Use of recombinant adeno-associated virus expressing RS1 protein in preparation of Anti-x-linked juvenile retinoschisis drug

Abstract

Provided is the use of a recombinant adeno-associated virus expressing RS1 protein in the preparation of an anti-X-linked juvenile retinoschisis (XLRS) drug, belonging to the technical field of biomedicine. In order to fill the clinical research gap of recombinant adeno-associated viruses capable of efficient tissue-specific expression of RS1 protein for preventing and / or treating XLRS patients, the use of the recombinant adeno-associated virus expressing RS1 protein in the preparation of an anti-XLRS drug is provided. Clinical data indicates that the provided recombinant adeno-associated virus expressing RS1 protein can effectively improve the eyesight of XLRS patients, and can also significantly improve the retinal structure of XLRS patients, so that macular retinoschisis cavities can completely disappear. Moreover, safety data shows that there is no adverse events related to the drug itself, which indicates controllable safety. Thus, the drug is expected to become a new-generation clinical candidate drug for treating and preventing XLRS.

Description

Application of recombinant adeno-associated virus expressing RS1 protein in the preparation of drugs against X-linked juvenile retinoschisis Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of recombinant adeno-associated virus expressing RS1 protein in the preparation of drugs against X-linked juvenile retinoschisis. Background Technology

[0002] X-linked juvenile retinoschisis (XLRS) is a rare, X-linked, inherited blinding eye disease. Currently, clinical treatment for XLRS mainly focuses on observation and managing complications, and there is no effective treatment. The final outcome for patients is often vision loss.

[0003] The pathogenic gene for XLRS is the Retinoschisin 1 (RS1) gene, located at Xp22.1 p22.2. This gene consists of six exons, encoding 224 amino acids with over 16,000 base pairs. Based on the retinal pathological progression described in XLRS patients, RS1 plays a crucial role in the normal development and maintenance of retinal structure. This protein is essential for maintaining the integrity of functional synapses, making gene replacement a promising treatment for XLRS. Adeno-associated virus (AAV) vector-mediated gene therapy technology has also become increasingly mature. Therefore, according to the existing patent CN113322281A, a recombinant adeno-associated virus that efficiently expresses RS1 protein in tissues has been disclosed. Animal experiments have shown that it has a good restorative effect on the retinal tissue morphology of RS1-KO disease model mice of different ages. Although current animal experimental results are promising, further research is needed on the effects of randomized, open-label, multicenter studies on the treatment, safety, tolerability, and pharmacokinetic characteristics of this type of adeno-associated virus on XLRS in clinical trials. Summary of the Invention

[0004] To address the technical gap in clinical research on the use of recombinant adeno-associated virus expressing RS1 protein with high efficiency in preventing and / or treating X-linked juvenile retinoschisis (XLRS), this invention provides the application of recombinant adeno-associated virus expressing RS1 protein in the preparation of anti-XLRS drugs.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] In a first aspect, the present invention provides the use of recombinant adeno-associated virus expressing RS1 protein in the preparation of medicaments for the prevention and / or treatment of XLRS.

[0007] In the above applications, the drug is prepared by adding pharmaceutically acceptable excipients to a recombinant adeno-associated virus expressing RS1 protein as the active ingredient.

[0008] Further, the pharmaceutically acceptable excipient is excipient 1, comprising: 0–20 mM Tris buffer system, 0–2 mM MgCl2, 100–200 mM NaCl, 0–0.005% poloxamer 188 or excipient 2, comprising: 0–20 mM phosphate buffer system, 100–200 mM NaCl, 0–0.2 mg / mL KCl, 0–0.22 mg / mL polysorbate-20, 0–50 mg / mL mannitol or sucrose.

[0009] In the above applications, the drug is administered via intravitreal or subretinal injection. Preferably, the subretinal injection volume is 100 μL to 300 μL per eye.

[0010] In the above applications, the dosage of the active ingredient in the drug is 1×10⁻⁶. 10 vg / eye ~3×10 11 vg / eye. Preferably, the dosage of the active ingredient in the drug is 7.5 × 10⁻⁶. 10 vg / eye ~1.5×10 11 vg / eye.

[0011] In the above applications, the recombinant adeno-associated virus expressing the RS1 protein contains an RS1 protein expression cassette, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0012] SEQ ID NO.1: RS1 protein expression cassette

[0013] Furthermore, the recombinant adeno-associated virus is a vector, specifically a double-stranded DNA AAV (Self-complementary AAV, scAAV), selected from any of the following recombinant adeno-associated virus vector serotypes: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10. Preferably, AAV2, AAV5, AAV8, or AAV9.

[0014] Most preferably, the nucleotide sequence of the recombinant adeno-associated virus expressing the RS1 protein is shown in SEQ ID NO.2.

[0015] SEQ ID NO.2:

[0016] In the above applications, the recombinant adeno-associated virus expressing the RS1 protein is prepared by co-transfecting host cells with a plasmid containing the adenovirus helper gene, Rep and Cap genes and a target gene plasmid containing the RS1 protein expression cassette using a transfection reagent.

[0017] Furthermore, the host cells are retinal cells and / or optic nerve cells.

[0018] Preferably, the host cell is selected from at least one of HEK293 cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as biganglionic cells), nerve cells, rod cells, cone cells, illuminated bipolar cells, deilocled bipolar cells, horizontal cells, ganglion cells, amacrine cells, or optic nerve cells.

[0019] In a second aspect, the present invention provides the use of a combination drug of recombinant adeno-associated virus expressing RS1 protein in the preparation of a drug for the prevention and / or treatment of XLRS.

[0020] In the above applications, the combined drugs are recombinant adeno-associated virus expressing RS1 protein and other anti-XLRS drugs administered separately or simultaneously.

[0021] In the above applications, the drug is administered via intravitreal or subretinal injection. Preferably, the subretinal injection volume is 100 μL to 300 μL per eye.

[0022] In the above applications, the effective dose of the recombinant adeno-associated virus expressing the RS1 protein in the drug is 1×10⁻⁶. 10 vg / eye ~3×10 11 vg / eye. Preferably, the dosage of the active ingredient in the drug is 7.5 × 10⁻⁶. 10 vg / eye ~1.5×10 11 vg / eye.

[0023] In the above applications, the recombinant adeno-associated virus expressing the RS1 protein contains an RS1 protein expression cassette, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0024] Furthermore, the recombinant adeno-associated virus is a vector, specifically a double-stranded DNA AAV (Self-complementary AAV, scAAV), selected from any of the following recombinant adeno-associated virus vector serotypes: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10. Preferably, AAV2, AAV5, AAV8, or AAV9.

[0025] Most preferably, the nucleotide sequence of the recombinant adeno-associated virus expressing the RS1 protein is shown in SEQ ID NO.2.

[0026] In the above applications, the recombinant adeno-associated virus expressing the RS1 protein is prepared by co-transfecting host cells with a plasmid containing the adenovirus helper gene, Rep and Cap genes and a target gene plasmid containing the RS1 protein expression cassette using a transfection reagent.

[0027] Furthermore, the host cells are retinal cells and / or optic nerve cells.

[0028] Preferably, the host cell is selected from at least one of HEK293 cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as biganglionic cells), nerve cells, rod cells, cone cells, illuminated bipolar cells, deilocled bipolar cells, horizontal cells, ganglion cells, amacrine cells, or optic nerve cells.

[0029] Beneficial Effects: This invention investigated the treatment, safety, and tolerability of recombinant adeno-associated virus (AAV) containing the RS1 protein expression cassette (nucleotide sequence as shown in SEQ ID NO. 1) for XLRS through clinical trials. Clinical data showed that subretinal injection into XLRS patients, with a preferred injection volume of 100 μL / eye, effectively improved visual acuity and significantly improved retinal structure, completely eliminating retinal schisis in the macular region. Furthermore, safety data showed no adverse events related to the drug itself, indicating manageable safety. Therefore, the recombinant AAV expressing the RS1 protein designed in this invention shows promise as a next-generation clinical candidate drug for the treatment and prevention of XLRS. Attached Figure Description

[0030] Figure 1 is a line graph of the mean change in best-corrected visual acuity (BCVA) from baseline in Experiment Example 1, showing the time-time variation.

[0031] Figure 2 shows the fundus image of the treated eye in Experiment Example 2 using optical coherence tomography (OCT).

[0032] Figure 3 shows the OCT fundus image of the non-treatment eye in Experiment Example 2;

[0033] Figure 4 is a line graph of the mean change in central subfield thickness (CST) from baseline in Experiment Example 2, over time.

[0034] Figure 5 shows the OCT fundus image of the treated eye of the subject in Experiment Example 2; dashed arrow: external membrane; solid arrow: high reflectivity layer outside the external membrane, including the ellipsoidal zone and the chimeric zone. Detailed Implementation

[0035] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0036] Two clinical trials of gene therapy for XLRS have been conducted globally. One was a Phase I / II clinical trial (NCT02416622) initiated by AGTC in the United States, investigating rAAV2tYF-CB-hRS1 for the treatment of XLRS. rAAV2tYF-CB-hRS1 uses AAV2tYF as a vector to express the hRS1 protein and is administered via intravitreal injection. The trial population in Phase I was male patients with XLRS aged ≥18 years. The other was a Phase I / II clinical trial (NCT02317887) initiated by the National Eye Institute (NEI) in the United States, investigating AAV8-scRS / IRBPhRS for the treatment of XLRS. AAV8-scRS / IRBPhRS uses AAV8 as a vector and is also administered via intravitreal injection. The trial population was also male patients with XLRS aged ≥18 years. Neither of these studies observed definitive efficacy, and the clinical trials have been terminated. Possible reasons include inappropriate choice of administration route, drug carrier design, treatment population, and / or inappropriate selection of the treatment window. The limited ability of AAV in the vitreous cavity to cross the internal limiting membrane and infect retinal cells results in low concentrations of the target therapeutic protein in the retina, hindering effective repair or prevention of retinal lesions. Therefore, selecting an appropriate administration route can improve the efficiency of AAV vector infection of retinal cells, thereby enhancing therapeutic efficacy. Through research, the applicant has discovered that subretinal injection of the recombinant adeno-associated virus containing the RS1 protein expression cassette (nucleotide sequence as shown in SEQ ID NO. 1) of this invention, combined with a suitable drug carrier, can significantly improve the efficiency of AAV vector infection of retinal cells, thus enhancing therapeutic efficacy.

[0037] Furthermore, studies have shown a positive correlation between visual acuity and the integrity of the outer retinal structures, including the length of the photoreceptor outer segment, the outer membrane, the ellipsoidal band, and the chimeric band. As the disease progresses, most patients experience outer retinal loss before the age of 20, significantly limiting the benefits of treatment for these patients. Therefore, patients in the early stages of the disease (or younger patients) will receive better treatment outcomes after receiving this type of gene therapy.

[0038] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0039] Example 1: Preparation of recombinant adeno-associated virus expressing RS1 protein

[0040] The drug formulation used in the examples is scAAV8-hRS1, whose active ingredient is a recombinant adeno-associated virus expressing the RS1 protein. The expression vector was constructed according to the scAAV8.RK.cohRS1.bGH virus packaging plasmid in Example 1 of patent CN113322281A. The full sequence of the plasmid is shown in SEQ ID NO.4 of patent CN113322281A. The specific preparation method of scAAV8-hRS1 is as follows:

[0041] The preparation of scAAV8-hRS1 was based on transient co-transfection of suspension HEK293.2sus cells with two plasmids, using the pAAVsc.RK.cohRS1.bGH plasmid and a plasmid containing adenovirus helper genes and Rep and Cap genes. Suspended human embryonic kidney epithelial cells (HEK293.2sus cells) were transiently transfected using transfection reagents. The viral fluid was harvested 96 hours after transfection and then subjected to deep filtration, recombinant nuclease treatment, salt treatment, ultrafiltration concentration, and POROS treatment. TM CaptureSelect TM AAVX affinity chromatography purification and QA anion exchange chromatography purification was performed, and the purified AAV virus (nucleotide sequence as shown in SEQ ID NO.2) was collected. The virus was then desalted by ultrafiltration using a 100KD hollow fiber column, dialyzed, and the medium was changed. Finally, the AAV virus was placed in 20mM Tris (pH 8.0), 1mM MgCl2, 200mM NaCl, and 0.001% poloxamer 188 preparation to obtain the final product.

[0042] The trial was designed to evaluate the safety, tolerability, and preliminary efficacy of scAAV8-hRS1 in pediatric patients with XLRS. The trial dose was 7.5 × 10⁻⁶. 10 The dose is administered via a single subretinal injection, with an injection volume of 100 μL per eye.

[0043] Key efficacy measures include: best-corrected visual acuity (BCVA), retinal structures including: central subfield thickness (CST), and optical coherence tomography (OCT) fundus images.

[0044] Safety checks include: vital signs, physical examination, 12-lead electrocardiogram, laboratory tests (complete blood count, blood biochemistry, coagulation function and urinalysis), ophthalmological examination (slit-lamp examination, intraocular pressure, fundus examination, fundus photography, fundus autofluorescence, optical coherence tomography) and immunogenicity.

[0045] BCVA test: The visual acuity chart is used to perform the test at a distance of 4 meters.

[0046] CST and OCT fundus images: The examination was performed using an ophthalmic optical coherence tomography scanner manufactured by Vision Micro Imaging (Henan) Technology Co., Ltd., with product model VG200D.

[0047] Subretinal injection: The core vitrectomy was performed using a standard three-incision transparnial vitrectomy. After creating the postvitrectomy detachment and removing the peripheral vitreous, the vitrectomy machine was adjusted to silicone oil injection mode, and the maximum injection pressure was set to 15-20 psi. One or two injection points were selected on the medial side of the infratemporal and / or superior temporal vascular arches, and a 41G retinal injection needle was used to perform subretinal injection. The total injection volume was 100 μL.

[0048] 7.5×10 10 Efficacy data from 6 subjects (subjects 1–6, aged 6–10 years) in the vg / ocular dose group 180 days after administration showed that after treatment with scAAV8-hRS1, the improvement in BCVA and CST in the treated eye was significantly better than that in the untreated eye. The retinal schisis cavity in the treated eye completely disappeared. scAAV8-hRS1 can improve visual acuity and retinal structure in patients with XLRS. Safety data showed that no adverse events related to scAAV8-hRS1 occurred, and adverse events related to subretinal injection did not affect patients' visual acuity. The safety of subretinal injection of scAAV8-hRS1 is manageable.

[0049] Experiment Example 1: Changes in Best Corrected Visual Acuity (BCVA)

[0050] As shown in Figure 1, at week 13 post-treatment, the BCVA in the treated eye improved by 8.5 letters from baseline (Mean, N=6), while the BCVA in the non-treated eye improved by 2.8 letters (Mean, N=6). At week 26 post-treatment, the BCVA in the treated eye improved further, with an improvement of 9.3 letters (Mean, N=6) from baseline. All subjects showed an improvement of 5 letters or more, with 3 showing an improvement of 10 letters or more. In contrast, the BCVA in the non-treated eye improved by only 1.5 letters (Mean, N=6), with no subjects showing an improvement of 5 letters or more. Overall, scAAV8-hRS1 improves visual acuity in patients with XLRS.

[0051] Experiment Example 2: Changes in Retinal Structure

[0052] As shown in Figures 2-4, at baseline, the macular retinal schisis was significant in the treated eyes of all subjects. By week 4 post-treatment, the macular retinal schisis in the treated eyes either completely disappeared (subjects 4 and 6), substantially disappeared (subjects 1, 2, and 3), or significantly decreased (subject 5). By week 8 post-treatment, the macular retinal structure continued to improve, with the schisis either completely disappearing or remaining completely eliminated. The mean CST (Mean, N=6) in the treated eyes decreased by an average of 481.5 μm from baseline. By week 26 post-treatment, the complete disappearance of the macular retinal schisis remained in all subjects, and the mean CST (Mean, N=6) in the treated eyes decreased by an average of 479.3 μm from baseline. At week 26, there was no significant change in the retinal structure of the untreated eyes compared to baseline, and the mean CST (Mean, N=6) in the untreated eyes decreased by an average of 12.7 μm from baseline. Overall, scAAV8-hRS1 significantly improved the retinal structure of XLRS patients, resulting in the complete disappearance of the macular retinal schisis.

[0053] As shown in Figure 5, at baseline, the continuity of the outer retinal structure in the subjects was poor, with unclear structures of the outer membrane, ellipsoidal band, and chimeric band, and some bands missing. By 26 weeks after drug administration, the outer retinal structure of most subjects (5 / 6 cases, 83.3%) had improved, the outer membrane was nearly intact, and the continuity of the ellipsoidal band and chimeric band showed varying degrees of recovery.

[0054] Test Example 3 Safety Test

[0055] 7.5×10 10A total of 24 adverse events occurred in the 6 subjects in the vg / ocular dose group. No adverse events related to scAAV8-hRS1 occurred. Thirteen adverse events related to subretinal injection occurred, including decreased intraocular pressure (4 events), conjunctival hyperemia (4 events), subconjunctival hemorrhage (3 events), ocular pain (1 event), and retinal atrophy (1 event). All of these events were mild to moderate in severity and did not affect the patients' visual acuity.

[0056] Table 1 List of adverse events in subject cases

Claims

1. Application of recombinant adeno-associated virus expressing RS1 protein in the preparation of drugs for the prevention and / or treatment of XLRS.

2. The application according to claim 1, characterized in that: The drug is prepared by adding pharmaceutically acceptable excipients to a recombinant adeno-associated virus expressing RS1 protein as the active ingredient.

3. The use of combination drugs of recombinant adeno-associated virus expressing RS1 protein in the preparation of drugs for the prevention and / or treatment of XLRS.

4. The application according to any one of claims 1 to 3, characterized in that: The drug is administered via intravitreal or subretinal injection; preferably, the subretinal injection volume is 100 μL to 300 μL per eye.

5. The application according to any one of claims 1 to 4, characterized in that: The dosage of the active ingredient in the drug is 1×10. 10 vg / eye ~3×10 11 vg / eye.

6. The application according to any one of claims 1 to 5, characterized in that: The recombinant adeno-associated virus expressing the RS1 protein contains an RS1 protein expression cassette, the nucleotide sequence of which is shown in SEQ ID NO.

1.

7. The application according to any one of claims 1 to 6, characterized in that: The recombinant adeno-associated virus is a vector, a double-stranded DNA adeno-associated virus, selected from any of the following recombinant adeno-associated virus vector serotypes: recombinant adeno-associated virus vector serotypes include AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10.

8. The application according to any one of claims 1 to 7, characterized in that: The nucleotide sequence of the recombinant adeno-associated virus expressing the RS1 protein is shown in SEQ ID NO.

2.

9. The application according to any one of claims 1 to 8, characterized in that: The recombinant adeno-associated virus expressing RS1 protein was prepared by co-transfecting host cells with a plasmid containing the adenovirus helper gene, Rep and Cap genes and a target gene plasmid containing the RS1 protein expression cassette using a transfection reagent.

10. The application according to claim 9, characterized in that: The host cell is selected from at least one of HEK293 cells, photoreceptor cells, other visual cells, nerve cells, rod cells, cone cells, illuminated bipolar cells, deilated bipolar cells, horizontal cells, ganglion cells, amacrine cells, or optic nerve cells.

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