WO2023011248A1 - 以iPSC来源的细胞为载体、基因治疗湿性年龄相关性黄斑变性 - Google Patents

以iPSC来源的细胞为载体、基因治疗湿性年龄相关性黄斑变性 Download PDF

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WO2023011248A1
WO2023011248A1 PCT/CN2022/107879 CN2022107879W WO2023011248A1 WO 2023011248 A1 WO2023011248 A1 WO 2023011248A1 CN 2022107879 W CN2022107879 W CN 2022107879W WO 2023011248 A1 WO2023011248 A1 WO 2023011248A1
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medium
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吴理达
顾雨春
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呈诺再生医学科技(珠海横琴新区)有限公司
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Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to gene therapy of wet age-related macular degeneration using iPSC-derived cells as carriers.
  • Age-related macular degeneration also known as age-related macular degeneration
  • AMD Age-related macular degeneration
  • WHO World Health Organization
  • AMD AMD is a multifactorial disease. Most patients are older than 50 years old. AMD occurs successively or simultaneously in both eyes.
  • AMD is divided into dry age-related macular degeneration (dAMD) and wet age-related macular degeneration (wAMD), Of these, most of the severe central vision loss was caused by wAMD.
  • wAMD also known as exudative or neovascular macular degeneration, causes severe visual impairment and seriously endangers the visual health of the elderly in my country.
  • wAMD is characterized by abnormal choroidal neovascularization (CNV) formation under the macula in the center of the retina. CNV may undergo pathological changes such as rupture, hemorrhage, and exudative edema.
  • CNV choroidal neovascularization
  • RPE retinal pigment epithelium
  • VEGF intraocular vascular endothelial growth factor
  • the main treatment for wAMD is intraocular injection of anti-VEGF drugs.
  • anti-VEGF therapy can alleviate the disease to a certain extent, it still cannot completely control the progression of the disease, and cannot fundamentally solve the vision loss caused by the damage of RPE cells.
  • Surgical removal of CNV is also one of the clinical methods for the treatment of wAMD. The surgical method is more direct than the treatment with anti-VEGF drugs. However, due to surgical reasons, the removal of CNV also removes the underlying RPE cells. Cause retinal choroidal atrophy, so the visual acuity of the patient can not be improved after the operation.
  • the present invention aims to use CRISPR technology to conduct targeted double-gene gene editing of iPSCs in the 19th chromosome nonsense region (AAVS region), express neurotrophic factors CNTF and miR-126 at the same time, and then differentiate the gene-edited iPSCs into RPE cells , and then transplant RPE cells to treat wet age-related macular degeneration, and fundamentally solve the vision loss caused by the damage of RPE cells.
  • AAVS region 19th chromosome nonsense region
  • the present invention uses iPSC-derived cells as carriers to treat wet age-related macular degeneration.
  • RPE cells the obtained RPE cells can express neurotrophic factors CNTF and miR-126, CNTF can promote the survival of rod photoreceptors, and CNTF can also provide neurotrophic functions for retinal ganglion cells (RGCs), in addition, CNTF also helps It can reduce retinal edema and help the retina reattach to the detached RPE by injecting more fluid from the retina into the choroid, thereby improving transplantation efficiency; miR-126 can inhibit the expression of VEGF in RPE cells and adjacent cells, thereby inhibiting the expression of choroid The generation of new blood vessels, and then achieve the purpose of effective treatment of wet age-related macular degeneration.
  • a first aspect of the invention provides a construct for engineering a cell to obtain a gene edited cell.
  • the construct comprises nucleotides encoding neurotrophic factors or their analog genes, and/or nucleotides of anti-angiogenic nucleic acids;
  • the neurotrophic factors or their analogs include CNTF, NGF, BDNF, NT-3, NT-4/5, NT-6, PEDF, GDNF, GMFB, NRG1, CHRNB3, NTR 368;
  • the anti-angiogenic nucleic acid includes miR-126, miR-15/107 family, miR-17-92 family, miR-21, miR-132, miR-296, miR-378, miR-519c, miR- 210, miR-222, miR-100, miR-23, miR-27, miR-31, miR-150, miR-146a, miR-23a;
  • the neurotrophic factor or its analogue is CNTF;
  • the anti-angiogenic nucleic acid is miR-126;
  • the construct comprises nucleotides encoding a CNTF gene, and nucleotides of miR-126;
  • nucleotide sequence of the gene encoding CNTF is shown in SEQ ID NO: 3;
  • nucleotide sequence of the miR-126 is shown in SEQ ID NO: 6;
  • the construct further comprises one or more exogenous promoters, and/or one or more endogenous promoters contained in selected sites;
  • nucleotides encoding the CNTF gene and the nucleotides of miR-126 are operably linked to one or more exogenous promoters contained in the construct, or to selected sites one or more endogenous promoters contained in;
  • nucleotides encoding the CNTF gene and the nucleotides of miR-126 are operably linked to one or more exogenous promoters contained in the construct;
  • nucleotides encoding the CNTF gene and the nucleotides of miR-126 are operably linked to the two exogenous promoters contained in the construct;
  • the exogenous promoters include CMV promoter, U6 promoter, EF1 ⁇ promoter, PGK promoter, CAG promoter, UBC promoter, SV40 promoter, Human beta actin promoter, TEF1 promoter, GDS Promoter, H1 promoter, U6 promoter, T7 promoter, TERT promoter, RSV promoter, PGK1 promoter;
  • the exogenous promoter is a CMV promoter, a U6 promoter;
  • nucleotide sequence of the CMV promoter is as shown in SEQ ID NO:2;
  • nucleotide sequence of the U6 promoter is as shown in SEQ ID NO:5;
  • the construct further comprises a pair of homology arms
  • said homology arms are specific for said selected site
  • the homology arms include a homology left arm HA-L and a homology right arm HA-R specifically targeting the selected site;
  • nucleotide sequence of the homologous left arm HA-L is shown in SEQ ID NO:1;
  • nucleotide sequence of the homologous right arm HA-R is shown in SEQ ID NO: 8;
  • the construct further comprises one or more exogenous terminators, and/or one or more endogenous terminators contained in selected sites;
  • nucleotides encoding the CNTF gene and the nucleotides of miR-126 are operably linked to one or more exogenous terminators contained in the construct, and/or selected sites one or more endogenous terminators contained in;
  • nucleotides encoding the CNTF gene and the nucleotides of miR-126 are operably linked to one or more exogenous terminators contained in the construct;
  • nucleotides encoding the CNTF gene and the nucleotides of miR-126 are operably linked to the two exogenous terminators contained in the construct;
  • the exogenous terminator includes PolyA terminator, NOS terminator, T7 transcription terminator, rrnB terminator, T0 terminator, SV40 terminator, hGH terminator, BGH terminator, rbGlob terminator;
  • the exogenous terminator is a PolyA terminator
  • the PolyA terminator includes PolyA element 1, PolyA element 2;
  • nucleotide sequence of the PolyA element 1 is shown in SEQ ID NO:4;
  • nucleotide sequence of the PolyA element 2 is shown in SEQ ID NO:7;
  • said selected locus is a safe harbor locus
  • said safe harbor loci include AAVS1, CCR5, ROSA26, HTRP, H11, TCR, RUNX1, beta-2 microglobulin, collagen, GAPDH, or a locus satisfying genomic safe harbor criteria;
  • said safe harbor locus is AAVS1;
  • the construct consists of homologous left arm HA-L, CMV promoter, nucleotides encoding CNTF gene, PolyA element 1, U6 promoter, nucleotides of miR-126, PolyA element 2, homologous
  • the source right arm HA-R is sequentially connected in series.
  • the second aspect of the present invention provides an expression vector or cloning vector.
  • the expression vector comprises the construct described in the first aspect of the present invention, and the cloning vector comprises nucleotides encoding neurotrophic factors or their analog genes, and/or nucleotides of anti-angiogenic nucleic acids;
  • the neurotrophic factors or their analogs include CNTF, NGF, BDNF, NT-3, NT-4/5, NT-6, PEDF, GDNF, GMFB, NRG1, CHRNB3, NTR 368;
  • the anti-angiogenic nucleic acid includes miR-126, miR-15/107 family, miR-17-92 family, miR-21, miR-132, miR-296, miR-378, miR-519c, miR- 210, miR-222, miR-100, miR-23, miR-27, miR-31, miR-150, miR-146a, miR-23a;
  • the neurotrophic factor or its analogue is CNTF;
  • the anti-angiogenic nucleic acid is miR-126;
  • the cloning vector comprises nucleotides encoding CNTF gene and nucleotides of miR-126;
  • nucleotide sequence of the gene encoding CNTF is shown in SEQ ID NO: 3;
  • nucleotide sequence of the miR-126 is shown in SEQ ID NO: 6;
  • the expression vector further comprises a vector
  • the vectors include dornor vectors, DNA vectors, viral vectors;
  • the DNA vectors include DNA plasmid vectors, liposomes bound to DNA plasmids, molecular coupling bodies bound to DNA plasmids, polymers bound to DNA plasmids;
  • the viral vectors include adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, herpes simplex virus vectors, baculovirus vectors, Sendai virus vectors, poxvirus vectors, geminivirus vectors;
  • the cloning vector further comprises a vector
  • the vectors include DNA plasmid vectors, phage vectors, yeast artificial chromosome vectors, and phage-plasmid hybrid vectors.
  • a third aspect of the invention provides an engineered host cell or population thereof.
  • engineered host cells or populations thereof express CNTF and/or miR-126;
  • the engineered host cell or population thereof overexpresses CNTF and/or miR-126;
  • said engineered host cells or populations thereof overexpress CNTF and miR-126;
  • the engineered host cell or population thereof comprises the construct described in the first aspect of the present invention, and/or the expression vector or cloning vector described in the second aspect of the present invention;
  • the host cells include iPSCs, embryonic stem cells, mesenchymal stem cells, adult stem cells, tissue-specific stem cells;
  • the host cell is iPSC.
  • a fourth aspect of the present invention provides a terminally differentiated cell or a precursor thereof, or a population thereof.
  • terminally differentiated cells or their precursor cells, or their populations express CNTF and/or miR-126;
  • the terminally differentiated cells or their precursor cells, or populations thereof overexpress CNTF and/or miR-126;
  • terminally differentiated cells or precursor cells thereof, or populations thereof overexpress CNTF and miR-126;
  • the terminally differentiated cells or their precursor cells or their populations are obtained by inducing differentiation of the engineered host cells or their populations according to the third aspect of the present invention.
  • the terminally differentiated cells or their precursor cells include retinal pigment epithelial cells, cone cells, rod cells, mesenchymal stem cells, photoreceptor progenitor cells, corneal epithelial cells, choroidal endothelial cells, retinal cells, keratocytes , lens cells, ganglion cells, optic nerve cells, choroid cells;
  • the terminally differentiated cells or their precursor cells are retinal pigment epithelial cells.
  • iPSC induced pluripotent stem cells
  • iPSC induced pluripotent stem cells
  • Programming refers to the process of obtaining induced pluripotent stem cells through exogenous gene expression, compound induction, and epigenetic modification.
  • terminal differentiated cells refer to iPSCs, embryonic stem cells, mesenchymal stem cells, adult stem cells, tissue-specific stem cells or fully differentiated cells obtained after differentiation of their cell populations.
  • the stem cells are iPSCs
  • the terminally differentiated cells include retinal pigment epithelial cells, cone cells, rod cells, photoreceptor progenitor cells, corneal epithelial cells, choroidal endothelial cells, retinal cells, keratocytes , lens cells, ganglion cells, optic nerve cells, choroid cells, preferably retinal pigment epithelial cells.
  • the "precursor cells” mentioned in the present invention refer to all intermediate cells in the process of inducing differentiation of iPSCs, embryonic stem cells, mesenchymal stem cells, adult stem cells, and tissue-specific stem cells to produce terminally differentiated cells
  • the intermediate cells include all cells involved in this induced differentiation process except initial cells (iPSCs, embryonic stem cells, mesenchymal stem cells, adult stem cells, tissue-specific stem cells) and terminal cells (terminal differentiated cells) .
  • the fifth aspect of the present invention provides a method for preparing the engineered host cell or population thereof described in the third aspect of the present invention.
  • the method includes the following steps: delivering the expression vector described in the second aspect of the present invention and the CRISPR/Cas vector targeting the selected site to the host cell;
  • said selected locus is a safe harbor locus
  • the safe harbor loci include AAVS1, CCR5, ROSA26, HTRP, H11, TCR, RUNX1, beta-2 microglobulin, collagen, GAPDH, or a locus that satisfies the genomic safe harbor criteria;
  • said safe harbor locus is AAVS1;
  • the CRISPR/Cas vector targeting the selected site is a CRISPR/Cas vector targeting AAVS1;
  • the CRISPR/Cas vector targeting AAVS1 is AAVS1 T2 CRIPR in pX330 vector;
  • the delivery is achieved by introducing the expression vector described in the second aspect of the present invention into host cells;
  • the introduction methods include electroporation, microinjection, ultrasound-mediated method, sonication, photoporation, magnetic transfer, heat shock, calcium phosphate, liposome and polymerization Physical method, nanoparticle method, virus transformation method;
  • the introduction method is electroporation
  • composition of the electroporation system in the electroporation method is as follows: 100 ⁇ L electroporation system, per 1 ⁇ 10 6 host cells, 1 g AAVS1 T2 CRIPR in pX330 vector, 1 ⁇ g the expression vector;
  • the voltage used in the electroporation method is 1200V;
  • the method further comprises culturing and screening the host cells after delivery of the expression vector described in the second aspect of the present invention and the CRISPR/Cas vector targeting the selected site;
  • the culture medium used in the cultivation is E8 medium, and the medium is changed every day;
  • the culture conditions are 37°C, 5% CO 2 ;
  • the culture time is 48-72h
  • the screening is performed with puromycin
  • the screening time is 5-7 days
  • the host cells include iPSCs, embryonic stem cells, mesenchymal stem cells, adult stem cells, tissue-specific stem cells;
  • the host cell is iPSC.
  • the sixth aspect of the present invention provides a method for preparing terminally differentiated cells or their precursor cells, or populations thereof according to the fourth aspect of the present invention.
  • the method includes the following steps: inducing differentiation of the engineered host cell or population thereof according to the third aspect of the present invention.
  • said induction of differentiation comprises the steps of:
  • the cell density of the engineered host cell or population thereof according to the third aspect of the present invention described in step (1) is 1.0 ⁇ 10 3 -5.0 ⁇ 10 5 /cm 2 ;
  • the cell density of the engineered host cell or population thereof described in the third aspect of the present invention in step (1) is 5.0 ⁇ 10 3 /cm 2 ;
  • the culture conditions described in step (1) are 37°C, 5% CO 2 ;
  • the RDM1 medium described in step (1) consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defned Lipid Concentrate, 1% L-glutamine;
  • the small molecule additives described in step (1) are noggin, XAV-939, LY2109761;
  • the RDM2 medium described in step (3) consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defned Lipid Concentrate, 1% L-glutamine;
  • the small molecule additive described in step (3) is 6-bromoindirubin-3'-oxime (BIO), SU5402, Thiazovivin;
  • the RDM3 medium described in step (4) consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defned Lipid Concentrate, 1% L-glutamine;
  • the small molecule additive described in step (4) is 6-bromoindirubin-3'-oxime (BIO), SU5402, Thiazovivin, Vitamin B3;
  • the RDM4 medium described in step (5) is made up of 89%DMEM/F12, 10%KSR, 1%N2 medium, 1%L-glutamine, 10mM Vitamin B3;
  • the RMM medium described in step (6) consists of 97% DMEM/F12, 2% B27 medium, 1% L-glutamine;
  • the Trypsin-EDTA described in step (7) is 0.25% Trypsin-EDTA preheated at 37°C;
  • the addition of Trypsin-EDTA described in step (7) is 1mL;
  • the culture conditions in step (7) are 37°C, 5% CO 2 ;
  • the culturing time in step (7) is 10 min.
  • the addition amount of REM medium described in step (8) is 3mL;
  • the terminally differentiated cells or their precursor cells include retinal pigment epithelial cells, cone cells, rod cells, mesenchymal stem cells, photoreceptor progenitor cells, corneal epithelial cells, choroidal endothelial cells, retinal cells, keratocytes , lens cells, ganglion cells, optic nerve cells, choroid cells;
  • the terminally differentiated cells or their precursor cells are retinal pigment epithelial cells.
  • a seventh aspect of the present invention provides a composition.
  • composition comprises the construct described in the first aspect of the present invention, and/or the expression vector or cloning vector described in the second aspect of the present invention, and/or the engineered host described in the third aspect of the present invention A cell or a population thereof, and/or a terminally differentiated cell or a precursor cell thereof, or a population thereof according to the fourth aspect of the present invention.
  • the eighth aspect of the present invention provides a pharmaceutical composition for treating and/or preventing wet age-related macular degeneration.
  • the pharmaceutical composition comprises the expression vector or cloning vector described in the second aspect of the present invention, and/or the engineered host cell or population thereof described in the third aspect of the present invention, and/or the fourth aspect of the present invention
  • the pharmaceutical composition further includes pharmaceutically acceptable carriers and/or excipients;
  • the pharmaceutical composition further includes one or more therapeutic agents;
  • the therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, miRNA, dsRNA, mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, antibodies, chemotherapy agents, immunomodulatory drugs.
  • a ninth aspect of the invention provides a kit for producing engineered host cells or populations thereof, terminally differentiated cells or precursors thereof, or populations thereof.
  • kit comprises the construct described in the first aspect of the present invention, the expression vector or cloning vector described in the second aspect of the present invention, a CRISPR/Cas vector targeting AAVS1, host cells, one or more culture base;
  • the CRISPR/Cas vector targeting AAVS1 is AAVS1 T2 CRIPR in pX330 vector;
  • the host cells include iPSCs, embryonic stem cells, mesenchymal stem cells, adult stem cells, tissue-specific stem cells;
  • the host cell is iPSC
  • the medium includes E8 medium, RDM1 medium, RDM2 medium, RDM3 medium, RDM4 medium, RMM medium, REM medium.
  • the tenth aspect of the present invention provides the application of any of the following aspects:
  • composition described in the seventh aspect of the present invention in the preparation of a medicament for treating and/or preventing wet age-related macular degeneration
  • composition described in the eighth aspect of the present invention is used in the treatment and/or prevention of wet age-related macular degeneration
  • the drug overexpresses CNTF and/or miR-126;
  • the drug overexpresses CNTF and miR-126;
  • the medicament comprises an agent for overexpressing CNTF and/or miR-126 expression
  • the medicament comprises an agent that overexpresses CNTF and miR-126 expression.
  • the present invention also provides a method for treating and/or preventing wet age-related macular degeneration.
  • the method includes administering to a subject in need an effective amount of the expression vector or cloning vector described in the second aspect of the present invention, and/or the engineered host cell or population thereof described in the third aspect of the present invention , and/or the terminally differentiated cell or its precursor cell described in the fourth aspect of the present invention, or its population, and/or the composition described in the seventh aspect of the present invention, and/or the composition described in the eighth aspect of the present invention pharmaceutical composition.
  • the present invention also provides a method of expressing CNTF and miR-126 in a subject in need thereof.
  • the method includes delivering an effective amount of the expression vector or cloning vector described in the second aspect of the present invention, and/or the engineered host cell described in the third aspect of the present invention to the subretina of a subject in need or a population thereof, and/or terminally differentiated cells or precursor cells thereof, or a population thereof according to the fourth aspect of the present invention.
  • the present invention prepares RPE by inducing differentiation of iPSCs subjected to site-directed double-gene gene editing, and the RPE cell transplantation can repair the damaged retinal pigment epithelial cells on CNV in patients with wet age-related macular degeneration;
  • CNTF retinal ganglion cells
  • miR-126 can inhibit the expression of VEGF in RPE cells and adjacent cells, thereby inhibiting the generation of choroidal neovascularization
  • the RPE obtained by the invention through site-specific double-gene gene editing iPSC differentiation can fundamentally treat wet age-related macular degeneration, and solve the current problem in the field that the intraocular injection of anti-VEGF drugs is expensive, Repeated injections are required, disease progression cannot be completely controlled, and the removal of CNV by surgery also removes the underlying RPE cells, leading to retinal choroidal atrophy, etc., which has a very good clinical application prospect.
  • Fig. 1 shows the schematic diagram of the core structure of the HA-CNTF-U6-MIR126-HA carrier constructed by the present invention
  • Figure 2 shows the results of iPSC stemness gene detection
  • Figure 3 shows the results of forming melanin-precipitating cells after iPSCs were differentiated into RPE
  • Fig. 4 shows the result figure of CNTF mRNA relative expression level in RPE cell
  • Figure 5 shows the result graph of the relative expression level of miR-126 in RPE cells
  • Figure 6 shows the results of the effect of RPE supernatant on the tube formation of HUVEC cells, wherein, panel a: the results of tube formation of HUVEC cells in the control group, panel b: the results of tube formation of HUVEC cells after adding RPE supernatant picture.
  • the reagent information used in the embodiment of the present invention is shown in Table 1.
  • AAVS is a nonsense region of human chromosome 19, which does not affect the stemness and differentiation of stem cells, and can be gene edited.
  • the iPSC cells used in this example are from Beijing Chengnuo Medical Technology Co., Ltd.
  • Electroporation method 100 ⁇ L electroporation system, for every 1 ⁇ 10 6 iPSC cells, 1 g AAVS1 T2 CRIPR in pX330 vector, 1 ⁇ g dornor vector, the voltage is 1200 V;
  • E8 medium (Thermo Fisher Scientific Co., Ltd., A1517001) to culture the cells, and change the medium every day, and the culture conditions are 37° C., 5% CO 2 ;
  • the monoclonal cells were gradually transferred from the 96-well plate to the 48-well plate, 24-well plate, 12-well plate, and 6-well plate for culture, expansion, and cryopreservation.
  • Example 2 of the present invention The iPSC cells obtained by gene editing technology in Example 2 of the present invention were constructed.
  • iPSC cells On day 0, seed iPSC cells at a density of 1.0 ⁇ 10 3 -5.0 ⁇ 10 5 /cm 2 .
  • cells are seeded at a density of 5.0 ⁇ 10 3 cells/cm 2 .
  • the medium used is RDM1
  • the basal medium of RDM1 consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defned Lipid Concentrate, 1% L-glutamine Amide composition, small molecule additives are noggin, XAV-939 and LY2109761;
  • the basal medium of RDM2 consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defned Lipid Concentrate, 1% L-glucose Aminoamide composition, small molecule additives are 6-bromoindirubin-3'-oxime (BIO), SU5402 and Thiazovivin;
  • the basal medium of RDM3 consists of 88% DMEM/F12, 10% KSR, 5mM Monothioglycerol Solution, 1% Chemically Defned Lipid Concentrate, 1% L-glucose Composition of aminoamide, small molecule additives are 6-bromoindirubin-3'-oxime (BIO), SU5402, Thiazovivin, Vitamin B3;
  • the basal medium of RDM4 consists of 89% DMEM/F12, 10% KSR, 1% N2 medium, 1% L-glutamine and 10mM Vitamin Composition of B3;
  • the basal medium of RMM is composed of 97% DMEM/F12, 2% B27 medium, and 1% L-glutamine;
  • Example 4 Immunofluorescence Detection of iPSC Cells Constructed by Gene Editing Technology in Example 2
  • Example 2 of the present invention The iPSC cells obtained by gene editing technology in Example 2 of the present invention were constructed.
  • Counterstaining nuclei add DAPI dropwise and incubate for 5 minutes in the dark, stain the specimens, wash with PBST 5 min ⁇ 4 times to remove excess DAPI;
  • Example 5 qPCR detection of expression levels of genes in RPE cells obtained from iPSC directional induction differentiation in Example 3
  • sequence of specific primer is as follows:
  • the forward primer is 5'-GAAGATTCGTTCAGACCTGACTG-3'(SEQ ID NO:9)
  • the reverse primer is 5'-AAGGTTCTCTTGGAGTCGCTC-3' (SEQ ID NO: 10)
  • the forward primer is 5'-TATGGTTGTTCTCGACTCCTTCAC-3' (SEQ ID NO: 11)
  • the reverse primer is 5'-TCGTCTGTCGTACCGTGAGTAAT-3' (SEQ ID NO: 12)
  • iPSC cells were directional induced to differentiate into RPE cells to form melanin-precipitating cells (see Figure 3), and the induced differentiated RPE cells expressed a large number of genes CNTF and miR-126 (see Figure 4 and Figure 5).
  • Example 6 Effect of RPE cell supernatant obtained from iPSC directional induction differentiation in Example 3 on tube formation of HUVEC cells
  • HUVEC medium promocell
  • RPE supernatant and HUVEC medium experimental group
  • the cell culture plate is placed in a 37° C., 5% CO 2 incubator, and cultured statically for 24 hours;

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Abstract

涉及以iPSC来源的细胞为载体，基因治疗湿性年龄相关性黄斑变性，利用CRISPR技术将iPSC进行定点双基因基因编辑，同时表达神经营养因子CNTF和miR-126，再将基因编辑后的iPSC诱导分化为RPE细胞，所述经诱导分化得到的RPE细胞能够修复已经损伤的CNV上的RPE细胞、抑制脉络膜新生血管的产生。此外，所述RPE细胞还能表达神经营养因子CNTF和miR-126，从根本上治疗湿性年龄相关性黄斑变性，具有非常好的临床应用前景。

Description

以iPSC来源的细胞为载体、基因治疗湿性年龄相关性黄斑变性 技术领域
本发明属于生物医学技术领域，具体而言，涉及以iPSC来源的细胞为载体、基因治疗湿性年龄相关性黄斑变性。
背景技术
年龄相关性黄斑变性(Age-related macular degeneration，AMD)，也称老年性黄斑变性，是一种常见的、慢性的、进行性的致盲性眼病。据世界卫生组织(WHO)的数据估计，全球约10％的视力下降是由AMD引起的。据预测，到2040年AMD患者将增加至近3亿人(Wong WL,Su X,Li X,et al.Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040:A systematic review and meta-analysis[J].Lancet Glob Health,2014,2:106-116.)。AMD是一种多因素疾病，患者多为50岁以上的老年人，双眼先后发病或同时发病，主要影响视网膜色素上皮、光感受器、Bruch’s膜和脉络膜毛细血管。吸烟、环境因素、家族遗传、代谢、营养失调以及视网膜黄斑长期慢性光损伤等因素也均与AMD相关。
根据疾病的临床表现和眼底病理改变的情况，AMD分为干性年龄相关性黄斑变性(Dry age-related macular degeneration，dAMD)和湿性年龄相关性黄斑变性(Wet age-related macular degeneration，wAMD)，其中，大部分的严重中心视力丧失是由wAMD引起的。wAMD又称渗出性或者新生血管性的黄斑变性，造成的视功能损伤也较为严重，严重危害我国老年人的视觉健康。wAMD的特点是视网膜的中心—黄斑下有异常的脉络膜新生血管(Choroidal neovascularizat ion，CNV)生成，CNV可发生破裂出血、渗出水肿等病理改变，这些病理改变损害了CNV上的视网膜色素上皮(Retinal pigment epithelium，RPE)细胞，使RPE细胞对其上的视网膜感光细胞的营养作用受到损害，进而破坏了视网膜感光细胞，导致视功能的损害。
wAMD的发病机制非常复杂，眼内血管内皮生长因子(Vascular endothelial growth factor，VEGF)升高是导致脉络膜新生血管产生的主要原因。所以目前wAMD最主要的治疗方法是抗VEGF药物眼内注射。但由于抗VEGF药物眼内注射需要多次重复治疗，并且价格昂贵，患者难以承受巨大的经济负担和精神负担。尽管抗VEGF治疗可以在一定程度上缓解病情，但是依然无法完全控制疾病的进展，无法从根本上解决因RPE细胞的损害而导致的视力下降。手术去除CNV也是临床上治疗wAMD的方法之一，手术方法要比采用抗VEGF药物进行治疗的方法更为直接，但因手术操作的原因，在去除CNV的同时也去除了其下的RPE细胞，导致视网膜脉络膜萎缩，所以手术后患者的视力并不能得到提高。
本发明旨在利用CRISPR技术将iPSC在第19对染色体无意义区域(AAVS区域)进行定点双基因基因编辑，同时表达神经营养因子CNTF和miR-126，之后将基因编辑后的iPSC分化为RPE细胞，然后将RPE细胞移植治疗湿性年龄相关性黄斑变性，从根本上解决因RPE细胞的损害而导致的视力下降。
发明内容
为了解决目前本领域面临的上述问题，本发明以iPSC来源的细胞为载体、基因治疗湿性年龄相关性黄斑变性，本发明提供了一种经定点双基因基因编辑后的iPSC，并将其分化为RPE细胞，得到的RPE细胞能够表达神经营养因子CNTF和miR-126，CNTF能够促进棒光感受器的存活，同时CNTF也能为视网膜神经节细胞(RGCs)提供神经营养功能，此外，CNTF还有助于减少视网膜水肿，并通过从视网膜向脉络膜注入更多的液体来帮助视网膜重新附着到视网膜脱离的RPE，进而提高移植效率；miR-126能够抑制RPE细胞以及临近细胞的VEGF的表达，进而抑制脉络膜新生血管的产生，进而达到有效治疗湿性年龄相关性黄斑变性的目的。
本发明的上述目的通过以下技术方案得以实现：
本发明的第一方面提供了一种用于工程改造细胞以获得基因编辑的细胞的构建体。
进一步，所述构建体包含编码神经营养因子或其类似物基因的核苷酸、和/或抗血管生成核酸的核苷酸；
优选地，所述神经营养因子或其类似物包括CNTF、NGF、BDNF、NT-3、NT-4/5、NT-6、PEDF、GDNF、GMFB、NRG1、CHRNB3、NTR 368；
优选地，所述抗血管生成核酸包括miR-126、miR-15/107家族、miR-17～92家族、miR-21、miR-132、miR-296、miR-378、miR-519c、miR-210、miR-222、miR-100、miR-23、miR-27、miR-31、miR-150、miR-146a、miR-23a；
更优选地，所述神经营养因子或其类似物为CNTF；
更优选地，所述抗血管生成核酸为miR-126；
最优选地，所述构建体包含编码CNTF基因的核苷酸、和miR-126的核苷酸；
最优选地，所述编码CNTF基因的核苷酸的序列如SEQ ID NO:3所示；
最优选地，所述miR-126的核苷酸的序列如SEQ ID NO:6所示；
优选地，所述构建体还包含一个或多个外源启动子、和/或所选位点中所含的一个或多个内源启动子；
更优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的一个或多个外源启动子、或连接到所选位点中所含的一个或多个内源启动子；
最优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的一个或多个外源启动子；
最优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的两个外源启动子；
最优选地，所述外源启动子包括CMV启动子、U6启动子、EF1α启动子、PGK启动子、CAG启动子、UBC启动子、SV40启动子、Human beta actin启动 子、TEF1启动子、GDS启动子、H1启动子、U6启动子、T7启动子、TERT启动子、RSV启动子、PGK1启动子；
最优选地，所述外源启动子为CMV启动子、U6启动子；
最优选地，所述CMV启动子的核苷酸序列如SEQ ID NO:2所示；
最优选地，所述U6启动子的核苷酸序列如SEQ ID NO:5所示；
优选地，所述构建体还包含一对同源臂；
更优选地，所述同源臂对所述所选位点具有特异性；
更优选地，所述同源臂包括特异性靶向所述所选位点的同源左臂HA-L、同源右臂HA-R；
最优选地，所述同源左臂HA-L的核苷酸序列如SEQ ID NO:1所示；
最优选地，所述同源右臂HA-R的核苷酸序列如SEQ ID NO:8所示；
优选地，所述构建体还包含一个或多个外源终止子、和/或所选位点中所含的一个或多个内源终止子；
更优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的一个或多个外源终止子、和/或所选位点中所含的一个或多个内源终止子；
最优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的一个或多个外源终止子；
最优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的两个外源终止子；
最优选地，所述外源终止子包括PolyA终止子、NOS终止子、T7转录终止子、rrnB终止子、T0终止子、SV40终止子、hGH终止子、BGH终止子、rbGlob终止子；
最优选地，所述外源终止子为PolyA终止子；
最优选地，所示PolyA终止子包括PolyA元件1、PolyA元件2；
最优选地，所述PolyA元件1的核苷酸序列如SEQ ID NO:4所示；
最优选地，所述PolyA元件2的核苷酸序列如SEQ ID NO:7所示；
最优选地，所述所选位点为安全港基因座；
最优选地，所述安全港基因座包括AAVS1、CCR5、ROSA26、HTRP、H11、TCR、RUNX1、β-2微球蛋白、胶原蛋白、GAPDH，或满足基因组安全港准则的基因座；
最优选地，所述安全港基因座为AAVS1；
最优选地，所述构建体由同源左臂HA-L、CMV启动子、编码CNTF基因的核苷酸、PolyA元件1、U6启动子、miR-126的核苷酸、PolyA元件2、同源右臂HA-R依次串联而成。
本发明的第二方面提供了一种表达载体或克隆载体。
进一步，所述表达载体包含本发明第一方面所述的构建体，所述克隆载体包含编码神经营养因子或其类似物基因的核苷酸、和/或抗血管生成核酸的核苷酸；
优选地，所述神经营养因子或其类似物包括CNTF、NGF、BDNF、NT-3、NT-4/5、NT-6、PEDF、GDNF、GMFB、NRG1、CHRNB3、NTR 368；
优选地，所述抗血管生成核酸包括miR-126、miR-15/107家族、miR-17～92 家族、miR-21、miR-132、miR-296、miR-378、miR-519c、miR-210、miR-222、miR-100、miR-23、miR-27、miR-31、miR-150、miR-146a、miR-23a；
更优选地，所述神经营养因子或其类似物为CNTF；
更优选地，所述抗血管生成核酸为miR-126；
最优选地，所述克隆载体包含编码CNTF基因的核苷酸、和miR-126的核苷酸；
最优选地，所述编码CNTF基因的核苷酸的序列如SEQ ID NO:3所示；
最优选地，所述miR-126的核苷酸的序列如SEQ ID NO:6所示；
优选地，所述表达载体还包含载体；
更优选地，所述载体包括dornor载体、DNA载体、病毒载体；
最优选地，所述DNA载体包括DNA质粒载体、结合DNA质粒的脂质体、结合DNA质粒的分子耦联体、结合DNA质粒的多聚物；
最优选地，所述病毒载体包括腺病毒载体、腺相关病毒载体、慢病毒载体、逆转录病毒载体、单纯疱疹病毒载体、杆状病毒载体、仙台病毒载体、痘病毒载体、双生病毒载体；
优选地，所述克隆载体还包含载体；
更优选地，所述载体包括DNA质粒载体、噬菌体载体、酵母人工染色体载体、噬菌体-质粒杂合载体。
本发明的第三方面提供了一种工程改造的宿主细胞或其群体。
进一步，所述工程改造的宿主细胞或其群体表达CNTF和/或miR-126；
优选地，所述工程改造的宿主细胞或其群体过表达CNTF和/或miR-126；
更优选地，所述工程改造的宿主细胞或其群体过表达CNTF和miR-126；
优选地，所述工程改造的宿主细胞或其群体包含本发明第一方面所述的构建体、和/或本发明第二方面所述的表达载体或克隆载体；
优选地，所述宿主细胞包括iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞；
更优选地，所述宿主细胞为iPSC。
本发明的第四方面提供了一种终端分化细胞或其前体细胞、或其群体。
进一步，所述终端分化细胞或其前体细胞、或其群体表达CNTF和/或miR-126；
优选地，所述终端分化细胞或其前体细胞、或其群体过表达CNTF和/或miR-126；
更优选地，所述终端分化细胞或其前体细胞、或其群体过表达CNTF和miR-126；
优选地，所述终端分化细胞或其前体细胞、或其群体为将本发明第三方面所述的工程改造的宿主细胞或其群体诱导分化得到的；
优选地，所述终端分化细胞或其前体细胞包括视网膜色素上皮细胞、视锥细胞、视杆细胞、间充质干细胞、光感受器祖细胞、角膜上皮细胞、脉络膜内皮细胞、视网膜细胞、角膜细胞、晶状体细胞、神经节细胞、视神经细胞、脉络膜细胞；
更优选地，所述终端分化细胞或其前体细胞为视网膜色素上皮细胞。
进一步，本发明中所述的“iPSC”，是指诱导多能干细胞，是一种由体细胞经重编程而获得的具有自我更新能力和向三胚层细胞分化潜能的一种干细胞，所述重编程是指通过外源基因表达、化合物诱导、表观遗传修饰等途径来获得诱导多能干细胞的过程。
进一步，本发明中所述的“终端分化细胞”，是指iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞或其细胞群体在经分化后得到的完全分化的细胞，在本发明的具体实施例中，所述干细胞为iPSC，所述终端分化细胞包括视网膜色素上皮细胞、视锥细胞、视杆细胞、光感受器祖细胞、角膜上皮细胞、脉络膜内皮细胞、视网膜细胞、角膜细胞、晶状体细胞、神经节细胞、视神经细胞、脉络膜细胞，优选为视网膜色素上皮细胞。
进一步，本发明中所述的“前体细胞”，是指在将iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞诱导分化产生终端分化细胞的过程中所有的中间态细胞，所述中间态细胞包括在该诱导分化过程中涉及的除起始细胞(iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞)和终端细胞(终端分化细胞)之外的所有细胞。
本发明的第五方面提供了一种本发明第三方面所述的工程改造的宿主细胞或其群体的制备方法。
进一步，所述方法包括如下步骤：向宿主细胞中递送本发明第二方面中所述的表达载体和靶向所述所选位点的CRISPR/Cas载体；
优选地，所述所选位点为安全港基因座；
更优选地，所述安全港基因座包括AAVS1、CCR5、ROSA26、HTRP、H11、TCR、RUNX1、β-2微球蛋白、胶原蛋白、GAPDH，或满足基因组安全港准则的基因座；
最优选地，所述安全港基因座为AAVS1；
最优选地，所述靶向所述所选位点的CRISPR/Cas载体为靶向AAVS1的CRISPR/Cas载体；
最优选地，所述靶向AAVS1的CRISPR/Cas载体为AAVS1 T2 CRIPR in pX330载体；
优选地，所述递送是通过将本发明第二方面中所述的表达载体导入到宿主细胞中实现的；
更优选地，所述导入的方式包括电转染法、显微注射法、超声波介导法、声穿孔法、光穿孔法、磁转法、热激法、磷酸钙法、脂质体和聚合物法、纳米粒子法、病毒转化法；
最优选地，所述导入的方式为电转染法；
最优选地，所述电转染法中的电转染体系的组成如下：100μL电转体系、每1×10 6个宿主细胞、1g AAVS1 T2 CRIPR in pX330载体、1μg本发明第二方面中所述的表达载体；
最优选地，所述电转染法中所采用的电压为1200V；
优选地，所述方法还包括对递送本发明第二方面中所述的表达载体和靶向所述所选位点的CRISPR/Cas载体后的宿主细胞进行培养和筛选；
更优选地，所述培养采用的培养基为E8培养基、每天换液；
更优选地，所述培养的条件为37℃、5％CO 2；
更优选地，所述培养的时间为48-72h；
更优选地，所述筛选采用嘌呤霉素进行筛选；
更优选地，所述筛选的时间为5-7天；
优选地，所述宿主细胞包括iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞；
更优选地，所述宿主细胞为iPSC。
本发明的第六方面提供了一种本发明第四方面所述的终端分化细胞或其前体细胞、或其群体的制备方法。
进一步，所述方法包括如下步骤：将本发明第三方面所述的工程改造的宿主细胞或其群体进行诱导分化；
优选地，所述诱导分化包括如下步骤：
(1)第0天，将本发明第三方面所述的工程改造的宿主细胞或其群体在添加有小分子添加物的RDM1培养基中进行培养；
(2)第1-6天，每日更换RDM1培养基；
(3)第7-12天，更换培养基为添加有小分子添加物的RDM2培养基，每日更换RDM2培养基；
(4)第13-17天，更换培养基为添加有小分子添加物的RDM3培养基，每日更换RDM3培养基；
(5)第18-24天，更换培养基为RDM4培养基，每日更换RDM4培养基；
(6)第25-36天，更换培养基为RMM培养基，每日更换RMM培养基；
(7)第37天以后，弃去原培养基，加入Trypsin-EDTA，进行培养；
(8)弃去Trypsin-EDTA，加入REM培养基终止消化；
优选地，步骤(1)中所述的本发明第三方面所述的工程改造的宿主细胞或其群体的细胞密度为1.0×10 3-5.0×10 5/cm 2；
更优选地，步骤(1)中所述的本发明第三方面所述的工程改造的宿主细胞或其群体的细胞密度为5.0×10 3/cm 2；
优选地，步骤(1)中所述的培养的条件为37℃、5％CO 2；
优选地，步骤(1)中所述的RDM1培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成；
优选地，步骤(1)中所述的小分子添加物为noggin、XAV-939、LY2109761；
优选地，步骤(3)中所述的RDM2培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成；
优选地，步骤(3)中所述的小分子添加物为6-bromoindirubin-3’-oxime(BIO)、SU5402、Thiazovivin；
优选地，步骤(4)中所述的RDM3培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成；
优选地，步骤(4)中所述的小分子添加物为6-bromoindirubin-3’-oxime(BIO)、 SU5402、Thiazovivin、Vitamin B3；
优选地，步骤(5)中所述的RDM4培养基由89％DMEM/F12、10％KSR、1％N2培养基、1％L-谷氨酰胺、10mM Vitamin B3组成；
优选地，步骤(6)中所述的RMM培养基由97％DMEM/F12、2％B27培养基、1％L-谷氨酰胺组成；
优选地，步骤(7)中所述的Trypsin-EDTA为37℃预热的0.25％Trypsin-EDTA；
优选地，步骤(7)中所述的Trypsin-EDTA的加入量为1mL；
优选地，步骤(7)中所述培养的条件为37℃、5％CO 2；
优选地，步骤(7)中所述培养的时间为10min。
优选地，步骤(8)中所述REM培养基的加入量为3mL；
优选地，所述终端分化细胞或其前体细胞包括视网膜色素上皮细胞、视锥细胞、视杆细胞、间充质干细胞、光感受器祖细胞、角膜上皮细胞、脉络膜内皮细胞、视网膜细胞、角膜细胞、晶状体细胞、神经节细胞、视神经细胞、脉络膜细胞；
更优选地，所述终端分化细胞或其前体细胞为视网膜色素上皮细胞。
本发明的第七方面提供了一种组合物。
进一步，所述组合物包含本发明第一方面所述的构建体、和/或本发明第二方面所述的表达载体或克隆载体、和/或本发明第三方面所述的工程改造的宿主细胞或其群体、和/或本发明第四方面所述的终端分化细胞或其前体细胞、或其群体。
本发明的第八方面提供了一种用于治疗和/或预防湿性年龄相关性黄斑变性的药物组合物。
进一步，所述药物组合物包含本发明第二方面所述的表达载体或克隆载体、和/或本发明第三方面所述的工程改造的宿主细胞或其群体、和/或本发明第四方面所述的终端分化细胞或其前体细胞、或其群体；
优选地，所述药物组合物还包括药学上可接受的载体和/或辅料；
优选地，所述药物组合物还包括一种或多种治疗剂；
更优选地，所述治疗剂包括肽、细胞因子、检查点抑制剂、丝裂原、生长因子、miRNA、dsRNA、单核血细胞、饲养细胞、饲养细胞组分或其置换因子、抗体、化学治疗剂、免疫调节药物。
本发明的第九方面提供了一种用于产生工程改造的宿主细胞或其群体、终端分化细胞或其前体细胞、或其群体的试剂盒。
进一步，所述试剂盒包含本发明第一方面所述的构建体、本发明第二方面所述的表达载体或克隆载体、靶向AAVS1的CRISPR/Cas载体、宿主细胞、一种或多种培养基；
优选地，所述靶向AAVS1的CRISPR/Cas载体为AAVS1 T2 CRIPR in pX330载体；
优选地，所述宿主细胞包括iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞；
更优选地，所述宿主细胞为iPSC；
优选地，所述培养基包括E8培养基、RDM1培养基、RDM2培养基、RDM3培养基、RDM4培养基、RMM培养基、REM培养基。
本发明的第十方面提供了如下任一方面的应用：
(1)本发明第一方面所述的构建体在制备表达载体或克隆载体中的应用；
(2)本发明第一方面所述的构建体在制备用于产生工程改造的宿主细胞或其群体、终端分化细胞或其前体细胞、或其群体的试剂盒中的应用；
(3)本发明第二方面所述的表达载体或克隆载体在制备工程改造的宿主细胞或其群体中的应用；
(4)本发明第二方面所述的表达载体或克隆载体在制备用于治疗和/或预防湿性年龄相关性黄斑变性的药物中的应用；
(5)本发明第二方面所述的表达载体或克隆载体在制备用于产生工程改造的宿主细胞或其群体、终端分化细胞或其前体细胞、或其群体的试剂盒中的应用；
(6)本发明第三方面所述的工程改造的宿主细胞或其群体在制备终端分化细胞或其前体细胞、或其群体中的应用；
(7)本发明第三方面所述的工程改造的宿主细胞或其群体在制备用于治疗和/或预防湿性年龄相关性黄斑变性的药物中的应用；
(8)本发明第四方面所述的终端分化细胞或其前体细胞、或其群体在制备用于治疗和/或预防湿性年龄相关性黄斑变性的药物中的应用；
(9)本发明第七方面所述的组合物在制备用于治疗和/或预防湿性年龄相关性黄斑变性的药物中的应用；
(10)本发明第八方面所述的药物组合物在治疗和/或预防湿性年龄相关性黄斑变性；
(11)本发明第九方面所述的试剂盒在产生工程改造的宿主细胞或其群体、终端分化细胞或其前体细胞、或其群体中的应用；
(12)CNTF和/或miR-126在制备治疗湿性年龄相关性黄斑变性的药物中的应用；
优选地，所述药物过表达CNTF和/或miR-126；
更优选地，所述药物过表达CNTF和miR-126；
优选地，所述药物包含过表达CNTF和/或miR-126表达的试剂；
优选地，所述药物包含过表达CNTF和miR-126表达的试剂。
本发明还提供了一种治疗和/或预防湿性年龄相关性黄斑变性的方法。
进一步，所述方法包括向有需要的受试者施用有效量的本发明第二方面所述的表达载体或克隆载体、和/或本发明第三方面所述的工程改造的宿主细胞或其群体、和/或本发明第四方面所述的终端分化细胞或其前体细胞、或其群体、和/或本发明第七方面所述的组合物、和/或本发明第八方面所述的药物组合物。
本发明还提供了一种在有需要的受试者的中表达CNTF和miR-126的方法。
进一步，所述方法包括向有需要的受试者的视网膜下递送有效量的本发明第二方面所述的表达载体或克隆载体、和/或本发明第三方面所述的工程改造的宿主细胞或其群体、和/或本发明第四方面所述的终端分化细胞或其前体细胞、或其群体。
相对于现有技术，本发明具有的优点和有益效果如下：
(1)本发明通过对经定点双基因基因编辑的iPSC进行诱导分化制备得到RPE，所述RPE进行细胞移植能够修复湿性年龄相关性黄斑变性患者已经损伤的CNV上的视网膜色素上皮细胞；
(2)基因编辑后的iPSC分化成RPE后，能够表达神经营养因子CNTF，CNTF不仅能够促进棒光感受器的存活，而且为视网膜神经节细胞(RGCs)提供了神经营养功能，此外，CNTF还有助于减少视网膜水肿，并通过从视网膜向脉络膜注入更多的液体来帮助视网膜重新附着到视网膜脱离的RPE，进而提高移植效率；
(3)基因编辑后的iPSC分化成RPE后，能够表达miR-126，miR-126能够抑制RPE细胞以及临近细胞的VEGF的表达，进而抑制脉络膜新生血管的产生；
综合上述3点，本发明制备得到的经定点双基因基因编辑的iPSC分化得到的RPE能够从根本上治疗湿性年龄相关性黄斑变性，解决了本领域目前面临的抗VEGF药物眼内注射剂价格昂贵、需多次重复注射使用、无法完全控制疾病进展，以及手术去除CNV的同时也去除了其下的RPE细胞、导致视网膜脉络膜萎缩等问题，具有非常好的临床应用前景。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示本发明构建的HA-CNTF-U6-MIR126-HA载体的核心结构示意图；
图2显示iPSC干性基因检测的结果图；
图3显示iPSC分化为RPE后形成黑色素沉淀细胞的结果图；
图4显示RPE细胞中CNTF mRNA相对表达水平的结果图；
图5显示RPE细胞中miR-126相对表达水平的结果图；
图6显示RPE上清对HUVEC细胞成管的影响的结果图，其中，a图：对照组的HUVEC细胞成管情况的结果图，b图：添加RPE上清后，HUVEC细胞成管情况的结果图。
具体实施方式
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1 HA-CNTF-U6-MIR126-HA载体的构建
1、实验材料
本发明实施例中所使用的试剂信息见表1。
表1本发明实施例中使用的试剂信息
Figure PCTCN2022107879-appb-000001
2、实验方法
(1)如图1所示，构建dornor载体；
(2)在dornor载体(pShuttle(addgene，Plasmid#16402))中插入AAVS1同源左臂HA-L(如SEQ ID NO:1所示)、CMV启动子(如SEQ ID NO:2所示)、CNTF基因序列(如SEQ ID NO:3所示)、polyA元件1(如SEQ ID NO:4所示)、U6启动子(如SEQ ID NO:5所示)、miR-126构架(如SEQ ID NO:6所示)、polyA元件2(如SEQ ID NO:7所示)、AAVS1同源右臂HA-R(如SEQ ID NO:8所示)，构建得到的dornor载体命名为HA-CNTF-U6-MIR126-HA；
其中，AAVS是人类19号染色体无意义区域，不影响干细胞干性以及分化的位点，均可进行基因编辑。
实施例2 iPSC细胞基因编辑
1、实验材料
本实施例中使用的iPSC细胞来源于北京呈诺医学科技有限公司。
2、实验方法
(1)将实施例1中构建得到的dornor载体与AAVS1 T2 CRIPR in pX330载体 (addgene,Plasmid#72833)通过电转的方式，导入到iPSC中；
(2)电转方法：100μL电转体系，每1×10 6个iPSC细胞、1g AAVS1 T2 CRIPR in pX330载体、1μg dornor载体，电压为1200V；
(3)电转后，使用E8培养基(赛默飞世尔科技有限公司，A1517001)培养细胞，并每天换液，培养条件为37℃，5％CO 2；
(4)培养48-72h后，使用嘌呤霉素筛选阳性细胞；
(5)抗性筛选5～7天后，待细胞稳定生长不再凋亡时，视克隆大小进行挑取单克隆置于96孔板中进行培养；
(6)视细胞生长状态，逐步将单克隆由96孔板转移至48孔板、24孔板、12孔板、6孔板中进行培养，扩增冻存等。
实施例3 iPSC定向诱导分化为RPE细胞
1、实验材料
本发明实施例2中通过基因编辑技术构建得到的iPSC细胞。
2、实验方法
(1)第0天，将iPSC细胞以1.0×10 3-5.0×10 5/cm 2铺种，本发明中以密度为5.0×10 3cells/cm 2铺种细胞，细胞培养在37℃、5％CO 2细胞培养箱中进行，使用的培养基为RDM1，RDM1的基础培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成，小分子添加物分别为noggin、XAV-939以及LY2109761；
(2)第1天到第6天，每日更换RDM1培养基；
(3)第7天到第12天，每日更换RDM2培养基，RDM2的基础培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成，小分子添加物分别为6-bromoindirubin-3’-oxime(BIO)、SU5402以及Thiazovivin；
(4)第13天到第17天，每日更换RDM3培养基，RDM3的基础培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成，小分子添加物分别为6-bromoindirubin-3’-oxime(BIO)、SU5402、Thiazovivin、Vitamin B3；
(5)第18天到第24天，每日更换RDM4培养基，RDM4的基础培养基由89％DMEM/F12、10％KSR、1％N2培养基、1％L-谷氨酰胺以及10mM Vitamin B3组成；
(6)第25天到第36天，每日更换RMM培养基，RMM的基础培养基由97％DMEM/F12、2％B27培养基、1％L-谷氨酰胺组成；
(7)第37天以后，吸去旧的培养基，用室温DPBS洗两遍，随后加入1mL 37℃预热过的0.25％Trypsin-EDTA，置于37℃、5％CO 2的细胞培养箱中培养10min，显微镜下观察单个细胞间出现空隙；
(8)弃去Trypsin-EDTA，加入3mL的REM培养基终止消化；
(9)使用35μM的过滤器过滤后，转移至15mL的离心管中，室温下1000rpm离心5min；
(10)弃去上清，用REM培养基轻轻吹打细胞，然后重悬，计数后铺板至matrigel包被的六孔板中；
(11)第38-51天，隔天更换一次REM培养基，直至收集细胞冻存。
实施例4免疫荧光检测实施例2中通过基因编辑技术构建得到的iPSC细胞
1、实验材料
本发明实施例2中通过基因编辑技术构建得到的iPSC细胞。
2、实验方法
(1)在24孔培养板中将已爬好细胞的玻片用PBS浸洗3次，每次浸洗的时间为3min；
(2)用4％的多聚甲醛室温下固定爬片5min，PBS浸洗玻片3次，每次浸洗的时间为3min；
(3)0.5％Triton X-100(5％BSA配制)室温通透20min(细胞膜上表达的抗原省略此步骤)；
(4)使用5％的BSA室温封闭1小时；
(5)去除封闭液，每孔添加400μL一抗，4℃过夜孵育；
(6)12小时后，加荧光二抗：PBST浸洗3次，每次浸洗的时间为5min，添加稀释好的荧光二抗，室温孵育1h，PBST浸洗3次，每次5min；
(7)复染核：滴加DAPI避光条件下孵育5min，对标本进行染核，PBST 5min×4次洗去多余的DAPI；
(8)添加500μL PBS，上机拍片。
3、实验结果
实验结果见图2，结果显示NANOG、OCT4、SOX2、SSEA4、TRA-1-60、均为阳性，表明了经基因编辑之后，iPSC的干性没有受到影响。
实施例5 qPCR检测实施例3中iPSC定向诱导分化得到的RPE细胞中基因的表达水平
1、实验材料
本发明实施例3中由iPSC定向诱导分化得到的RPE细胞。
2、实验方法
(1)收集200W细胞加1mL TRIZOL，提取RNA并测定RNA浓度，取1μg RNA逆转录为cDNA，按如表2所述的体系进行预混；
其中，具体引物的序列如下：
CNTF基因：
正向引物为5’-GAAGATTCGTTCAGACCTGACTG-3’(SEQ ID NO:9)
反向引物为5’-AAGGTTCTCTTGGAGTCGCTC-3’(SEQ ID NO:10)
miR-126基因：
正向引物为5’-TATGGTTGTTCTCGACTCCTTCAC-3’(SEQ ID NO:11)
反向引物为5’-TCGTCTGTCGTACCGTGAGTAAT-3’(SEQ ID NO:12)
表2体系构成
Figure PCTCN2022107879-appb-000002
(2)将上述体系放入Light cycler仪器中按照3步法进行反应，循环数为45，反应体系见表3。
表3反应体系
Figure PCTCN2022107879-appb-000003
3、实验结果
结果显示，iPSC细胞定向诱导分化为RPE细胞后形成黑色素沉淀细胞(见图3)，诱导分化得到的RPE细胞大量表达基因CNTF和miR-126(见图4和图5)。
实施例6实施例3中iPSC定向诱导分化得到的RPE细胞上清对HUVEC细胞成管的影响
1、实验材料
本发明实施例3中由iPSC定向诱导分化得到的RPE细胞。
2、实验方法
(1)取适量的HUVEC细胞(promocell)悬液，调整细胞密度为5×10 5/mL；
(2)用200μL的枪头，以每孔100μL的细胞悬液，加入到matrigel包被的96孔板中；
(3)一半细胞使用HUVEC培养基(promocell)(对照组)，另一半细胞使用1：1的RPE上清与HUVEC培养基(实验组)；
(4)细胞培养板置于37℃、5％CO 2培养箱中，静置培养24h；
(5)24h后，观察成管结果。
3、实验结果
结果显示，对照组的HUVEC细胞正常成管(见图6a)，而添加本发明实施例3中由iPSC定向诱导分化得到的RPE细胞上清后，HUVEC细胞不能成管(见图6b)，表明了RPE细胞上清抑制内皮细胞成管，进一步证明了本发明由iPSC定向诱导分化得到的RPE细胞具有抑制细胞血管形成的能力。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (10)

  1. 一种用于工程改造细胞以获得基因编辑的细胞的构建体，其特征在于，所述构建体包含编码神经营养因子或其类似物基因的核苷酸、和/或抗血管生成核酸的核苷酸；
    优选地，所述神经营养因子或其类似物包括CNTF、NGF、BDNF、NT-3、NT-4/5、NT-6、PEDF、GDNF、GMFB、NRG1、CHRNB3、NTR 368；
    优选地，所述抗血管生成核酸包括miR-126、miR-15/107家族、miR-17～92家族、miR-21、miR-132、miR-296、miR-378、miR-519c、miR-210、miR-222、miR-100、miR-23、miR-27、miR-31、miR-150、miR-146a、miR-23a；
    更优选地，所述神经营养因子或其类似物为CNTF；
    更优选地，所述抗血管生成核酸为miR-126；
    最优选地，所述构建体包含编码CNTF基因的核苷酸、和miR-126的核苷酸；
    最优选地，所述编码CNTF基因的核苷酸的序列如SEQ ID NO:3所示；
    最优选地，所述miR-126的核苷酸的序列如SEQ ID NO:6所示；
    优选地，所述构建体还包含一个或多个外源启动子、和/或所选位点中所含的一个或多个内源启动子；
    更优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的一个或多个外源启动子、或连接到所选位点中所含的一个或多个内源启动子；
    最优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的一个或多个外源启动子；
    最优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的两个外源启动子；
    最优选地，所述外源启动子包括CMV启动子、U6启动子、EF1α启动子、PGK启动子、CAG启动子、UBC启动子、SV40启动子、Human beta actin启动子、TEF1启动子、GDS启动子、H1启动子、U6启动子、T7启动子、TERT启动子、RSV启动子、PGK1启动子；
    最优选地，所述外源启动子为CMV启动子、U6启动子；
    最优选地，所述CMV启动子的核苷酸序列如SEQ ID NO:2所示；
    最优选地，所述U6启动子的核苷酸序列如SEQ ID NO:5所示；
    优选地，所述构建体还包含一对同源臂；
    更优选地，所述同源臂对所述所选位点具有特异性；
    更优选地，所述同源臂包括特异性靶向所述所选位点的同源左臂HA-L、同源右臂HA-R；
    最优选地，所述同源左臂HA-L的核苷酸序列如SEQ ID NO:1所示；
    最优选地，所述同源右臂HA-R的核苷酸序列如SEQ ID NO:8所示；
    优选地，所述构建体还包含一个或多个外源终止子、和/或所选位点中所含的一个或多个内源终止子；
    更优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的一个或多个外源终止子、和/或所选位点中所含的一个或多个内源终止子；
    最优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的一个或多个外源终止子；
    最优选地，所述编码CNTF基因的核苷酸和miR-126的核苷酸可操作地连接到所述构建体中所含的两个外源终止子；
    最优选地，所述外源终止子包括PolyA终止子、NOS终止子、T7转录终止子、rrnB终止子、T0终止子、SV40终止子、hGH终止子、BGH终止子、rbGlob终止子；
    最优选地，所述外源终止子为PolyA终止子；
    最优选地，所示PolyA终止子包括PolyA元件1、PolyA元件2；
    最优选地，所述PolyA元件1的核苷酸序列如SEQ ID NO:4所示；
    最优选地，所述PolyA元件2的核苷酸序列如SEQ ID NO:7所示；
    最优选地，所述所选位点为安全港基因座；
    最优选地，所述安全港基因座包括AAVS1、CCR5、ROSA26、HTRP、H11、TCR、RUNX1、β-2微球蛋白、胶原蛋白、GAPDH，或满足基因组安全港准则的基因座；
    最优选地，所述安全港基因座为AAVS1；
    最优选地，所述构建体由同源左臂HA-L、CMV启动子、编码CNTF基因的核苷酸、PolyA元件1、U6启动子、miR-126的核苷酸、PolyA元件2、同源右臂HA-R依次串联而成。
  2. 一种表达载体或克隆载体，其特征在于，所述表达载体包含权利要求1所述的构建体，所述克隆载体包含编码神经营养因子或其类似物基因的核苷酸、和/或抗血管生成核酸的核苷酸；
    优选地，所述神经营养因子或其类似物包括CNTF、NGF、BDNF、NT-3、NT-4/5、NT-6、PEDF、GDNF、GMFB、NRG1、CHRNB3、NTR 368；
    优选地，所述抗血管生成核酸包括miR-126、miR-15/107家族、miR-17～92家族、miR-21、miR-132、miR-296、miR-378、miR-519c、miR-210、miR-222、miR-100、miR-23、miR-27、miR-31、miR-150、miR-146a、miR-23a；
    更优选地，所述神经营养因子或其类似物为CNTF；
    更优选地，所述抗血管生成核酸为miR-126；
    最优选地，所述克隆载体包含编码CNTF基因的核苷酸、和miR-126的核苷酸；
    最优选地，所述编码CNTF基因的核苷酸的序列如SEQ ID NO:3所示；
    最优选地，所述miR-126的核苷酸的序列如SEQ ID NO:6所示；
    优选地，所述表达载体还包含载体；
    更优选地，所述载体包括dornor载体、DNA载体、病毒载体；
    最优选地，所述DNA载体包括DNA质粒载体、结合DNA质粒的脂质体、结合DNA质粒的分子耦联体、结合DNA质粒的多聚物；
    最优选地，所述病毒载体包括腺病毒载体、腺相关病毒载体、慢病毒载体、逆转录病毒载体、单纯疱疹病毒载体、杆状病毒载体、仙台病毒载体、痘病毒载体、双生病毒载体；
    优选地，所述克隆载体还包含载体；
    更优选地，所述载体包括DNA质粒载体、噬菌体载体、酵母人工染色体载体、噬菌体-质粒杂合载体。
  3. 一种工程改造的宿主细胞或其群体，其特征在于，所述工程改造的宿主细胞或其群体表达CNTF和/或miR-126；
    优选地，所述工程改造的宿主细胞或其群体过表达CNTF和/或miR-126；
    更优选地，所述工程改造的宿主细胞或其群体过表达CNTF和miR-126；
    优选地，所述工程改造的宿主细胞或其群体包含权利要求1所述的构建体、和/或权利要求2所述的表达载体或克隆载体；
    优选地，所述宿主细胞包括iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞；
    更优选地，所述宿主细胞为iPSC。
  4. 一种终端分化细胞或其前体细胞、或其群体，其特征在于，所述终端分化细胞或其前体细胞、或其群体表达CNTF和/或miR-126；
    优选地，所述终端分化细胞或其前体细胞、或其群体过表达CNTF和/或miR-126；
    更优选地，所述终端分化细胞或其前体细胞、或其群体过表达CNTF和miR-126；
    优选地，所述终端分化细胞或其前体细胞、或其群体为将权利要求3所述的工程改造的宿主细胞或其群体诱导分化得到的；
    优选地，所述终端分化细胞或其前体细胞包括视网膜色素上皮细胞、视锥细胞、视杆细胞、间充质干细胞、光感受器祖细胞、角膜上皮细胞、脉络膜内皮细胞、视网膜细胞、角膜细胞、晶状体细胞、神经节细胞、视神经细胞、脉络膜细胞；
    更优选地，所述终端分化细胞或其前体细胞为视网膜色素上皮细胞。
  5. 一种权利要求3所述的工程改造的宿主细胞或其群体的制备方法，其特征在于，所述方法包括如下步骤：向宿主细胞中递送权利要求2中所述的表达载体和靶向所述所选位点的CRISPR/Cas载体；
    优选地，所述所选位点为安全港基因座；
    更优选地，所述安全港基因座包括AAVS1、CCR5、ROSA26、HTRP、H11、TCR、RUNX1、β-2微球蛋白、胶原蛋白、GAPDH，或满足基因组安全港准则的基因座；
    最优选地，所述安全港基因座为AAVS1；
    最优选地，所述靶向所述所选位点的CRISPR/Cas载体为靶向AAVS1的CRISPR/Cas载体；
    最优选地，所述靶向AAVS1的CRISPR/Cas载体为AAVS1 T2 CRIPR in pX330载体；
    优选地，所述递送是通过将权利要求2中所述的表达载体导入到宿主细胞中实现的；
    更优选地，所述导入的方式包括电转染法、显微注射法、超声波介导法、声穿孔法、光穿孔法、磁转法、热激法、磷酸钙法、脂质体和聚合物法、纳米粒子法、病毒转化法；
    最优选地，所述导入的方式为电转染法；
    最优选地，所述电转染法中的电转染体系的组成如下：100μL电转体系、每1×10 6个宿主细胞、1g AAVS1 T2 CRIPR in pX330载体、1μg权利要求2中所述的表达载体；
    最优选地，所述电转染法中所采用的电压为1200V；
    优选地，所述方法还包括对递送权利要求2中所述的表达载体和靶向所述所选位点的CRISPR/Cas载体后的宿主细胞进行培养和筛选；
    更优选地，所述培养采用的培养基为E8培养基、每天换液；
    更优选地，所述培养的条件为37℃、5％CO 2；
    更优选地，所述培养的时间为48-72h；
    更优选地，所述筛选采用嘌呤霉素进行筛选；
    更优选地，所述筛选的时间为5-7天；
    优选地，所述宿主细胞包括iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞；
    更优选地，所述宿主细胞为iPSC。
  6. 一种权利要求4所述的终端分化细胞或其前体细胞、或其群体的制备方法，其特征在于，所述方法包括如下步骤：将权利要求3所述的工程改造的宿主细胞或其群体进行诱导分化；
    优选地，所述诱导分化包括如下步骤：
    (1)第0天，将权利要求3所述的工程改造的宿主细胞或其群体在添加有小分子添加物的RDM1培养基中进行培养；
    (2)第1-6天，每日更换RDM1培养基；
    (3)第7-12天，更换培养基为添加有小分子添加物的RDM2培养基，每日 更换RDM2培养基；
    (4)第13-17天，更换培养基为添加有小分子添加物的RDM3培养基，每日更换RDM3培养基；
    (5)第18-24天，更换培养基为RDM4培养基，每日更换RDM4培养基；
    (6)第25-36天，更换培养基为RMM培养基，每日更换RMM培养基；
    (7)第37天以后，弃去原培养基，加入Trypsin-EDTA，进行培养；
    (8)弃去Trypsin-EDTA，加入REM培养基终止消化；
    优选地，步骤(1)中所述的权利要求3所述的工程改造的宿主细胞或其群体的细胞密度为1.0×10 3-5.0×10 5/cm 2；
    更优选地，步骤(1)中所述的权利要求3所述的工程改造的宿主细胞或其群体的细胞密度为5.0×10 3/cm 2；
    优选地，步骤(1)中所述的培养的条件为37℃、5％CO 2；
    优选地，步骤(1)中所述的RDM1培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成；
    优选地，步骤(1)中所述的小分子添加物为noggin、XAV-939、LY2109761；
    优选地，步骤(3)中所述的RDM2培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成；
    优选地，步骤(3)中所述的小分子添加物为6-bromoindirubin-3’-oxime(BIO)、SU5402、Thiazovivin；
    优选地，步骤(4)中所述的RDM3培养基由88％DMEM/F12、10％KSR、5mM Monothioglycerol Solution、1％Chemically Defned Lipid Concentrate、1％L-谷氨酰胺组成；
    优选地，步骤(4)中所述的小分子添加物为6-bromoindirubin-3’-oxime(BIO)、SU5402、Thiazovivin、Vitamin B3；
    优选地，步骤(5)中所述的RDM4培养基由89％DMEM/F12、10％KSR、1％N2培养基、1％L-谷氨酰胺、10mM Vitamin B3组成；
    优选地，步骤(6)中所述的RMM培养基由97％DMEM/F12、2％B27培养基、1％L-谷氨酰胺组成；
    优选地，步骤(7)中所述的Trypsin-EDTA为37℃预热的0.25％Trypsin-EDTA；
    优选地，步骤(7)中所述的Trypsin-EDTA的加入量为1mL；
    优选地，步骤(7)中所述培养的条件为37℃、5％CO 2；
    优选地，步骤(7)中所述培养的时间为10min。
    优选地，步骤(8)中所述REM培养基的加入量为3mL；
    优选地，所述终端分化细胞或其前体细胞包括视网膜色素上皮细胞、视锥细 胞、视杆细胞、间充质干细胞、光感受器祖细胞、角膜上皮细胞、脉络膜内皮细胞、视网膜细胞、角膜细胞、晶状体细胞、神经节细胞、视神经细胞、脉络膜细胞；
    更优选地，所述终端分化细胞或其前体细胞为视网膜色素上皮细胞。
  7. 一种组合物，其特征在于，所述组合物包含权利要求1所述的构建体、和/或权利要求2所述的表达载体或克隆载体、和/或权利要求3所述的工程改造的宿主细胞或其群体、和/或权利要求4所述的终端分化细胞或其前体细胞、或其群体。
  8. 一种用于治疗和/或预防湿性年龄相关性黄斑变性的药物组合物，其特征在于，所述药物组合物包含权利要求2所述的表达载体或克隆载体、和/或权利要求3所述的工程改造的宿主细胞或其群体、和/或权利要求4所述的终端分化细胞或其前体细胞、或其群体；
    优选地，所述药物组合物还包括药学上可接受的载体和/或辅料；
    优选地，所述药物组合物还包括一种或多种治疗剂；
    更优选地，所述治疗剂包括肽、细胞因子、检查点抑制剂、丝裂原、生长因子、miRNA、dsRNA、单核血细胞、饲养细胞、饲养细胞组分或其置换因子、抗体、化学治疗剂、免疫调节药物。
  9. 一种用于产生工程改造的宿主细胞或其群体、终端分化细胞或其前体细胞、或其群体的试剂盒，其特征在于，所述试剂盒包含权利要求1所述的构建体、权利要求2所述的表达载体或克隆载体、靶向AAVS1的CRISPR/Cas载体、宿主细胞、一种或多种培养基；
    优选地，所述靶向AAVS1的CRISPR/Cas载体为AAVS1 T2 CRIPR in pX330载体；
    优选地，所述宿主细胞包括iPSC、胚胎干细胞、间充质干细胞、成体干细胞、组织特异性干细胞；
    更优选地，所述宿主细胞为iPSC；
    优选地，所述培养基包括E8培养基、RDM1培养基、RDM2培养基、RDM3培养基、RDM4培养基、RMM培养基、REM培养基。
  10. 如下任一方面的应用，其特征在于，所述应用包括：
    (1)权利要求1所述的构建体在制备表达载体或克隆载体中的应用；
    (2)权利要求1所述的构建体在制备用于产生工程改造的宿主细胞或其群体、终端分化细胞或其前体细胞、或其群体的试剂盒中的应用；
    (3)权利要求2所述的表达载体或克隆载体在制备工程改造的宿主细胞或其群体中的应用；
    (4)权利要求2所述的表达载体或克隆载体在制备用于治疗和/或预防湿性年龄相关性黄斑变性的药物中的应用；
    (5)权利要求2所述的表达载体或克隆载体在制备用于产生工程改造的宿主 细胞或其群体、终端分化细胞或其前体细胞、或其群体的试剂盒中的应用；
    (6)权利要求3所述的工程改造的宿主细胞或其群体在制备终端分化细胞或其前体细胞、或其群体中的应用；
    (7)权利要求3所述的工程改造的宿主细胞或其群体在制备用于治疗和/或预防湿性年龄相关性黄斑变性的药物中的应用；
    (8)权利要求4所述的终端分化细胞或其前体细胞、或其群体在制备用于治疗和/或预防湿性年龄相关性黄斑变性的药物中的应用；
    (9)权利要求7所述的组合物在制备用于治疗和/或预防湿性年龄相关性黄斑变性的药物中的应用；
    (10)权利要求8所述的药物组合物在治疗和/或预防湿性年龄相关性黄斑变性；
    (11)权利要求9所述的试剂盒在产生工程改造的宿主细胞或其群体、终端分化细胞或其前体细胞、或其群体中的应用；
    (12)CNTF和/或miR-126在制备治疗湿性年龄相关性黄斑变性的药物中的应用；
    优选地，所述药物过表达CNTF和/或miR-126；
    更优选地，所述药物过表达CNTF和miR-126；
    优选地，所述药物包含过表达CNTF和/或miR-126表达的试剂；
    优选地，所述药物包含过表达CNTF和miR-126表达的试剂。
PCT/CN2022/107879 2021-08-05 2022-07-26 以iPSC来源的细胞为载体、基因治疗湿性年龄相关性黄斑变性 WO2023011248A1 (zh)

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