WO2023227043A1 - 编码rpgr的核酸及其应用 - Google Patents

编码rpgr的核酸及其应用 Download PDF

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WO2023227043A1
WO2023227043A1 PCT/CN2023/096146 CN2023096146W WO2023227043A1 WO 2023227043 A1 WO2023227043 A1 WO 2023227043A1 CN 2023096146 W CN2023096146 W CN 2023096146W WO 2023227043 A1 WO2023227043 A1 WO 2023227043A1
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rpgr
nucleic acid
vector
plasmid
adeno
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French (fr)
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李斌
李秋棠
程超
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武汉纽福斯生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the present invention relates to the field of genetic engineering technology, and in particular to nucleic acid encoding RPGR and its application.
  • Retinitis pigmentosa is a phenotypic linkage group of hereditary retinal dystrophies. This disease will cause the patient's vision to gradually decrease. The incidence of RP is 1/4000 to 1/3000. Early symptoms of RP include decreased night vision and peripheral vision. As the disease progresses, central vision and color vision may also be affected. The age of onset of symptoms of RP is variable, but usually occurs between the ages of 10 and 30, and the rate of progression varies from person to person.
  • RP is caused by mutations in one or several genes related to eye health and function. Among all single genes that cause RP, X-linked lesions due to defects in the retinitis pigmentosa GTPase regulatory gene (RPGR) are the most common. X-linked retinitis pigmentosa (XLRP) is considered the most severe form of retinitis pigmentosa. About 70% of XLRP are caused by RPGR mutations. RPGR is located in the connecting cilia of photoreceptor cells and plays a role in protein transport. More than 300 RPGR mutations have been discovered so far. RPGR has multiple splicing isoforms. Among them, RPGR-ORF15 is mainly expressed in the photoreceptor cells of the retina. RPGR gene mutations cause the cone and rod photoreceptor cells of patients to begin to develop in childhood. Degeneration and degeneration.
  • RPGR X-linked lesions due to defects in the retinitis pigmentosa GTP
  • the RPGR gene is highly mutagenic, and mutations produced in the body increase the likelihood of disease. Therefore, the nature of this mutation also makes it more difficult to clone the sequence encoding the RPGR protein into a vector in gene therapy. In fact, previously developed strategies for XLRP gene replacement therapy have also been hampered by such factors.
  • the technical problem to be solved by the present invention is to provide nucleic acids encoding RPGR and their applications.
  • the nucleic acid encoding RPGR provided by the invention includes at least one of I) to IV):
  • nucleic acid that replaces, deletes or adds one or more nucleotides in the fragment described in I);
  • nucleic acid that has at least 90% homology with the sequence of the nucleic acid described in I) and encodes RPGR;
  • nucleic acid sequence encoding RPGR is shown in SEQ ID NO: 1.
  • the present invention also provides a recombinant vector, which includes a skeleton vector and the nucleic acid of the present invention.
  • the backbone vector in the recombinant vector is a viral vector.
  • the virus The vector is selected from at least one of a lentiviral vector, an adenovirus vector, and an adeno-associated virus vector; wherein, the serotypes of the adeno-associated virus vector are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV2.7M8 or AAV2-TYF mutant.
  • the backbone vector contains the RK1 promoter and/or the SV40 intron.
  • the recombinant vector includes the sequentially connected RK1 promoter, SV40 intron, nucleic acid shown in SEQ ID NO: 1, and SV40 poly(A) signal.
  • the recombinant vector includes sequentially connected RK1 promoter, SV40 intron, the nucleic acid shown in SEQ ID NO: 1, and SV40 poly(A) signal.
  • the nucleic acid sequence of the RK1 promoter is shown in SEQ ID NO: 4, and the nucleic acid sequence of the SV40 intron is shown in SEQ ID NO: 5.
  • the present invention also provides a plasmid combination, which includes the recombinant vector, an auxiliary functional plasmid and an accessory functional plasmid.
  • the auxiliary function plasmid is pAdHelper; the accessory function plasmid is pAAV-r2c5.
  • the present invention also provides a method for preparing an adeno-associated virus expressing RPGR, which includes: transfecting the plasmid combination into a host cell, and obtaining an adeno-associated virus expressing RPGR through purification.
  • the host cell is 293 cells or 293T cells.
  • the adeno-associated virus expressing RPGR is prepared by the preparation method of the present invention.
  • the eye disease is a disease caused by RPGR mutation.
  • the eye disease is retinitis pigmentosa.
  • the prevention and treatment includes repairing retinal structure, increasing the number of visual cells and/or improving eye function.
  • the present invention also provides a medicine, which includes the recombinant vector of the present invention, or plasmid combination, or adeno-associated virus.
  • the medicine of the present invention also includes pharmaceutically acceptable carriers and excipients.
  • the excipients are nanocarriers and/or liposomes
  • the dosage form of the medicine of the present invention is an injection, wherein the content of the adeno-associated virus is 1 ⁇ 10 9 to 1 ⁇ 10 16 viruses/ml.
  • the content of the adeno-associated virus is 1 ⁇ 10 12 to 1 ⁇ 10 14 viruses/ml.
  • the titer of the adeno-associated virus is 1 ⁇ 10 13 vg/mL.
  • the drug administration methods of the present invention include subretinal injection, intravitreal injection, anterior chamber injection or subconjunctival injection.
  • the medicines described in the present invention also include other medicines with the activity of improving retinitis pigmentosa.
  • the present invention also provides a method for preventing and treating retinitis pigmentosa, which involves administering the medicine described in the present invention.
  • the present invention provides a nucleic acid encoding RPGR, and a vector and adeno-associated virus containing the nucleic acid.
  • the coding nucleic acid has been specially optimized to significantly increase the expression of RPGR, which can be used to treat X-linked retinitis pigmentosa caused by RPGR mutations.
  • AAV-RPGR drugs can significantly improve the eye lesions of mice with X-linked retinitis pigmentosa caused by RPGR mutations.
  • Figure 1A ?? Figure 1D is an alignment of the codon-optimized RPGR ORF15 and wild-type sequences. The differential codon sequences after optimization are bolded and underlined;
  • Figure 2 is the AAV-RPGR vector map:
  • A is a schematic diagram of the codon-optimized RPGR ORF15 plasmid vector.
  • the vector contains AAV2 5'ITR, RK1 promoter, SV40 intron, and after codon optimization, RPGR ORF15, SV40 polyA sequence and AAV2 3'ITR;
  • B is a schematic diagram of the wild-type RPGR ORF15 plasmid vector.
  • the vector contains AAV2 5'ITR, RK1 promoter, SV40 intron, wild-type RPGR ORF15, SV40 polyA sequence and AAV2 3'ITR;
  • Figure 3 shows the cloning and enzyme digestion verification of the AAV-RPGR vector
  • A shows the constructed optimized AAV-RPGR ORF15 plasmid transformed into Stbl3 E. coli strain
  • B shows the constructed wild-type AAV-RPGR ORF15 plasmid transformed into Stbl3 large intestine Bacillus strain
  • C is to select a single clone and extract the plasmid and identify it by double enzyme digestion with HindIII and XhoI to verify the transformation efficiency and sequence integrity of the codon-optimized and wild-type vectors
  • 1-5 Codon-optimized plasmid enzyme Digestion results
  • M 10kb Marker
  • 6-10 Wild-type plasmid digestion results
  • Figure 5 shows the verification of AAV-mediated RPGRopt1 protein expression levels in mice
  • Figure 6 is a comparison of RPGR expression efficiency in mice. Immunofluorescence staining was used to observe the distribution of RPGR protein in the retina. A is the staining of RPGR antibody after retinal spreading to observe the distribution and density of RPGR positive signals in the retina. B is RPGR positive cells. The overall proportion of the retina;
  • Figure 7 shows the tissue localization of RPGR in mice
  • Figure 8 shows the therapeutic effect of AAV5-RPGRopt gene therapy on X-linked retinal pigment abnormalities caused by RPGR mutations.
  • B is the drug injection of RPGR knockout mice 18 months after injection.
  • the present invention provides nucleic acids encoding RPGR and their applications. Those skilled in the art can learn from the contents of this article and appropriately improve the process parameters for implementation. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the present invention. Invent technology.
  • RPGR refers to the retinitis pigmentosa GTPase regulatory gene, which is the most common causative gene of As shown in ID NO: 2, this sequence is from human origin.
  • the present invention is directed to the product shown in SEQ ID NO: 2
  • the sequence was optimized to significantly improve the ocular lesions of mice deficient in X-linked retinitis pigmentosa caused by RPGR mutation.
  • the nucleic acid encoding RPGR provided by the present invention is shown in SEQ ID NO: 1 (opt1) or SEQ ID NO: 3 (opt2).
  • the nucleotide sequence shown in SEQ ID NO: 1 is:
  • the results show that compared with other optimized sequences, the nucleic acid shown in SEQ ID NO: 1 has an improved transcription level and a significantly increased expression level, which can more effectively treat retinitis pigmentosa. therapeutic effect.
  • the effect of wild-type or other optimized sequences is not as good as the nucleic acid shown in SEQ ID NO: 1.
  • the present invention uses adeno-associated virus (AAV) vectors for gene replacement therapy, which has been proven to be effective in rescuing retinal function and structure in many animal models of retinal degeneration.
  • AAV adeno-associated virus
  • the plasmid was extracted and sequenced for identification to verify the efficiency of RPGR sequence cloning and the integrity and consistency of the RPGR sequence; secondly, the plasmid was transfected into small cells In the mouse cone cell line 661W, the expression efficiency of the RPGR protein was higher than that of the wild-type protein; the RPGR protein controlled by the RK1 promoter was packaged with the virus and infected 661W cells, and its expression increased with the increase of the virus dose.
  • test materials used in this invention are all common commercial products and can be purchased in the market.
  • the present invention will be further described below in conjunction with the examples:
  • the wild-type RPGR ORF15 coding sequence was codon-optimized.
  • the two optimizations were designated RPGRopt1 and RPGRopt2 respectively.
  • the comparison between RPGRopt1 and wild-type is shown in Figure 1A- Figure 1D, and the corresponding AAV vector plasmid was constructed ( Figure 2) .
  • Plasmids (200ng/ ⁇ L) containing codon-optimized RPGR ORF15 sequences and wild-type RPGR ORF15 sequences were transformed into E. coli Stbl3 strains and spread on LB plates added with corresponding antibiotics. After overnight culture, the growth of single colonies was observed.
  • the plasmids were extracted separately and the band size was observed by electrophoresis after double enzyme digestion. It was found that the optimized plasmid digestion result RPGR ORF15 maintained the full size, while the wild-type plasmid digestion result showed that RPGR ORF15 failed to maintain the full size (C in Figure 3), indicating that the codon The optimized RPGR ORF15 has higher fidelity during cloning than the wild type.
  • Example 2 AAV-mediated expression of RPGR in cells in vitro and in mice
  • the plasmids containing the codon-optimized RPGR ORF15 sequence and the wild-type RPGR ORF15 sequence were 661W cells were transfected, cells were lysed 48 hours later, and protein expression was confirmed by Western Blot after protein extraction.
  • ECL reagents to detect proteins. Take 1 ml of each and mix well, drop it on the surface of the protein membrane, and incubate in the dark for 1-2 minutes. Use tweezers to place the protein film neatly on the plastic paper and place it on the gel imager for exposure.
  • the optimized AAV-RPGRopt1 vector packaged the AAV5 serotype virus, and infected 661W cells at different multiplicities of infection (MOI). After 72 hours, the cells were lysed to extract RNA, and qPCR was used to confirm the mRNA expression. The results showed that the expression level of RPGRopt1 mRNA in vitro varied with the virus. It increased with increasing dose, and there was an obvious dose-dependent effect (B in Figure 4, Table 2).
  • mice 4. Virus packaging, virus drug injection into mice:
  • HEK293T cells with a polymerization degree of more than 90% are transferred to the plate at a ratio of 1:3.
  • the AAV5-RPGRopt1 viral drug was administered into the eyes of 6-8 week old C57 mice by subretinal injection, and then the retina was removed 4 weeks after the administration, and the protein in the tissue was extracted for Western Blot detection to observe the expression of RPGR in the mice. Expression in retinal tissue.
  • the expression of RPGR protein can be detected in the treated eyes of knockout mice, but the corresponding protein expression cannot be detected in the control eye tissue, and the protein expression level also has a linear growth relationship with the dosage.
  • the retinal tiling results show that the optimized RPGRopt1 drug has a larger distribution area in the retina, and the density of positive signals per unit area is higher than that of the wild-type RPGR drug and the optimized sequence RPGRopt2 (A in Figure 6). It means that the protein abundance is higher; after calculation, the expression ratio of the wild-type drug in the retina is 7.82%, the expression ratio of the optimized drug RPGRopt2 in the retina is 15.7%, and the expression ratio of the optimized drug RPGRopt1 in the retina is The proportion is 36.3% (B in Figure 6, Table 3).
  • the sectioning results show that the RPGR protein (red fluorescence signal, indicated by the arrow) can be correctly positioned in the inner segment (IS) of the photoreceptor cell, and the optimized drug opt1 signal is stronger than the optimized drug opt2 and the unoptimized drug. .
  • Example 3 AAV-RPGRopt1 gene therapy improves ocular function and repairs retinal structure in RPGR knockout mice
  • Example 1-2 confirms the correct expression of the codon-optimized RPGRORF15 coding sequence controlled by the RK1 promoter in vitro and in vivo, in order to further prove the AAV5-RPGRopt1 gene therapy drug for the treatment of X-chromosome-linked retinitis pigmentosa caused by RPGR mutations. Effect, in vivo experiments were conducted using the RPGR knockout mouse model, and the improvement of eye lesions in mice after drug treatment was observed 18 months after injection.
  • mice Under dark adaptation conditions, let the mice adapt to darkness overnight. Use LED lights to give brief flash stimulation with an intensity of -2 to +3 log sc cd.s/m 2. Record the dark adaptation ERG. Depending on the intensity of the stimulation, 3 to 60 Responses were recorded at intervals of seconds.
  • the LED light gives a brief flash stimulus of -0.5 to +2 log sc cd.s/m 2 intensity, and the light adaptation ERG is recorded, and the response is recorded at 2-minute intervals.
  • the mouse eye tissue was then used for OCT analysis.
  • the measured thickness of this layer of retina of different mice was sorted into EXCEL, the average value was calculated and analyzed.
  • the AAV-RPGRopt1 gene therapy drug can compensate for the adverse effects of gene deletion on eye lesions by providing a protein that can function normally in RPGR knockout mice.
  • the AAV-RPGRopt1 gene therapy drug has an effect on RPGR mutations.
  • the therapeutic effect of X-linked retinitis pigmentosa caused by this disease lays the foundation for further clinical application development.

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Abstract

提供了一种编码RPGR的核酸、含有该核酸的载体及腺相关病毒。该核酸经过特殊优化而使RPGR的表达量显著提高，从而可以用于治疗RPGR突变引起的X染色体连锁视网膜色素变性。

Description

编码RPGR的核酸及其应用
本申请要求于2022年05月27日提交中国专利局、申请号为202210586471.X、发明名称为“编码RPGR的核酸及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因工程技术领域，尤其涉及编码RPGR的核酸及其应用。
背景技术
视网膜色素变性(RP)是遗传性视网膜营养不良的一个表型连锁群，该病症会导致病人的视力逐渐下降，RP的发病率为1/4000到1/3000。RP的早期症状包括夜间视力和周边视力下降。随着疾病的进一步发展，中央视觉和彩色视觉也可能受到影响。RP发病年龄症状是可变的，但通常在10岁到30岁之间，恶化的速度也因人而异。
RP是由一种或几种与眼睛的健康和功能相关的基因突变引起的。在所有导致RP的单基因中，由于视网膜色素变性GTPase调节基因(RPGR)的缺陷导致的X染色体连锁病变是最常见的。X染色体连锁视网膜色素变性(XLRP)被认为是最严重的视网膜色素变性。XLRP中有约70％由RPGR突变引起。RPGR定位在感光器细胞的连接纤毛，在蛋白运输中发挥作用。目前已发现超过300种的RPGR突变，RPGR有多种剪接异构体，其中RPGR-ORF15主要表达在视网膜的感光器细胞中，RPGR基因突变导致患者在儿童时期视锥细胞和视杆细胞即开始变性和退化。
RPGR基因具有高度的突变性，这种体内产生的突变增加了致病的可能性。由此，这种突变性质也导致在基因治疗中将编码RPGR蛋白的序列克隆到载体上的难度增大。实际上，以前开发的XLRP基因替代疗法的策略也受到了这类因素的阻碍。
因此，开发一种高效、特异性的药物是满足当下在治疗RPGR突变引起的X染色体连锁视网膜色素变性方面的迫切需求的。
发明内容
有鉴于此，本发明要解决的技术问题在于提供编码RPGR的核酸及其应用。
本发明提供的编码RPGR的核酸，包括I)～IV)中至少一种：
I)、具有如SEQ ID NO：1所示核苷酸序列的核酸；
II)、在I)所述的片段中取代、缺失或添加一个或多个核苷酸的核酸；
III)、与I)所述核酸的序列至少具有90％的同源性，且编码RPGR的核酸；
IV)、与I)～III)中任一项部分互补或完全互补的核酸。
一些实施例中，编码RPGR的核酸序列如SEQ ID NO：1所示。
本发明还提供了一种重组载体，其包括骨架载体和本发明所述的核酸。
本发明中，所述的重组载体中的骨架载体为病毒载体。一些实施例中，所述病毒 载体选自慢病毒载体、腺病毒载体、腺相关病毒载体中至少一种；其中，腺相关病毒载体的血清型为AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV2.7M8或AAV2-TYF突变型。一些具体实施例中，其骨架载体上含有RK1启动子和/或SV40内含子。在本发明实施例中，所述重组载体上包括顺序连接的RK1启动子、SV40内含子、SEQ ID NO：1所示的核酸、SV40poly(A)signal。具体的，所述重组载体上包括顺序连接RK1启动子、SV40内含子、SEQ ID NO：1所示的核酸、SV40poly(A)signal。其中，RK1启动子的核酸序列如SEQ ID NO：4所示，所述SV40内含子的核酸序列如SEQ ID NO：5所示。
本发明还提供了一种质粒组合，其包括所述的重组载体、辅助功能质粒和附属功能质粒。所述辅助功能质粒为pAdHelper；所述附属功能质粒为pAAV-r2c5。
本发明还提供了表达RPGR的腺相关病毒的制备方法，其包括：将所述的质粒组合转染宿主细胞，经纯化获得RPGR的腺相关病毒。所述宿主细胞为293细胞或293T细胞。
本发明所述制备方法制备获得的表达RPGR的腺相关病毒。
本发明所述的重组载体、所述的质粒组合或所述的腺相关病毒在制备防治眼部疾病的药物中的应用。本发明中，所述眼部疾病为RPGR突变所致疾病。一些实施例中，所述眼部疾病为视网膜色素变性。一些实施例中，所述防治包括修复视网膜结构，提高视细胞数量和/或改善眼部功能。
本发明还提供了一种药物，其包括本发明所述的重组载体，或质粒组合、或腺相关病毒。
本发明所述的药物中，还包括药学上可以接受的载体和赋形剂。在某些实施方式中，所述赋形剂为纳米载体和/或脂质体、本发明所述药物的剂型为注射液剂，其中所述的腺相关病毒的含量为1×109～1×1016个病毒/毫升，一些实施例中，所述腺相关病毒的含量为1×1012～1×1014个病毒/毫升。一些具体实施例中，所述的腺相关病毒的滴度为1×1013vg/mL。
本发明所述药物的给药方式包括视网膜下注射、玻璃体腔注射、前房注射或者结膜下注射。
本发明所述的药物中，还包括其他具有改善视网膜色素变性活性的药物。
本发明还提供了一种防治视网膜色素变性的方法，其为给予本发明所述的药物。
本发明提供了编码RPGR的核酸，并提供了含有该核酸的载体、腺相关病毒。该编码核酸经过特殊优化从而使RPGR的表达量显著提高，从而可以用于用于治疗RPGR突变引起X染色体连锁视网膜色素变性。实验表明，AAV-RPGR药物能够显著改善RPGR突变引起X染色体连锁视网膜色素变性的缺陷小鼠的眼部病变。
附图说明
图1A～图1D是密码子优化后的RPGR ORF15与野生型序列比对，优化后差异性的密码子序列加粗并用下划线标注；
图2是AAV-RPGR载体图谱：其中，A是密码子优化的RPGR ORF15质粒载体示意图，载体包含AAV2 5’ITR，RK1启动子，SV40内含子，密码子优化后RPGR ORF15，SV40 polyA序列和AAV2 3’ITR；B是野生型的RPGR ORF15质粒载体示意图，载体包含AAV2 5’ITR，RK1启动子，SV40内含子，野生型RPGR ORF15，SV40 polyA序列和AAV2 3’ITR；
图3显示AAV-RPGR载体的克隆与酶切验证；其中，A为将构建好的优化AAV-RPGR ORF15质粒转化Stbl3大肠杆菌菌株；B为将构建好的野生型AAV-RPGR ORF15质粒转化Stbl3大肠杆菌菌株；C为挑取单克隆抽提质粒用HindIII与XhoI双酶切鉴定，对密码子优化后与野生型的载体转化效率与序列的完整性进行验证；1-5：密码子优化质粒酶切结果；M：10kb Marker；6-10：野生型质粒酶切结果；
图4显示优化后的RPGR序列编码蛋白在体外细胞的表达验证；其中，A是在小鼠视锥细胞系661W中转染AAV-RPGR质粒，48小时后裂解细胞检测RPGR蛋白表达水平，检测密码子优化后opt1，opt2和野生型的RPGR蛋白表达差异，B为将AAV5-RPGR-opt1病毒用不同的感染复数(MOI＝1E4，3E4，1E5)对661W细胞进行感染，72小时后裂解细胞利用qPCR检测RPGR mRNA表达水平；
图5显示小鼠体内AAV介导的RPGRopt1蛋白表达水平验证；
图6是小鼠体内RPGR表达效率比较，利用免疫荧光染色观察RPGR蛋白在视网膜的分布，其中A为视网膜铺片后染色RPGR抗体，观察RPGR阳性信号在视网膜的分布和密度，B为RPGR阳性细胞在视网膜的整体占比；
图7显示小鼠体内RPGR组织定位；
图8显示AAV5-RPGRopt基因治疗药物对RPGR突变引起X染色体连锁视网膜色素异常的治疗作用，其中，A为注射后18个月RPGR敲除小鼠的药物注射眼和对照眼视网膜电图分析，光照条件下给予逐渐增强的光刺激，记录不同强度光刺激下各小鼠(n＝20)药物注射眼和对照眼的b波振幅；B为注射后18个月RPGR敲除小鼠的的药物注射眼和对照眼视网膜电图分析，黑暗条件下给予逐渐增强的光刺激，记录不同强度光刺激下各小鼠(n＝20)药物注射眼和对照眼的b波振幅；C示注射后18个月RPGR敲除小鼠的药物注射眼和对照眼视网膜外核层厚度(n＝20)的定量分析。
具体实施方式
本发明提供了编码RPGR的核酸及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中，RPGR是指视网膜色素变性GTPase调节基因，是X连锁视网膜色素变性(RP)最常见的致病基因，RPGR有多种剪接异构体，其中RPGR-ORF15的野生型编码序列如SEQ ID NO：2所示，该序列来自人源。本发明对SEQ ID NO：2所示的 序列进行优化，使其能够显著改善RPGR突变引起X染色体连锁视网膜色素变性的缺陷小鼠的眼部病变。本发明提供的编码RPGR的核酸如SEQ ID NO：1(opt1)或SEQ ID NO：3(opt2)所示，SEQ ID NO：1所示核苷酸序列为：

通过分析和试验筛选，结果表明，相对于其他优化序列而言，如SEQ ID NO：1所示的核酸，转录水平得到提高，表达量显著提高，从而能够更显著的对视网膜色素变性起到良好的治疗作用。而野生型或其他优化序列的效果不及SEQ ID NO：1所示的核酸。
本发明利用腺相关病毒(AAV)载体进行基因置换治疗，在许多视网膜变性动物模型中，已证明对视网膜功能和结构的挽救是有效的。通过提供一种重组载体，其中RPGR ORF15编码区被置于组织特异性的启动子的控制之下，同时其序列进行了密码子优化，为由RPGR突变引起X染色体连锁视网膜色素变性提供具有活性的RPGR蛋白并达到接近野生型水平。首先将构建好的AAV-RPGR质粒转化大肠杆菌，挑取单菌落扩增后提取质粒酶切与测序鉴定，验证RPGR序列克隆的效率以及RPGR序列的完整性与一致性；其次将质粒转染小鼠视锥细胞系661W，检测RPGR蛋白的表达效率高于野生型蛋白；由RK1启动子控制的RPGR蛋白包装病毒后感染661W细胞，其表达随病毒剂量提高而升高。随后对C57小鼠进行视网膜下药物注射，比较RPGR优化前后的体内表达效率，同时检测RPGR蛋白在视网膜组织中正确定位表达；最后对RPGR敲除小鼠药物治疗后的疗效进行评估，视网膜电图分析表明小鼠接受治疗眼功能得到改善；同时OCT结果显示治疗眼视网膜组织外核层厚度明显大于未治疗眼，表明视细胞整体存活数量显著提升。总而言之，体外实验证明了AAV5-RPGRopt1药物的效率，小鼠体内实验证明了AAV介导的基因治疗药物能有效的治疗RPGR突变引起的X染色体连锁视网膜色素变性。
本发明采用的试材皆为普通市售品，皆可于市场购得。下面结合实施例，进一步阐述本发明：
实施例1 AAV-RPGR载体的克隆与酶切验证
将野生型RPGR ORF15编码序列进行了密码子优化，两种优化分别记做RPGRopt1和RPGRopt2，其中RPGRopt1与野生型的比对如图1A-图1D，并构建了相应的AAV载体质粒(图2)。将包含有密码子优化的RPGR ORF15序列和野生型RPGR ORF15序列的质粒(200ng/μL)分别转化大肠杆菌Stbl3菌株并涂布添加了对应抗生素的LB平板，过夜培养后观察单菌落生长情况。
质粒转化大肠杆菌：
按以下反应体系混合均匀，于冰上放置20min，室温放置10min，加入500μL无 抗LB 37℃，200rpm摇床培养40min，然后5000rpm离心3min，吸弃500μL上清，将余下液体重悬菌体沉淀并于相应抗性LB固体平板均匀涂布。将平板置于37℃培养箱培养过夜。
如图3所示，相同浓度的质粒DNA，优化质粒平板的单菌落数量要高于野生型质粒平板(图3中的A、图3中的B)，说明密码子优化后的RPGR ORF15序列的克隆效率要高于野生型。
质粒DNA的小量提取：
1.挑取单菌落于适量的LA(添加的抗生素取决于质粒抗性)液体培养基中，37℃，200rpm摇过夜(16-20h)；
2.将培养液分装于1.5mLEP管中，每管分装1mL，12000rpm离心3min，颠倒EP管弃去上清；
3.加入100μL溶液I，涡旋混匀至菌体充分悬浮；加入200μL溶液II，轻柔颠倒混匀，至液体澄清而粘稠；加入150μL溶液III，迅速上下颠倒混匀，冰上放置5min后，12000rpm离心10min；
4.吸取上清于新的EP管中，加入2倍体积的无水乙醇沉淀DNA，颠倒混匀，在-20℃放置30min；12000rpm离心10min，弃上清，加入70％酒精180μL，12000rpm离心2-3min，弃上清；
5.室温晾干质粒DNA，加入TE溶解，可室温放置，短期保存置于4℃，长期保存置于-20℃。
质粒DNA的酶切验证：
按照以下体系配制反应溶液，置于37℃孵育1-4h，取适量体积上样电泳检测。
分别提取质粒双酶切后电泳观察条带大小，发现优化质粒酶切结果RPGR ORF15保持完整大小，而野生型质粒酶切结果显示RPGR ORF15未能保持完整大小(图3中C)，说明密码子优化后的RPGR ORF15在克隆过程中的保真度高于野生型。
实施例2 AAV介导的RPGR在体外细胞以及小鼠体内的表达
将包含密码子优化的RPGR ORF15序列和野生型RPGR ORF15序列的质粒分别 转染661W细胞，48小时后裂解细胞，提取蛋白后利用Western Blot确认蛋白表达量。
一、细胞转染：
1.转染前一天，胰酶消化细胞并计数，细胞铺板，使其在转染日密度为90％。
2.对于每孔细胞，使用50μl无血清DMEM培养基稀释DNA。
3.混合稀释的DNA和稀释的LIPOFECTAMINE 2000在室温保温20分钟。
4.直接将复合物加入到每孔中，摇动培养板，轻轻混匀。
5.在37℃，5％的CO2中培养，在细胞中加入复合物24-72小时后，分析细胞抽提物或进行原位细胞染色，检测报告基因活性。
二、病毒感染细胞：
1.消化HEK293T细胞，铺板6孔板。
2.培养过夜后，取部分孔的细胞以合适的MOI感染目的病毒和其对照病毒。
3.感染36-48h后，收取细胞进行后续实验。
三、qPCR测定mRNA含量：经总RNA抽提、反转录、定量PCR等步骤，Western Blot检测蛋白表达：
1.蛋白样品制备，按1∶100的比例在裂解液中加入PMSF(按用量现配现用)。
2.使用强裂解液裂解细胞。
3.使用BCA法测定蛋白浓度。
4.电泳
a、根据所检测蛋白大小配制相应的分离胶(5ml/块)，待分离胶凝固。
b、配制5％的浓缩胶(2ml/块)，加满玻璃板，插入梳子。
c、将5μl预染蛋白质分子marker SDS-PAGE加入加样孔内，使用1x的SDS-PAGE蛋白上样缓冲液10μl上样到样品孔边上的空白加样孔内。
5.转膜
将转膜白夹子上面放上湿的垫层，垫层上面铺三张叠在一起的湿滤纸，滤纸上面依次放置湿pvdf膜、胶、滤纸、垫层、黑夹板，将装好的夹板放入装有转膜缓冲液的电泳槽，把转膜槽放置在冰浴中进行转膜。
6.封闭
转膜完毕后，漂洗1-2分钟，用滴管吸尽缓冲液，加入5％的脱脂奶粉，在侧摆摇床上缓慢摇动，室温封闭15-60min。加入TBS洗涤液洗涤5分钟。共洗涤3次。
7.一抗孵育
按照比例使用5％的脱脂奶粉/PBS+2％BSA稀释适量的一抗，4℃缓慢摇动孵育过夜或室温在侧摆摇床上缓慢摇动孵育2h。孵育后洗涤。
8.二抗孵育
加入稀释好的二抗，室温侧摆摇床上缓慢摇动孵育40min-1h。孵育后洗涤。
9.蛋白检测
使用ECL类试剂来检测蛋白，各取1ml混匀后，滴加在蛋白膜表面，避光孵育1-2min。用镊子将蛋白膜整齐的摆放在塑料纸上，放入凝胶成像仪上曝光。
如图4所示，发现密码子优化的RPGRopt1蛋白表达量要高于野生型和优化序列RPGRopt2(图4中的A)，优化后的RPGRopt1蛋白体外表达量相对于野生型序列提高了3.6倍(表1)。
表1蛋白条带相对灰度值
优化的AAV-RPGRopt1载体包装AAV5血清型病毒，以不同感染复数(MOI)对661W细胞进行感染，72小时后裂解细胞提取RNA，利用qPCR确认mRNA表达量，结果显示RPGRopt1mRNA在体外的表达水平随病毒剂量的增加而升高，存在明显的剂量依赖效应(图4中的B，表2)。
表2不同剂量病毒感染661W细胞后RPGRmRNA的表达水平
四、病毒包装，病毒药物注射小鼠：
1.聚合度90％以上的HEK293T细胞按1∶3比例传盘。
2.转质粒前1-2h左右，换成无血清培养基，用转染试剂将目的基因质粒和辅助质粒转入HEK293T中。
3质粒转化24h后，换新的无血清培养基。
4.转染72h收毒。带着培养基，吹下细胞，离心；然后分别收获培养基上清与细胞沉淀。用PEG8000沉淀培养基上清中的病毒，沉淀过夜后收集病毒沉淀。
5.将病毒的混合液用碘克沙醇密度梯度离心进行纯化，然后用超滤管进行浓缩。
6.构建人源化的RPGR敲除小鼠。
7.准备好1×1013vg/ml的AAV-RPGRopt1药物，进行不同剂量的稀释。
8.将1μl/眼的AAV-RPGRopt1药物和PBS通过视网膜下注射进小鼠视网膜区域。
将AAV5-RPGRopt1病毒药物以视网膜下注射的方式给药到6-8周龄的C57小鼠眼部，然后在给药4周后取视网膜，提取组织中蛋白进行WesternBlot检测，观察RPGR在小鼠视网膜组织中的表达。
如图5所示，敲除小鼠的治疗眼能够检测到RPGR蛋白的表达，而对照眼组织中未能检测到相应的蛋白表达，且蛋白的表达水平与给药剂量同样存在线性增长关系。
五、免疫荧光染色：
1、将切片或样品铺片用PBS洗2遍，每次5min。
2、弃去PBS，每孔加入200μl的1％Triton打孔15min。打孔结束后用0.05％Triton洗3遍。
3、每个样品加入30μl的碧云天封闭液，室温静置封闭2小时。
4、在每片样品上滴加30μl的合适浓度的一抗(1∶200)。整个封口膜放入湿盒内，4℃静置过夜。
5、用添加0.05％Triton的PBS清洗3遍，每遍5min。
6、在每片样品上滴加40-50μl的合适浓度的二抗(1∶1000)抗体。整个封口膜放入湿盒内，室温静置1小时。
7、用添加0.05％Triton的PBS清洗3遍。
8、每个样品加入30μl的DAPI染液，染色15min。
9、用PBS清洗3遍。
在盖玻片上滴加15μl的抗荧光淬灭封片液，小心的将盖玻片封到载玻片上，指甲油封片。
如图6所示，视网膜铺片结果显示优化的RPGRopt1药物在视网膜的分布面积更大，且单位面积内阳性信号的密度高于野生型的RPGR药物和优化序列RPGRopt2(图6中的A)，意味着蛋白丰度更高；经过计算，野生型的药物在视网膜中的表达占比为7.82％，优化的药物RPGRopt2在视网膜中的表达占比为15.7％，优化的药物RPGRopt1在视网膜中的表达占比为36.3％(图6中的B，表3)。
表3阳性表达细胞在整体视网膜中的占比
如图7所示，切片结果显示RPGR蛋白(红色荧光信号，箭头所示)能够正确地定位于感光细胞内段(IS)，且优化的药物opt1信号强于优化的药物opt2和未优化的药物。
以上结果表明AAV-RPGRopt1病毒药物在体内能够正确地表达蛋白，且优化的药物在体内的表达效率相对于未优化的药物有显著的提升。
实施例3 AAV-RPGRopt1基因治疗药物改善RPGR敲除小鼠眼部功能，修复视网膜结构
实施例1-2证实了RK1启动子控制的密码子优化的RPGRORF15编码序列在体外的和体内的正确表达，为了进一步证明AAV5-RPGRopt1基因治疗药物对RPGR突变引起的X染色体连锁视网膜色素变性的治疗作用，利用RPGR敲除小鼠模型进行了体内实验，注射后18个月观察药物治疗对小鼠眼部病变的改善情况。
首先，我们利用视网膜电图分析对药物治疗眼和对照眼的功能进行评估。
病毒包装、病毒药物注射小鼠的方法同前所述。
一、视网膜电图分析：
1.对小鼠进行麻醉和瞳孔扩张处理，同时向眼部滴入包含电极的2.5％羟丙甲纤维素液体，记录角膜电位反应。
2.暗适应条件下让小鼠适应黑暗过夜，用LED灯给予-2到+3log sc cd.s/m2强度的短暂闪光刺激，记录暗适应ERG，根据刺激强度的不同，以3到60秒的间隔对反应进行记录。
3.光适应条件下，LED灯给予-0.5到+2log sc cd.s/m2强度的短暂闪光刺激，记录光适应ERG，以2分钟的间隔对反应进行记录。
如图8所示，发现光照条件下，随着光刺激强度增大，治疗眼的b波振幅逐渐高于对照眼(图8中的A，表4)；黑暗条件下，治疗眼的b波振幅在不同光刺激强度下均显著高于对照眼(P＜0.5)，与野生型接近(图8中的B，表5)，这说明药物治疗对眼部功能起到了明显的改善作用。
表4光照条件下小鼠ERG眼部b波振幅
表5黑暗条件下小鼠ERG眼部b波振幅
随后小鼠的眼部组织被用来进行OCT分析。
二、小鼠OCT检测
1.动物准备
动物麻醉后，滴加扩瞳保持2～3分钟，后用棉签轻轻擦去扩瞳液，涂布卡波姆凝 胶，使动物眼部保持湿润；
检查左眼时，将小鼠放在摄像机前平台中心偏右的位置，调节小鼠位置，使左眼正对摄像机镜头；同理，检查右眼时，将小鼠放在摄像机前平台中心偏左的位置，调节小鼠位置，使左眼正对摄像机镜头。
2.图像采集
在本实验，选择IR及IR+OCT两种模式：
选择IR模式(30°镜头)，定位到小鼠视盘中央区域，图像采集并保存
3.外核层图像分析
将所要测量厚度的层次用两条水平红线标记出来，移动绿色竖线至45°、90°、135°、225°、270°和315°的位置，将绿色竖线右侧显示的数字记录下来。
将所测量不同小鼠视网膜的该层厚度整理至EXCEL中，计算平均值并分析。
对外核层厚度的定量分析表明，治疗眼的外核层厚度明显大于对照眼(图8中的C，表6)，说明药物注射后治疗眼视网膜中得以保留更多的视细胞，同时基本维持了视网膜的结构。
表6小鼠视网膜外核层(ONL)厚度
综合以上结果，AAV-RPGRopt1基因治疗药物通过在RPGR敲除小鼠体内提供能正常发挥功能的蛋白，弥补基因缺失对眼部病变造成的不利影响，我们证明了AAV-RPGRopt1基因治疗药物对RPGR突变引起的X染色体连锁视网膜色素变性的治疗作用，为进一步的临床应用开发奠定了基础。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (15)

  1. 编码RPGR的核酸，包括I)～IV)中至少一种：
    I)、具有如SEQ ID NO：1所示核苷酸序列的核酸；
    II)、在I)所述的片段中取代、缺失或添加一个或多个核苷酸的核酸；
    III)、与I)所述核酸的序列至少具有90％的同源性，且编码RPGR的核酸；
    IV)、与I)～III)中任一项部分互补或完全互补的核酸。
  2. 根据权利要求1所述的核酸，其特征在于，其序列如SEQ ID NO：1所示。
  3. 重组载体，其包括骨架载体和权利要求1所述的核酸。
  4. 根据权利要求4所述的重组载体，其特征在于，其为病毒载体；
    所述病毒载体选自慢病毒载体、腺病毒载体、腺相关病毒载体中至少一种；其中，腺相关病毒载体的血清型为AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV2.7M8或AAV2-TYF突变型。
  5. 根据权利要求3或4所述的重组载体，其特征在于，其骨架载体上含有RK1启动子和/或SV40内含子。
  6. 一种质粒组合，其特征在于，包括权利要求3～5任一项所述的重组载体、辅助功能质粒和附属功能质粒。
  7. 根据权利要求6所述的质粒组合，其特征在于，所述辅助功能质粒为pAdHelper；所述附属功能质粒为pAAV-r2c5。
  8. 表达RPGR的腺相关病毒的制备方法，其包括：将权利要求6所述的质粒组合转染宿主细胞，经纯化获得RPGR的腺相关病毒。
  9. 权利要求8所述制备方法制备获得的表达RPGR的腺相关病毒。
  10. 权利要求3～5任一项所述的重组载体，或权利要求6或7所述的质粒组合、或权利要求9所述的腺相关病毒在制备防治眼部疾病的药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述眼部疾病为视网膜色素变性。
  12. 根据权利要10或11所述的应用，其特征在于，所述防治包括修复视网膜结构，提高视细胞数量和/或改善眼部功能。
  13. 一种药物，其特征在于，包括权利要求3～5任一项所述的重组载体，或权利要求6或7所述的质粒组合、或权利要求9所述的腺相关病毒。
  14. 根据权利要求13所述的药物，其特征在于，其剂型为注射液剂，其中权利要求10所述的腺相关病毒的滴度为1×1013vg/mL。
  15. 根据权利要求13或14所述的药物，其特征在于，其给药方式包括视网膜下注射、玻璃体腔注射、前房注射或者结膜下注射。
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