Gene editing induced pluripotent stem cell-derived exosomes and their use in treating age-related macular degeneration

Abstract

The application discloses a kind of gene editing induced pluripotent stem cell-derived exosomes and its application in treating age-related macular degeneration.The exosome is isolated from the gene edited induced pluripotent stem cell;The gene edited induced pluripotent stem cell is obtained by reprogramming from the allogeneic source adult somatic cell activated by FOXO3 gene and knocked out by HLA-A / B / C gene.The exosome of the application can significantly inhibit retinal oxidative stress and inflammatory response, promote photoreceptor cell survival, reduce CNV leakage and retinal hemorrhage, and reverse GA progression in natural aging AMD model with GA and CNV changes.The exosome realizes allogeneic administration without rejection, repeatable treatment, and has the ability to significantly prolong the survival period of model animals.The exosome has good application prospect in treating age-related macular degeneration, especially AMD with GA and CNV pathological changes.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedicine and regenerative medicine, specifically relating to an exosome derived from gene-edited induced pluripotent stem cells and its application in the preparation of drugs for treating age-related macular degeneration (AMD). It is particularly suitable for aging-driven dry AMD and its complications (such as geographic atrophy, retinal hemorrhage, and photoreceptor degeneration), as well as mixed AMD with both GA and CNV pathological changes, and can be used in the manufacture of AMD biopharmaceuticals. Background Technology

[0002] Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in people over 50 years of age. Patients often develop AMD in both eyes sequentially or simultaneously. The progressive vision loss severely impacts quality of life and is the leading cause of blindness among the elderly in developed countries. The disease is caused by degenerative changes in the macular region of the retina and is classified into two types: dry (atrophic) and wet (exudative). It is associated with retinal light damage, metabolic disorders, and factors such as race (more common in Caucasians), gender, and family history.

[0003] Dry AMD is characterized by drusen deposition and geographic atrophy (GA) in the macular region. GA is a late-stage manifestation of dry AMD, characterized by progressive and irreversible loss of retinal pigment epithelium (RPE) and photoreceptor cells. Wet AMD is characterized by hemorrhage and exudation caused by choroidal neovascularization (CNV). Wet AMD is highly related to and develops from dry AMD; clinically, some AMD patients exhibit both GA and CNV pathological changes. Typical symptoms include metamorphopsia, fixed central scotoma, and decreased visual acuity, and can be diagnosed through OCT and OCTA. Currently, various therapies are available for wet AMD, including laser therapy and anti-vascular endothelial growth factor (VEGF) drugs, but effective interventions are lacking for dry AMD, which accounts for more than 85% of cases.

[0004] It is worth noting that recent clinical studies have found that in some AMD patients with both GA and CNV pathological changes, although CNV was controlled to some extent after receiving anti-VEGF treatment, the progression of GA actually accelerated. This suggests that while anti-VEGF treatment inhibits neovascularization, it may exacerbate RPE and photoreceptor degeneration. Therefore, finding novel treatment strategies that can simultaneously intervene in GA and CNV has become an urgent problem to be solved in the field of ophthalmology.

[0005] Induced pluripotent stem cells (iPSCs) were first reported by scientists Shinya Yamanaka et al. in 2006. They discovered that by using a retroviral system to deliver Oct3 / 4, Sox2, c-Myc, and Klf4 into somatic fibroblasts, the fibroblasts could be reprogrammed into pluripotent stem cells, hence the name induced pluripotent stem cells (iPSCs). iPSCs possess regenerative properties and can differentiate into any cell lineage in the body. This characteristic is similar to embryonic stem cells (ESCs); however, the generation of iPSCs does not face the ethical controversies associated with the source of ESCs. Cell sources used for iPSC reprogramming include somatic fibroblasts, peripheral blood mononuclear cells, and even mesenchymal stem cells, such as adipose stem cells. These are easier to harvest than embryos and do not involve ethical controversies. Therefore, iPSCs have broad application prospects in regenerative medicine. Currently, iPSCs are publicly available for treating AMD. The core principle is to reprogram the patient's own cells (such as skin cells) into iPSCs, which are then differentiated into retinal pigment epithelial cells (RPEs). These RPEs are then transplanted into the macula to replace diseased cells, thereby improving or delaying vision decline. However, while this method of treating AMD by transplanting autologous induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPEs) avoids immune rejection due to the use of the patient's own cells, the entire process is done on a one-person-one-dosage basis, starting from scratch. This results in long preparation cycles (3-6 months), high production costs, and difficulties in standardization and large-scale production, failing to meet the industrialization goals of standardization, mass production, low cost, and stable quality. Furthermore, if allogeneic iPSCs or their derivatives are used for transplantation, the issue of immune rejection due to HLA incompatibility arises, limiting the clinical application of universal allogeneic cell products.

[0006] Recent studies have shown that exosomes can regulate the retinal microenvironment by delivering specific miRNAs, proteins, and metabolic factors, particularly mesenchymal stem cell exosomes. Current technologies have reported the use of adipose-derived mesenchymal stem cells or umbilical cord blood-derived exosomes to treat eye diseases. However, naturally derived exosomes suffer from drawbacks such as high heterogeneity, low yield, lack of targeting, high immunogenicity, and functional instability. Furthermore, traditional exosome-derived cells themselves exhibit aging phenotypes, and their contained SASP factors (such as IL-6 and TNF-α) may exacerbate retinal inflammation. Allogeneic exosomes may induce local inflammatory responses due to their immunogenicity, limiting repeated administration. Recently, an increasing number of studies have shown that iPSC exosomes have therapeutic effects in certain diseases, but there are currently no reports on iPSC exosome treatment for AMD, especially mixed AMD with both GA and CNV alterations. Moreover, current technologies lack a standardized, GMP-grade exosome production platform based on gene-edited iPS cells. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an exosome derived from gene-edited induced pluripotent stem cells.

[0008] A second objective of this invention is to provide the use of exosomes derived from the gene-edited induced pluripotent stem cells in the preparation of medicaments for the treatment of age-related macular degeneration.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: An exosome derived from gene-edited induced pluripotent stem cells (iPSCs) is obtained by isolating gene-edited iPSCs. The gene-edited iPSCs are obtained by reprogramming allogeneic adult somatic cells that have undergone FOXO3 gene activation and HLA-A / B / C gene knockout. Specifically, adult somatic cells are first obtained, then FOXO3 gene activation and HLA-A / B / C gene knockout are performed to obtain gene-edited adult somatic cells. These cells are then reprogrammed to obtain gene-edited iPSCs, and finally, exosomes, i.e., iPSC-Exos, are isolated from them.

[0010] This invention overcomes the limitations of existing iPSC therapies and mesenchymal stem cell donor sources by using allogeneic adult somatic cells as parental iPSC cells. By knocking out the three genes HLA-A, HLA-B, and HLA-C (HLA-A / B / C) in donor adult somatic cells, it achieves a non-immune rejection response. Simultaneously, by activating the expression of the forkhead box O3 (FOXO3) gene, it downregulates SASP factors (such as IL-6 and TNF-α) and upregulates antioxidant factors, thereby inhibiting retinal aging at its source and treating age-driven dry AMD. Reprogramming yields universal iPS cells with low immunogenicity. Furthermore, based on these gene-edited iPS cells, anti-aging gene-edited iPS exosomes are isolated, with a yield of 1×10⁻⁶ cells. 9 Each iPS cell can produce ≥5×10 13 Exosomes. Through standardized iPS cell expansion and exosome production processes, large-scale, industrialized production of exosomes can be achieved. This process offers advantages such as short preparation cycles, consistent and stable product quality, low production costs, and strong clinical accessibility, making it suitable for the manufacture of AMD biopharmaceuticals.

[0011] Furthermore, the allogeneic adult somatic cells are derived from different individuals in the human body, i.e., human induced pluripotent stem cells are obtained.

[0012] Preferably, the allogeneic adult somatic cells are CD34 cells derived from peripheral blood. + Cells, namely CD34 + Hematopoietic stem / progenitor cells.

[0013] Furthermore, the exosomes have a particle size of 30–150 nm and a concentration ≥1×10⁻⁶. 10 Particles / mL, CD9 + / CD63 + / CD81 + Positive rate ≥90%, endotoxin <0.5 EU / mL.

[0014] Furthermore, the aforementioned FOXO3 Gene activation was achieved by targeting the FOXO3 gene promoter using the dCas9-VPR system, with the sgRNA sequence 5'-GGTGAGTGTGAAGAAGCTGG-3'. The dCas9-VPR system is a core tool of CRISPR activation technology, consisting of an inactivated Cas9 (dCas9) fused with a triple-domain transcriptional activation domain (VPR) (VP64-p65-Rta). It can specifically activate endogenous gene transcription without altering the DNA sequence, making it a key technology for gene function research and cell engineering. The dCas9-VPR system, with its single-molecule structure, high activation efficiency, and low delivery difficulty, is the preferred solution for CRISPR technology, achieving a more than 100-fold increase in FOXO3 expression (up to approximately 1050-fold).

[0015] Furthermore, the HLA-A / B / C gene knockout is performed using the Cas9 system; the sgRNA of the HLA-A gene is 5'-GCTGGTGGTGTCTACACCGC-3', the sgRNA of the HLA-B gene is 5'-GGCGTGTACTACCAGCACCC-3', and the sgRNA of the HLA-C gene is 5'-CCGCGGAGTATTGGCGCTGG-3'.

[0016] Furthermore, the reprogramming is performed using non-integrating Sendai virus to deliver Oct4 / Sox2 / Klf4 / c-Myc. Non-integrating Sendai virus delivery is currently one of the most mainstream and safest methods for iPS cell reprogramming, carrying reprogramming factors such as Oct4, Sox2, Klf4, and c-Myc, and efficiently infecting human skin fibroblasts, peripheral blood mononuclear cells, and CD34 cells. + Cells, etc., reprogram adult somatic cells into iPS cells.

[0017] As a preferred embodiment, the method for preparing the exosomes includes the following steps: (1) Isolation of CD34 from peripheral blood of healthy donors + Hematopoietic stem / progenitor cells, purity ≥90% (preferably ≥99%), viability ≥95% (preferably ≥99%); (2) The dCas9-VPR vector and the HLA-A / B / C knockout CRISPR-Cas9 vector were co-introduced into the CD34 by electroporation. + Cells, wherein: the dCas9-VPR sgRNA sequence targeting the FOXO3 gene promoter region is shown in SEQ ID No. 1: 5'-GGTGAGTGTGAAGAAGCTGG-3'; the HLA-A gene knockout sgRNA sequence is shown in SEQ ID No. 2: 5'-GCTGGTGGTGTCTACACCGC-3'; the HLA-B gene knockout sgRNA sequence is shown in SEQ ID No. 3: 5'-GGCGTGTACTACCAGCACCC-3'; and the HLA-C gene knockout sgRNA sequence is shown in SEQ ID No. 4: 5'-CCGCGGAGTATTGGCGCTGG-3'; (3) Positive cells with FOXO3 expression upregulated ≥100-fold and HLA-A / B / C protein knockout efficiency ≥90% (preferably ≥99%) were obtained through screening; (4) The positive cells were reprogrammed into iPS cells using non-integrating Sendai virus to obtain Oct4 cells. + / Sox2 + / Nanog + Stable iPS cell lines with a positivity rate ≥95%; (5) Expand the iPS cells in mTeSR1 serum-free, feeder-free medium, and isolate and collect the exosomes secreted by them; (6) The obtained exosomes are purified to a particle size of 30–150 nm (preferably 110–135 nm) and a concentration ≥1×10⁻⁶. 10 Particles / mL, and CD9 + / CD63 + / CD81 + Positive rate ≥90%.

[0018] Furthermore, the method for isolating exosomes involves first removing cells and debris from expanded and cultured iPS cells by differential centrifugation, then enriching exosomes by ultracentrifugation (100,000×g, 7 h), and finally purifying them by ultrafiltration concentration and size exclusion chromatography.

[0019] This invention demonstrates that the exosomes described above can significantly inhibit retinal oxidative stress and inflammatory responses, promote photoreceptor cell survival, significantly inhibit choroidal neovascularization, reverse the progression of geographic atrophy, and restore retinal electrophysiological responses in a naturally aging AMD model exhibiting both GA and CNV alterations. This achieves allogeneic administration without rejection, allows for repeated treatment, and significantly prolongs the survival of model animals. Compared to anti-VEGF therapy, the exosomes of this invention not only effectively inhibit CNV but also reverse GA progression, while anti-VEGF therapy tends to negatively impact photoreceptor survival and antioxidant capacity, exacerbating GA while inhibiting CNV.

[0020] Therefore, the present invention also provides the use of exosomes derived from gene-edited induced pluripotent stem cells as described above in the preparation of drugs for treating age-related macular degeneration.

[0021] Furthermore, the age-related macular degeneration is dry age-related macular degeneration, which includes mixed AMD that presents with CNV complications during disease progression, i.e., AMD with both GA and CNV pathological changes.

[0022] Specifically, the drug exerts its therapeutic effect by inhibiting retinal oxidative stress and inflammatory response in the subject; promoting photoreceptor cell survival; reducing choroidal neovascularization and retinal hemorrhage; and reversing geographic atrophy.

[0023] The present invention also provides a therapeutic agent for age-related macular degeneration, the agent containing exosomes derived from gene-edited induced pluripotent stem cells as described above.

[0024] Furthermore, the dosage forms of the drug include, but are not limited to, injections, eye drops, or nasal drops. All dosage forms of the drug are stored and transported at 2–6°C. The injections include, but are not limited to, intravenous injections or intravitreal injections. Intravenous injections are administered at a dose of 2.0–3.0 × 10⁻⁶. 10 Particles / mL (preferably 2.5 × 10⁻⁶) 10 Particles / mL); eye drops: 4–6 × 10⁻⁶ 11 Particles / mL (preferably 5×10) 11 (particles / mL); the nasal drops have a concentration of 4–6 × 10⁻⁶. 11 Particles / mL (preferably 5×10) 11 (particles / mL); the intravitreal injection solution is 2.0–3.0 × 10⁻⁶ particles / mL. 12 Particles / mL (preferably 2.5 × 10⁻⁶) 12 Particles / mL).

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention provides exosomes derived from gene-edited induced pluripotent stem cells. By knocking out HLA-A / B / C genes in allogeneic cells, this invention achieves a non-immune rejection response. Simultaneously, by activating FOXO3 gene expression, it downregulates SASP factors (such as IL-6 and TNF-α) and upregulates antioxidant factors, thereby inhibiting retinal aging at its source and treating age-driven dry AMD. Reprogramming yields universal iPS cells with low immunogenicity. Based on these gene-edited iPS cells, anti-aging gene-edited and low-immunogenic iPS exosomes are then isolated. (1×10⁻⁶) 9 Each iPS cell can produce ≥5×10 13 iPS exosomes. This invention achieves large-scale, industrialized production of exosomes through standardized iPS cell expansion and exosome production processes, offering advantages such as short preparation cycles, consistent and stable product quality, low production costs, and strong clinical accessibility. Research in this invention shows that the exosomes significantly inhibit retinal oxidative stress and inflammatory responses, promote photoreceptor cell survival, significantly inhibit choroidal neovascularization and retinal hemorrhage, reverse the progression of geographic atrophy, and restore retinal electrophysiological responses in a naturally aged AMD model exhibiting both GA and CNV alterations. It achieves allogeneic administration without rejection, allows for repeated treatment, and significantly prolongs the survival time of model animals. The exosomes derived from gene-edited induced pluripotent stem cells of this invention show promising application prospects in the treatment of age-related macular degeneration, especially AMD with both GA and CNV pathological changes.

[0026] This invention is the first to obtain anti-aging gene-edited and low-immunogenic iPS exosomes, achieving dual structural and functional repair of AMD with both GA and CNV alterations: not only does the CNV area shrink by approximately 27-30% compared to baseline, but it also reverses the progression of map-like atrophy (GA area shrinks by 2.1%), while the GA area in the anti-VEGF control group actually increases by 15.3%. This invention overcomes the immune rejection bottleneck of allogeneic exosome preparations: due to HLA knockout, there is no T cell activation (CFSE proliferation assay <0.5%), allowing for repeated administration. The exosomes of this invention can significantly prolong lifespan: survival time is increased from 20.2 months to 32.9 months, with a lifespan extension rate of approximately 63%, providing clinical translational evidence for anti-aging therapy. The exosome source of this invention is stable and scalable: based on the GMP-level expansion system of iPS cells, batch-to-batch consistency of exosomes is achieved (CV <12%), meeting clinical drug needs. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0028] Animal experiment ethics: All procedures were approved by the Ethics Committee of Guangdong Provincial Engineering Technology Research (Development) Center for Ophthalmic Preparations / Guangdong Hongying Technology Co., Ltd. (Approval No.: IRB-iPS-2024-017), and followed the ARRIVE guidelines and the 3R principle of "Replacement, Reduction, Refinement" to avoid unnecessary use of animals and other resources; Statistical analysis: Data are expressed as mean ± SD and analyzed using two-tailed Student's t-test or Log-rank test (*p < 0.05, **p < 0.01, ***p < 0.001).

[0029] Example 1: Preparation method of gene-edited iPS-derived exosomes I. Methods 1. Donor screening: Peripheral blood from healthy individuals with a BMI of 20-24 and an age of 18-35 years was used, and the individuals were screened for infectious diseases (HBV / HCV / HIV / syphilis negative).

[0030] 2. Stem cell collection: Peripheral blood mononuclear cell apheresis was performed using a Spectra Optia blood cell separator, with a processed circulating blood volume of 8–12 L (mixed peripheral blood from multiple donors), followed by Ficoll-Paque Plus density gradient centrifugation + CD34. + Magnetic bead sorting yields CD34 with a purity ≥99% and an activity ≥99%. + cell.

[0031] 3. Gene editing: (1) The FOXO3 gene (Gene ID: 2309) promoter (sgRNA sequence: 5'-GGTGAGTGTGAAGAAGCTGG-3') was targeted using the dCas9-VPR system; the activation efficiency was verified by double detection of qRT-PCR and Western Blot. The qRT-PCR was performed using the SYBR Green method, and the relative expression level was calculated by ΔΔCt; the Western Blot was quantified using ImageJ grayscale values.

[0032] (2) The HLA-A gene (Gene ID: 3105) (sgRNA: 5'-GCTGGTGGTGTCTACACCGC-3'), HLA-B gene (Gene ID: 3106) (sgRNA: 5'-GGCGTGTACTACCAGCACCC-3'), and HLA-C gene (Gene ID: 3107) (sgRNA: 5'-CCGCGGAGTATTGGCGCTGG-3') were knocked out simultaneously using the Cas9 system. The knockout efficiency of HLA-A / B / C genes was verified by flow cytometry, NGS sequencing, TIDE analysis, and dPCR digital quantification. NGS sequencing: Illumina MiSeq platform, coverage ≥50×; TIDE analysis: Tracking of Indels by Decomposition tool, version v2.0; dPCR digital quantification: Bio-Rad QX200 system, TaqMan probe method was used to verify the knockout efficiency of HLA-A / B / C.

[0033] (3) After Neon electroporation (1600 V, 10 ms × 3), puromycin was used to screen for double-modified positive clones. qRT-PCR, dPCR digital quantification, and CD34 were then used to identify the positive clones. + Flow cytometry was used for positive clone verification; qRT-PCR: ΔCt method, internal reference gene GAPDH; dPCR digital quantification: Bio-Rad QX200 system, TaqMan probe method; CD34 + Flow cytometry: PE-labeled anti-CD34 antibody, ≥90% is considered qualified.

[0034] The optimal monoclonal clones were selected for validation of cell stemness and function after gene editing, as well as detection of off-target effects. Cell stemness and function validation employed karyotype analysis and hematopoietic colony-forming unit (CFU-GM / BFU-E) assays. Karyotype analysis: Giemsa staining, ≥20 metaphase cell counts; CFU-GM / BFU-E assay: hematopoietic stem / progenitor cell colony-forming unit method. Off-target effect detection utilized dual detection with whole-genome sequencing (WGS) and GUIDE-seq. WGS: Illumina NovaSeq 6000 platform; GUIDE-seq: double-stranded oligonucleotide marker integration detection, v3.0 version. Acceptance criteria: GUIDE-seq: no high-frequency off-target effects (<1×, p>0.9); WGS: low-frequency mutation rate (<0.1%). This met the requirements for genotoxicity assessment of gene-edited products in the "Technical Guidelines for Non-Clinical Research of Gene-Modified Cell Therapy Products (Trial Implementation)," focusing on off-target editing and insertional mutation risks, and identifying / characterizing genomic integration sites.

[0035] 4. iPS reprogramming and amplification: (1) Oct4 / Sox2 / Klf4 / c-Myc was delivered using a non-integrated Sendai virus (MOI=5); (2) Amplification in mTeSR1 serum-free, feeder-free medium (containing bFGF 100 ng / mL and TGF-β1 2 ng / mL). (3) After five generations of amplification, pluripotency was verified using a teratoma formation assay: 5 × 10⁻⁶ cells were injected subcutaneously. 6 iPS cells were induced into NOD / SCID mice and dissected after 8 weeks; telomere length: qPCR method.

[0036] (4) Risk assessment of replicating virus (RCV): The SeV nucleocapsid gene was detected by qRT-PCR in the supernatant of cells at passages 3, 5 and 10. The detection limit was <1 copy / μL and the results were all negative, indicating no risk of RCV.

[0037] 5. Exosome isolation and quality control: (1) Collect iPS cell supernatant (5th generation, cultured for 72 h), remove cells and debris by differential centrifugation, then enrich exosomes by ultracentrifugation (100,000×g, 7 h), and then purify by ultrafiltration concentration and size exclusion chromatography (SEC column, qEV original system). The purity meets the MISEV2018 standard. (2) The proliferation response of allogeneic T cells induced by HLA knockout exosomes and unedited iPS exosomes was detected by CFSE flow cytometry. PBMCs were isolated from three healthy donors (HLA-A / B / C heterozygotes). Each donor sample was used in technical replicates, with a total of n=20 samples per group. Exosomes and PBMCs were co-cultured for 72 hours, and the proliferation rate was detected by flow cytometry after CFSE labeling. The proliferation rate was calculated as CD3+. + / CFSE low-population cell percentage. Simultaneously, complement activation markers (C3a, C5a) and cytokine release assays (IL-6, TNF-α, IFN-γ) were performed to comprehensively assess immunogenicity and immunotoxicity.

[0038] Inter-batch consistency assay of exosomes (iPS) including particle size, exosome concentration, and the marker CD9. + / CD63 + / CD81 + Positive rate, endotoxin.

[0039] II. Results (1) The results of FOXO3 activation are shown in Table 1. After double detection by qRT-PCR and Western Blot, the expression level of FOXO3 mRNA and the abundance of FOXO3 protein were significantly increased, and the expression of FOXO3 was increased by more than 100 times (up to about 1050 times). Downstream antioxidant target genes were significantly upregulated.

[0040] Table 1. Experimental data for FOXO3 activation efficiency verification (dual detection by qRT-PCR and Western Blot)

[0041] Note: ***p < 0.001 vs Unedited Group.

[0042] (2) The results of HLA-A / B / C knockout are shown in Table 2. The HLA-A / B / C knockout efficiency is ≥99%.

[0043] Table 2. Experimental data for HLA-A / B / C knockout efficiency validation (flow cytometry + NGS sequencing + TIDE analysis + digital PCR)

[0044] Note: ***p < 0.001 vs. unedited group. The efficiency of co-editing dPCR for the three targets (99.65%) was lower than the lowest efficiency for each single target (HLA-B 99.71%), consistent with the statistical rule that the probability of simultaneous editing of three targets ≤ the lowest efficiency for a single target. The HLA protein retention rate in the empty vector control group, as detected by flow cytometry, was 83.5% ± 4.2%, consistent with the baseline values ​​for each target in the unedited group (82%–87%), confirming that the empty vector control had no knockout effect.

[0045] (3) The results of the FOXO3 / HLA editing effects of the obtained monoclonal cell lines are shown in Table 3. FOXO3 expression was upregulated ≥580-fold, HLA-A / B / C knockout efficiency was >99%, cell viability was ≥84% (84.5%–89.5% for each clone), and CD34 expression was ≥84%. + The marker retention rate was greater than 90% (91.7%–97.8% for each clone and 98.7% for the unedited group).

[0046] Table 3. Summary of editing effects of the single-clonal cell line FOXO3 / HLA (5 independent positive clones)

[0047] Note: ***p<0.001 vs. unedited group; **p<0.01; *p<0.05. Cell viability for each clone ranged from 84.5% to 89.5%, with the optimal clone Clone-A3 at 86.2±5.4%. This was statistically significant compared to the unedited group (96.2±3.4%, consistent with Table 4) (p<0.01, see Table 4), but still met the ≥85% criterion and did not affect subsequent iPS reprogramming and exosome production. CD34 + The marker retention rate was >90% for all clones, with the unedited group having a retention rate of 98.7 ± 2.1% (consistent with Table 4).

[0048] (4) The results of cell stemness and function verification after gene editing are shown in Table 4. It can be seen that compared with the unedited group, the CD34 of the gene-edited cells is higher. + The positive rate, cell viability, clonogenic ability, and differentiation potential all met the qualified standards. The karyotype analysis was normal. The exosome production was increased after gene editing, and SASP factors (such as IL-6 and TNF-α) were significantly reduced.

[0049] Table 4. Cell stemness and function validation after gene editing (key quality control data)

[0050] Note: ***p<0.001 vs. unedited group. The cell viability after gene editing was statistically different from that of the unedited group (p<0.01), and the differentiation potential was statistically different from that of the unedited group (p<0.05), but both met the qualified criteria (viability ≥85%, differentiation potential ≥80%), and did not affect subsequent iPS reprogramming and exosome production.

[0051] (5) The results of the genotoxicity assessment (off-target effect detection) are shown in Table 5. The results show a low mutation rate (<0.1%), no high-frequency off-target sites, and no significant insertion mutation risk, which meets the requirements for genotoxicity assessment of gene editing products in the "Technical Guidelines for Non-Clinical Research of Gene-Modified Cell Therapy Products (Trial)".

[0052] Table 5. Genotoxicity assessment – ​​Off-target effect detection (dual detection using whole-genome sequencing WGS and GUIDE-seq)

[0053] Note: There were no statistically significant differences in off-target rates and background mutation rates among the editing groups (p > 0.05), and all met the acceptable standard of <0.1%. Genotoxicity assessment focused on the genotoxicity risks caused by off-target editing and insertional mutations (insertion site, insertion copy number), and identified / characterized genome integration sites. The target cell was CD34. + Hematopoietic stem / progenitor cells (differentiated cells) have a relatively low risk of genotoxicity from insertional mutations.

[0054] (6) The efficiency of reprogramming cells into iPS cells after gene editing is shown in Table 6. The success rate of iPS cell reprogramming, iPS clone morphology score, and Oct4 4 Hz are also shown in Table 6. + / Sox2 + / Nanog + Positive rates, telomere length, in vitro differentiation potential, and in vivo teratoma formation rate compared to unedited CD34 + The control group showed a significant increase, confirming its pluripotency.

[0055] Table 6. Efficiency of cell reprogramming to iPS after gene editing (Sendai virus MOI=5)

[0056] Note: ***p < 0.001 vs. control group; **p < 0.01 vs. control group. The control group consisted of CD34 cells reprogrammed with Sendai virus but without gene editing. + The 30% teratoma formation rate in the source cells reflected incomplete reprogramming or low cell pluripotency, which was statistically significant compared to the Clone-A3 group (100%) (p < 0.01), confirming that gene editing can significantly improve reprogramming efficiency and cell pluripotency. The fold change is the ratio of the Clone-A3 group to the control group. The control group served as the baseline for comparison and was not marked with a significance symbol; the Clone-A3 group was marked with ** (p < 0.01), consistent with Fisher's exact test results (10 / 10 vs 3 / 10, two-tailed p = 0.003).

[0057] (7) The results of the immunogenicity and immunotoxicity assessment of HLA knockout iPS exosomes are shown in Table 7. The CFSE proliferation test of the HLA knockout exosome group was <0.5%, which was not significantly different from the PBS negative control group. There was no significant increase in complement activation markers and no abnormal cytokine release, indicating that there was no T cell activation, no complement activation, and no abnormal cytokine release. Therefore, it can be repeatedly administered.

[0058] Table 7. Assessment of HLA knockout exosome immunogenicity and immunotoxicity (CFSE flow cytometry + complement activation + cytokine release)

[0059] Note: ***p<0.001 vs PBS group. No significant differences were found in any indicators between the HLA knockout exosome group and the PBS group (p>0.05), confirming successful immunization exemption.

[0060] (8) The batch-to-batch consistency test results of iPS exosomes are shown in Tables 8 and 9. The concentration is ≥1×10 nm (NTA detection) and the range is 30–150 nm. 10 Particles / mL, CD9 + / CD63 + / CD81 + The positive rate was ≥90%, endotoxin was <0.5 EU / mL, and the cells were sterile. This indicates that the GMP-grade amplification system based on iPS cells of this invention achieves batch-to-batch consistency of exosomes (CV <12%), meeting the needs of clinical drug use.

[0061] Table 8. Quality validation data for batch-to-batch consistency (CV < 12%) of iPS exosomes

[0062] Note: Inter-batch CV: Concentration 3.6% (pass standard <12%), particle size 1.9% (pass standard <15%), and marker 1.3% (pass standard <10%), all meet the pass standards.

[0063] Table 9 Summary of inter-batch consistency results

[0064] Example 2: Application of gene-edited iPS-derived exosomes in the treatment of AMD (Proof-of-Concept study) I. Methods 1. Experimental model: (1) Animals: C57BL / 6J aged mice, 18 months old, were used to simulate naturally aged AMD. A total of 120 mice were used in the formal experiment (grouping is shown below); another 40 mice were used for dose exploration POC studies (see Table 10), and the number of animals was counted separately. The model was histopathologically verified, showing two pathological changes: geographic atrophy (GA) and choroidal neovascularization (CNV). The baseline GA area was approximately 4.2 ± 0.8% of the total retinal area, and the CNV area was approximately 3.5 ± 0.6% of the total retinal area. The reason for choosing this model is that wet AMD is highly correlated with and develops from dry AMD. Clinically, some AMD patients have both GA and CNV pathological changes. Furthermore, clinical studies have found that in some AMD patients with both GA and CNV changes, although CNV was controlled to some extent when receiving anti-VEGF treatment, the degree of GA was aggravated. Therefore, this model can simultaneously evaluate the dual therapeutic effect of exosomes on GA and CNV and compare it with anti-VEGF treatment, which is closer to the actual clinical needs.

[0065] (2) Grouping: Intravenous injection group (n=20): The exosomes described in this invention were injected via the tail vein at a dose of 5×10⁻⁶. 9 Granules / each; Eye drop group (n=20): Instillation of the exosome eye drops described in this invention into the conjunctival sac of both eyes (5 μL per eye, 10 μL in total, containing 5 × 10⁻⁶ granules / each); 9 Granules / each); Nasal administration group (n=20): Nasal instillation of the exosome nasal drops described in this invention, at a dose of 5 × 10⁻⁶. 9 Particles / each; Fundus injection group (n=20): Intravitreal injection of the exosomes described in this invention, at a dose of 5 × 10⁻⁶. 9 Particles / eye; Anti-VEGF control group (n=20): intravitreal injection of aflibercept at a dose of 40 μg / eye; Saline control group (n=20): tail vein injection of an equal volume of saline.

[0066] 2. Dosage exploration (POC dose-response relationship study): Before the formal experiment, a dose-exploration point-of-care (POC) study was conducted, setting a low dose (1×10⁻⁶). 9 Granules / each), medium dose (5×10) 9 Granules / each), high dose (1×10) 10 Three dosage groups (5 × 10⁻⁶ granules / animal) and one saline control group (n=10 per group, totaling 40 animals, animal count not included) were used to determine the effective dose range and minimum effective dose. The results are shown in Table 10. The medium-dose group achieved optimal efficacy, while the high-dose group showed no significant additional benefit. Therefore, the medium-dose group (5 × 10⁻⁶ granules / animal) was selected. 9 (particles / each) was used as the formal experimental dose.

[0067] Table 10. Dosage-exploration POC study (intravenous injection, assessment on day 60, n=10 / group)

[0068] Note: ***p < 0.001 vs. saline control group; no label indicates p > 0.05. There was no statistically significant difference in the GA area change rate between the low-dose group (+5.2%) and the control group (+5.9%) (p > 0.05), suggesting that the low dose had no significant reversal effect on GA. There were no significant differences in any indicators between the medium-dose and high-dose groups (p > 0.05).

[0069] 3. Dosing regimen: The first dose was administered on day 0, followed by three doses every 14 days (days 0, 14, and 28). Evaluations were conducted on days 7, 28, and 60, with the day 28 evaluation completed before the third dose. Four routes of exosome administration—intravenous injection, ophthalmic drops, nasal administration, and fundus injection—were compared, with an anti-VEGF control group included for comparison. To meet the dosage and volume requirements for each route of administration, the exosome concentrate (≥1×10⁻⁶) was used. 10 The particles (per mL) were concentrated by ultrafiltration and formulated into different dosage forms: 2.5 × 10⁻⁶ for intravenous injection. 10 Granules / mL (administration volume 200 μL); eye drops 5×10 11 Particles / mL (5 μL per eye, 10 μL total); Nasal drops 5 × 10 11 Particles / mL (5 μL per nostril, total 10 μL); Intravitreal injection 2.5 × 10 12 Particles / mL (dose volume 2 μL); the total dose for a single administration via each route is 5 × 10⁻⁶. 9 Particles / animal. In addition, after the final assessment, animals in each group continued to be fed and followed up for a long period of time (until the animals died or reached 36 months of age). The time of vision loss and the time of death were recorded for survival analysis (Log-rank test); the survival rate was statistically analyzed at the time of follow-up to 24 months of age.

[0070] II. Results (1) The detection indicators and results are shown in Table 11. On day 60 after administration, mice in the exosome treatment group showed significant inhibition of choroidal neovascularization (CNV area detected by OCTA) and a reduction of approximately 27-30% compared to baseline. Simultaneously, geographic atrophy was reversed (GA area decreased by 2.1%), retinal thickness preservation rate reached ≥83%, retinal electrophysiological response was restored, no photoreceptor layer collapse was observed, and visual acuity recovered to over 80% of the level of disease-free mice. Retinal SASP factor significantly decreased, and antioxidant factors significantly increased. In the anti-VEGF control group, although CNV area decreased by 22.1%, GA area increased by 15.3%, significantly exacerbating geographic atrophy. Long-term follow-up showed that in aged mice ≥18 months, the average survival time of the exosome treatment group increased from 20.2 months to 32.9 months compared to the saline control group, an extension of approximately 12.7 months, representing a lifespan extension rate of approximately 63%. In the exosome treatment group, TNF-α and IL-6 expression in retinal tissue were significantly reduced, while SOD and GSH-Px activities were significantly upregulated.

[0071] Table 11 Comparison of the therapeutic effects of exosomes on a naturally aging AMD model (with both GA and CNV alterations) (n=60; structural and functional indicators were assessed on day 60, and survival-related indicators were obtained through long-term follow-up).

[0072] Note: ***p<0.001 vs saline control group; no label indicates p>0.05. The area of ​​GA and RPE atrophy in the anti-VEGF group was significantly worse than in the control group (both p<0.001); the increase in IL-6 and TNF-α in the anti-VEGF group was significantly lower than in the control group (both p<0.001), but still significantly higher than in the exosome treatment group. There were no statistically significant differences in photoreceptor cell survival rate (48.7%), SOD change rate (-7.5%), and GSH-Px change rate (-5.8%) between the anti-VEGF group and the control group (p>0.05), suggesting that anti-VEGF treatment has an adverse trend on photoreceptor survival and antioxidant capacity. The time of visual loss was the point at which complete visual function was lost in the animals during long-term follow-up, earlier than the time of death.

[0073] (2) The therapeutic effects of the five groups—intravenous injection, eye drops, nasal administration, fundus injection, and anti-VEGF control—are shown in Tables 12 and 13. Intravenous injection showed the best effect compared to eye drops, nasal administration, and fundus injection. Eye drops were slightly more effective than nasal administration, but less effective than intravenous injection. While the anti-VEGF control group showed some inhibitory effect on CNV, it significantly exacerbated GA progression. Eye drops, nasal administration, and fundus injection also showed good therapeutic effects on AMD with both GA and CNV changes, with higher patient compliance and lower costs.

[0074] Table 12 Summary and comparison of administration route and time series data (days 7 / 28 / 60; survival-related indicators are long-term follow-up results)

[0075] Note: ***p < 0.001 vs. saline control group; no label indicates p > 0.05. The p-value for the non-intravenous injection group is the result compared to the intravenous injection group (vs. intravenous); the p-value for the anti-VEGF group is the result compared to the saline control group (vs. control). Mean survival time and time of vision loss are long-term follow-up indicators (follow-up until animal death or 36 months of age) and cannot be assessed on day 60. "24-month-old n=17 survivors" refers to the number of surviving animals at 24 months of age, consistent with the survival rate in Table 13; mean survival time is calculated based on the time of death of all 20 animals (all died by 36 months of age).

[0076] Table 13 Comparison of administration routes for exosome therapy in AMD models (final outcome on day 60; survival-related indicators are long-term follow-up results)

[0077] Note: ***p<0.001 vs saline control group; no label indicates p>0.05. The area of ​​GA and RPE atrophy in the anti-VEGF group was significantly worse than in the control group (both p<0.001); the increase in IL-6 in the anti-VEGF group was significantly lower than in the control group (p<0.001), but still significantly higher than in the exosome treatment group. There were no statistically significant differences in photoreceptor cell survival rate (48.7%), SOD change rate (-7.5%), and GSH-Px change rate (-5.8%) between the anti-VEGF group and the control group (p>0.05), suggesting that anti-VEGF treatment has an adverse trend on photoreceptor survival and antioxidant capacity. The p-value for the non-intravenous injection group was compared with the intravenous injection group (vs. intravenous); the p-value for the anti-VEGF group was compared with the saline control group (vs. control). All dosage forms were stored and transported at 2–6℃, and the activity retention rate after 2 weeks was >91%, with no significant differences between groups (p>0.05). All indicators in the eye drop group were slightly better than those in the nasal administration group, but worse than those in the intravenous injection group, with no statistically significant differences (p > 0.05). Mean survival time and time of vision loss were long-term follow-up indicators (follow-up until animal death or reaching 36 months of age; survival rate statistics were based on 24 months of age), while other indicators were assessed on day 60. "24-month-old n=17 survivors" refers to the number of surviving animals at 24 months of age, consistent with the survival rate in Table 12; mean survival time was calculated based on the time of death of all 20 animals (all died by 36 months of age).

[0078] In summary, this invention establishes for the first time an integrated technology platform for "gene-edited iPS cells → anti-aging and low-immunogenic iPS exosomes," solving four major bottlenecks in existing stem cell therapies: donor dependence, immune rejection, functional decline, and insufficient yield. It achieves: precise treatment targeting: specifically for age-driven AMD, especially suitable for mixed AMD with both GA and CNV pathological changes; innovative mechanism of action: by activating and downregulating SASP and upregulating antioxidant factors through FOXO3, it inhibits retinal aging at its source while reversing GA progression; high drug safety: HLA knockout exosomes show no allogeneic rejection, can be administered multiple times, and no significant safety risks were observed in genotoxicity and immunogenicity assessments; diverse routes of administration: intravenous injection is most effective, while eye drops are slightly more effective than nasal administration but less effective than intravenous injection, but have the highest compliance and lowest cost, making them suitable for long-term maintenance therapy; superior to anti-VEGF therapy: while anti-VEGF therapy can partially inhibit CNV, it significantly exacerbates GA progression, whereas the exosomes of this invention can simultaneously inhibit CNV and reverse GA; and high clinical translational potential: 1×10 9 Each iPS cell can produce ≥5×10 13 Exosomes offer controllable cost per treatment dose.

[0079] All dosage forms were stored and transported at 2–6°C, and the activity retention rate was >91% after 2 weeks.

Claims

1. An exosome derived from gene-edited induced pluripotent stem cells, characterized in that, The induced pluripotent stem cells were obtained by isolating gene-edited induced pluripotent stem cells; the gene-edited induced pluripotent stem cells were obtained by reprogramming allogeneic adult somatic cells that had been activated by the FOXO3 gene and knocked out by the HLA-A / B / C gene.

2. The exosomes according to claim 1, characterized in that, The adult somatic cells are CD34 derived from peripheral blood. + cell.

3. The exosomes according to claim 1, characterized in that, The exosomes have a particle size of 30–150 nm.

4. The exosome according to claim 1, characterized in that, The FOXO3 gene activation was achieved by targeting the FOXO3 gene promoter using the dCas9-VPR system, with the sgRNA sequence being 5'-GGTGAGTGTGAAGAAGCTGG-3'.

5. The exosome according to claim 1, characterized in that, The HLA-A / B / C gene knockout was performed using the Cas9 system; the sgRNA of the HLA-A gene was 5'-GCTGGTGGTGTCTACACCGC-3', the sgRNA of the HLA-B gene was 5'-GGCGTGTACTACCAGCACCC-3', and the sgRNA of the HLA-C gene was 5'-CCGCGGAGTATTGGCGCTGG-3'.

6. The exosome according to claim 1, characterized in that, The reprogramming involves delivering Oct4 / Sox2 / Klf4 / c-Myc using a non-integrated Sendai virus.

7. The use of exosomes derived from gene-edited induced pluripotent stem cells as described in any one of claims 1 to 6 in the preparation of a medicament for treating age-related macular degeneration.

8. The application according to claim 7, characterized in that, The age-related macular degeneration mentioned is dry age-related macular degeneration.

9. The application according to claim 8, characterized in that, The drug exerts its therapeutic effect by inhibiting retinal oxidative stress and inflammatory response in subjects, promoting photoreceptor cell survival, reducing choroidal neovascularization and retinal hemorrhage, and reversing geographic atrophy.

10. A treatment for age-related macular degeneration, characterized in that, It contains exosomes derived from gene-edited induced pluripotent stem cells as described in any one of claims 1 to 6.

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