CN109136266A - Genophore and application thereof for treating or preventing fluorescent angiography in patients with crystalline retinitis pigmentosa - Google Patents

Abstract

The present invention relates to it is a kind of for treat or prevent the genophore of fluorescent angiography in patients with crystalline retinitis pigmentosa (BCD) with and application thereof.The genophore of the present invention for being used to treat or prevent fluorescent angiography in patients with crystalline retinitis pigmentosa, comprising: packaging plasmid is the AAV2 packaging plasmid for inserting the gene order of anti-CD59 small peptide C9；And vector plasmid, be the gene order for inserting Human cytochrome P450 superfamily proteins CYP4V2, the gene order of anti-vegf small peptide HRH, promoter sequence AAV vector plasmid.The AAV carrier can the anti-CD59 small peptide of amalgamation and expression C9 to virus coat protein；It can be realized CYP4V2 and HRH coexpression, protein function caused by CYP4V2 gene mutation in RPE cell is compensated to lack, can effectively repair the normal function of RPE cell, and can specific antagonist vascular endothelial growth factor VEGF, delay neurodeatrophia caused by choroid hyperplasia.The present invention also provides the genophore is used to prepare to the purposes for treating or preventing the drug of fluorescent angiography in patients with crystalline retinitis pigmentosa (BCD).

Description

Genophore and application thereof for treating or preventing fluorescent angiography in patients with crystalline retinitis pigmentosa

Technical field

The invention belongs to gene engineering technology fields, are related to a kind of genophore, more particularly, to one kind for treating Or prevention fluorescent angiography in patients with crystalline retinitis pigmentosa (Bietti's crystalline dystrophy, BCD) genophore and Its purposes.

Background technique

Fluorescent angiography in patients with crystalline retinitis pigmentosa (Bietti's crystalline dystrophy, BCD) is a kind of heritable Retina degeneration eye disease.Generally there is yctalopia in teenager in the stage in patient；With progression of the disease, patient is on cornea and eyeground There is the rouge crystallization of yellow-white crystalline shape, and is lacked with the visual field；Terminal stage of a disease, patient's neurodeatrophia, and finally blind 1. The disease belongs to rare disease in west, but in Asia, particularly in China, there is higher incidence (1/20000) (Hu, D.-N. (1983).Ophthalmic genetics in China.Ophthalmic Paediatrics and Genetics,2(1), 39-45.).Regrettably, currently without any effective treatment means for the disease.Clinical observation surface, BCD blindness It is retinal pigment epithelium (the Retinal pigment as caused by the CYP4V2 gene mutation of Cytochrome P450 family Epithelium, RPE) degenerate (Xiaodong Jiao (2001) .Genetic caused by the neurodeatrophia and train of thought hyperplasia caused Linkage of Bietti Crystallin Corneoretinal Dystrophy to Chromosome 4q35.Am J Hum Genet.2000Nov；67(5):1309–1313.).And AAV2 has been widely used in treating monogenic inheritance disease Disease, and obtain more achievement (Naso MF (2017) .Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.BioDrugs.2017Aug；31(4):317-334.).

Summary of the invention

The purpose of the present invention is to provide a kind of genophores, can obtain certain curative effect in the prevention and treatment of BCD.

The genophore of the present invention for being used to treat or prevent fluorescent angiography in patients with crystalline retinitis pigmentosa, comprising: packaging matter Grain, is the AAV2 packaging plasmid for inserting the gene order of anti-CD59 small peptide C9；And vector plasmid, it is to insert human cell The gene order of cytochrome p 450 superfamily proteins CYP4V2, the gene order of anti-vegf small peptide HRH, the AAV load of promoter sequence Constitution grain.

The further feature of genophore according to the present invention, the gene order of the CYP4V2 albumen such as SEQ ID Shown in NO:1；The gene order of the HRH is as shown in SEQ ID NO:2；The gene order of the C9 such as SEQ ID NO:3 institute Show.

The further feature of genophore according to the present invention, the promoter are consensus promoter or retina The specificity promoter of pigment epithelial cell.

The further feature of genophore according to the present invention, the consensus promoter are to be selected from: CBh promoter, CAG promoter, PGK promoter, EF1 α promoter.

The further feature of genophore according to the present invention, the nucleotide sequence such as SEQ of the CBh promoter Shown in ID NO:4；The nucleotide sequence of the CAG promoter is as shown in SEQ ID NO:5；The nucleotide of the PGK promoter Sequence is as shown in SEQ ID NO:6；The nucleotide sequence of the EF1 α promoter is as shown in SEQ ID NO:7.

The further feature of genophore according to the present invention, the specificity promoter are that RPE65 specificity opens Mover.

The further feature of genophore according to the present invention, the nucleotide of the RPE65 specificity promoter tool Sequence is as shown in SEQ ID NO:8；

CD59 surface antigen has been found wide expression on mankind's RPE cell membrane, if the AAV2 virus of eyeground injection The surface antigen can be specifically bound, it will increase substantially AAV2 virus transfection efficiency (Yang P, Tyrrell J, Han I,Jaffe G(2009)50(7):3473-81.Expression and modulation of RPE cell membrane complement regulatory proteins.Invest Ophthalmol Vis Sci.2009,50(7): 3473-81.).Document report, small peptide C9 efficient identification and can specifically bind CD59 surface antigen (J Biol Chem.2006Sep 15；281(37):27398-404.Epub 2006Jul 14.Defining the CD59-C9binding interaction.Huang Y,Qiao F,Abagyan R,Hazard S,Tomlinson S.).The present invention passes through fusion table Up to C9 and AAV2 pack case plasmid capsin albumen, it can be achieved that AAV2 virus and CD59 surface antigen combination, to improve Virus infection efficiency.

The difference of the promoter of mediate foreign gene expression also influences whether the therapeutic effect of AAV.Present invention employs one A little consensus promoters, including CBh, CAG, hPGK and EF1 α, they have been widely used for mediating cell-free difference foreign gene High efficient expression.The present invention additionally uses some cell specificity promotors, they are used for specific expressed foreign gene Mr. Yu One specific cells.RPE65 promoter is the specificity promoter of RPE cell.The exogenous gene expression mediated using the promoter It will be confined to this specific cells of RPE.

The VEGF of RPE secretion plays key player in view membrane degradation and train of thought atrophy, and HRH small peptide can pass through knot It closes VEGF and effectively inhibits anti-vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) living Property.The present invention constructs AAV2-CYP4V2-HRH coexpression vector, utilizes T2A autothermic cracking peptide (MAbs.2015；7(2):403- 12.doi:10.1080/19420862.2015.1008351.Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.Chng J,Wang T,Nian R,Lau A, Hoi KM, Ho SC, Gagnon P, Bi X, Yang Y.) small peptide connection CYP4V2 and HRH, it can effectively realize their total table It reaches, and does not intervene its function, to be finally reached while mending endogenous CYP4V2 protein delation, expression HRH small peptide comes Inhibit VEGF, to delay view membrane degradation.

Therefore, it can be specifically bound with retina R PE cell surface antigen CD59 the present invention provides a kind of, while mistake Express the gland relevant viral vector of CYP4V2-HRH albumen.The carrier is made of two parts.It was transformed first part AAV2 packaging plasmid, the plasmid can the anti-CD59 small peptide of amalgamation and expression C9 to virus coat protein；Second part carrier is able to achieve people The coexpression of cytochromoid P450 superfamily proteins CYP4V2 and HRH anti-vegf small peptide.The exogenous gene expression is by general starting The gene order of mover or retinal pigment epithelium RPE65 specificity promoter mediates.It is produced using the two plasmids The adenovirus come can amalgamation and expression C9 to virus coat protein, and by specific binding RPE cell surface antigen CD59, from And AAV virus is effectively improved to the infection ability of RPE cell.Meanwhile the AAV carrier can be realized CYP4V2 and HRH coexpression, The former compensates protein function caused by CYP4V2 gene mutation in RPE cell and lacks, and can effectively repair the normal function of RPE cell； The latter can specific antagonist vascular endothelial growth factor VEGF, delay neurodeatrophia caused by choroid hyperplasia.

A second object of the present invention is to provide a kind of compositions, it includes genophore as described in the present invention, and Pharmaceutically acceptable excipient.For example, the genophore to be made to the composition for being used for intraocular injection, can be added suitable Excipient for intraocular injection agent.Preferably, the intraocular injection refers to subretinal space injection or intravitreal.

Third object of the present invention is to provide the purposes of genophore of the present invention.

A purposes of the present invention is that the genophore is used to prepare treatment or prevention crystalline retinal The purposes of the drug of pigmental degeneration (BCD).

Another purposes of the present invention is used to prepare the genophore with crystalline retinal color The purposes of the drug of CYP4V2 and HRH coded sequence is co-expressed in the retinal pigment epithelium of the object of element denaturation.

It, can be in the retinal pigment epithelium of the object with fluorescent angiography in patients with crystalline retinitis pigmentosa according to such use The middle expression for restoring normal CYP4V2 albumen.

Detailed description of the invention

Fig. 1 is AAV2/2-C9 plasmid characteristic pattern constructed by the present invention.

Fig. 2 is AAV-pCBh-CYP4V2-HRH plasmid characteristic pattern constructed by the present invention.

Fig. 3 is AAV-pCAG-CYP4V2-HRH plasmid characteristic pattern constructed by the present invention.

Fig. 4 is AAV-pPGK-CYP4V2-HRH plasmid characteristic pattern constructed by the present invention.

Fig. 5 is AAV-pEF1 α-CYP4V2-HRH plasmid characteristic pattern constructed by the present invention.

Fig. 6 is AAV-pRPE65-CYP4V2-HRH plasmid characteristic pattern constructed by the present invention.

Fig. 7 is external CYP4V2 overexpression figure.In figure: 1 is the blank group of uninfecting virus；2 be AAV2-pCAG- HCYP4V2-HRH control group；3 be AAV2-C9-pCAG-hCYP4V2-HRH group；4 be AAV2-pRPE65-hCYP4V2-HRH pairs According to group；5 be AAV2-C9-pRPE65-hCYP4V2-HRH.

Fig. 8 is patient's BCD RPE cell AAV treatment figure.In figure: i is iPSC (graduation mark is 200 microns)；Ii is differentiation RPE (graduation mark is 50 microns)；The fat drips dyeing that iii is normal person RPE (graduation mark is 50 microns)；Iv is patient BCD RPE rouge Drop dyeing (graduation mark is 15 microns)；V is that patient's BCD RPE fat drips of AAV2-pCAG-CYP4V2 virus infection dye (graduation mark It is 15 microns)；Patient's BCD RPE fat drips dyeing of viAAV2-pRPE65-CYP4V2 virus infection (graduation mark is 15 microns).

Specific embodiment

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as document (for example, J. Pehanorm Brooker is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, 2002) in the industry Described in condition, or according to the normal condition proposed by manufacturer.

Unless otherwise defined, all scientific and technical terminologies used herein all have and the technical field of the invention The identical meaning of the meaning that those of ordinary skill is generally understood.

Embodiment 1: it the building of adenovirus vector and isolates and purifies

1. expanding the coded sequence of CYP4V2-HRH gene.

1) NCBI inquiry obtains the cDNA sequence (https: //www.ncbi.nlm.nih.gov/ of people CYP4V2 gene Nuccore/NM_207352.3), and the sequence (SEQ ID NO:1) is synthesized.

2) according to above-mentioned sequence, HRH small peptide nucleotide sequence is merged using T2A, and company is sent to synthesize CYP4V2-HRH base Because of segment (SEQ ID NO:2).

3) primer is designed and synthesized, the High fidelity PCR clonal expansion CYP4V2-HRH sequence is utilized.

CYP4V2-F:CGCGGATCCATGGCGGGGCTCTGGCTGG

CYP4V2-R:GGTAAGCTTTTAGCGCGGTATGGCGC

2. expanding many general promoter

1) inquiry Addgene (the non-commercial plasmid resources bank in Cambridge) obtains CBh, CAG, PGK and EF1 α promoter sequence, Plasmid ID:42230,16664,21217 and 26797.

2) using inquiry gained promoter sequence, its DNA sequence dna (SEQ ID NO:4 to SEQ ID NO:7) is synthesized.

3) primer of synthesis carrying restriction enzyme site as follows, utilizes these promoters of high-fidelity PCR amplification.

CBh-F primer: GTCAACGCGTCGTTACATAACTTACGGTA；

CBh-R primer: TCACGGTACCGCTTGAGTTGAGGCCTG；

CAG-F primer: GTCAACGCGTGCGTTACATAACTTACGGTAAAT；

CAG-R primer: TCACGGTACCGGCACGGGGCGAAGGCAG；

PGK-F primer: GTCAACGCGTGGTTGGGGTTGCGCCTTTTC；

PGK-R primer: TCACGGTACCCCTGGGGAGAGAGGTCGGTG；

EF1-F primer: GTCAACGCGCGTGAGGCTCCGGTGCCC；

EF1-R primer: TCACGGTACCCACGACACCTGAAATGGAA.

3. expanding RPE65 promoter

1) people's complete genome DNA is extracted, for expanding the template of RPE65 promoter.

2) according to the literature (Yang P, Tyrrell J, Han I, Jaffe G (2009) 50 (7): 3473- 81.Expression and modulation of RPE cell membrane complement regulatory Proteins.Invest Ophthalmol Vis Sci.2009,50 (7): 3473-81.), design synthetic primer:

RPE65-F primer: GTCAACGCGTATTGCTGGTTTAAGAAGATTTGG；

RPE65-R primer: TCACGGTACCTTTCTTCCAGTTCAGGATCCAGA.

3) high-fidelity PCR amplification RPE65 upstream region of gene promoter, overall length about 1500bp (SEQ ID NO:8).

4. constructing the AAV expression plasmid that different promoters mediate

1) MluI and KpnI is utilized, 37 ° overnight digestion AAV shuttle plasmid (Addgene, 60958)；37 ° of overnight enzymes simultaneously Cut the promoter of amplification, including CMV, CAG, PGK, EF1 α and RPE65 promoter.

2) by glue recovery experiment, above-mentioned plasmid backbone and digestion products (promoter) are purified.

3) enzyme even reacts: utilizing T4 ligase, in 4 ° of connections overnight, each promoter is cloned into AAV shuttle plasmid respectively.

4) competent escherichia coli cell is converted, and utilizes ampicillin screening and cloning.

5) picking monoclonal, sequencing identification.

5. constructing the AAV2-C9-hCYP4V2-HRH shuttle plasmid (as shown in Figures 2 to 6) that different promoters mediate.

1) BamH1 and HindIII, 37 ° of overnight above-mentioned AAV2 shuttle plasmids of digestion are utilized；37 ° of overnight digestion amplifications simultaneously CYP4V2-HRH gene cDNA sequence.

2) by glue recovery experiment, above-mentioned plasmid backbone and digestion products (CYP4V2-HRH cDNA sequence) are purified.

3) enzyme even reacts: utilizing T4 ligase, in 4 ° of connections overnight, the cDNA sequence of CYP4V2 gene is cloned into respectively AAV2 expression plasmid.

4) competent escherichia coli cell is converted, and utilizes ampicillin screening and cloning.

5) picking monoclonal, sequencing identification.

6. constructing AAV2/2-C9 chitin grain (as shown in Figure 1)

1) rAAV2-retro chitin grain Cap egg is obtained from Addgene (https: //www.addgene.org/81070/) White nucleus nucleotide sequence.

2) C9 small peptide nucleotide sequence is merged according to the sequence, and company is sent to synthesize subsidiary restriction enzyme site NotI and BmgBI Genetic fragment.

3) the above-mentioned synthesis genetic fragment of NotI and BmgBI double digestion and rAAV2-retro chitin grain.

4) enzyme even reacts: utilizing T4 ligase, in 4 DEG C of connections overnight, converts competent escherichia coli cell.

5) picking monoclonal, sequencing identification.

7. packing the AAV2-C9-hCYP4V2-HRH virus that different promoters mediate

1) 293 cell of 10cm culture dish culture (Sai Mofei genome company), it is long to 80% saturation state.

2) liposome Lipo2000 transfects CYP4V2-AAV2 expression vector and its packaging plasmid (including AAV2/2-C9 chitin Grain).

AAV-pCAG-hCY4VP2-HRH plasmid: 10ug；

AAV2/2-C9 chitin grain: 5ug；

Helper plasmid: 5ug；

Liposome: 60ul；

Add 750ul ultrapure water, mixes, be stored at room temperature 20 minutes, be added in 293 cells.

3) after 3 days, culture medium supernatant is collected, which contains a large amount of AAV2-C9-pCAG-hCYP4V2-HRH viruses.

8.AAV2-C9-hCYP4V2-HRH viral purification

After iodine gram butanol preliminary purification, by the fast protein liquid using 5ml-Hitrp Q Ago-Gel as filler It (is Pharmacia AKTA FPLC system using instrument that chromatography is further purified by ion-exchange chromatography (AmershamBiosciences, Piscataway, NJ, the U.S.)).PH8.0 is used later, and the NaCl elution agarose of 215mM is solidifying Rubber column gel column is collected the recombinant viral vector of peak value.The liquid of collection by inspissator (100K concentrater, Millipore after), recombinant viral vector is concentrated with the polysorbas20 elution inspissator containing 0.014%.DNase I is used again DNA other than virion is digested, and determines the titre of virus by the method for real-time fluorescence quantitative PCR.Finally use silver nitrate Dyeing-SDS polyacrylamide gel electrophoresis method ensures that vector particles are contaminated and are free of endotoxin, and dispenses subzero 80 DEG C storage.

Embodiment 2: the external overexpression verifying of adenovirus vector

1. ARPE-19 cell is cultivated in 12 porocyte culture plates, it is long to 70% saturation state.

2. 10000MOI AAV2-C9-pCAG-hCYP4V2-HRH and AAV2-C9-pRPE65-hCYP4V2-HRH disease is added The corresponding AAV2-pCAG-hCYP4V2-HRH and AAV2- that poison is incubated overnight, and is packed with the AAV2 chitin grain of not engineered mistake PRPE65-hCYP4V2-HRH virus is control.

3. changing fresh culture to continue to cultivate.

4. after 3 days, extracting total protein of cell after collecting cell.

5.western blot verifies CYP4V2 and expresses situation.

6. result is as shown in Figure 7: 1 is the blank group of uninfecting virus, only expresses the CYP4V2 albumen of very low amounts；2 are AAV2-pCAG-hCYP4V2-HRH control group, CYP4V2 protein expression increase, and are able to detect that a certain amount of HRH expression；3 be AAV2-C9-pCAG-hCYP4V2-HRH group, and relative to its control group, CYP4V2 and HRH expressing quantity is aobvious It writes and improves, show that C9 can significantly improve AAV2 virus to the efficiency of infection of ARPE-19 cell；4 be AAV2-pRPE65- HCYP4V2-HRH control group, CYP4V2 protein expression increase, and are able to detect that a certain amount of HRH expression；5 are AAV2-C9-pRPE65-hCYP4V2-HRH, relative to its control group, CYP4V2 and HRH expressing quantity is equally significantly improved, Again show that C9 can significantly improve AAV2 virus to the efficiency of infection of ARPE-19 cell.Finally, it is demonstrated experimentally that being transformed AAV2/2-C9 plasmid and CYP4V2-HRH expression plasmid can effectively improve AAV2 to the infection ability of RPE and realize CYP4V2 And the efficient coexpression of HRH.

Embodiment 3: the functional verification of adenovirus vector

1. establishing patient's BCD iPSC cell line

Technology (Cell.2006Aug 25 is reprogrammed using iPSC；126(4):663-76.Epub 2006Aug 10.Induction of pluripotent stem cells from mouse embryonic and adult Fibroblast cultures by defined factors.Takahashi K1, Yamanaka S.) extract normal person and Patient's BCD peripheral blood cells, after cultivating 10 days in CD34+ serum free medium, electricity turns tetra- factor plasmid of Yamanaka, adds nothing Serum free culture system continues culture two days later, goes to the processed tissue culture plate of Geltrex, continues to train in stem cell media E8 It supports to iPSC to clone and occur.Usual iPSC occurs after two weeks, when the clone grow to naked eyes it is visible when, amplification can be passed on, use In next step cell differentiation experimentation.

Patient 2.BCD and normal person's RPE cell differentiation

RPE cell differentiation carries out (PLoS One.2015Jul 1 according to announced method；10(7): e0131288.doi:10.1371/journal.pone.0131288.eCollection 2015.Human Ocular Epithelial Cells Endogenously Expressing SOX2and OCT4Yield High Efficiency of Pluripotency Reprogramming.Poon MW,He J,Fang X,Zhang Z,Wang W,Wang J,Qiu F, Tse HF, Li W, Liu Z, Lian Q.), it is specific as follows.Patient BCD and normal person iPSC are cultivated in E8 culture medium to full With, then be added have serum differentiation culture medium A continue culture two days, then in serum-free differentiation media culture continue to train It supports, after three weeks i.e. it can be seen that typical cobble shaped and the RPE cell containing pigementation, which can pass on amplification, and be used for Subsequent experimental (specific culture medium prescription, see citation).

3.AAV2 infection experiment

It is inoculated with RPE cell and 10000MOI AAV2 disease is added when cell grows to 70% saturation degree to tissue culture plate Poison changes liquid for second day, cultivates cell to saturation state, to subsequent analysis.Fluorescent dye Bodipy-493/503 dyeing display Occurs fat drips accumulation in patient BCD RPE.In order to verify the function of adenovirus vector of the present invention, AAV2-pCAG- is utilized HCYP4V2 and AAV2-pRPE65-hCYP4V2 virus infection patient BCD RPE cell (method is consistent with infection ARPE19 cell), MOI is 10000.

Final result is as shown in Figure 8: i is the iPSC of patient BCD；Ii is the RPE cell of patient BCD of iPSC differentiation； Iii is the fat drips accumulation of normal person RPE cell；Iv is the fat drips accumulation of patient BCD RPE cell, fat drips accumulation Degree is significantly larger than the normal person RPE of iii；V is after patient BCD RPE receives AAV2-C9-pCAG-hCYP4V2-HRH treatment, carefully The fat drips of born of the same parents' accumulation are obviously reduced；After vi receives AAV2-C9-pRPE65-hCYP4V2-HRH treatment for patient BCD RPE, The fat drips of Cellular Accumulation are obviously reduced.These results illustrate that AAV packaged by the plasmid of above-mentioned offer is viral, Neng Gouyou Effect improves the lipoidosis of patient BCD RPE cell, to achieve the purpose that treat BCD.

Embodiment 4: the application of genophore

Genophore of the present invention is virus expression carrier, can be used for the gene therapy of BCD.

Recombinant viral vector can be generated according to standard technique.For example, recombined glandulae correlation viral vectors can be in 293 cell of people It is propagated in (it provides trans- E1A and E1B characteristic), to reach 10⁷~10¹³Titre within the scope of a virion/mL.

Before applying in vivo, viral vectors can carry out desalination by gel filtration method (for example, agarose column), and lead to Subsequent filtering is crossed to be purified.Purifying reduces potential illeffects in the main body of drug administration carrier.The virus being administered is basic It is upper viral without wild-type virus and replication competent type.Suitable method, such as sodium dodecyl sulfate-polypropylene amide can be passed through Gel electrophoresis (SDS-PAGE), then carries out silver staining, to prove the purity of virus.The suitable dose of AAV for people about 1 × 10¹⁰~1 × 10¹⁴In the range of a virion.

Gene therapy vector is intraocular injection administration, can be administered using subretinal space or intravitreal.

For the ease of clinical application, pharmaceutical composition of the invention may be embodied in injection delivery device (such as pumping needle) In, in the injection delivery device, it may include the pharmaceutical composition of single administration amount.The injection administration Device can be contained in medicine box, to facilitate storage, use.It needs to place the small container equipped with drug suspension when transport In dry ice.It should usually be stored in -80C refrigerator.In medicine box or kit of the present invention, it may also include operation instruction Book, so that those skilled in the art use according to correct mode.As used herein, the ingredient of " pharmaceutically acceptable " is Suitable for people and/or mammal without excessive bad side reaction (such as toxicity), that is, there is the object of reasonable benefit/risk ratio Matter.Term " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and diluent.The art Language refers to medicament carriers some in this way: themselves not being necessary active constituent, and does not have excessive toxicity after applying.Properly Pharmaceutically acceptable carrier be well known to those of ordinary skill in the art.In Remington ' sPharmaceutical Absolutely proving about pharmaceutically acceptable carrier can be found in Sciences (Mack Pub.Co., N.J., 1991).In group Liquid can be contained by closing acceptable carrier in object Chinese pharmacology, such as water, BBS (Balanced SaltSolution) phosphate-buffered Liquid, ringer solution, physiological saline, balanced salt solution, glycerol or sorbierite etc..In addition, in these carriers, there is likely to be auxiliary The substance of helping property, such as lubricant, glidant, wetting agent or emulsifier, pH buffer substance and stabilizer.

SEQUENCE LISTING

<110>Shenzhen Hong Xi biotechnology Development Co., Ltd

<120>for treating or preventing genophore of fluorescent angiography in patients with crystalline retinitis pigmentosa and application thereof

<130>

<160> 8

<170> PatentIn version 3.5

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tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 120

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 180

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 240

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 300

ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 360

ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 420

ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 480

gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 540

ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcg 584

<210> 6

<211> 513

<212> DNA

<213>artificial synthesized

<400> 6

cggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct 60

ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt 120

ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc 180

ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa 240

cggaagccgc acgactcact agtaccctcg cagacggaca gcgccaggga gcaatggcag 300

cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcggggcgc gccgagagca 360

gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc 420

tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc 480

tcgttgaccg aatcaccgac ctctctcccc agg 513

<210> 7

<211> 1188

<212> DNA

<213>artificial synthesized

<400> 7

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120

aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180

gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300

gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 360

agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 420

gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 480

ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 540

tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 600

tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 660

ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct 720

ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 780

tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 840

aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg 900

gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 960

cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 1020

tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 1080

ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 1140

cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtga 1188

<210> 8

<211> 1533

<212> DNA

<213>people

<400> 8

attgctggtt taagaagatt tggattatcc ttgtactttg aggagaagtt tcttatttga 60

aatattttgg aaacaggtct tttaatgtgg aaagatagat attaatctcc tcttctatta 120

ctctccaaga tccaacaaaa gtgattatac cccccaaaat atgatggtag tatcttatac 180

taccatcatt ttataggcat agggctctta gctgcaaata atggaactaa ctctaataaa 240

gcagaacgca aatattgtaa atattagaga gctaacaatc tctgggatgg ctaaaggatg 300

gagcttggag gctacccagc cagtaacaat attccgggct ccactgttga atggagacac 360

tacaactgcc ttggatgggc agagatatta tggatgctaa gccccaggtg ctaccattag 420

gacttctacc actgtcccta acgggtggag cccatcacat gcctatgccc tcactgtaag 480

gaaatgaagc tactgttgta tatcttggga agcacttgga ttaattgtta tacagttttg 540

ttgaagaaga cccctagggt aagtagccat aactgcacac taaatttaaa attgttaatg 600

agtttctcaa aaaaaatgtt aaggttgtta gctggtatag tatatatctt gcctgttttc 660

caaggacttc tttgggcagt accttgtctg tgctggcaag caactgagac ttaatgaaag 720

agtattggag atatgaatga attgatgctg tatactctca gagtgccaaa catataccaa 780

tggacaagaa ggtgaggcag agagcagaca ggcattagtg acaagcaaag atatgcagaa 840

tttcattctc agcaaatcaa aagtcctcaa cctggttgga agaatattgg cactgaatgg 900

tatcaataag gttgctagag agggttagag gtgcacaatg tgcttccata acattttata 960

cttctccaat cttagcacta atcaaacatg gttgaatact ttgtttacta taactcttac 1020

agagttataa gatctgtgaa gacagggaca gggacaatac ccatctctgt ctggttcata 1080

ggtggtatgt aatagatatt tttaaaaata agtgagttaa tgaatgaggg tgagaatgaa 1140

ggcacagagg tattaggggg aggtgggccc cagagaatgg tgccaaggtc cagtggggtg 1200

actgggatca gctcaggcct gacgctggcc actcccacct agctcctttc tttctaatct 1260

gttctcattc tccttgggaa ggattgaggt ctctggaaaa cagccaaaca actgttatgg 1320

gaacagcaag cccaaataaa gccaagcatc agggggatct gagagctgaa agcaacttct 1380

gttccccctc cctcagctga aggggtgggg aagggctccc aaagccataa ctccttttaa 1440

gggatttaga aggcataaaa aggcccctgg ctgagaactt ccttcttcat tctgcagttg 1500

gtgccagaac tctggatcct gaactggaag aaa 1533

Claims (10)

1. a kind of for treating or preventing the genophore of fluorescent angiography in patients with crystalline retinitis pigmentosa characterized by comprising packaging matter Grain, is the AAV2 packaging plasmid for inserting the gene order of anti-CD59 small peptide C9；And

Vector plasmid is the gene order for inserting Human cytochrome P450 superfamily proteins CYP4V2, anti-vegf small peptide HRH Gene order, promoter sequence AAV vector plasmid.

2. genophore according to claim 1, it is characterised in that: the gene order of the CYP4V2 albumen such as SEQ ID Shown in NO:1；The gene order of the HRH is as shown in SEQ ID NO:2；The gene order of the C9 such as SEQ ID NO:3 institute Show.

3. genophore according to claim 1, which is characterized in that the promoter is consensus promoter or retina The specificity promoter of pigment epithelial cell.

4. genophore according to claim 3, which is characterized in that the consensus promoter is to be selected from: CBh promoter, CAG promoter, PGK promoter, EF1 α promoter.

5. genophore according to claim 4, it is characterised in that: the nucleotide sequence such as SEQ of the CBh promoter Shown in ID NO:4；The nucleotide sequence of the CAG promoter is as shown in SEQ ID NO:5；The nucleotide of the PGK promoter Sequence is as shown in SEQ ID NO:6；The nucleotide sequence of the EF1 α promoter is as shown in SEQ ID NO:7.

6. genophore according to claim 3, which is characterized in that the specificity promoter is that RPE65 specificity opens Mover.

7. genophore according to claim 6, it is characterised in that: the nucleotide of the RPE65 specificity promoter tool Sequence is as shown in SEQ ID NO:8.

8. a kind of composition, including genophore as described in claim 1 and pharmaceutically acceptable excipient.

9. genophore as described in claim 1 is used to prepare the drug for treating or preventing fluorescent angiography in patients with crystalline retinitis pigmentosa Purposes.

10. genophore as described in claim 1 is used to prepare the view in the object with fluorescent angiography in patients with crystalline retinitis pigmentosa The purposes of the drug of CYP4V2 and HRH coded sequence is co-expressed in retinal pigment epithelial cell.