Method for treating or preventing eye disease

Abstract

The present invention relates to the technical field of gene therapy, and in particular to a method for treating or preventing an eye disease. The method comprises: administering to the eye of a subject in need thereof a recombinant adeno-associated virus (rAAV) comprising a nucleotide sequence encoding a therapeutic product. The rAAV comprises a nucleic acid, the nucleic acid comprises a coding sequence, and the coding sequence is a nucleotide sequence encoding an anti-vascular endothelial growth factor (VEGF) fusion protein or an anti-VEGF antibody. Delivery of the therapeutic product is achieved by means of gene therapy to continuously supply the therapeutic product in the eye.

Description

Methods for treating or preventing eye diseases

[0001] This application claims priority to Chinese patent applications filed on December 3, 2024, No. 2024117640070 and August 15, 2025, No. 2025111477968, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure belongs to the field of gene therapy technology, specifically relating to a method for treating or preventing eye diseases. Background Technology

[0003] Age-related macular degeneration (AMD) is a leading cause of severe, irreversible vision loss. Also known as age-related macular degeneration, it primarily affects people over 50 years of age, with the incidence increasing with age, severely impacting their physical and mental health and quality of life.

[0004] The main clinical features of AMD include drusen, retinal pigment epithelium (RPE) atrophy, and pigmentary changes in the macular region. Based on different pathological features and clinical manifestations, AMD is divided into dry age-related macular degeneration (dry-AMD) and neovascular age-related macular degeneration (nAMD). nAMD accounts for approximately 10%–20% of AMD patients. Its main pathological changes are the formation of submacular choroidal neovascularization (CNV), vascular leakage, hemorrhage, and edema, ultimately leading to fibrous scarring. Clinically, it is characterized by rapidly developing visual distortions and progressive central vision loss within weeks to months, resulting in severe blindness.

[0005] Factors contributing to AMD include age, lifestyle (smoking and diet), family history, and genetic polymorphism. While the pathogenesis of AMD is not fully understood, it is known that high expression of vascular endothelial growth factor (VEGF) promotes choroidal neovascularization (CNV) formation in the macular region, a major cause of visual impairment in AMD patients. VEGF, a member of the platelet-derived growth factor family, is highly specific for inducing angiogenesis and permeability in vascular endothelial cells. VEGF promotes angiogenesis, increases microvascular permeability, and causes vascular leakage. Anti-VEGF therapy has become a first-line treatment for nAMD, but its efficacy is often compromised due to poor adherence, requiring long-term, repeated intravitreal injections and frequent clinical monitoring. This inadequate treatment modality can lead to vision loss in real-world clinical settings.

[0006] There is an urgent need for new treatment methods in clinical practice to meet the long-term treatment needs of nAMD patients. Gene therapy offers the hope of a one-time administration for lifelong improvement or even a cure. Summary of the Invention

[0007] This disclosure describes a method for treating ocular conditions by delivering therapeutic products (such as therapeutic proteins (e.g., antibodies) and therapeutic aptamers) to the retina of a human subject's eye, involving, for example, recombinant adeno-associated virus (rAAV). The therapeutic product may be a fusion protein antibody against a human protein. Delivery of the therapeutic product is accomplished via gene therapy, for example by administering a recombinant adeno-associated virus encoding the therapeutic product or a recombinant DNA expression construct to the suprachoroidal or subretinal space of a human patient's eye to continuously supply the therapeutic product to the eye.

[0008] A first aspect of this disclosure provides a method for treating or preventing an eye disease, the method comprising administering a recombinant adeno-associated virus (rAAV) containing a nucleotide sequence encoding a therapeutic product to the subretinal space of the eye of a subject in need;

[0009] The rAAV contains nucleic acid, which comprises, in a 5' to 3' sequence:

[0010] Cytomegalovirus (CMV) enhancer, wherein the CMV enhancer comprises the sequence shown in SEQ ID NO:2 or SEQ ID NO:14, or a sequence having 85%, 90%, 95%, or 99% or more of identity with it; in some embodiments, the CMV enhancer is the sequence shown in SEQ ID NO:2 or SEQ ID NO:14, or a sequence having 85%, 90%, 95%, or 99% or more of identity with it;

[0011] A cytomegalovirus (CMV) promoter comprising the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; in some embodiments, the cytomegalovirus (CMV) promoter is the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity;

[0012] bGlob introns, wherein the bGlob introns comprise the sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; in some embodiments, the bGlob introns are the sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity;

[0013] The coding sequence is a nucleotide sequence encoding a VEGF fusion protein or an anti-VEGF antibody; in some embodiments, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein shown in SEQ ID NO:5, or a sequence having 85%, 90%, 95%, or 99% or more identity with it; in some embodiments, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein shown in SEQ ID NO:5; wherein the amino acid sequence of the C11 protein is as shown in SEQ ID NO:6; and

[0014] The human growth hormone (HGH or hGH) polyA sequence comprises either the sequence shown in SEQ ID NO:4 or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; in some embodiments, the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO:4 or a sequence having 85%, 90%, 95%, or 99% or more of the same identity.

[0015] In some embodiments, the method includes administering the rAAV to a subject in need at a dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, in some implementations, is approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 11 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, in some implementations, is approximately 1.0 × 10 9vg up to 1.3×10 11 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, in some implementations, is approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 vg, approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 In some embodiments, the rAAV described in vg is applied at the dose once, twice, three times, or four times each time. In other embodiments, the rAAV is applied at the dose once, twice, three times, or four times each time, wherein the dose applied each time is independently the same or different, and the application site is the same or different each time.

[0016] In some embodiments, the method includes administering a dose of the rAAV to one eye of a subject in need of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, in some implementations, is approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 11 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, in some implementations, is approximately 1.0 × 109 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 vg, approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 In some embodiments, the rAAV described in vg is administered to a subject in need at the dose once, twice, three times, or four times in one eye at a time. In some embodiments, the rAAV is administered to a subject in need at the dose once, twice, three times, or four times in one eye at a time, wherein the dose administered each time is independently the same or different. In some embodiments, each administration is performed by multi-site injection.

[0017] In some implementations, the method includes administering approximately 1.5 × 10¹² ppm to a single, monocular dose to a subject in need. 6 vg up to 1.5×10 12 vg, in some implementations approximately 1.5 × 10 6 vg up to 1.5×10 11 vg, in some implementations approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, in some implementations approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, in some implementations approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, in some implementations approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, in some implementations approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, in some implementations approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 vg, approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 109 vg or approximately 1.0 × 10 10 vg describes rAAV;

[0018] In some embodiments, the method includes administering the rAAV independently to each eye of a subject in need at a single, monocular dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, in some implementations approximately 1.5 × 10 6 vg up to 1.5×10 11 vg, in some implementations approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, in some implementations approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, in some implementations approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, in some implementations approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, in some implementations approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, in some implementations approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 vg, approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 vg, in some embodiments, the method includes administering the rAAV to each eye of a subject in need independently, at a dose for each eye, more than once, more than twice, more than three times, or more than four times; in some embodiments, the rAAV is administered once, twice, three times, or four times, wherein the dose administered to each eye each time is independently the same or different; in some embodiments, each eye is administered via multi-site injection.

[0019] In some embodiments, the method includes administering the rAAV to one or both eyes of a subject in need at a dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12vg / eye, in some implementations, approximately 1.5 × 10 6 vg / eye up to 1.5×10 11 vg / eye, approximately 1.0 × 10 in some implementations. 9 vg / eye to 1.0×10 12 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye up to 1.3×10 11 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 1.0×10 11 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 1.0×10 10 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 3.0×10 9 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye, approximately 2.0×10 9 vg / eye, approximately 3.0×10 9 vg / eye, approximately 4.0×10 9 vg / eye, approximately 5.0×10 9 vg / eye, approximately 6.0×10 9 vg / eye, approximately 7.0×10 9 vg / eye, approximately 8.0×10 9 vg / eye, approximately 9.0×10 9 vg / eye or approximately 1.0 × 10 10 vg / eye; In some embodiments, the rAAV is administered to each eye or one eye of the subject in need at the dose independently once, twice, three times or four times each time, and in some embodiments, the rAAV is administered once, twice, three times or four times, wherein the dose administered each time is independently the same or different, and the site of administration is the same or different each time.

[0020] In some embodiments, the therapeutic product is a VEGF fusion protein; in some embodiments, the VEGF fusion protein has an amino acid sequence as shown in SEQ ID NO:6.

[0021] In some embodiments, the rAAV further comprises an AAV 5' inverted terminal repeat (L-ITR) upstream of the 5' end of the nucleic acid and an AAV 3' inverted terminal repeat (R-ITR) downstream of the 3' end of the expression cassette nucleotide sequence; in some embodiments, the AAV 5' inverted terminal repeat (L-ITR) comprises either the sequence shown in SEQ ID NO:8 or a sequence having 85%, 90%, 95%, or 99% or more of identity with it, and the AAV 3' inverted terminal repeat (R-ITR) comprises either the sequence shown in SEQ ID NO:9 or a sequence having 85%, 90%, 95%, or 99% or more of identity with it; in some embodiments, the nucleotide sequence of the nucleic acid contained in the rAAV is as shown in SEQ ID NO:7;

[0022] In some embodiments, the rAAV further comprises an AAV capsid; in some embodiments, the rAAV is selected from AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV; in some embodiments, the rAAV is AAV type 6 (AAV-6) or AAV type 8 (AAV-8).

[0023] In some embodiments, the eye disease is angiogenesis-related eye disease; in some embodiments, the eye disease is selected from age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization, and diabetic retinopathy; in some embodiments, the age-related macular degeneration is wet AMD or dry AMD; in some embodiments, it is wet AMD.

[0024] In some implementations, the rAAV is administered ocularly to the subject in need via intraocular injection, intravitreal injection, subretinal injection, or suprachoroidal injection.

[0025] In some implementations, the injection is a single-site injection or a multi-site injection.

[0026] In some embodiments, the rAAV is administered to the eye of the subject in need via the subretinal space and / or the suprachoroidal space, at a dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, in some implementations, is approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 Approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 vg, in some embodiments, the rAAV is administered at the stated dose once, twice, three times, or four times; in some embodiments, the rAAV is administered at the stated dose once, twice, three times, or four times, wherein the dose administered each time is independently the same or different; in some embodiments, the administered dose is about 1.0 × 10 9 vg / eye up to 1.3×10 11 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 1.0×10 11 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 1.0×10 10 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye, approximately 2.0×10 9 vg / eye, approximately 3.0×10 9 / eye, approximately 4.0×10 9 vg / eye, approximately 5.0×10 9 vg / eye, approximately 6.0×10 9 vg / eye, approximately 7.0×10 9 vg / eye, approximately 8.0×10 9 vg / eye, approximately 9.0×10 9 vg / eye or approximately 1.0 × 10 10In some embodiments, the rAAV is administered at the dose once, twice, three times, or four times at a time; in some embodiments, the dose is administered once or twice at a time, with the doses being the same or different.

[0027] In some implementations, the following dose of rAAV is administered independently to each eye or one eye of the subject in need: approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg / eye, in some implementations, approximately 1.5 × 10 6 vg / eye up to 1.5×10 11 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 1.0×10 12 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye up to 1.3×10 11 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 1.0×10 11 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 1.0×10 10 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye to 3.0×10 9 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye, approximately 2.0×10 9 vg / eye, approximately 3.0×10 9 vg / eye, approximately 4.0×10 9 vg / eye, approximately 5.0×10 9 vg / eye, approximately 6.0×10 9 vg / eye, approximately 7.0×10 9 vg / eye, approximately 8.0×10 9 vg / eye, approximately 9.0×10 9 vg / eye or approximately 1.0 × 10 10 vg / eye; In some embodiments, the rAAV is administered to each eye or one eye of the subject in need at the dose independently once, twice, three times or four times each time, and in some embodiments, the rAAV is administered once, twice, three times or four times, wherein the dose administered each time is independently the same or different, and the site of administration is the same or different each time.

[0028] In some embodiments, the rAAV is administered to the eye of the subject in need via the suprachoroidal space, at a dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, in some implementations, is approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, in some implementations, is approximately 1.0 × 10 10 vg up to 1.0×10 12 vg, in some implementations, is approximately 1.0 × 10 10 vg up to 1.0×10 11 vg or approximately 1.0 × 10 11 vg up to 1.0×10 12 vg, in some embodiments, the rAAV is applied at the dose once, twice, three times, or four times each time, and in some embodiments, the dose is applied once, twice, three times, or four times each time, wherein the dose applied each time is independently the same or different.

[0029] In some implementations, the required subjects are subjects aged 45 years or older; in some implementations, subjects aged 50 years or older; in some implementations, subjects aged 50 years or younger and 80 years or older; and in some implementations, subjects aged 65 years or younger and 80 years or older.

[0030] In some implementations, the subjects in need suffer from age-related macular degeneration; in some implementations, the subjects in need exhibit the following conditions:

[0031] drusen;

[0032] In some implementations, retinal pigment epithelial (RPE) cells are abnormal;

[0033] In some implementations, there is map-like atrophy of the retinal pigment epithelial cells and the inner choroidal layer (including the fovea);

[0034] In some implementations, neovascular (exudative) macular degeneration;

[0035] In some implementations, the subject in need suffers from submacular choroidal neovascularization (CNV), vascular leakage, hemorrhage, and / or edema.

[0036] In some embodiments, the best-corrected visual acuity (BCVA) of the subject's eye to be treated is 20 / 800 to 20 / 40 on the Snellen chart, and further in some embodiments, it is 20 / 400 to 20 / 40 on the Snellen chart; or the best-corrected visual acuity (BCVA) of the subject's eye to be treated is approximately 5-80 letters when using the ETDRS chart, approximately 5-73 letters in some embodiments, approximately 19-80 letters in some embodiments, approximately 20-80 letters in some embodiments, and approximately 50-74 letters in some embodiments.

[0037] In some embodiments, the subject's eyes have previously received and responded to anti-VEGF drug treatment; in some embodiments, the anti-VEGF drug is Conbercept, Faricimab, Bevacizumab, Ranibizumab, or Aflibercept, and in some embodiments, Aflibercept;

[0038] In some embodiments, the method further includes administering a VEGF fusion protein to the subject in need at least 14 days prior to administering the rAAV to the eye of the subject in need, or in some embodiments, 14-21 days prior, wherein the VEGF fusion protein is, in some embodiments, an intraocular injection solution of aflibercept. The application should be carried out according to its drug instructions; in some implementation schemes, the application... The dosage is 1-2 mg, and in some implementations, 2 mg;

[0039] In some embodiments, the method further includes: administering a glucocorticoid to the subject in need 1 to 7 days prior to administering the rAAV to the eye of the subject in need, wherein the glucocorticoid is prednisone tablets in some embodiments; in some embodiments, the dose of the glucocorticoid is initially 0.5 mg / kg / day to 2 mg / kg / day, and not exceeding 40 mg / day, for 5 to 15 days; in some embodiments, initially 1 mg / kg / day; and in some embodiments, for 10 days; optionally, after the initial administration of the glucocorticoid, the dose is reduced to 0.3 mg / kg / day to 1 mg / kg / day, and not exceeding 20 mg / day, for 5 to 10 days; in some embodiments, reduced to 0.5 mg / kg / day; and in some embodiments, for 7 days.

[0040] In some embodiments, the method further includes: administering the rAAV to the eye of the subject in need for at least 28 days, followed by supplemental administration of a VEGF fusion protein to the subject in need, wherein the VEGF fusion protein, in some embodiments, is... The supplemental administration is performed according to its product instructions; in some embodiments, the subject's eyes do not require supplemental treatment with VEGF fusion protein after drug administration (in some embodiments, after 20 or 52 weeks of administration); in some embodiments, supplemental administration of VEGF fusion protein is not required after administration of rAAV to the eyes of the subject in need; in some embodiments, supplemental administration is not required after administration of rAAV to the eyes of the subject in need.

[0041] In some implementations, the recipient of the medication is a patient with wet age-related macular degeneration, and the method includes administering approximately 1.0 × 10¹² ppm to the recipient's eye in a single, unilateral dose. 9 vg up to 1.0×10 10 vg, in some implementations, is approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 Approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 The rAAV described in vg; in some embodiments, the method includes administering the rAAV independently to each eye of a subject in need at a dose of approximately 1.0 × 10⁻⁶ each time. 9 vg / eye to 1.0×10 10 vg / eye, in some implementations, approximately 1.0 × 10 9 vg / eye, approximately 2.0×10 9 vg / eye, approximately 3.0×10 9 vg / eye, approximately 4.0×10 9 vg / eye, approximately 5.0×10 9 vg / eye, approximately 6.0×10 9 vg / eye, approximately 7.0×10 9 vg / eye, approximately 8.0×10 9 vg / eye, approximately 9.0×10 9 vg / eye or approximately 1.0 × 10 10vg / eye.

[0042] Another aspect of this disclosure provides a method for treating the eye of a human subject suffering from an eye disease, said human subject's eye having previously received intravitreal injection of aflibercept, the method comprising:

[0043] In some embodiments, the pharmaceutical composition is administered to the eye of a human subject as a single unit dose. The composition comprises a pharmaceutically acceptable carrier and the aforementioned recombinant adeno-associated virus (rAAV) containing a nucleic acid sequence encoding an anti-VEGF protein, wherein the single unit dose comprises approximately 1.0 × 10⁻⁶ units. 9 vg to approximately 1.0 × 10 11 In some implementations, the recombinant adeno-associated virus (vg) contains approximately 1.0 × 10⁻⁶ cells / mL. 9 vg to approximately 1.0 × 10 10 vg, in some implementations, is approximately 1.0 × 10 9 vg to approximately 3.0×10 9 vg, in some implementations, contains approximately 1.0 × 10 9 vg, approximately 3.0 × 10 9 vg or approximately 1.0 × 10 10 VG recombinant adeno-associated virus, and when measured after at least 20 weeks, 52 weeks, 6 months, 9 months or one year after administration of the drug composition, in some embodiments, after 9 months, sufficient to cause an increase in aflibercept levels in the eyes of the human subjects;

[0044] In some implementations, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, and diabetic retinopathy;

[0045] In some implementations, the injection is administered into the subretinal space of the human subject.

[0046] In some embodiments, the pharmaceutical composition comprises:

[0047] a. A vector genome of recombinant adeno-associated virus type 8 (AAV 8), which contains nucleic acids comprising, in the 5' to 3' sequence:

[0048] The upstream AAV 5' inverted terminal repeat (L-ITR) sequence at the 5' end contains or is the sequence shown in SEQ ID NO:8;

[0049] CMV enhancer, wherein the CMV enhancer comprises the sequence shown in SEQ ID NO:2 or SEQ ID NO:14, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; in some embodiments, the CMV enhancer is the sequence shown in SEQ ID NO:2 or SEQ ID NO:14, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity;

[0050] A cytomegalovirus (CMV) promoter comprising the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; in some embodiments, the cytomegalovirus (CMV) promoter is the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity;

[0051] bGlob introns, wherein the bGlob introns comprise the sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; in some embodiments, the bGlob introns are the sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity;

[0052] The coding sequence is a nucleotide sequence encoding a VEGF fusion protein or an anti-VEGF antibody; in some embodiments, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein shown in SEQ ID NO:5, or a sequence having 85%, 90%, 95%, or 99% or more identity with it; in some embodiments, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein shown in SEQ ID NO:5; wherein the amino acid sequence of the C11 protein is as shown in SEQ ID NO:6; and

[0053] The human growth hormone (HGH or hGH) polyA sequence comprises either the sequence shown in SEQ ID NO:4 or a sequence having 85%, 90%, 95%, or 99% or more identity with it; in some embodiments, the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO:4 or a sequence having 85%, 90%, 95%, or 99% or more identity with it; and

[0054] The downstream AAV 3' inverted terminal repeat (R-ITR) sequence (3' end) includes the sequence shown in SEQ ID NO:9;

[0055] In some embodiments, the pharmaceutical composition further comprises:

[0056] b. about 0.001%, about 0.005% or about 0.01% of poloxamer 188, in some embodiments about 0.01% of poloxamer 188;

[0057] c. Approximately 2.67 mM potassium chloride;

[0058] d. Approximately 1.47 mM potassium dihydrogen phosphate;

[0059] e. about 100 mM, about 138 mM, about 150 mM, or about 180 mM sodium chloride, in some embodiments, about 150 mM sodium chloride; and

[0060] f. Approximately 8.06 mM disodium hydrogen phosphate;

[0061] The pH of the pharmaceutical composition is approximately 7.0 to 7.5.

[0062] In some implementations, the subject has the following characteristics:

[0063] 1) Age 50 ≤ Age ≤ 80; in some implementation plans, age 65 ≤ Age ≤ 80.

[0064] 2) The eye to be treated is diagnosed with choroidal neovascularization (CNV) secondary to neovascular age-related macular degeneration (nAMD) or wet AMD;

[0065] 3) The best corrected visual acuity (BCVA) of the eye to be treated is 20 / 800 to 20 / 40 on the Snellen chart, in some embodiments it is 20 / 400 to 20 / 40 on the Snellen chart, or the best corrected visual acuity (BCVA) of the eye to be treated is about 5-80 letters when using the ETDRS chart, in some embodiments it is about 5-73 letters, in some embodiments it is about 19-80 letters, in some embodiments it is about 20-80 letters, and in some embodiments it is about 50-74 letters.

[0066] In some implementations, the subject's BCVA increased by at least about 3, about 3.1, about 3.5, about 4, about 4.5, about 5, about 10, or about 15 letters from baseline; in some implementations, about 3-30 letters; in some implementations, about 3-25 letters; in some implementations, about 3-20 letters; and in some implementations, about 3.1, about 3.2, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 letters.

[0067] In some implementation schemes, a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After more than 4 weeks of administration of the vg / eye dose, the subjects’ mean BCVA increased by at least approximately 4.5 letters from baseline;

[0068] In some implementation schemes, a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After more than 52 weeks of administration of the vg / ocular dose, the subjects' mean BCVA increased by at least approximately 15 letters from baseline; and / or

[0069] With a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After administration of the vg / ocular dose for 4, 8, 20 or 52 weeks or more, the mean central retinal thickness (CRT) of the subjects decreased from baseline by at least about 20 μm, in some embodiments about 40-180 μm, in some embodiments about 60-160 μm, in some embodiments about 90-150 μm, and in some embodiments about 100 μm, about 110 μm, about 120 μm, about 125 μm, about 130 μm, about 135 μm, about 140 μm, about 141 μm, about 142 μm, about 143 μm, about 144 μm or about 145 μm;

[0070] In some implementation schemes, a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After administration of vg / eye dose for more than 4 weeks, the central macular retinal thickness (CRT) of the subjects was reduced by at least about 90 μm from baseline, and in some implementations by about 100 μm, about 120 μm, about 140 μm, about 141.83 μm, about 142 μm, about 143 μm, about 144 μm, or about 145 μm;

[0071] In some implementation schemes, a single dose of 1.0 × 10 9vg / eye or 3.0×10 9 After administration of vg / eye dose for more than 8 weeks, the central macular retinal thickness (CRT) of subjects decreased by an average of at least about 90 μm from baseline, and in some implementations by about 100 μm, about 120 μm, about 140 μm, about 141 μm, about 142 μm, about 143 μm, about 144 μm, or about 145 μm.

[0072] In some implementations, the aforementioned recombinant adeno-associated virus is contained in a packaging material, said packaging material including printed matter illustrating the method described above. Attached Figure Description

[0073] Figure 1 shows the expression level of C11 protein after #3 AAV cells were infected with 293T and HeLa cells, respectively.

[0074] Figure 2 shows the injections of hRHO-hVEGFA mice. Result of fluorescein fundus angiography (FFA) after RGX-314 and #3 AAV.

[0075] Figure 3 shows the results of laser-induced mice injected with different doses of... #3 FFA results after AAV.

[0076] Figure 4 shows the results of laser-induced mice injected with different doses of... #3 Statistical chart of FFA results after AAV.

[0077] Figure 5 shows the laser imaging results of cynomolgus monkeys after being injected with solvent, RGX-314, and different doses of #3 AAV.

[0078] Figure 6 shows the pharmacokinetic results of laser-induced cynomolgus monkeys after injection of different doses of #3 AAV. The horizontal axis represents time (0 days, 15 days, 29 days, 59 days, 75 days, 89 days), and the vertical axis represents concentration (ng / ml).

[0079] Figure 7 shows the laser contrast imaging results of macaques after laser-induced injection of solvent, low-dose #3 AAV, medium-dose #3 AAV, and high-dose #3 AAV.

[0080] Figure 8 shows the pharmacokinetic results of mice in each group after being given different doses of #3 AAV. The horizontal axis represents time (3, 7, 14 days, and 4, 8, 12, 24 weeks), and the vertical axis represents concentration (ng / eye).

[0081] Figure 9 shows the statistical results of mouse body weight and organ weight after each group of mice were injected with solvent and #3 AAV injection solution, respectively.

[0082] Figure 10 shows the statistical results of serum antibody titers in mice after each group was injected with the solvent and #3 AAV injection solution, respectively. The horizontal axis represents the group and the vertical axis represents the antibody titer (dilution factor).

[0083] Figure 11 shows the fundus images of mice in each group after they were injected with the solvent and #3 AAV injection solution, respectively.

[0084] Figure 12 shows the electroretinography (ERG) results of mice in each group after being injected with the solvent and #3 AAV injection solution, respectively.

[0085] Figures 13a-13c show the protein concentrations of the three vector structures encoding the C11 protein, transfected into 293T cells, HeLa cells, and ARPE-19 cells, respectively. The vertical axis represents protein concentration (μg / ml), and the horizontal axis represents the vector number.

[0086] Figure 14 shows the results of C11 protein activity expressed in #3AAV-infected cells and the activity of commercial Eylea®. The horizontal axis represents protein concentration (pM), and the vertical axis represents relative light units.

[0087] Figure 15a shows the expression levels of C11 protein and V3 gene expression in the whole eye of C57BL / 6J wild-type mice after injection of #3 AAV and positive control drug RGX-314 into the subretinal space. Figure 15b shows the expression levels of C11 protein and V3 gene expression in the vitreous body of C57BL / 6J wild-type mice after injection of #3 AAV and positive control drug RGX-314 into the subretinal space. The horizontal axis represents the type of drug, and the vertical axis represents the protein concentration (μg / ml).

[0088] Figure 16 shows the statistical results of OCT and CRT of the subjects in the clinical study. Detailed Implementation

[0089] I. Definitions and Explanations

[0090] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0091] As used herein and unless otherwise stated, the terms “comprising,” “including,” and “having” include their grammatical equivalents and should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0092] In this application, when referring to specifically listed numerical values ​​or ranges, the term “about” as used herein generally means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0093] In this document, administration of "rAAV containing a nucleotide sequence encoding a therapeutic product" includes administration of its pharmaceutical composition and host cells. The pharmaceutical composition comprises the rAAV described herein and a pharmaceutically acceptable excipient, sterile carrier, or diluent. The host cells are isolated host cells transfected or transduced with the rAAV described herein.

[0094] To provide injectable aqueous solutions, buffer solutions can be prepared as needed.

[0095] The sterile injection solution is prepared by mixing the required amount of active rAAV with various other components listed in this invention in a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier, which contains the dispersion medium and the desired other components.

[0096] In some embodiments, the pharmaceutical composition is prepared as a formulation suitable for subretinal or suprachoroidal injection.

[0097] In this article, "angiogenesis-related eye diseases" include, but are not limited to, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma and retrolental fibrosis, epidemic keratoconjunctivitis, vitamin A deficiency, excessive contact lens wear, atopic keratitis, bacterial keratitis, ulcers, primary keratosis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stephen Johnson's disease, and pemphigoid radial keratotomy. Opening surgery, corneal transplant rejection, sickle cell anemia, sarcoidosis, pseudoxanthoma elastica, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusive disease, chronic uveitis / glaucoma, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales' disease, Behcet's disease, retinitis or choroiditis infection, presumed ocular histoplasmosis, Bests' disease, myopia, fovea, Stargardt's disease, pars plana, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, complications after trauma or laser treatment.

[0098] The term "vector" refers to a nucleic acid medium in which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. This vector can be used to express the carried genetic material elements in host cells through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, artificial chromosomes such as yeast artificial chromosomes, bacterial artificial chromosomes, and viruses; bacteriophages such as λ phage or M13 phage. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40). Vectors may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication.

[0099] The term "host cell" refers to a cellular system that can be engineered to produce a target protein, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term covers not only the specific test cell but also its progeny. Because certain modifications can occur in subsequent generations due to mutations or environmental influences, such progeny may differ from the parent cell but are still included within the scope of the term "host cell."

[0100] The term "transfection" refers to the absorption of foreign or exogenous DNA by a cell, which is "transfected" when the exogenous DNA is introduced into the cell membrane. Various transfection techniques are well known in the art. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. These techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0101] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term covers all subtypes, as well as naturally occurring and recombinant forms, unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals, etc.

[0102] A "viral AAV," "viral AAV particle," or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically composed of all capsid proteins of wild-type AAV) and capsidated polynucleotides. If the particle includes heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to mammalian cells), it is usually referred to as a recombinant AAV vector or rAAV. Typically, the heterologous polynucleotides are flanked by AAV inverted terminal repeat (ITR) sequences.

[0103] The term "packaging" refers to a series of intracellular events that lead to the assembly and shell formation of AAV particles.

[0104] The term "triple plasmid packaging system" is a technique commonly used in genetic engineering research that converts foreign DNA into plasmids and expresses them through intracellular biosynthetic mechanisms. This system includes transfer plasmids, helper plasmids, and adHelper plasmids. Transfer plasmids (rAAV plasmids) contain the target gene sequence to be transferred, typically located between two AAV inverted terminal repeats (ITRs). ITRs are the only sequences borrowed from wild-type AAV, and these sequences are crucial for rAAV packaging and integration into the host cell's genome. Helper plasmids (Rep-Cap plasmids) carry the essential rep and cap genes, which encode AAV replication (Rep) and capsid (Cap) proteins. Rep proteins are responsible for replicating the rAAV plasmid, while Cap proteins are responsible for forming the viral capsid that encapsulates the rAAV plasmid DNA. AdHelper plasmids provide additional Adenovirus proteins that are essential during packaging but are not included in the final rAAV particle. These proteins function to enhance viral replication, assembly, and cell release processes.

[0105] The term "coding sequence" refers to the nucleotide sequence that encodes a gene product, either in vitro or in vivo. In some cases, a gene consists of or is essentially composed of a coding sequence, that is, the sequence that encodes the gene product. In other cases, a gene includes additional non-coding sequences.

[0106] The term "therapeutic product" includes, for example, "therapeutic protein," referring to a protein intended for human or veterinary treatment, which may be administered acutely or chronically. In particular, "therapeutic protein" is a protein intended for the prevention and / or treatment of mammals suffering from a disease or pathological condition. Therapeutic proteins include, but are not limited to, any polypeptide, protein, antibody, or fusion protein that can be administered to mammals.

[0107] The term "promoter" is a DNA sequence that guides the binding of RNA polymerase and thereby promotes RNA synthesis. Promoters and their corresponding protein or polypeptide expression can be ubiquitous (meaning they are highly active in a wide range of cells, tissues, and species) or cell-type specific, tissue-specific, or species-specific. Promoters can be "constitutive" (meaning they are continuously active) or "inducible" (meaning they can be activated or inactivated by the presence or absence of biological or abiotic factors). Common promoters include, for example, the cytomegalovirus (CMV) promoter, actin promoter, elongation factor 1α (EF1α) promoter, CB7 promoter, ubiquitin promoter, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter.

[0108] The term "enhancer" encompasses cis-acting elements that stimulate or repress transcription of adjacent genes. Enhancers that repress transcription are also known as "silencers." Enhancers can act in either orientation at a distance of several thousand base pairs (kb) downstream of the coding sequence and transcribed region (i.e., they can be associated with the coding sequence). The enhancer sequence may or may not be adjacent to the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' region of the native gene.

[0109] The term "intron," also known as a spacer sequence, refers to a non-coding segment within a gene or mRNA molecule. They are intercalation sequences in eukaryotic cell DNA. These sequences are transcribed into precursor RNA, removed through splicing, and ultimately not present in the mature RNA molecule. The alternation of introns and exons constitutes split genes. Introns in precursor RNA are often called "intercalation sequences." They undergo more mutations than exons during post-transcriptional processing. An intron is a special DNA sequence. Here, bGlob_int represents the bGlob intron, and hGH / polyA signal represents the human growth hormone (HGH or hGH) polyA sequence.

[0110] The term "polyA" or "polyA signal sequence" encompasses the recognition region required by endonucleases to cleave RNA transcripts, followed by the polyA concordant sequence AATAAA. The polyA signal sequence provides a "polyA site," a location on the RNA transcript where adenine residues are added post-transcriptionally via polyA conversion. A representative example of a "polyA" region is the human growth hormone (HGH) or hGH polyA sequence, which can also be represented as hGH / polyA signal.

[0111] The term "operably linked" refers to the juxtaposition of genetic elements (e.g., promoters, enhancers, termination signal sequences, polyadenylation sequences, etc.) in a relationship that allows them to operate in a intended manner. For example, if a promoter helps initiate transcription of a coding sequence, then the promoter is operably linked to the coding region. Intercalation residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0112] The term "identity" or "homology" refers to the sequence similarity between two nucleotide sequences or two polypeptides. Two compared sequences are homologous at positions occupied by the same base or amino acid monomer subunit; for example, if every position in two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the total number of positions compared × 100. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous.

[0113] The term "fusion protein" refers to a protein product obtained by linking the coding regions of two or more genes through gene recombination, chemical methods, or other appropriate methods, and expressing the gene recombination under the control of the same regulatory sequence. Unless otherwise specified, the first block polypeptide at the N-terminus of the fusion protein is linked to the N-terminus of the next block (or linker) polypeptide, and so on. Therefore, the N-terminus of the polypeptide located in the N-terminal block of the fusion protein is the N-terminus of the fusion protein, and the C-terminus of the polypeptide located in the C-terminal block of the fusion protein is the C-terminus of the fusion protein.

[0114] The term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains: two heavy chains (H) and two light chains (L) linked by disulfide bonds.

[0115] The term "eye disease" refers to a disease, ailment, or condition that affects or involves the eye or one or more parts or areas of the eye. Therefore, eye diseases include retinal diseases or diseases affecting the light-sensitive layer of the tissues at the back of the eye. The eye comprises the eyeball and the tissues and fluids that make up the eyeball, the periocular muscles (such as the oblique and rectus muscles), and the portion of the optic nerve within or near the eyeball.

[0116] The term "multi-site injection" refers to the injection of a recombinant adeno-associated virus vector carrying the therapeutic gene into multiple different locations within the same target tissue or organ during a single treatment or administration process.

[0117] The term "packaging material" refers to packaging boxes, bags, or bottles (e.g., medicine bottles) used to package products.

[0118] The term "treatment" includes therapeutic treatment, preventative treatment, and applications that reduce the risk of a subject developing a disease or other risk factors. Treatment includes, but is not limited to, a complete cure of a disease, as well as the relief of symptoms or reduction of potential risks.

[0119] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably in this document and refer to mammals, including but not limited to: human and non-human primates, including apes and humans; mammalian locomotion animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0120] The term "adverse event (AE)" refers to all adverse medical events that occur after a subject receives the investigational drug. These events may manifest as symptoms, signs, illnesses, or abnormal laboratory tests, but are not necessarily causally related to the investigational drug.

[0121] The term "Severe Adverse Event (SAE)" refers to adverse medical events that occur to a subject after receiving the investigational drug, such as death, life-threatening events, permanent or severe disability or loss of function, hospitalization or prolonged hospitalization, or congenital abnormalities or birth defects.

[0122] II. Examples

[0123] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0124] Abbreviations:

[0125] PBS refers to phosphate buffer solution.

[0126] ELISA stands for enzyme-linked immunosorbent assay.

[0127] GFP stands for green fluorescent protein.

[0128] PEI stands for Polyethylenimine.

[0129] CDS stands for protein-coding region or coding sequence.

[0130] FFA stands for fundus fluorescein angiography.

[0131] MOI stands for Multiplicity of Infection.

[0132] OCT stands for Optical Coherence Tomograph.

[0133] ERG stands for Electroretinogram.

[0134] BCVA is the best corrected visual acuity.

[0135] DLT stands for dose-limiting toxicity.

[0136] Example 1: Construction of expression vector or expression cassette and detection of target protein

[0137] 1.1 Construction of expression vectors or expression cassettes

[0138] This disclosure constructs three exemplary expression vectors, and the order of the expression vectors from 5' to 3' is shown in Table 1-1.

[0139] The coding sequence in the exemplary AAV expression vector is selected from the gene sequence of the VEGF fusion protein (C11 protein).

[0140] Table 1-1 The combination of sequence elements used in the exemplary AAV expression vector of the present invention

[0141] The exemplary AAV expression vector and its exemplary elements, as well as the sequence information of the encoded protein disclosed herein, are shown in Tables 1-2:

[0142] Table 1-2: Sequence Information

[0143] 1.2 Protein Expression

[0144] The nucleotide sequence encoding protein C11 (SEQ ID NO:5) was loaded into the three vectors mentioned above, and then transiently transfected into three cell lines. After 48 hours of transfection, the cell culture supernatant and total cell protein were harvested. The C11 protein in the supernatant of 293T cells, HeLa cells, and ARPE-19 cells was analyzed by ELISA.

[0145] Elisa testing

[0146] (1) Coating: At 4°C, coat the ELISA plate overnight with 100 μL of human VEGF-A165 (1 μg / mL) in carbonate / bicarbonate buffer. The next day, discard the solution in the wells and wash the plate four times with PBST (0.1% Tween in PBST).

[0147] (2) Blocking: Add 200 μL of blocking solution to each well and incubate at 37°C for 21 h;

[0148] (3) Washing: Remove the sealing film, put it into the plate washer, and wash it 3-5 times;

[0149] (4) Sample addition: Add 100 μL of the appropriately diluted sample to the above-mentioned coated reaction wells;

[0150] (5) Incubation: After sealing the plate with sealing film, incubate at 37°C for 1 hour;

[0151] (6) Washing: Same as step 3;

[0152] (7) Add antibody: Add 100 μL of the corresponding diluted (1:15,000) antibody working solution to each well;

[0153] (8) Incubation: After sealing the plate with sealing film, incubate at 37°C for 1 hour;

[0154] (9) Washing: Same as step 3.

[0155] (10) Add colorimetric substrate: Add 100 μL of TMB substrate solution to each well and develop color at room temperature for 6-7 min;

[0156] (11) Termination of reaction: Add 100 μL of ELISA termination solution to each reaction well;

[0157] (12) Results determination: Read the plate at 450nm using an ELISA reader within 10 min.

[0158] The ELISA results are shown in Figures 13a-13c. In 293T cells, HeLa cells, and ARPE-19 cells, the #3 vector structure plasmid showed the highest efficiency in expressing C11 protein.

[0159] 1.3 Virus infection of cells

[0160] The vector sequence containing the gene sequence of the C11 protein was packaged in vitro as #3 AAV and infected 293T cells and HeLa cells.

[0161] A three-plasmid packaging system and purified recombinant AAV8 virus were used. A phelper plasmid, AAV Cap and Rep protein expression plasmids, and the target expression vector plasmid were mixed with PEI transfection agent (purchased from Polysciences) at a 2:1:1 mass ratio to form a transfection complex. This complex was then transfected into HEK293T cells (purchased from ATCC) and HeLa cells (purchased from ATCC) to package the AAV virus containing the coding sequence. Cells were collected 60 hours post-transfection and repeatedly freeze-thawed to obtain AAV virus particles containing the coding sequence. Density gradient centrifugation with different concentrations of iodixanol (15%, 25%, 40%, and 60%) was performed to obtain purified AAV virus. The purified AAV virus was then used to infect cells using the following method:

[0162] (1) Cells were seeded in 24-well plates at a density of 293 cells / well: 2.5 × 10⁻⁶ cells / well. 5 6 × 10 cells; HeLa: 6 × 10 4 One cell;

[0163] (2) After 24 hours, virus infection was performed. The virus sample was diluted with complete culture medium according to the required MOI (multiplicity of infection). The old culture medium in the well was discarded and 500 μL of diluted sample was added.

[0164] (3) Add 500 μL of fresh complete culture medium after 12 h;

[0165] (4) Collect the cell supernatant 72 hours after infection, which can be used for subsequent ELISA detection;

[0166] The concentration of C11 protein in the cell supernatant was detected by ELISA, and the results are shown in Figure 1. The results indicate that C11 protein was successfully expressed in both 293T cells and HeLa cells after infection with the #3 AAV vector.

[0167] 1.4 C11 protein activity assay

[0168] #3AAV vector was used to infect the supernatant of 293T cells to express C11 protein. Serial dilutions of VEGF Trap were prepared in antibiotic-resistant medium. Protein activity was detected using HEK293 cells overexpressing VEGFR2 for commercial use. As a control, a Luciferase experiment was performed. The experimental steps are as follows:

[0169] (1) VEGFR2 cells were seeded in 96-well plates, with 3 replicates per sample. 100 μL of 5 × 10⁶ cells were seeded in each well. 4 Cells were plated and Luciferase assay was performed 24 hours later.

[0170] (2) Prepare C11 and 11 in the resistance medium. A series of diluents, with concentrations ranging from 13 pM to 5000 pM;

[0171] (3) Remove the old culture medium from the 96-well plate, and add 80 μL of culture medium containing different concentrations of C11 and different concentrations of [unclear text - possibly a continuation of the previous sentence] to each well. The culture medium was prepared. Simultaneously, 80 μL of blank culture medium was added to the control wells. C11 and... Immediately after adding to the VEGFR2 cell line, add 20 μL of VEGF165 working solution (8 ng / mL);

[0172] (4) Equilibrate the cell lysis buffer and firefly luciferase detection reagent to room temperature in advance. It is recommended to thaw at room temperature and avoid light.

[0173] Incubate at 37℃, 5% CO2 for 4 hours, equilibrate to room temperature for 30 minutes, centrifuge at 500g for 5 minutes at room temperature, and discard the supernatant in the well plate.

[0174] (5) After thoroughly mixing the reporter gene cell lysis buffer, add 100 μL of lysis buffer to each well, shake the microplate at 600 rpm for 5 min to completely lyse the cells, pipette evenly, and then add 70 μL to an opaque white plate.

[0175] (6) Add 100 μL of firefly luciferase detection reagent to each well, shake the microplate at 600 rpm for 1 min, mix well, and then measure the relative light unit (RLU).

[0176] (7) Use the Tecan multifunctional enzyme-linked immunosorbent assay (ELISA) reader, use the detection module for detecting firefly luciferase, select an opaque white plate for the well plate, set the detection time to 2000ms, and take the reading.

[0177] The activity results are shown in Figure 14. #3AAV-infected cells showed increased C11 protein activity and... There was no difference in activity.

[0178] 1.5 Intraocular injection of virus, detection of C11 protein expression.

[0179] Experimental subjects: C57BL / 6J wild-type mice

[0180] The positive control drug RGX-314 and #3 AAV virus were injected into the subretinal space of mice, with an injection volume of 1 μL and a concentration of 1E+9 vg / μL. Two weeks after injection, whole eye proteins or vitreous fluid were extracted, and the expression level of VEGF protein was detected. The positive control drug RGX-314 was prepared in the laboratory, and #3 AAV was prepared according to the method in Example 1.

[0181] The ELISA results are shown in Figures 15a-15b. The results show that in whole-eye protein detection, the expression level of the target protein C11 in the #3AAV vector was significantly higher than that of ranibizumab in RGX-314. Calculations showed that the concentration of protein C11 was 157 times higher than that of the protein expressed by the V3 gene. In vitreous detection, the expression level of viral protein C11 in the #3 vector was significantly higher than that of the protein expressed by the V3 gene in RGX-314. Calculations showed that the concentration of C11 protein was 25 times higher than that of the protein expressed by the V3 gene in the positive control drug RGX-314. Comparing the two, the expression level of the target protein C11 in the #3AAV viral vector of this invention is significantly better than the expression level of the protein expressed by the V3 gene in the positive control drug RGX-314.

[0182] Example 2: Pharmacodynamic Study

[0183] 2.1#3 Inhibitory effect of AAV on angiogenesis in hRHO-hVEGFA mice

[0184] Experimental subjects: hRHO-hVEGFA mice (source: Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). Human VEGF165 mice were expressed using the human rhodopsin promoter as a vector promoter. Without induction, they naturally develop choroidal angiogenesis, making them a good model for studying ophthalmic diseases caused by angiogenesis in newborn mice. They can be used to screen and evaluate the efficacy and mechanisms of drugs for related diseases.

[0185] The positive control drug RGX-314 was prepared in the laboratory (coding the nucleic acid sequence of ranibizumab in CN114502197A; other steps were the same as the preparation method for the C11 protein). This anti-VEGF protein drug, purchased from Bayer, is for the treatment of age-related macular degeneration and diabetic macular edema. #3 AAV was prepared using conventional methods.

[0186] Experimental Groups:

[0187] hRHO-hVEGFA mice were divided into three groups of five mice each. The left eye of each mouse was injected with PBS (Gibco) as a negative control, while the right eye was injected with 1 μL of a different test or control substance. Grouping and administration information are shown in Table 2. Fourteen days after injection, the fundus vascular status of the mice was observed by fluorescein angiography: fluorescein sodium (100 mg / ml, 0.02 ml / mouse) was injected intraperitoneally, and fundus images were acquired and recorded 3 minutes after injection.

[0188] Table 2: Dosing Regimen

[0189] The FFA results, as shown in Figure 2, indicate that at the time points captured, in Group A, there was no significant difference in vascular leakage and angiogenesis between the right and left eyes; in Group B, there was no significant difference in retinal vascular leakage and angiogenesis between the right and left eyes; and in Group C, the number of neovascularized plaques was significantly reduced in the right eye compared to the left eye, indicating that retinal vascular leakage was significantly inhibited. These results demonstrate that the test sample #3 AAV and the positive control drug... Compared with RGX-314, it has outstanding efficacy and obvious advantages in inhibiting angiogenesis and abnormalities.

[0190] 2.2#3 Inhibitory effect of AAV on angiogenesis in C57BL / 6J mice

[0191] Experimental subject: C57BL / 6J mice (Source: Beijing Vital River Laboratory Animal Technology Co., Ltd.)

[0192] Experimental Groups

[0193] C57BL / 6J mice were divided into 9 groups, with 6-11 mice in each group. On day 1 of the experiment, the corresponding drug dosage for the subretinal space of mice in experimental groups 1, 2, 3, 4, and 5 was 1 μL. Grouping and drug administration information are shown in Table 3. On day 14 of the experiment, mice in the negative control group were injected intravitreally with PBS, while mice in the positive control groups 1 and 2 were injected intravitreally with 2.5 μg / μL and 25 μg / μL of PBS, respectively. 1 μL of laser (power: 300 Mv, duration: 100 ms) was used to irradiate the retinas of all mice, penetrating Brunch's membrane in the superior, inferior, temporal, and nasal quadrants centered on the optic disc, thus inducing choroidal angiogenesis. Fundus angiogenesis was assessed by FFA on day 21 of the experiment.

[0194] Table 3: Dosing Regimen

[0195] One week after laser injury, fundus imaging was performed on mice. The results are shown in Figures 3 and 4. In the negative control group, approximately 90% of the laser-irradiated sites showed definite neovascularization, of which approximately 43% were progressive neovascularization. In the positive control group 1, the number of definite and progressive neovascularizations in the eyes was not different from that in the PBS group, indicating that... Low dose (2.5 μg / μL) had no inhibitory effect on angiogenesis induced by laser damage; in the positive control group 2, approximately 20% of the laser-irradiated sites showed definite and progressive angiogenesis plaques, and approximately 80% of the laser-irradiated sites showed suspected angiogenesis or no angiogenesis plaques, indicating that... High doses (25 μg / μL) significantly inhibited angiogenesis caused by laser damage.

[0196] #3 AAV experimental groups 2, 3, 4, and 5 all showed positive therapeutic effects on laser-induced damage. In experimental group 2, approximately 20% of the mice developed clearly defined and progressive neovascularization plaques in their eyes, compared to high-dose (25 μg / μL) AAV. The therapeutic effects were comparable. In the eyes of mice in experimental groups 3-5, the treatment completely inhibited the development of well-defined and progressive angiogenesis plaques following laser-induced damage. These results indicate that the #3 AAV drug of this invention is superior to the positive control drug in inhibiting angiogenesis and abnormalities.

[0197] 2.3#3 Inhibitory effect of AAV on choroidal angiogenesis in cynomolgus monkeys

[0198] Laboratory animal: Crab-eating macaque (Source: Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd.)

[0199] Before the experiment, the cynomolgus monkeys were divided into two groups: Group 1 (RGX-314 group) and Group 2 (#3 AAV group), with one cynomolgus monkey in each group. On the first day of the experiment, subretinal injections were administered in both eyes, as shown in Table 4. Continuous observation followed administration. An immunomodulator (prednisolone acetate injection) was administered once daily for 3 days prior to and 3 days after administration, at a dose of 1 mg / kg.

[0200] Table 4: Dosing Regimen Note: *The left eye of the RGX-314 group was given the same volume of solvent.

[0201] Laser photocoagulation was performed 45 days after the experimental drug administration; fundus imaging was performed before the experiment, 2 days before laser photocoagulation, and 2 weeks after laser photocoagulation.

[0202] The macular region was photocoagulated via panretinal microscopy, and the results are shown in Figure 5. Photocoagulation avoided the fovea centralis, and nine points were irradiated in each eye.

[0203] Significant leakage was observed at the laser irradiation point in the solvent-treated eyes. In the RGX-314-injected eyes (1.3E+9vg / eye), there was no significant difference in leakage at the laser irradiation point compared to the control eyes, indicating that this dose of RGX-314 had no inhibitory effect on choroidal angiogenesis in monkeys. In the low-dose eyes injected with the same dose (1.3E+9vg / eye) of #3 AAV, no significant leakage was observed at the laser irradiation point, indicating that #3 AAV at a dose of 1.3E+9vg / eye already had a significant inhibitory effect on choroidal angiogenesis, significantly better than RGX-314. In the high-dose eyes of #3 AAV (1.3E+11vg / eye), the leakage at the laser irradiation point was further reduced compared to the low-dose eyes.

[0204] The above test results show that #3 AAV can significantly and effectively inhibit choroidal angiogenesis in the cynomolgus monkey model.

[0205] Aqueous humor samples were collected on days 15, 29, 59, 75, and 89 after drug administration. The concentration of C11 protein in the aqueous humor was detected by ELISA. The C11 protein expression results in the #3 AAV group are shown in Figure 6. In the #3 AAV low-dose eye (1.3E+9vg / eye), the C11 protein content in the aqueous humor at each time point was 5.65 ng / ml, 5.06 ng / ml, 9.1 ng / ml, 7.84 ng / ml, and 9.50 ng / ml, respectively. In the #3 AAV high-dose eye (1.3E+11vg / eye), the C11 protein content in the aqueous humor at days 15 and 29 was 605 ng / ml, 492 ng / ml, 698 ng / ml, 499 ng / ml, and 868 ng / ml, respectively.

[0206] The above test results show that #3 AAV can express the target protein normally in situ and enter the intraocular circulation after subretinal injection; 15 days after injection, the intraocular concentration of C11 protein has remained stable, and the change in C11 protein concentration in aqueous humor has a dose-dependent relationship with #3 AAV.

[0207] 2.4#3 Inhibitory effect of AAV on choroidal angiogenesis in macaques

[0208] Laboratory animals: Rhesus monkeys (Source: Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd.)

[0209] Before the experiment, the macaques were divided into four groups: Group 1 was the solvent group, Group 2 was the low-dose #3 AAV group, Group 3 was the medium-dose #3 AAV group, and Group 4 was the high-dose #3 AAV group, with two macaques in each group. On the first day of the experiment, subretinal injections were administered in both eyes, and the administration regimen is shown in Table 5. The macaques were observed for 8 weeks after administration.

[0210] Table 5: Dosing Regimen Note: *The same volume of solvent was administered to both eyes.

[0211] Laser photocoagulation was performed 30 days after drug administration; fundus photography and fluorescein angiography were performed before the experiment, 29 days after drug administration, and at 6 and 8 weeks after drug administration.

[0212] The macular region was irradiated via panretinal photocoagulation, and the results are shown in Figure 7. Nine points were irradiated in each eye.

[0213] Significant leakage was observed at the laser-guided eye in the solvent group; no significant leakage was observed at the laser-guided eye in any of the #3 AAV dose groups, indicating that #3 AAV at a dose of 1E+9vg / eye already has a significant inhibitory effect on choroidal angiogenesis.

[0214] The above test results show that #3 AAV can significantly and effectively inhibit choroidal angiogenesis in the macaque model.

[0215] Example 3 Pharmacokinetic Study

[0216] Experimental subjects: C57BL / 6J mice (source: Beijing Vital River Laboratory Animal Technology Co., Ltd.).

[0217] Experimental grouping: C57BL / 6J mice were randomly divided into 4 groups according to their body weight. Group 1 was the solvent group, with 3 animals each. Groups 2, 3, and 4 were the drug administration groups, with 24 animals each. The drug administration regimens for each group are shown in Table 6. The drugs were administered via subretinal injection in one eye (left eye). After a single dose, the animals were observed for up to one year. The day of drug administration was defined as day 1 of the experimental period (D1).

[0218] Table 6: Dosing Regimen

[0219] After drug administration, a single-line OCT scan of the injection site was performed using a small animal retinal imaging system to confirm successful injection and locate the injection area. The criteria for successful drug administration were: a noticeable local retinal bulge observed under a microscope during the injection process, and OCT scan showing retinal neuroepithelial detachment accompanied by subretinal fluid accumulation at the injection site.

[0220] Drug concentration samples were collected at 3, 7, and 14 days after administration, and at 4, 8, 12, and 24 weeks. The method was as follows: mice were euthanized with carbon dioxide, the injection eye was removed, the mice were ground and crushed at low temperature, the supernatant was collected by high-speed centrifugation, and the samples were transported to storage below -60°C.

[0221] After 24 weeks of observation, the results for each group are shown in Figure 8, as detailed below:

[0222] 1.5E+6vg dose group: C11 protein expression was detectable 7 days after intraocular injection of #3 AAV; the intraocular C11 protein content tended to stabilize after 2 weeks, with the concentration in the mouse eye ranging from about 0.2 ng / eye to 1.5 ng / eye at various time points; at the 24th week, the intraocular C11 protein content in the mouse eye was 1.3 ng / eye;

[0223] 1.5E+7vg dose group: C11 protein expression was detectable 3 days after intraocular injection of #3 AAV; after 2 weeks, the intraocular protein content tended to stabilize, with the concentration in the mouse eye ranging from approximately 13ng / eye to 42ng / eye at various time points; at the 24th week, the intraocular protein content in the mouse eye was 35ng / eye;

[0224] In the 1.5E+9vg dose group: C11 protein expression was detectable 3 days after intraocular injection of #3 AAV. The intraocular protein concentration tended to stabilize after 2 weeks, with the concentration in the mouse eye ranging from approximately 1795 ng / eye to 3495 ng / eye at various time points; at week 24, the intraocular C11 protein content in the mouse eye was 1873 ng / eye.

[0225] The above test results show that in the mouse model, #3 AAV can express the target protein normally in situ after subretinal injection, and there is a dose-dependent relationship with #3 AAV; the intraocular concentration of the target protein can remain stable 24 weeks after injection.

[0226] Example 4: Toxicological Study

[0227] 4.1 Safety testing of mice

[0228] Six groups were established: solvent group, 1.5×10⁻⁶ group, and 1.5×10⁻⁶ group. 6 Group, 1.5×10 7 Group, 1.5×10 8 Group, 1.5×10 9 The group consisted of 6 animals, and each group received a subretinal injection in one eye.

[0229] Body weight and tissue weight of mice after subretinal injection of #3 AAV.

[0230] Mice were injected subretinally with PBS at a dose of 1.5 × 10⁻⁶. 6 vg / eye ~1.5×10 9 Mice were injected with Vg / eye #3 AAV and euthanized one month later. Heart, liver, spleen, lung, kidney, brain, stomach, and ovarian tissues were harvested, and their weights were measured and recorded. There were no statistically significant differences in body weight and organ weight between the #3 AAV experimental groups and the PBS group (Figure 9).

[0231] 4.2 Detection of blood IFNγ concentration after subretinal intracavitary injection of #3 AAV

[0232] Mice were injected subretinally with PBS at a dose of 1.5 × 10⁻⁶. 6 vg / eye~1.5×10 9Mice were injected with VG / eye#3 AAV and euthanized one month later. Serum was collected and the IFNγ level in the serum was examined by ELISA. The IFNγ levels in the serum of mice in both the #3 AAV experimental group and the PBS group were below the detection limit.

[0233] 4.3 Detection of neutralizing antibody titers after subretinal injection of #3 AAV

[0234] Flow cytometry can determine the proportion of GFP-positive cells in each sample well, representing the proportion of cells that escape neutralizing antibodies (Nabs) and enter to induce the expression of the GFP transgene. The proportion of GFP-positive cells in the experimental sample wells minus the proportion of GFP-positive cells in the positive control wells without serum is inferred as the remaining proportion due to antibody capture and neutralization. The neutralizing antibody titer is defined as the reciprocal of the serum dilution ratio at which the proportion of GFP-positive cells is reduced to below 50%. PBS and 1.5 × 10⁻⁶ cells were also tested. 7 1.5×10 9 Three groups of serum neutralizing antibody titers were collected. Statistical results showed that #3 AAV 1.5×10 7 Dosage group and 1.5×10 9 The neutralizing antibody titers in both the dosage group and the PBS group were less than 1 / 200 and showed no statistical difference. The neutralizing antibody titers were not dose-dependent with respect to #3 AAV (Figure 10).

[0235] 4.4 Fundus image examination of mice after subretinal injection of #3 AAV

[0236] Mice were injected subretinally with PBS at a dose of 1.5 × 10⁻⁶. 6 vg / eye, 1.5×10 7 vg / eye, 1.5×10 8 vg / eye, 1.5×10 9 VG / eye #3AAV, fundus imaging was performed 3 weeks after injection under anesthesia.

[0237] As shown in Figure 11, the retinas of all mice were clearly visible, and the vitreous fluid was not cloudy. (1.5 × 10⁻⁶) 6 Dosage group, 1.5 × 10 7 The dosage group was consistent with the PBS group, with only some mice showing mild pigmentary degeneration in the subretinal injection area, while the fundus in the non-injection area was normal; 1.5×10 8 The vg / eye dose group and 1.5×10 9 In the vg / ocular dose group, one mouse eye showed pigmentary degeneration in the non-injection area. This may be related to physical damage caused by subretinal injection.

[0238] 4.5 Electrophysiological (ERG) Detection of the Retinal Receptor in Mice After Subretinal Injection of #3 AAV

[0239] Mice were injected subretinally with PBS at a dose of 1.5 × 10⁻⁶. 6 vg / eye ~1.5×10 9 VG / eye #3 AAV, anesthesia was administered 4 weeks later for electrophysiological data acquisition.

[0240] ERG results showed that the a-wave and b-wave in the injected eyes of mice in all dose groups of #3 AAV were normal, with no abnormal waveforms observed. There was no significant difference in the amplitude of the a-wave and b-wave in the injected eyes of mice in all dose groups of #3 AAV compared with the non-injected eyes of the same mouse (Figure 12).

[0241] Example 5#3 Preparation of AAV drug composition

[0242] The preparation of the formulation is carried out according to the following steps: 1) Take the #3 AAV after Q chromatography and replace the buffer with a 50kD ultrafiltration concentration tube. The replacement buffer is 2.67mM potassium chloride (KCl), 1.47mM potassium dihydrogen phosphate (KH2PO4), and 8.06mM disodium hydrogen phosphate (Na2HPO4).

[0243] 2) Perform genomic titer analysis on the #3 AAV obtained from ultrafiltration concentration in step 1. Based on the genomic titer results, dilute the AAV to 1.0 × 10⁻⁶. 12 The concentration of vg / mL was dispensed into 1.5mL EP tubes. The final formulation of #3 AAV injection solution was: 1.0 × 10⁻⁶ g / mL. 12 Vg / mL #3 AAV, 150mM sodium chloride (NaCl), 0.01% poloxamer 188, adjust pH to 7.3.

[0244] Example 6: Safety and efficacy trial of #3 AAV injection in subjects with wet age-related macular degeneration.

[0245] 6.1 Test Plan

[0246] This was a single-center, single-arm, open-label, dose-exploration clinical trial aimed at evaluating the preliminary safety and tolerability of subretinal injection of the disclosed #3 AAV solution, its preliminary efficacy in treating wet age-related macular degeneration (nAMD), immunogenicity, vector shedding and distribution characteristics, and long-term safety and efficacy of subretinal injection of the disclosed #3 AAV solution through long-term follow-up. A total of 6 subjects were enrolled; the detailed experimental protocol is shown in Table 7.

[0247] Primary endpoint:

[0248] To assess the occurrence of DLT (dose-limiting toxicity) in each dose group within 28 days after administration of #3 AAV injection.

[0249] Assess the occurrence of adverse events within 52 weeks following treatment with #3 AAV injection.

[0250] Secondary endpoint:

[0251] The study investigated the number of times the eye received aflibercept adjuvant therapy and the time since the first adjuvant therapy.

[0252] The study investigated the changes in BCVA (best corrected visual acuity) from baseline at various time points within 52 weeks after the eyes received #3 AAV injection treatment using the ETDRS visual acuity chart.

[0253] OCT scans at various time points within 52 weeks after the study eyes received #3 AAV injection showed changes in central macular retinal thickness (CRT) from baseline.

[0254] Fluorescein angiography (FFA) at various time points within 52 weeks after the study eyes received #3 AAV injection showed changes in the area of ​​choroidal neovascularization (CNV) and the total area of ​​fluorescein leakage from baseline.

[0255] To assess the immunogenicity (humoral and cellular immunity), vector shedding, and distribution characteristics of subjects after treatment with #3 AAV injection.

[0256] Table 7: Scheme Design Note: Store the injection solution at ≤-60°C, protected from light. Before injection, thaw and dilute according to the drug preparation procedure. The safe dose should not exceed 1.3 × 10⁻⁶. 11 vg / eye (genomic titer assay). The injection solution formulation for dilution is: containing 1.0 × 10 12 vg / mL#3 AAV, 150mM NaCl, 2.67mM KCl, 1.47mM KH2PO4, 8.06mM Na2HPO4 and 0.01% (w / v) poloxamer 188, with a pH of 7.0-7.5.

[0257] Dosage change rules:

[0258] If no DLT occurs after the first subject in the low-dose group completes the DLT assessment, the study can continue with enrollment in the low-dose group or the enrollment of the first subject in the high-dose group, subject to a decision by the SRC (Security Review Commission); if DLT occurs, a lower dose should be explored or the study should be suspended.

[0259] If the first subject in the high-dose group does not experience DLT after completing the DLT assessment, the SRC can decide whether to continue enrollment in the high-dose group; if DLT occurs, the subject will be returned to the low-dose group or an intermediate dose will be explored; if no DLT occurs in any subject in the high-dose group, the SRC will decide whether to proceed with enrollment at a higher dose.

[0260] Dose-limiting toxicity refers to any of the following adverse events (AEs) that the investigator determined to be related to the injection solution and occurred within the observation window from injection to day 28:

[0261] 1) BCVA continues to decrease by ≥15 letters from baseline and has not recovered (“continued decrease” is defined as a decrease that lasts for at least 48 hours before visual recovery, and “visual recovery” is defined as a decrease in BCVA to within 10 letters from baseline;

[0262] 2) Intraocular pressure remains above 25 mmHg for 2 weeks or below 6.5 mmHg for 1 week after drug treatment;

[0263] 3) Intraocular inflammation (aqueous humor flare / cells, vitreous cells) indicators ≥ grade 3 (see Tables 8-9 for details);

[0264] 4) Vitreous hemorrhage ≥ grade 3 (see Table 10 for details);

[0265] 5) Liver function tests show ALT or AST > 3 × pre-treatment value (ULN) (normal before treatment), or ALT or AST > 3 × pre-treatment value (abnormal before treatment), and the value has not decreased to the pre-treatment value after 14 days.

[0266] 6) Grade 3 or higher ocular and non-ocular adverse events (AEs) as per CTCAE 5.0.

[0267] 7) Other adverse events as determined by the researchers.

[0268] Table 8: Grading of Aqueous Fluid Shimmer / Cell Count Note: a: Field of view is a 1mm slit beam.

[0269] Table 9: Grading of Vitreous Opacity / Cell Count

[0270] Source: Nussenblatt et al. Am J Ophthalmol 92(4)467-1.

[0271] Table 10: Grading of Vitreous Hemorrhage Severity

[0272] Source: Zhang Chengfen, Dong Fangtian, et al. Fundus Diseases (Second Edition). People's Medical Publishing House. 2010, 460 pages.

[0273] Nussenblatt, Whitcup SM, Palestine AG. Uveitis: Basic Principles and Clinical Practice. Second Revised Edition, New York: Mosby, 1996, 64 pages.

[0274] 6.2 Pre-injection and assessment of aflibercept

[0275] After the subjects complete the screening and enrollment, the study eye will first receive a 2 mg intravitreal pre-injection of aflibercept (used according to the dosage and administration instructions in the drug package insert). Seven days after the aflibercept pre-injection and before the subretinal injection of #3 AAV injection, the researchers will conduct one or more efficacy assessments based on the individual subject's condition, including optical coherence tomography (OCT), best corrected visual acuity (BCVA), fundus condition, etc. The researchers may add examinations as needed based on the subject's condition.

[0276] 1) Changes in central macular retinal thickness (CRT) and subretinal or intraretinal fluid accumulation compared to baseline;

[0277] 2) Changes in best-corrected visual acuity (BCVA) compared to baseline;

[0278] 3) Researchers make a comprehensive judgment based on the condition of the subject's fundus.

[0279] 6.3 Glucocorticoid administration

[0280] To reduce inflammation, oral corticosteroids (such as prednisone) should be started 3 days before subretinal injection of #3 AAV solution, initially at 1 mg / kg / day (maximum dose 40 mg / day) for 10 days, then reduced to 0.5 mg / kg / day (maximum dose 20 mg / day) for 7 days. Researchers may adjust the corticosteroid regimen according to the patient's condition.

[0281] 6.4 Subretinal injection

[0282] Within 14–21 days after the pre-injection of aflibercept, the study eyes will be given a single subretinal injection of #3 AAV solution (day 0), with an injection volume of 100 μL and multiple injection sites.

[0283] 6.5 Aflibercept supplementary therapy

[0284] If a study eye exhibits any of the following three criteria for supplemental treatment due to choroidal neovascularization (CNV) activity, the investigator may consider administering aflibercept as supplemental treatment to the subject from week 4 (D28) after administration of #3 AAV injection until the end of the trial follow-up period (dosage and administration as per the drug instructions).

[0285] Supplementary treatment criteria:

[0286] 1) BCVA loss of more than 10 letters from baseline due to intraretinal or subretinal fluid;

[0287] 2) OCT examination confirmed that the central retinal thickness (CRT) increased by more than 75 μm from baseline;

[0288] 3) There is retinal hemorrhage that seriously affects vision.

[0289] 4) Situations where researchers believe supplementary treatment is necessary.

[0290] While conducting safety trials, immunogenicity studies and vector shedding and distribution studies will be carried out simultaneously by collecting tears, blood, and aqueous humor from the subjects.

[0291] 6.6 Inclusion and exclusion criteria for subjects:

[0292] Subjects must meet all of the following criteria to be enrolled:

[0293] 1) The subjects were able to understand and voluntarily sign the written informed consent form and were willing to comply with the long-term follow-up protocol;

[0294] 2) Age 50 ≤ age ≤ 80;

[0295] 3) The study confirmed the presence of CNV secondary to nAMD in the eye;

[0296] 4) Using the ETDRS visual acuity chart, the best corrected visual acuity (BCVA) of the study eye is ≥5 letters and ≤73 letters (approximately equivalent to 20 / 800 to 20 / 40 on the Snellen visual acuity chart), and the best corrected visual acuity (BCVA) of the contralateral eye of the study eye is ≥19 letters (approximately equivalent to 20 / 400 on the Snellen visual acuity chart).

[0297] 5) Within 12 months prior to screening, the study eye had received ocular anti-VEGF treatment and the investigator believed that the subject had responded to the treatment (e.g., clinically significant changes in visual acuity or OCT, etc.).

[0298] Subjects must not meet any of the following criteria to be eligible for enrollment:

[0299] Studying eye condition:

[0300] 1) The study investigated the presence of CNV or macular edema secondary to causes other than AMD in the eye.

[0301] 2) In the researcher's opinion, any accompanying eye disease in the study eye may lead to non-response to the study treatment or confound the interpretation of the study results. Examples include polypoid choroidal vascular disease (PCV), diabetic retinopathy (severe or proliferative DR), retinal vein occlusion, and uncontrolled glaucoma in the study eye (defined as intraocular pressure ≥25 mmHg after standard treatment).

[0302] 3) If, as determined by the researchers, the study eye has any accompanying ocular disease that may prevent the subject from undergoing subretinal injection or increase the risk of subretinal injection, such as high-risk hypotension (defined as intraocular pressure <6.5 mmHg after standard treatment), retinal detachment, macular hole, vitreomacular traction affecting central vision, epiretinal membrane involving the fovea or destroying the macular structure, or an equivalent spherical lens ≤-8.00D in the study eye, etc.

[0303] 4) The study eye has undergone prior ocular surgery that may not respond to the study treatment, as assessed by the investigator. Examples include retinal detachment repair, trabecular meshwork resection or other filtration surgery, and retinal grid or panretinal photocoagulation.

[0304] 5) The presence of implants (excluding artificial lenses), severe turbidity of the refractive media, or insufficient pupil dilation in the study eye may affect BCVA, surgical procedures, and the acquisition of sufficiently clear ocular imaging data (such as OCT, FFA, and fundus photography), thus affecting the researchers' observation of safety and efficacy.

[0305] 6) The study eye underwent cataract surgery within one month prior to enrollment or was deemed by the investigator to be likely to require cataract surgery within three months after injection of the study drug.

[0306] 7) The eye has received intravitreal injections of medication other than anti-VEGF drugs within 6 months prior to the screening, such as intraocular corticosteroids (e.g., Omeprazole) or any investigational drugs.

[0307] Any eye scenario:

[0308] 8) Active intraocular or periocular inflammation or infection exists in any eye.

[0309] 9) Any eye has received gene therapy.

[0310] 10) Poor blood pressure control (defined as systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥110 mmHg after antihypertensive drug treatment).

[0311] 11) Diabetic patients who meet any of the following conditions:

[0312] ① Known macrovascular complications; ② Baseline glycated hemoglobin (HbA1c) > 7.5%.

[0313] 12) Any other pre-existing medical conditions, metabolic disorders, physical examination results, or clinical laboratory test results that reasonably suggest the presence of a disease or condition that could contraindicate the use of the investigational drug, affect the interpretation of study results, or place the subject at high risk of treatment complications. Examples include coagulation disorders, cerebrovascular accidents or transient ischemic attacks or myocardial infarctions within the 6 months prior to screening, or a history of treated or untreated malignant tumors within the past 5 years.

[0314] 13) Any of the following abnormalities are present:

[0315] ① Platelet count < 90 × 10 9 / L, hemoglobin (Hb) <10g / dL (male) or <9g / dL (female); ② aspartate aminotransferase (AST) or alanine aminotransferase (ALT) ≥2×ULN, total bilirubin (TBil) ≥1.5×ULN; ③ serum creatinine or urea >1.5×ULN.

[0316] 14) Those who have undergone major or medium-sized surgery or have had serious trauma within 3 months prior to screening.

[0317] 15) Within 3 months prior to screening (or within 5 half-lives for drugs, whichever is longer), participated in any other clinical study (excluding vitamins and minerals, or anti-VEGF drug treatment in non-study eyes), or attempted to participate in other clinical trials during the study period.

[0318] 16) The screening results showed positive for human immunodeficiency virus (HIV) antibody, hepatitis C (HCV) antibody, or syphilis-specific antibody.

[0319] 17) Acute or chronic active hepatitis B was present at the time of screening (defined as positive for hepatitis B surface antigen and / or hepatitis B core antibody, and HBV-DNA viral load ≥2000 IU / mL).

[0320] 18) Pregnant or breastfeeding women or individuals of reproductive potential who are unwilling to use effective contraception during the study period.

[0321] 19) Subjects with rAAV8 neutralizing antibody titers >1:1000.

[0322] 20) Other situations where researchers deem the study unsuitable for inclusion.

[0323] Studying the choice of eye:

[0324] All participants in the study received the drug in one eye, i.e., the study eye.

[0325] If both eyes of the subject meet the inclusion criteria but do not meet the exclusion criteria, the researcher will select the eye with the worse BCVA as the study eye; if the BCVA of both eyes of the subject are similar, the eye with the worse fundus condition will be selected as the study eye; if the BCVA and fundus condition of both eyes of the subject are similar, the right eye will be selected as the study eye; or the researcher will determine which eye is more suitable for subretinal injection as the study eye.

[0326] 6.7 Results Analysis

[0327] This includes the FAS (full analysis set), EAS (efficacy analysis set), SS (safety analysis set), IMS (immunogenicity analysis set), and DLTS (dose-limiting toxicity analysis set). Baseline values ​​were the results of the most recent examination prior to aflibercept adjuvant therapy injection.

[0328] Validity endpoint analysis:

[0329] A descriptive summary was made of the BCVA, CRT, FFA and their changes from the baseline at each time point.

[0330] Security Analysis:

[0331] Safety endpoints include direct retinal latency (DLT) within 28 days after subretinal administration, ocular adverse events (AEs) (study eye AE and contralateral eye AE), non-ocular AEs, subretinal acute exacerbations (SAE), as well as abnormalities in vital signs, physical examination, electrocardiogram, and laboratory tests. The severity of adverse events will be classified using the NCI CTCAE version 5.0 standard.

[0332] This study recruited six nAMD patients who met the inclusion and exclusion criteria, aged 65-80 years, with active choroidal neovascularization, best-corrected visual acuity (BCVA) between 50 and 74 letters, and who had responded to previous anti-VEGF therapy. All six subjects received a single subretinal injection of #3 AAV injection (1 subject was assigned to a 1.0 × 10⁻⁶ dredge). 9 In the vg / eye dose group, 5 subjects were assigned to receive 3.0 × 10 9 (vg / ocular dose group). The primary outcome was an assessment of safety and tolerability. Secondary outcomes included efficacy, immunogenicity, vector distribution, and vector clearance. Long-term follow-up results were continuously evaluated for safety and efficacy.

[0333] According to the study schedule, subjects underwent bilateral ocular safety testing at each follow-up visit, including: best corrected visual acuity (BCVA); intraocular pressure (IOP); color fundus photography (CFP); optical coherence tomography (OCT); fundus fluorescein angiography (FFA); and routine ophthalmological examinations, including slit-lamp examination. Subject safety was monitored through regular physical examinations, vital sign assessments, and routine clinical laboratory tests (complete blood count, blood biochemistry, urinalysis, coagulation tests, etc.). No dose-limiting toxicity (DLT) or drug-related or potentially related adverse events occurred in any subject.

[0334] As described above, participants received one of two dose levels of #3 AAV subretinal injection: low dose (vector genome 1.0 × 10⁻⁶). 9 (vg / eye) or high dose (3.0×10) 9 vg / eye).

[0335] One patient in the low-dose group (1.0×10) 9 The patient (vg / eye) completed a 52-week follow-up, while 5 patients in the high-dose group (3.0 × 10⁻⁶) completed the follow-up. 9 (vg / eye) Follow-up for at least 20 weeks. Single dose or single injection of #3 AAV showed clinically significant visual improvement. At 20 weeks post-treatment, all 6 patients showed clinically significant improvement without supplemental injections: complete resolution of macular edema in 5 patients and significant reduction in macular edema in 1 patient. Notably, these benefits were achieved without any supplemental anti-VEGF injections, highlighting the potential of the #3 AAV of this disclosure as a durable treatment option.

[0336] OCT results showed that the submacular fluid in 6 subjects had been partially or completely absorbed. (1.0×10) 9 vg / eye group, 3.0×10 9 In the vg / eye group, after 4 weeks of administration of #3 AAV injection, the central macular retinal thickness (CRT) decreased by an average of 141.83 μm from baseline (6 cases); after 8 weeks of administration, the CRT decreased by an average of 143.43 μm from baseline (6 cases); after 24 weeks of administration, the CRT decreased by an average of 143.8 μm from baseline (5 cases); and after 52 weeks of administration, the CRT increased by 19 μm from baseline (1 case) (with clear retinal structure and no submacular effusion). See Figure 16 for detailed results of OCT and changes in central macular retinal thickness (CRT).

[0337] The BCVA results showed 1.0 × 109 vg / eye group, 3.0×10 9 In the vg / eye group, after 4 weeks of administration, the mean BCVA increased by 4.5 letters from baseline (6 cases); after 8 weeks of administration, the mean BCVA increased by 3.1 letters from baseline (6 cases); after 20 weeks of administration, the mean BCVA increased by 0.3 letters from baseline (6 cases) and the mean BCVA increased by 5.4 letters from baseline (5 cases); after 24 weeks of administration, the mean BCVA increased by 3.5 letters from baseline (5 cases); and after 52 weeks of administration, the mean BCVA increased by 15 letters from baseline (1 case). No subjects received aflibercept supplemental therapy.

[0338] As shown above, #3 AAV injection provided significant clinical benefits to all six nAMD patients. Retinal structure was significantly repaired in all six patients, with a significant reduction in central macular retinal thickness and submacular fluid accumulation; correspondingly, overall visual function showed an improving trend, which continued to improve over time. None of the six patients received supplemental anti-VEGF therapy.

[0339] Therefore, a single subretinal administration of #3 AAV via the dosing regimen disclosed herein results in improved ocular structure and visual function, and is well-tolerated. #3 AAV holds promise as a novel single-dose treatment strategy for nAMD, addressing a critical unmet need in the management of this disease.

[0340] This disclosure relates to the following sequences:

[0341] SEQ ID NO: 1CMV promoter 1

[0342] SEQ ID NO:2.CMV enhancer 1

[0343] SEQ ID NO:3.bGlob intron

[0344] SEQ ID NO:4hGH / polyA

[0345] SEQ ID NO:5. Nucleotide sequence encoding C11 protein

[0346] Amino acid sequence of SEQ ID NO:6.C11 protein

[0347] SEQ ID NO:7. Vector sequence: The AAV vector sequence disclosed herein (containing the nucleotide sequence encoding the C11 protein)

[0348] The sequence of SEQ ID NO:8.5'ITR

[0349] The sequence of SEQ ID NO:9.3'ITR

[0350] SEQ ID NO:10.GFP

[0351] SEQ ID NO:11.EES

[0352] SEQ ID NO:12.WPRE

[0353] SEQ ID NO:13CMV promoter 2

[0354] SEQ ID NO:14CMV enhancer 2

[0355] SEQ ID NO:15GFP

[0356] The present invention has been described through the above embodiments. However, it should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and many variations and modifications can be made based on the teachings of the present invention, all of which fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

A method for treating or preventing an eye disease, the method comprising administering to the eye of a subject in need a recombinant adeno-associated virus (rAAV) containing a nucleotide sequence encoding a therapeutic product; in, The rAAV contains nucleic acid, which comprises, in a 5' to 3' sequence: Cytomegalovirus (CMV) enhancer, wherein the CMV enhancer comprises the sequence shown in SEQ ID NO:2 or SEQ ID NO:14, or a sequence having 85%, 90%, 95%, or 99% or more of identity with it; preferably, the CMV enhancer is the sequence shown in SEQ ID NO:2 or SEQ ID NO:14, or a sequence having 85%, 90%, 95%, or 99% or more of identity with it; A cytomegalovirus (CMV) promoter comprising the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; preferably, the cytomegalovirus (CMV) promoter is the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; bGlob introns, wherein the bGlob introns comprise the sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; preferably, the bGlob introns are the sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; The coding sequence is a nucleotide sequence encoding a VEGF fusion protein or an anti-VEGF antibody; preferably, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein shown in SEQ ID NO:5, or a sequence having 85%, 90%, 95%, or 99% or more identity with it; preferably, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein shown in SEQ ID NO:5; wherein the amino acid sequence of the C11 protein is as shown in SEQ ID NO:6; and The human growth hormone (HGH or hGH) polyA sequence comprises either the sequence shown in SEQ ID NO:4 or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; preferably, the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO:4 or a sequence having 85%, 90%, 95%, or 99% or more of the same identity. According to the method of claim 1, wherein, The method includes administering the rAAV to a subject in need at a dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, preferably about 1.5 × 10 6 vg up to 1.5×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, preferably approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, preferably approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, preferably approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 vg, approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 The rAAV described in vg is preferably applied at the dose once, twice, three times, or four times each time. More preferably, the dose of rAAV is applied at the dose once, twice, three times, or four times each time, wherein the dose applied each time is independently the same or different, and the application site is the same or different each time. Preferably, the method comprises administering a dose of the rAAV to one eye of a subject in need of the following dosage: approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, preferably about 1.5 × 10 6 vg up to 1.5×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, preferably approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, preferably approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, preferably approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 vg, approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 The rAAV described in vg is preferably administered to a subject in need at the dose once, twice, three times, or four times in a single eye each time. More preferably, the rAAV is administered to a subject in need at the dose once, twice, three times, or four times in a single eye each time. The dose administered each time may be the same or different independently. More preferably, each administration is performed by multi-site injection. Preferably, the method includes administering approximately 1.5 × 10⁻⁶ ppm to a single, monocular eye of a subject in need. 6 vg up to 1.5×10 12 vg, preferably about 1.5 × 10 6 vg up to 1.5×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, preferably approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, preferably approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, preferably approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 vg, approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 vg describes rAAV; Preferably, the method comprises administering the rAAV independently to each eye of a subject in need at a single, monocular dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, preferably about 1.5 × 10 6 vg up to 1.5×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, preferably approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, preferably approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, preferably approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 vg, approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 vg, preferably, the method includes administering the rAAV to each eye of the subject in need independently at a dose for each eye, more than once, more than twice, more than three times, or more than four times, preferably administering the rAAV once, twice, three times, or four times, wherein the dose administered to each eye each time is independently the same or different, preferably administered to each eye in a multi-site injection manner. Preferably, the method comprises administering the rAAV to one or both eyes of a subject in need at a dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg / eye, preferably about 1.5×10 6 vg / eye up to 1.5×10 11 vg / eye, preferably about 1.0×10 9 vg / eye to 1.0×10 12 vg / eye, preferably about 1.0×10 9 vg / eye up to 1.3×10 11 vg / eye, preferably about 1.0×10 9 vg / eye to 1.0×10 11 vg / eye, preferably about 1.0×10 9 vg / eye to 1.0×10 10 vg / eye, preferably about 1.0×10 9 vg / eye to 3.0×10 9 vg / eye, preferably about 1.0×10 9 vg / eye, approximately 2.0×10 9 vg / eye, approximately 3.0×10 9 vg / eye, approximately 4.0×10 9 vg / eye, approximately 5.0×10 9 vg / eye, approximately 6.0×10 9 vg / eye, approximately 7.0×10 9 vg / eye, approximately 8.0×10 9 vg / eye, approximately 9.0×10 9 vg / eye or approximately 1.0 × 10 10 vg / eye; preferably, the rAAV is administered to each eye or one eye of the subject in need at the dose independently once, twice, three times or four times, preferably once, twice, three times or four times, wherein the dose administered each time is independently the same or different, and the site of administration is the same or different each time. The method according to claim 1 or 2, wherein, The therapeutic product is a VEGF fusion protein; preferably, the VEGF fusion protein has the amino acid sequence shown in SEQ ID NO:

6. The method according to any one of claims 1-3, wherein, The rAAV further comprises an AAV 5' inverted terminal repeat (L-ITR) upstream of the 5' end of the nucleic acid and an AAV 3' inverted terminal repeat (R-ITR) downstream of the 3' end of the expression cassette nucleotide sequence; preferably, the AAV 5' inverted terminal repeat (L-ITR) comprises or has 85%, 90%, 95%, or 99% or more identity with the sequence shown in SEQ ID NO:8, and the AAV 3' inverted terminal repeat (R-ITR) comprises or has 85%, 90%, 95%, or 99% or more identity with the sequence shown in SEQ ID NO:9; preferably, the nucleotide sequence of the nucleic acid contained in the rAAV is as shown in SEQ ID NO:7; Preferably, the rAAV further comprises an AAV capsid; preferably, the rAAV is selected from AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV; preferably, the rAAV is AAV type 6 (AAV-6) or AAV type 8 (AAV-8). The method according to any one of claims 1-4, wherein, The eye disease is an angiogenesis-related eye disease. Preferably, the eye disease is selected from age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization, and diabetic retinopathy. Preferably, the age-related macular degeneration is wet AMD or dry AMD, and more preferably wet AMD. The method according to any one of claims 1-5, wherein, The rAAV is administered ocularly to the subject in need via intraocular injection, intravitreal injection, subretinal injection, or suprachoroidal injection; and / or The injection can be a single-point injection or a multi-point injection. The method according to any one of claims 1-6, wherein, The rAAV is administered to the eye of the subject in need via the subretinal space and / or the suprachoroidal space, at a dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, preferably approximately 1.0 × 10 9 vg up to 1.3×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 11 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 10 vg, preferably approximately 1.0 × 10 9 vg up to 3.0×10 9 vg, preferably approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 Approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 vg, preferably, the rAAV is administered at the stated dose once, twice, three times, or four times each time, preferably at the stated dose once, twice, three times, or four times each time, wherein the dose administered each time is independently the same or different; preferably, the administered dose is about 1.0 × 10 9 vg / eye up to 1.3×10 11 vg / eye, preferably about 1.0×10 9 vg / eye to 1.0×10 11 vg / eye, preferably about 1.0×10 9 vg / eye to 1.0×10 10 vg / eye, preferably about 1.0×10 9 vg / eye, approximately 2.0×10 9 vg / eye, approximately 3.0×10 9 / eye, approximately 4.0×10 9 vg / eye, approximately 5.0×10 9 vg / eye, approximately 6.0×10 9 vg / eye, approximately 7.0×10 9 vg / eye, approximately 8.0×10 9 vg / eye, approximately 9.0×10 9 vg / eye or approximately 1.0 × 10 10 vg / eye, preferably the rAAV is applied once, twice, three times or four times each time, preferably once or twice each time, with the same or different doses applied in the two applications; Preferably, the following dose of rAAV is administered independently to each eye or one eye of the subject in need: approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg / eye, preferably about 1.5×10 6 vg / eye up to 1.5×10 11 vg / eye, preferably about 1.0×10 9 vg / eye to 1.0×10 12 vg / eye, preferably about 1.0×10 9 vg / eye up to 1.3×10 11 vg / eye, preferably about 1.0×10 9 vg / eye to 1.0×10 11 vg / eye, preferably about 1.0×10 9 vg / eye to 1.0×10 10 vg / eye, preferably about 1.0×10 9 vg / eye to 3.0×10 9 vg / eye, preferably about 1.0×10 9 vg / eye, approximately 2.0×10 9 vg / eye, approximately 3.0×10 9 vg / eye, approximately 4.0×10 9 vg / eye, approximately 5.0×10 9 vg / eye, approximately 6.0×10 9 vg / eye, approximately 7.0×10 9 vg / eye, approximately 8.0×10 9 vg / eye, approximately 9.0×10 9 vg / eye or approximately 1.0 × 10 10 vg / eye; preferably, the rAAV is administered independently to each eye or unilateral eye of the subject in need, once, twice, three times, or four times, preferably once, twice, three times, or four times, wherein the dose administered each time is independently the same or different, and the application site is the same or different each time. and / or The rAAV is administered to the eye of the recipient via the suprachoroidal space at a dose of approximately 1.5 × 10⁻⁶. 6 vg up to 1.5×10 12 vg, preferably approximately 1.0 × 10 9 vg up to 1.0×10 12 vg, preferably approximately 1.0 × 10 10 vg up to 1.0×10 12 vg, preferably approximately 1.0 × 10 10 vg up to 1.0×10 11 vg or approximately 1.0 × 10 11 vg up to 1.0×10 12 vg, preferably, the rAAV is applied once, twice, three times or four times at the dose said, preferably once, twice, three times or four times at the dose said, wherein the dose applied each time is independently the same or different. The method according to any one of claims 1-7, wherein, The required subjects are those aged 45 years or older, preferably those aged 50 years or older, more preferably those aged 50 to 80 years, and even more preferably those aged 65 to 80 years; and / or The subjects in need suffer from age-related macular degeneration; preferably, the subjects in need exhibit the following conditions: drusen; Abnormalities in retinal pigment epithelial cells (RPE); Map-like atrophy of retinal pigment epithelial cells and the inner choroidal layer (including the fovea); and / or Neovascular (exudative) macular degeneration; Preferably, the subject in need suffers from submacular choroidal neovascularization (CNV), vascular leakage, hemorrhage, and / or edema. The method according to any one of claims 1-8, wherein, The best corrected visual acuity (BCVA) of the subject's eye to be treated is 20 / 800 to 20 / 40 on the Snellen visual acuity chart, more preferably 20 / 400 to 20 / 40 on the Snellen visual acuity chart; or the best corrected visual acuity (BCVA) of the subject's eye to be treated is approximately 5-80 letters, preferably approximately 5-73 letters, preferably approximately 19-80 letters, preferably approximately 20-80 letters, more preferably approximately 50-74 letters when using the ETDRS visual acuity chart. According to any one of claims 1-9, the subject's eye has previously received and responded to an anti-VEGF drug; preferably, the anti-VEGF drug is Conbercept, Faricimab, Bevacizumab, Ranibizumab, or Aflibercept, more preferably Aflibercept; and / or The method further includes: At least 14 days, preferably 14-21 days, before administering the rAAV to the eye of the desired subject, the VEGF fusion protein, preferably aflibercept intraocular injection solution, shall be administered to the desired subject. The application should be carried out according to its drug instructions; preferably, the application... The dosage is 1-2 mg, preferably 2 mg; and / or The method further includes: administering a glucocorticoid, preferably prednisone tablets, to the subject in need 1 to 7 days prior to applying the rAAV to the eye; preferably, the dose of the glucocorticoid is initially 0.5 mg / kg / day to 2 mg / kg / day, and not exceeding 40 mg / day, for 5 to 15 days, preferably initially 1 mg / kg / day, and preferably for 10 days; optionally, after the initial glucocorticoid administration, the dose is reduced to 0.3 mg / kg / day to 1 mg / kg / day, and not exceeding 20 mg / day, for 5 to 10 days, preferably reduced to 0.5 mg / kg / day, and preferably for 7 days; and / or The method further includes: administering the rAAV to the eye of the subject in need for at least 28 days, followed by supplemental administration of a VEGF fusion protein to the subject in need, wherein the VEGF fusion protein is preferably... The supplemental administration is performed according to its drug instructions; preferably, after drug administration (preferably 20 or 52 weeks later), the subject's eyes do not require supplemental treatment with VEGF fusion protein; preferably, no supplemental administration of VEGF fusion protein is required after administering rAAV to the eyes of the subject in need; preferably, no supplemental administration is required after administering rAAV to the eyes of the subject in need. The method according to any one of claims 1-10, wherein, The subjects in need are patients with wet age-related macular degeneration, and the method involves administering approximately 1.0 × 10¹² of the solution to each eye in a single dose. 9 vg up to 1.0×10 10 vg, preferably approximately 1.0 × 10 9 vg, approximately 2.0 × 10 9 vg, approximately 3.0 × 10 9 Approximately 4.0 × 10 9 vg, approximately 5.0 × 10 9 vg, approximately 6.0 × 10 9 vg, approximately 7.0 × 10 9 vg, approximately 8.0 × 10 9 vg, approximately 9.0 × 10 9 vg or approximately 1.0 × 10 10 vg describes the rAAV; preferably, the method comprises administering the rAAV independently to each eye of the subject in need at a dose of approximately 1.0 × 10⁻⁶ each time. 9 vg / eye to 1.0×10 10 vg / eye, preferably about 1.0×10 9 vg / eye, approximately 2.0×10 9 vg / eye, approximately 3.0×10 9 vg / eye, approximately 4.0×10 9 vg / eye, approximately 5.0×10 9 vg / eye, approximately 6.0×10 9 vg / eye, approximately 7.0×10 9 vg / eye, approximately 8.0×10 9 vg / eye, approximately 9.0×10 9 vg / eye or approximately 1.0 × 10 10 vg / eye. A method for treating the eye of a human subject suffering from an eye disease, said human subject's eye having previously received intravitreal injection of aflibercept, the method comprising: A single unit dose of a pharmaceutical composition is administered to the eye of a human subject, preferably the pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant adeno-associated virus (rAAV) comprising a nucleic acid sequence encoding an anti-VEGF protein as defined in any one of claims 1-11, wherein the single unit dose comprises about 1.0 × 10⁻⁶ units. 9 vg to approximately 1.0 × 10 11 VG recombinant adeno-associated virus, preferably containing about 1.0 × 10 9 vg to approximately 1.0 × 10 10 vg, further optimized to approximately 1.0 × 10 9 vg to approximately 3.0×10 9 vg, further preferably containing approximately 1.0 × 10 9 vg, approximately 3.0 × 10 9 vg or approximately 1.0 × 10 10 The recombinant adeno-associated virus (vg) was sufficient to cause an increase in aflibercept levels in the eyes of the human subjects when measured at least 20 weeks, 52 weeks, 6 months, 9 months or 1 year after administration of the drug composition, preferably after 9 months. Preferably, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, and diabetic retinopathy; Preferably, the injection is administered into the subretinal space of the human subject. The method according to claim 12, wherein the pharmaceutical composition comprises: a. A vector genome of recombinant adeno-associated virus type 8 (AAV 8), which contains nucleic acids comprising, in the 5' to 3' sequence: The upstream AAV 5' inverted terminal repeat (L-ITR) sequence at the 5' end contains or is the sequence shown in SEQ ID NO:8; CMV enhancer, wherein the CMV enhancer comprises the sequence shown in SEQ ID NO:2 or SEQ ID NO:14, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; preferably, the CMV enhancer is the sequence shown in SEQ ID NO:2 or SEQ ID NO:14, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; A cytomegalovirus (CMV) promoter comprising the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; preferably, the cytomegalovirus (CMV) promoter is the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; bGlob introns, wherein the bGlob introns comprise the sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; preferably, the bGlob introns are the sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95%, or 99% or more of the same identity; The coding sequence is a nucleotide sequence encoding a VEGF fusion protein or an anti-VEGF antibody; preferably, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein shown in SEQ ID NO:5, or a sequence having 85%, 90%, 95%, or 99% or more identity with it; preferably, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein shown in SEQ ID NO:5; wherein, The amino acid sequence of the C11 protein is shown in SEQ ID NO:6; and The human growth hormone (HGH or hGH) polyA sequence, wherein the human growth hormone (HGH or hGH) polyA sequence comprises either the sequence shown in SEQ ID NO:4, or a sequence having 85%, 90%, 95%, or 99% or more identity with it; preferably, the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO:4, or a sequence having 85%, 90%, 95%, or 99% or more identity with it; and The downstream AAV 3' inverted terminal repeat (R-ITR) sequence (3' end) includes the sequence shown in SEQ ID NO:9; Preferably, the pharmaceutical composition further comprises: b. about 0.001%, about 0.005% or about 0.01% of poloxamer 188, preferably about 0.01% of poloxamer 188; c. Approximately 2.67 mM potassium chloride; d. Approximately 1.47 mM potassium dihydrogen phosphate; e. Sodium chloride of about 100 mM, about 138 mM, about 150 mM, or about 180 mM, preferably about 150 mM sodium chloride; and f. Approximately 8.06 mM disodium hydrogen phosphate; The pH of the pharmaceutical composition is approximately 7.0 to 7.

5. The method according to claim 12 or 13, wherein, The subjects mentioned have the following characteristics: 1) Age 50 ≤ Age ≤ 80, preferably 65 ≤ Age ≤ 80; 2) The eye to be treated is diagnosed with choroidal neovascularization (CNV) secondary to neovascular age-related macular degeneration (nAMD) or wet AMD; and / or 3) The best corrected visual acuity (BCVA) of the eye to be treated is 20 / 800 to 20 / 40 on the Snellen visual acuity chart, more preferably 20 / 400 to 20 / 40 on the Snellen visual acuity chart, or the best corrected visual acuity (BCVA) of the eye to be treated is about 5-80 letters, preferably about 5-73 letters, preferably about 19-80 letters, preferably about 20-80 letters, more preferably about 50-74 letters when using the ETDRS visual acuity chart. According to any one of claims 1-14, wherein the subject's BCVA increased by at least about 3, about 3.1, about 3.5, about 4, about 4.5, about 5, about 10, or about 15 letters from baseline, preferably about 3 to 30 letters, preferably about 3 to 25 letters, preferably about 3 to 20 letters, preferably about 3.1, about 3.2, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 letters. The method according to claim 15, wherein, With a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After administration of the vg / ocular dose for more than 4 weeks, the subjects' mean BCVA increased by at least approximately 4.5 letters from baseline; and / or With a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After more than 52 weeks of administration of the vg / ocular dose, the subjects' mean BCVA increased by at least approximately 15 letters from baseline; and / or With a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After administration of the vg / ocular dose for 4, 8, 20, or 52 weeks or more, the mean central retinal thickness (CRT) of the subjects decreased by at least about 20 μm from baseline, preferably about 40-180 μm, preferably about 60-160 μm, preferably about 90-150 μm, more preferably about 100 μm, about 110 μm, about 120 μm, about 125 μm, about 130 μm, about 135 μm, about 140 μm, about 141 μm, about 142 μm, about 143 μm, about 144 μm, or about 145 μm; and / or With a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After administration of the vg / ocular dose for more than 4 weeks, the central macular retinal thickness (CRT) of the subjects decreased by an average of at least about 90 μm from baseline, preferably about 100 μm, about 120 μm, about 140 μm, about 141.83 μm, about 142 μm, about 143 μm, about 144 μm, or about 145 μm; and / or With a single dose of 1.0 × 10 9 vg / eye or 3.0×10 9 After administration of vg / eye dose for more than 8 weeks, the central macular retinal thickness (CRT) of the subjects decreased by an average of at least about 90 μm from baseline, preferably about 100 μm, about 120 μm, about 140 μm, about 141 μm, about 142 μm, about 143 μm, about 144 μm, or about 145 μm. The method according to any one of claims 1-16, wherein, The recombinant adeno-associated virus is contained in a packaging material, the packaging material including printed matter, the printed matter illustrating... The method according to any one of claims 1-16.

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