, about 40 mg to about 45 mg, about 45 mg to about 50 mg, about 50 mg to about 60 mg, about 60 mg to about 70 mg, about 70 mg to about 75 mg, about 75 mg to about 80 mg, about 80 mg to about 90 mg, about 90 mg to about 100 mg, about 100 mg to about 125 mg, about 125 mg to about 150 mg, about 150 mg to about 175 mg, or about 175 mg to about 200 mg.In certain embodiments, triamcinolone acetonide is administered in a volume of about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, about 1.5 mL, about 1.6 mL, about 1.7 mL, about 1.8 mL, about 1.9 mL, or about 2 mL. In certain embodiments, triamcinolone acetonide is administered in a volume of about 1 mL. In certain embodiments, triamcinolone acetonide is administered in a volume of between about 0.1 mL to about 0.2 mL, about 0.2 mL to about 0.3 mL, about 0.3 mL to 0.4 mL, about 0.4 mL to 0.5 mL, about 0.5 mL to about 0.6 mL, about 0.6 mL to about 0.7 mL, about 0.7 mL to 0.8 mL, about 0.8 mL to about 0.9 mL, about 0.9 mL to about 1.0 mL, about 1 mL to about 1.1 mL, about 1.1 mL to about 1.2 mL, about 1.2 mL to 1.3 mL, about 1.3 mL to about 1.4 mL, about 1.4 mL to about 1.5 mL, about 1.5 mL to about 1.6 mL, about 1.6 mL to about 1.7 mL, about 1.7 mL to about 1.8 mL, about 1.8 mL to about 1.9 mL, or about 1.9 mL to about 2 mL.

[0254] In certain embodiments provided herein, the anti-hVEGF treatment comprises administering a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye, and the steroid treatment comprises administering difluprednate to the subject's eye. In certain embodiments, difluprednate is administered daily to the subject's eye. In certain embodiments, the steroid treatment comprises administering difluprednate four times daily. In certain embodiments, difluprednate is administered four times daily for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. In certain embodiments, difluprednate is administered four times daily for about 4 weeks. In certain embodiments, the steroid treatment comprises administering difluprednate three times daily. In certain embodiments, difluprednate is administered three times daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week. In certain embodiments, difluprednate is administered three times daily for about one week. In certain embodiments, the steroid treatment comprises administering difluprednate twice daily. In certain embodiments, difluprednate is administered twice daily for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least one week. In certain embodiments, difluprednate is administered twice daily for about one week. In certain embodiments, the steroid treatment comprises administering difluprednate once daily. In certain embodiments, difluprednate is administered once daily for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least one week. In certain embodiments, difluprednate is administered once daily for about one week. In certain embodiments, difluprednate is administered to the subject's eye for a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or at least 7 weeks. In certain embodiments, difluprednate is administered to the subject's eye for a period of about 7 weeks.In another specific embodiment, the steroid treatment comprises administering difluprednate once on the first day of steroid treatment, followed by four times a day for about four weeks, followed by three times a day for about one week, followed by twice a day for about one week, followed by once a day for about one week. In yet another specific embodiment, the steroid treatment comprises administering difluprednate once on the first day of steroid treatment, followed by four times a day for about four weeks, followed by three times a day for about one week, followed by twice a day for about one week, followed by once a day for about one week. In certain embodiments, the difluprednate is administered in the form of an ophthalmic emulsion. In certain embodiments, the ophthalmic emulsion comprises 0.5 mg / mL (0.05%) difluprednate. In certain embodiments, each administration of difluprednate comprises instilling one drop of ophthalmic emulsion into the subject's eye. In certain embodiments, each administration of difluprednate consists of instilling one drop of ophthalmic emulsion into the subject's eye. In certain embodiments, difluprednate is first administered to the subject's eye within about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day after administration of the recombinant viral vector. In certain embodiments, difluprednate is first administered to the subject's eye on the same day that the recombinant viral vector is administered. In certain embodiments, the first administration of difluprednate occurs after the first administration of the recombinant viral vector.

[0255] In certain embodiments, a method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; The anti-hVEGF treatment comprises administering a therapeutically effective amount of a recombinant viral vector provided herein comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye, wherein the recombinant viral vector is administered in an amount of at least about 5.0 x 10 per eye. 11 administered at a dose of 1 genome copy, The steroid treatment comprises administering a therapeutically effective amount of a topical steroid to the subject's eye. A method is provided herein.

[0256] In certain embodiments, the recombinant viral vector is administered to about 5.0 x 10 eyes. 11 Genome copies ~ approximately 1.0 x 10 per eye 12 In certain embodiments, the recombinant viral vector is administered at a dose of at least about 1.0 x 10 genome copies per eye. 12 In certain embodiments, the recombinant viral vector is administered at a dose of about 1.0 x 10 genome copies per eye. 12 In certain embodiments, the recombinant viral vector is administered in a dose of 1000 genome copies. In certain embodiments, the recombinant viral vector is administered by multiple suprachoroidal injections. In certain embodiments, the recombinant viral vector is administered by triple suprachoroidal injections. In certain embodiments, the recombinant viral vector is administered by double suprachoroidal injections. In certain embodiments, the recombinant viral vector is administered by a single suprachoroidal injection. In certain embodiments, the administration of a steroid reduces or prevents intraocular inflammation.

[0257] In certain embodiments, administration of a steroid reduces or prevents intraocular inflammation associated with the dose of the recombinant viral vector, the number of suprachoroidal injections, and / or the location of the suprachoroidal injections. In certain embodiments, the steroid treatment comprises administering a therapeutically effective amount of a corticosteroid. In certain embodiments, the steroid treatment comprises topically administering a therapeutically effective amount of a steroid, such as a corticosteroid. In certain embodiments, the steroid treatment comprises administering a therapeutically effective amount of a steroid, such as a corticosteroid, sub-Tenon's administration of the steroid. In certain embodiments, the corticosteroid is selected from the group consisting of cortisone, hydrocortisone, fludrocortisone acetate, prednisolone, prednisone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, triamcinolone acetonide, difluprednate, and fluorometholone. In certain embodiments, the corticosteroid is difluprednate. In certain embodiments, the corticosteroid is triamcinolone acetonide.

[0258] In certain embodiments, provided herein are methods for treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment, wherein the anti-hVEGF treatment comprises administering a therapeutically effective amount of a recombinant viral vector provided herein comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye, and the steroid treatment comprises administering a therapeutically effective amount of a steroid topically to the subject's eye. In certain embodiments, the recombinant viral vector is administered in a concentration of at least about 5.0 x 10 per eye. 11 In certain embodiments, the recombinant viral vector is administered at a dose of about 5.0 x 10 genome copies per eye. 11 Genome copies ~ approximately 1.0 x 10 per eye 12In certain embodiments, the recombinant viral vector is administered at a dose of at least about 1.0 x 10 genome copies per eye. 12 In certain embodiments, the recombinant viral vector is administered at a dose of about 1.0 x 10 genome copies per eye. 12 In certain embodiments, the recombinant viral vector is administered in a dose of 100 genome copies. In certain embodiments, the recombinant viral vector is administered by multiple suprachoroidal injections. In certain embodiments, the recombinant viral vector is administered by triple suprachoroidal injections. In certain embodiments, the recombinant viral vector is administered by double suprachoroidal injections. In certain embodiments, the recombinant viral vector is administered by a single suprachoroidal injection. In certain embodiments, topical administration of a steroid reduces or prevents intraocular inflammation. In certain embodiments, topical administration of a steroid reduces or prevents intraocular inflammation associated with the dose of the recombinant viral vector, the number of suprachoroidal injections, and / or the location of the suprachoroidal injections. In certain embodiments, the steroid treatment comprises topically administering a therapeutically effective amount of a corticosteroid. In certain embodiments, the steroid treatment comprises administering a therapeutically effective amount of a steroid, for example, a corticosteroid, sub-Tenon to the eye. In certain embodiments, the topical corticosteroid is selected from the group consisting of cortisone, hydrocortisone, fludrocortisone acetate, prednisolone, prednisone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, triamcinolone acetonide, difluprednate, and fluorometholone. In certain embodiments, the topical corticosteroid is difluprednate. In certain embodiments, the corticosteroid administered sub-Tenon's in the eye is triamcinolone acetonide.

[0259] 5.5 Combination Therapy The treatment methods provided herein can be combined with one or more additional therapies.In one embodiment, the treatment methods provided herein are administered together with laser photocoagulation.In one embodiment, the treatment methods provided herein are administered together with photodynamic therapy using verteporfin.

[0260] In one aspect, the methods of treatment provided herein are administered in conjunction with intravitreal (IVT) injection with an anti-VEGF agent, including, but not limited to, HuPTMFabVEGFi, e.g., HuGlyFabVEGFi produced in a human cell line (Dumont et al., 2015, supra), or other anti-VEGF agents such as pegaptanib, ranibizumab, aflibercept, or bevacizumab.

[0261] The additional therapy may be administered prior to, concurrently with, or following the gene therapy treatment.

[0262] The efficacy of gene therapy treatment can be demonstrated by the elimination or reduction in the number of rescue treatments using standard of care, for example, intravitreal injections with anti-VEGF agents, including, but not limited to, HuP™FabVEGFi, e.g., HuGlyFabVEGFi produced in human cell lines, or other anti-VEGF agents, such as pegaptanib, ranibizumab, aflibercept, or bevacizumab.

[0263] [Table 5] TIFF2025534347000011.tif253169TIFF2025534347000012.tif247169TIFF2025534347000013.tif249168TIFF2025534347000014.tif246169 TIFF2025534347000015.tif247168TIFF2025534347000016.tif248168TIFF2025534347000017.tif250168TIFF2025534347000018.tif233168 [Example]

[0264] 6. Working Example 6.1 [Example 1] Bevacizumab Fab cDNA-based vector A bevacizumab Fab cDNA-based vector is constructed containing a transgene comprising the bevacizumab Fab portion of the light and heavy chain cDNA sequences (SEQ ID NOs: 10 and 11, respectively). The transgene also contains a nucleic acid comprising a signal peptide selected from the group listed in Table 1. The nucleotide sequences encoding the light and heavy chains are separated by an IRES element or a 2A cleavage site, resulting in a bicistronic vector. Optionally, the vector additionally contains a hypoxia-inducible promoter.

[0265] 6.2 [Example 2] Ranibizumab cDNA-based vector A ranibizumab Fab cDNA-based vector is constructed, containing a transgene comprising ranibizumab Fab light and heavy chain cDNAs (the portions of SEQ ID NOs: 12 and 13, respectively, that do not encode the signal peptide). The transgene also contains a nucleic acid comprising a signal peptide selected from the group listed in Table 1. The nucleotide sequences encoding the light and heavy chains are separated by an IRES element or a 2A cleavage site, resulting in a bicistronic vector. Optionally, the vector additionally comprises a hypoxia-inducible promoter.

[0266] 6.3 [Example 3] Hyperglycosylated bevacizumab Fab cDNA-based vector A hyperglycosylated bevacizumab Fab cDNA-based vector is constructed, comprising a transgene containing the bevacizumab Fab portion of the light and heavy chain cDNA sequences (SEQ ID NOs: 10 and 11, respectively) with mutations in the sequences encoding one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S (light chain). The transgene also contains a nucleic acid comprising a signal peptide selected from the group listed in Table 1. The nucleotide sequences encoding the light and heavy chains are separated by an IRES element or a 2A cleavage site, resulting in a bicistronic vector. Optionally, the vector additionally contains a hypoxia-inducible promoter.

[0267] 6.4 [Example 4] Hyperglycosylated ranibizumab cDNA-based vector A hyperglycosylated ranibizumab Fab cDNA-based vector is constructed, comprising a transgene containing ranibizumab Fab light and heavy chain cDNAs (the signal peptide-non-encoding portions of SEQ ID NOs: 12 and 13, respectively) with mutations in the sequences encoding one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S (light chain). The transgene also contains a nucleic acid comprising a signal peptide selected from the group listed in Table 1. The nucleotide sequences encoding the light and heavy chains are separated by an IRES element or a 2A cleavage site, resulting in a bicistronic vector. Optionally, the vector additionally contains a hypoxia-inducible promoter.

[0268] 6.5 [Example 5] Ranibizumab-based HuGlyFabVEGFi A ranibizumab Fab cDNA-based vector (see Example 2) was expressed in the PER.C6® cell line (Lonza) in an AAV8 background. The resulting product, ranibizumab-based HuGlyFabVEGFi, was determined to be stably produced. N-glycosylation of HuGlyFabVEGFi was confirmed by hydrazinolysis and MS / MS analysis. See, e.g., Bondt et al., Mol. & Cell. Proteomics 13.11:3029-3039. Based on glycan analysis, HuGlyFabVEGFi was confirmed to be N-glycosylated with 2,6 sialic acid as the predominant modification. The advantageous properties of N-glycosylated HuGlyFabVEGFi were determined using methods known in the art. HuGlyFabVEGFi was found to have increased stability and increased affinity for its antigen (VEGF). For methods of assessing stability, see Sola and Griebenow, 2009, J Pharm Sci., 98(4):1223-1245, and for methods of assessing affinity, see Wright et al., 1991, EMBO J. 10:2717-2723 and Leibiger et al., 1999, Biochem. J. 338:529-538.

[0269] 6.6 [Example 6] Treatment of Wet AMD with Ranibizumab-Based HuGlyFabVEGFi by Peripheral Injection Based on the determination of the advantageous characteristics of ranibizumab-based HuGlyFabVEGFi (see Example 5), ranibizumab Fab cDNA-based vectors are considered useful for treating wet AMD when expressed as transgenes. Subjects with wet AMD are administered AAV8 encoding ranibizumab Fab at a dose sufficient to produce a transgene product concentration of at least 0.330 μg / mL Cmin in the vitreous humor over a 3-month period. Administration is carried out by subretinal administration via a peripheral injection into the retina (i.e., the optic disc, fovea, and macula located at the back of the eye), which is achieved by intravitreal injection. After treatment, subjects are evaluated for improvement in the symptoms of wet AMD.

[0270] 6.7 Example 7: A Randomized, Partially Blinded, Controlled Phase 2b Clinical Study to Evaluate the Safety and Efficacy of Construct II Gene Therapy in Participants with nAMD 6.7.1 Overview Primary objective.

[0271] To assess the mean change in best-corrected visual acuity (BCVA) for Construct II compared with ranibizumab at week 50.

[0272] Secondary objective.

[0273] To evaluate the safety and tolerability of Construct II through 102 weeks. To evaluate the effect of Construct II on BCVA. To evaluate the effect of Construct II on central retinal thickness (CRT) as measured by spectral domain optical coherence tomography (SD-OCT). To evaluate the need for supplemental anti-vascular endothelial growth factor (VEGF) therapy in the Construct II treatment group. To evaluate aqueous humor protein concentrations of Construct II. To evaluate the immunogenicity of Construct II. 【027...

Claims

1. 1. A method of treating neovascular age-related macular degeneration (nAMD) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; a. the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; b. The steroid treatment comprises administering a therapeutically effective amount of a steroid to the subject's eye; method.

2. 1. A method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; a. The anti-hVEGF treatment comprises administering a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye; b. The steroid treatment comprises administering a therapeutically effective amount of a steroid to the subject's eye; method.

3. 1. A method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; a. the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; b. The steroid treatment comprises administering a therapeutically effective amount of triamcinolone acetonide to the subject's eye; method.

4. 1. A method of treating neovascular age-related macular degeneration (nAMD) or diabetic retinopathy (DR) in a subject in need thereof, comprising administering an anti-hVEGF treatment and a steroid treatment; a. the anti-hVEGF treatment comprises administering to the eye of the subject a therapeutically effective amount of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; b. The steroid treatment comprises administering a therapeutically effective amount of difluprednate to the eye of the subject; method.

5. The method of any one of claims 2 to 4, which is a method for treating neovascular age-related macular degeneration (nAMD).

6. The method according to any one of claims 2 to 4, which is a method for treating diabetic retinopathy (DR).

7. The method of any one of claims 1 or 3 to 6, wherein the recombinant viral vector is administered to the suprachoroidal space of the subject's eye.

8. The method of any one of claims 1 to 7, wherein the recombinant viral vector is administered by injection into the suprachoroidal space of the eye using a suprachoroidal drug delivery device.

9. 9. The method of claim 8, wherein the suprachoroidal drug delivery device is a microinjector.

10. 10. The method of any one of claims 1 or 3-6, wherein the recombinant viral vector is administered into the subretinal space of the subject's eye.

11. 11. The method of claim 10, which does not include performing a vitrectomy on the subject's eye.

12. 11. The method of claim 10, wherein the subretinal administration comprises performing a vitrectomy on the subject's eye.

13. 13. The method of claim 12, wherein the vitrectomy is a partial vitrectomy.

14. 12. The method of claim 10 or 11, wherein the recombinant viral vector is administered to the subretinal space via the suprachoroidal space of the subject's eye.

15. 15. The method of claim 14, wherein the recombinant viral vector is administered using a subretinal drug delivery device comprising a catheter that can be inserted and tunneled through the suprachoroidal space toward the posterior pole, wherein a thin needle injects into the subretinal space at the posterior pole.

16. 16. The method of claim 15, wherein the anti-hVEGF treatment comprises catheterizing and tunneling a subretinal drug delivery device through the suprachoroidal space to administer a recombinant viral vector.

17. 17. The method of any one of claims 1 to 16, wherein the steroid treatment reduces or prevents intraocular inflammation.

18. The method of any one of claims 1 to 17, wherein the steroid treatment ameliorates or prevents intraocular inflammation associated with the dose of the recombinant viral vector, the number of suprachoroidal injections, and / or the location of the suprachoroidal injections.

19. 19. The method of any one of claims 1, 2 and 4-18, wherein the steroid is administered topically.

20. 20. The method of any one of claims 1, 2 and 5 to 19, wherein the steroid is a corticosteroid.

21. 21. The method of any one of claims 1, 2 and 5 to 20, wherein the steroid is triamcinolone acetonide.

22. 21. The method of any one of claims 1, 2 and 5 to 20, wherein the steroid is difluprednate.

23. a. The anti-hVEGF treatment comprises administering a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye; b. The method of any one of claims 1-3, 5-9, and 17-21, wherein the steroid treatment comprises administering triamcinolone acetonide to the subject's eye.

24. 24. The method of any one of claims 3, 5-21 and 23, wherein triamcinolone acetonide is administered after administering the recombinant viral vector.

25. 24. The method of any one of claims 3, 5-21 and 23, wherein triamcinolone acetonide is administered prior to administering the recombinant viral vector.

26. 26. The method of any one of claims 3, 5-21, and 23-25, wherein triamcinolone acetonide is administered to the eye of the subject within about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute of administration of the recombinant viral vector.

27. 27. The method of any one of claims 3, 5-18, 20, 21 and 23-26, wherein triamcinolone acetonide is administered by injection into the subject's eye.

28. 28. The method of claim 27, wherein triamcinolone acetonide is administered by a single injection into the subject's eye.

29. 29. The method of any one of claims 3, 5-18, 20, 21 and 23-28, wherein the steroid treatment consists of a single injection of triamcinolone acetonide into the subject's eye.

30. 30. The method of any one of claims 3, 5-18, 20, 21 and 23-29, wherein the triamcinolone acetonide is administered to a different quadrant of the eye than the recombinant viral vector.

31. 31. The method of any one of claims 3, 5-18, 20, 21 and 23-30, wherein triamcinolone acetonide is administered sub-Tenon's capsule of the eye.

32. 32. The method of any one of claims 3, 5-18, 20, 21 and 23-31, wherein triamcinolone acetonide is administered at a dose of about 40 mg.

33. 33. The method of any one of claims 3, 5-18, 20, 21 and 23-32, wherein triamcinolone acetonide is administered in a volume of about 1 mL.

34. a. The anti-hVEGF treatment comprises administering a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment to the suprachoroidal space of the subject's eye; b. The steroid treatment comprises administering difluprednate to the subject's eye; The method according to any one of claims 1, 2, 4 to 9, 17 to 20 and 22.

35. 35. The method of any one of claims 4 to 20, 22 and 34, wherein difluprednate is administered daily to the subject's eye.

36. 36. The method of claim 35, wherein the steroid treatment comprises difluprednate administered four times daily.

37. 37. The method of claim 36, wherein difluprednate is administered four times daily for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks.

38. 38. The method of claim 37, wherein difluprednate is administered four times daily for about four weeks.

39. 39. The method of any one of claims 35 to 38, wherein the steroid treatment comprises difluprednate administered three times daily.

40. 40. The method of claim 39, wherein difluprednate is administered three times daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week.

41. 41. The method of claim 40, wherein difluprednate is administered three times daily for about one week.

42. 42. The method of any one of claims 35 to 41, wherein the steroid treatment comprises twice-daily administration of difluprednate.

43. 43. The method of claim 42, wherein difluprednate is administered twice daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week.

44. 45. The method of claim 44, wherein difluprednate is administered twice daily for about one week.

45. 45. The method of any one of claims 35 to 44, wherein the steroid treatment comprises once-daily administration of difluprednate.

46. 46. ​​The method of claim 45, wherein difluprednate is administered once daily for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week.

47. 47. The method of claim 46, wherein difluprednate is administered once daily for about one week.

48. 48. The method of any one of claims 4-20, 22 and 34-47, wherein difluprednate is administered to the subject's eye for a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or at least 7 weeks.

49. 49. The method of claim 48, wherein difluprednate is administered to the subject's eye for a period of about 7 weeks.

50. 50. The method of claim 49, wherein the steroid treatment comprises administering difluprednate once on the first day of steroid treatment, followed by four times daily for about four weeks, followed by three times daily for about one week, followed by twice daily for about one week, followed by once daily for about one week.

51. 50. The method of claim 49, wherein the steroid treatment comprises administering difluprednate once on the first day of steroid treatment, followed by four times daily for about four weeks, followed by three times daily for about one week, followed by twice daily for about one week, followed by once daily for about one week.

52. 52. The method of any one of claims 4 to 20, 22 and 34 to 51, wherein difluprednate is administered in the form of an ophthalmic emulsion.

53. 53. The method of claim 52, wherein the ophthalmic emulsion comprises 0.5 mg / mL (0.05%) difluprednate.

54. 54. The method of claim 52 or claim 53, wherein each administration of difluprednate comprises instilling one drop of the ophthalmic emulsion into the subject's eye.

55. 54. The method of claim 52 or claim 53, wherein each administration of difluprednate consists of instilling one drop of the ophthalmic emulsion into the subject's eye.

56. 56. The method of any one of claims 4-20, 22, and 34-55, wherein difluprednate is first administered to the subject's eye within about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day of administration of the recombinant viral vector.

57. 57. The method of claim 56, wherein difluprednate is first administered to the subject's eye on the same day that the recombinant viral vector is administered.

58. 58. The method of claim 56 or claim 57, wherein the first administration of difluprednate occurs after the first administration of the recombinant viral vector.

59. The anti-hVEGF antigen-binding fragment may be Fab, F(ab') ２ or a single chain variable fragment (scFv).

60. 60. The method of any one of claims 1 to 59, wherein the anti-hVEGF antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 and a light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

3.

61. The method of any one of claims 1 to 60, wherein the anti-hVEGF antigen-binding fragment comprises (a) a heavy chain comprising heavy chain CDR1 to CDR3 of the amino acid sequence of SEQ ID NO: 2 and (b) a light chain comprising light chain CDR1 to CDR3 of the amino acid sequence of SEQ ID NO:

1.

62. The method of any one of claims 1 to 60, wherein the anti-hVEGF antigen-binding fragment comprises (a) a heavy chain comprising heavy chain CDR1 to CDR3 of the amino acid sequence of SEQ ID NO: 4 and (b) a light chain comprising light chain CDR1 to CDR3 of the amino acid sequence of SEQ ID NO:

3.

63. 63. The method of any one of claims 1 to 62, wherein the anti-hVEGF antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 or SEQ ID NOs: 14, 15, and 63 and heavy chain CDR1-3 of SEQ ID NOs: 17-19 or SEQ ID NOs: 20, 18, and 21.

64. 64. The method of any one of claims 1 to 63, wherein administration of the recombinant viral vector delivers a therapeutically effective amount of the anti-hVEGF antigen-binding fragment to the retina of the human subject.

65. 66. The method of claim 65, wherein the therapeutically effective amount of the anti-hVEGF antigen-binding fragment is produced by retinal cells of the subject.

66. The method of any one of claims 1 to 65, wherein the recombinant viral vector is an rAAV vector.

67. The method of any one of claims 1 to 66, wherein the recombinant viral vector is an rAAV8 vector.

68. The recombinant viral vector comprises an expression cassette encoding an anti-hVEGF antigen-binding fragment, the expression cassette being flanked by AAV2 inverted terminal repeats (ITRs), the expression cassette comprising: a. CB7 promoter consisting of chicken β-actin promoter and CMV enhancer; b. Chicken β-actin intron, c. i. IL-2 signal peptide, ii. a heavy chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2; iii. a self-cleaving furin (F) / F2A linker; iv. a second IL-2 signal peptide, and v. The light chain of an anti-hVEGF antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding d. Rabbit β-globin poly(A) signal 68. The method of any one of claims 1 to 67, comprising:

69. 69. The method of any one of claims 1 to 68, wherein the recombinant viral vector comprises the nucleotide sequence of SEQ ID NO:

56.

70. The recombinant viral vector was administered to approximately 2.5 x 10 １１ 70. The method of any one of claims 1 to 69, wherein the method is administered in a dose of genome copies.

71. The recombinant viral vector was administered to approximately 5.0 x 10 １１ 70. The method of any one of claims 1 to 69, wherein the method is administered in a dose of genome copies.

72. The recombinant viral vector was administered to approximately 1.0 x 10 １２ 70. The method of any one of claims 1 to 69, wherein the method is administered in a dose of genome copies.

73. The method of any one of claims 1 to 72, wherein the recombinant viral vector is administered by double suprachoroidal injection.

74. The method of any one of claims 1 to 72, wherein the recombinant viral vector is administered by a single suprachoroidal injection.

75. A kit for use in a method of treating neovascular age-related macular degeneration (nAMD) according to any one of claims 1 to 5 and 7 to 74, comprising: a. a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; and b. Steroids Kit including:

76. A kit for use in the method of treating diabetic retinopathy (DR) according to any one of claims 2 to 4 and 6 to 74, comprising: a. a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment; and b. Steroids Kit including:

77. 77. The kit of claim 75 or claim 76, wherein the recombinant viral vector is formulated to be suitable for administration to the suprachoroidal space of the subject's eye.

78. 77. The kit of claim 75 or claim 76, wherein the recombinant viral vector is formulated to be suitable for administration to the subretinal space of the subject's eye.

79. 79. The kit of any one of claims 75 to 78, wherein the steroid is triamcinolone acetonide.

80. 79. The kit of any one of claims 75 to 78, wherein the steroid is difluprednate.

81. 75. Use of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment and a steroid in the manufacture of a medicament for the treatment of neovascular age-related macular degeneration (nAMD) according to any one of claims 1 to 5 and 7 to 74.

82. 75. Use of a recombinant viral vector comprising a nucleotide sequence encoding an anti-hVEGF antigen-binding fragment and a steroid in the manufacture of a medicament for the treatment of diabetic retinopathy (DR) according to any one of claims 2 to 4 and 6 to 74.

83. 83. The use of claim 81 or 82, wherein the recombinant viral vector is formulated to be suitable for administration to the suprachoroidal space of the subject's eye.

84. 83. The use of claim 81 or claim 82, wherein the recombinant viral vector is formulated to be suitable for administration to the subretinal space of the subject's eye.

85. 85. The use according to any one of claims 81 to 84, wherein the steroid is triamcinolone acetonide.

86. 85. The use according to any one of claims 81 to 84, wherein the steroid is difluprednate.

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