CN102872464A - Novel choroidal neovascularization gene therapeutic medicine and use thereof - Google Patents

Abstract

The invention discloses a novel choroidal neovascularization gene therapeutic medicine and use thereof. The medicine Epo-siRNA (erythropoietin-short interfering ribose nucleic acid), by taking an RNA interference technology as base, acquires cDNA sequences of the Epo gene from GenBank, and is the siRNA with 21 nucleotide according to basic principles selected by siRAN target sequence and designed aiming at Epo gene; two deoxyribonucleotide at 5' ends of the medicine Epo-siRNA are in a single strand suspension state; and the medicine Epo-siRNA is subjected to methylation. By the Epo-siRNA, the target mRNA and target protein levels can be efficiently and specifically reduced; the inhibition ratio can be up to 40%; and formation of choroidal neovascularization can be effectively restrained. The invention further discloses the use of the choroidal neovascularization gene therapeutic medicine Epo-siRNA for preparing the medicine for treating the choroidal neovascularization disease.

Description

A kind of novel choroidal neovascularization gene therapy medicament and uses thereof

Technical field

The present invention relates to a kind of short interfering ribonucleic acid (short interfering RNA, siRNA) medicine, produce inhibiting gene therapy medicament to the Epo expression silencing and to choroidal neovascularization on especially a kind of in vivo RNA and the protein level.

Background technology

Choroidal neovascularization refers to from choroidal vascular endothelial cell proliferation, migration, passes Bruch ' s film and enters under subretinal space or the retinal pigment epithelium, forms neovascular tissue, causes bleeding, and forms at last cicatrix.This pathological changes betides macular area especially easily, and namely therefore the sharpest position of human eyesight has a strong impact on vision.Choroidal neovascularization is found in the diseases such as age-related macular degeneration (Age related macular degeneration, AMD), idiopathic choroidal neovascularization, pathologic myopia, angioid streak, and wherein AMD is modal reason.In developed country, AMD is the modal irreversibility causes of blindness of old people.Epidemiology statistics according to the U.S., early stage and late period, 5 annual morbidities of AMD were respectively 6.9% and 0.6% in 60-70 year crowd, be 12.2% and 2.9% among the 70-80 year crowd, be 22.4% among the 80-90 year crowd and be 50% and 22.2% among the crowd more than 6.8%, 90 years old.In China, along with socioeconomic development, the population structure aging, the sickness rate of AMD is also more and more higher, becomes day by day serious public health problem.Showing that according to the Epidemiological study in Shanghai the AMD prevalence is 5.7% among 50-59 year crowd, is 13.5% among 60-69 year crowd, is to be 23.5% among the crowd more than 20.2%, 80 years old among 70-79 year crowd.Clinically, choroidal neovascularization is very thorny disease, does not have desirable Therapeutic Method, usually causes serious visual deterioration even blind, has a strong impact on Quality of Life and ability to work, brings white elephant for patient and family thereof and society.

At present, the pathogenesis of choroidal neovascularization is not yet fully clear, and existing multiple theory thinks that anoxia, heredity, inflammatory reaction, photo-oxidation product, the traction of vitreous body macula lutea, bone marrow vascular endothelial precursor cell etc. may work in the genesis of choroidal neovascularization.In fact, any single theory all can not be explained whole pathophysiological change.Therefore, choroidal neovascularization is that synergism by number of mechanisms causes.The unbalance of new vessels stimulating factor and inhibitive factor is the process of a key, but concrete mechanism wherein is very complicated, relates to participation and the interaction of cytokine profiles.Clinically, the treatment of choroidal neovascularization disease is quite thorny.Operation and laser therapy have been proved poor effect, form the treatment as the therapeutic strategy for the treatment of target as the choroidal neovascularization disease of stimulating factor and inhibitive factor take new vessels and bring new hope.Vascular endothelial cell growth factor (Vascular Endothelial Growth Factor, VEGF) is one of them important new vessels stimulating factor, and anti-VEGF treatment can suppress choroidal neovascularization.But, still there is following problem:

1.VEGF only be that multiple new vessels forms a kind of in the stimulating factor, simple anti-VEGF treatment can not suppress choroidal neovascularization fully.

2. anti-VEGF treatment need to be carried out intraocular injection repeatedly, has increased the chance of occurrence of endophthalmitis.

3. anti-VEGF medicine all is to be researched and developed by the pharmaceutical factory of the U.S., and China lacks the medicine of independent intellectual property right.

Therefore, research to other new vessels stimulating factors or inhibitive factor can make us understand better the pathogenesis of choroidal neovascularization disease, and find more treatment target, also may obtain by drug combination the safety of better curative effect and Geng Gao.Simultaneously, also can reduce by the medicine of China's independent intellectual property right patient's financial burden.Erythropoietin (Erythropoietin, EPO) is the cytokine of " ancient ", and the Main Function recognized of people is to stimulate erythropoiesis at the beginning.In recent years, along with going deep into of research, find that EPO also participates in new vessels and forms.EPO and receptor thereof have expression in vascular endothelial cell and smooth muscle cell.EPO can stimulate that new vessels forms in the aortic arch model of Embryo Gallus domesticus and In vitro culture.In animal model, exogenous EPO can promote wound healing, the reparation of ischemia myocardial damage and brain injury, and promote that tumor growth, its mechanism of action are to promote the new vessels in these models to form.

EPO also participates in the morbidity of retinal neovascularization disease.Proliferative diabetic retinopathy (Proliferative diabetic retinopathy, PDR) concentration of EPO is significantly higher than matched group in patient's vitreous body, and vascular endothelial cell and the stromal cell of the PDR patient's who takes out in operation preretinal membrane have EPO receptor strongly expressed.In the retinal neovascularization mouse model that oxygen is induced, the gene expression of EPO, EPO receptor and VEGF all significantly raises, and adopts EPO soluble recepter and siRNA blocking-up EPO signal can suppress retinal neovascularization at anaerobic phase and forms.Clinically, recombinant epo albumen has been used for the treatment of anemia, and is particularly common in diabetic nephropathy patient, also is used for premature infant's Supporting Therapy, but have report EPO may accelerate the progress of proliferative diabetic retinopathy and retinopathy of prematurity.Last year, our research finds that a single nucleotide polymorphism rs1617640 T allele that is arranged in the EPO gene promoter is significantly higher than matched group in 1 type and the concurrent PDR patient's of type 2 diabetes mellitus frequency, and T allele has increased the expression of EPO, the T allele that measures with Luciferase reporter gene method can make the expression of EPO raise 25 times, and the genotypic individuality of epo protein concentration ratio GG in the individual vitreous body of TT genotype has exceeded 7.5 times.

The mechanism that EPO participates in new vessels formation may relate to whole body and local two aspects.EPO has the biological effect of whole body, can promote erythropoiesis, also can Effects of Bone Marrow Stem Cells Mobilization enter blood circulation, exogenous EPO treatment can increase the quantity of blood circulation Vascular Endothelial precursor, and propagation, the transfer ability of these cells have been strengthened, and the new vessels formation of this promotion ischemic myocardial pathological changes, improve myocardial ischemia.Our result in early stage shows that EPO can promote human umbilical vein endothelial cell migration and the sleeve pipe of In vitro culture to form, and the VEGF of its effect and same molar ratio is suitable, also has bibliographical information EPO can promote the retinal endothelial cell propagation of cultivating.Other mechanism that EPO participates in new vessels also may comprise the expression of regulating the cytokines such as VEGF, the apoptosis of vascular endothelial cell that the inhibition many reasons causes etc.Research in EPO signaling system and the ocular angiogenesis disease mainly concentrates on the retinal neovascularization disease.Also there are report EPO and EPO receptor in pterygia, to express increase, are higher than the healthy conjunctiva tissue.But the relation between EPO signaling system and the choroidal neovascularization there is not yet report.Choroidal neovascularization and retinal neovascularization may have some common pathogenesis.Anoxia plays an important role in choroidal neovascularization forms equally, and the expression of hypoxia inducible factor (Hypoxia inducible factor, HIF) in choroidal neovascularization also increases, and HIF can raise the expression of EPO.Our early-stage Study has found that also EPO expresses increase in the choroidal neovascularization of induced with laser.On the other hand, the vascular endothelial precursor cell of bone marrow derived has also participated in the formation of choroidal neovascularization, blocks the area that can reduce choroidal neovascularization that sticks of precursor and new vessels.

In sum, the EPO signaling system may work in the choroidal neovascularization disease; Its mechanism may promote the approach such as vascular endothelial precursor cell release by local direct stimulation to vascular endothelial cell and whole body; Therefore, theoretically, the treatment of blocking-up EPO signal path also can suppress choroidal neovascularization.

And the RNA that found in 1998 disturbs (RNAi) phenomenon, is ubiquitous a kind of physiological mechanism of defending Exogenous Nucleic Acid to invade and harass all from the unicellular lower eukaryote to the higher organism, and it is the PTGS effect by the high special of siRNA mediation.Bring into play silencing complex (the RNA-induced silencing complex that guide effect comes activator RNA to induce by siRNA, RISC), come high special ground cutting said target mrna o RNA perturbation technique to develop into an effective tool that suppresses expression of target gene by wherein RNase III.

Although the research of RNAi still is in the imperfection stage, oneself is considered to the intrinsic genome of a kind of organism " immune system " for it, so that it has the incomparable advantage of many antisensenucleic acidses.SiRNA has the following characteristics: the former is played a role in cell by double stranded rna molecule, owing to may have certain stabiliser in the cell, so that the stability of siRNA is higher; The positive-sense strand of siRNA and antisense strand make RNAi compare with traditional antisense technology and have high efficiency, the characteristics of high specific just because of these characteristics.Oneself has a large amount of reports to show at present, imports siRNA in mammal and the human cell and can cause the efficient special degraded of said target mrna molecule.

Therefore, the RNA interference mechanism that itself exists take this organism is the basis, manually design and synthesize for the special siRNA conduct of Epo gene and bring out the precursor that RNA disturbs, the mRNA of the efficient Epo gene of degrading specifically, reduce the Epo protein level, reduce the generation of choroidal neovascularization, can provide more efficiently medicine for the gene therapy of the choroidal neovascularization diseases such as AMD.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art part and provides a kind of and can play reticent effect, efficiently reduce specifically the novel choroidal neovascularization gene therapy medicament Epo-siRNA of said target mrna and target protein level the expression of Epo; Simultaneously, the present invention also provides described medicine Epo-siRNA for the preparation of the purposes in the medicine for the treatment of choroidal neovascularization disease.

For achieving the above object, the technical scheme that the present invention takes is: a kind of novel choroidal neovascularization gene therapy medicament Epo-siRNA, described medicine Epo-siRNA is as the basis take the RNA perturbation technique, the basic principle that from GenBank, obtains the cDNA sequence of Epo gene, selects according to the siRNA target sequence, be the siRNA of 21 nucleotide for the length of Epo gene design, it is the single catenary suspension state that the 5 ' end of described medicine Epo-siRNA has two deoxyribonucleotides, and described medicine Epo-siRNA has carried out the modification that methylates.

The present invention is take the RNA perturbation technique as the basis, from GenBank, obtain the cDNA sequence of Epo gene, basic principle according to the selection of siRNA target sequence, for the Epo gene design length be the siRNA of 21 nucleotide, 3 ' the end of siRNA has two deoxyribose nuclear former times acid (dTdT) to be the single catenary suspension state, and siRNA carried out the modification that methylates, with this strengthen siRNA in vivo with external toleration to nuclease (RNase).Employed siRNA is by inventor's designed, designed, entrusts Wuhan Biomic company synthetic by chemical method among the present invention.

Preferred implementation as novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention, 5 ' end of the antisense strand of described medicine Epo-siRNA is A/U, have 5 A/U at least in 7 bases of 5 ' end of described antisense strand, 5 ' end of the positive-sense strand of described medicine Epo-siRNA does not have continuous GC fragment more than 9 for G/C in the described siRNA sequence.

As the preferred implementation of novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention, the oligonucleotide sequence of described medicine Epo siRNA is:

Positive-sense strand: 5'GACCCUUCAGCUUCAUAUATT3';

Antisense strand: 5'UAUAUGAAGCUGAAGGGUCTT3'.

As the preferred implementation of novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention, described medicine Epo-siRNA can play the silence effect that Epo is expressed.Utilize the siRNA of 5g/l that induced with laser choroidal neovascularization mice is carried out intravitreal.At first retina is spread sheet and carry out the new vessels analysis, the result shows, negative control-the siRNA that any gene is not produced reticent effect to the choroidal neovascularization of mice induced with laser without obvious inhibitory action, and Epo-siRNA can produce fairly obvious inhibitory action to it, illustrate that Epo-siRNA can play the silence effect that Epo is expressed, thereby suppress Epo signal path and the further generation of minimizing choroidal neovascularization.

As the preferred implementation of novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention, described medicine Epo-siRNA is inhibited to the mrna expression of Epo gene.Adopt real-time quantitative PCR (realtime-PCR) method to detect Epo-siRNA to the impact of Epo mRNA.After with siRNA induced with laser choroidal neovascularization mice being carried out intravitreal, extract the mRNA of tela chorioidea, the step of going forward side by side is carried out reverse transcription and realtime-PCR.The result shows that the trend that the mrna expression of the mice Epo of its tela chorioidea gene of induced with laser new vessels rises after the Epo-siRNA injection is obviously suppressed, and the mRNA level of the then Epo gene of processing through negative control siRNA obviously rises.

As the preferred implementation of novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention, described medicine Epo-siRNA is inhibited to the Epo protein level.Detect the silence effect of Epo-siRNA with Western Blotting method.Western Blotting result shows that the Epo-siRNA of its tela chorioidea of mice of induced with laser new vessels presents obvious reticent effect to the impact of Epo protein level after the Epo-siRNA injection.The body weight of matched group and administration group nude mice approaches in the experiment, and each group is all without animal dead.

In addition, the present invention also provides a kind of choroidal neovascularization gene therapy medicament Epo-siRNA described above for the preparation of the purposes in the medicine for the treatment of choroidal neovascularization disease.Preferably, described choroidal neovascularization disease comprises degeneration of macula, idiopathic choroidal neovascularization, pathologic myopia and angioid streak.

Choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention is take the RNA interference as technology platform, a kind of brand-new choroidal neovascularization gene therapy medicament is provided, has comprised the reticent effect detection of siRNA design, the detection of choroidal neovascularization form, mRNA and protein level etc.The Epo-siRNA of gained can efficiently reduce said target mrna and target protein level specifically, and suppression ratio reaches 40%, the formation of establishment choroidal neovascularization.Therefore, described Epo-siRNA has a wide range of applications to the gene therapy of the choroidal neovascularization diseases such as AMD.

Description of drawings

Fig. 1 does the time spent for adopting Western blot to detect novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention to the silence of epo protein, and right and left eyes is injected respectively Epo siRNA and the negative control situation that Epo expresses after one week in the mice of three induced with laser choroidal neovascularization.

Fig. 2 is the as a result figure to Epo siRNA and negative control Western blot quantitative analysis.

When Fig. 3 is novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention to the inhibitory action of the choroid/retinal neovascularization of induced with laser, the right and left eyes injection situation of Epo siRNA and negative control retinal neovascularization after one week respectively in the mice of induced with laser choroidal neovascularization.

Fig. 4 is the as a result figure to Epo siRNA and the analysis of negative control retinal neovascularization area quantitative.

The specific embodiment

Embodiment 1siRNA design

From GenBank, obtain the Epo gene order, its AccessionNumber:Epo (Acc.No.NM_007942.2) in GenBank.Following 4 design principles are satisfied in Epo siRNA design simultaneously, in order to can reach higher Gene silencing efficacy:

1.antisense 5 ' end for A/U;

2.sense 5 ' end for G/C;

3.antisense 5 ' end 7 bases in have 5 A/U at least;

4.siRNA there is not continuous GC fragment more than 9 in the sequence.

The Epo-siRNA oligonucleotide sequence of design is as follows:

Positive-sense strand: 5'GACCCUUCAGCUUCAUAUATT3';

Antisense strand: 5'UAUAUGAAGCUGAAGGGUCTT3 ',

The negative control siRNA sequence that adopts in the experiment is:

Positive-sense strand 5'-UUCUCCGAACGUGUCACGUTT3';

Antisense strand 5'ACGUGACACGUUCGGAGAATT3'.

The choroidal neovascularization mouse model of embodiment 2 experimental construction induced with laser

Get the C57BL/6 mice at 2-3 monthly age, with compound tropicamide eye drop mydriasis, general anesthesia, with 532nm laser machine (Quantel medical, VITRA, France) and slit lamp microscope (Haag-Streit, BQ900, Germany) carry out light and coagulate, with the cornea applanation sheet, just can under slit lamp, observe the mice optical fundus with coverslip.Looking 2 disc diameter places around the nipple, 3,6,9 o'clock, to carry out 3 light solidifying in the position, and parameter is as follows: energy 120mW, and time of exposure 0.1s, spot diameter 75nm has Bubble formation as the sign of success when solidifying with light.

Embodiment 3 choroid spread the measurement of sheet and new vessels area

Induced with laser is after one week, and disconnected neck is put to death mice, extracts eyeball, reduces intraocular pressure with 30G syringe needle paracentesis of anterior chamber, wipes out a fritter cornea tissue with microscissors, places 4% paraformaldehyde to fix 2 hours.Wipe out anterior chamber of eye under stereoscopic microscope (Leica, M Z9.5), take out crystal, blunt separation retina and choroid discard choroid/sclera, get complete retina.Retina is carried out Isolectin GS-IB4Alexa Flour488conjugate immunofluorescence dyeing.Fluorescence immunoassay system: cumulative volume 25ul(0.2mM MgCl2 solution+0.2%Triton X-100 solution+10ul1ug/ul Isolectin GS-IB4Alexa Flour488conjugate) spend the night in 4 ℃ of shaking tables.The shop sheet operates on the microscopically microscope slide retina is become four radial incisions, drips upper an amount of mountant covered.In the lower observation of fluorescence microscope (Nikon, E80I, Japan), take a picture, with image analysis software Image J(NIH, the U.S.) measurement new vessels area.

Embodiment 4 intravitreal siRNA:

Routine disinfection assists to open the mice eyelid with left hand, and is rear to optic nerve direction inserting needle in angle Gong Yuan puncture with the 30G syringe needle, withdraws from the rear microinjector (Hamilton, the U.S.) of using and contains 0.5 ~ 5ug siRNA to intravitreal 1ul.Process eye injection EPO-siRNA, the negative control siRNA of Second eye injection equivalent.

It is reticent that embodiment 5Real Time PCR detects siRNA

From retina, tela chorioidea, use Nucleo Spin test kit (Clontech) to extract total RNA, using SuperScript III First Strand Synthesize System(Invitrogen) reverse transcription is cDNA, carry out Real Time PCR reaction with Qiagen QuantiTect SYBR Green test kit, used RT-PCR instrument is Bio-Rad iQ5.The primer such as the following table that adopt with β-actin(Actb).The parameter of RT-PCR reaction is as follows: first 50 ℃ of 2min that circulate, 95 ℃ of 15min carry out 35 circulations subsequently, 94 ℃ 15 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds.Cycle threshold (CT) value that reaction finishes rear gained Epo siRNA and negative control siRNA is organized data adopts Δ Δ CT relative quantification method, the change of calculating the Epo expression according to following formula.

Annotate: E is amplification efficiency, is defaulted as 100%

Embodiment 6Western blot detects the reticent effect of siRNA

Induced with laser is after one week, and disconnected neck is put to death mice, extracts eyeball, wipes out anterior chamber of eye under stereoscopic microscope (Leica, M Z9.5), takes out crystal, and blunt separation retina and choroid discard choroid/sclera, get complete retinal tissue.Carry out following experiment:

1, the choroid total protein extracts: tissue is put adding RIPA Lysis Buffer100ul(MILLIPORE in the 1.5mlep pipe), contain protease inhibitor 1ul(MERCR--protease inhibitor cocktail set III, EDTA-Free), in homogenate on ice, to particle suspension, react 30mi on ice, 1,6000rcf/min, 20min, draw the albumen supernatant, packing.

2, total protein concentration is measured: formulate standard curve with 2mg/ml calf serum (BSA) 0,0.5,2.5,5,10,20,40,200ng/ml.With 100 times of dilutions of specimen, behind the adding developer (Protein assay kit, PIERCE), on microplate reader, compare by the OD value that records, calculate the concentration of specimen.

3, in the protein sample behind 6 * loading buffer, 90-100 ℃ of heating 10min, the cooling rear electrophoresis, applied sample amount is 50ug.

4, electrophoresis: preparation double focusing acrylamide gel, concentrated glue 60V, electrophoresis 30min; Separation gel 100V, electrophoresis 90min.

5, transfer printing: under the effect of electric current, protein is transferred to solid phase carrier 0.45um PVDF(MILLIPORE from glue) on the film.100mA on ice is with the wet 60min that turns of electrophresis apparatus (Bio-rad).After Ponceau S (worker is given birth in Shanghai) dyed in advance, protein band was high-visible.

6, sealing: pvdf membrane is put into 5% defatted milk powder (disposing with phosphate buffer) room temperature 1h.

7, primary antibodie is hatched (SANTACRUZ): with antibody 1:100 dilution, pvdf membrane is immersed in the antibody diluent, 4 degree spend the night.TBST(contains 50mM Tris-HCL, 150mM NaCl, 0.05%Tween20, pH7.6) washing 10min * 3 time.

8, two anti-hatch (SANTACRUZ):, pvdf membrane is immersed in the antibody diluent room temperature 2h, TBST washing 10min * 3 time with antibody 1:1000 dilution.

9, develop: pvdf membrane is lain on gel imaging system instrument (Bio-rad) platform, drip developer (SANTA CRUZ), to covering whole film, behind the lucifuge reaction 5min, take pictures under the dark ground (Quantity One), analysis image (ImageJ).

Experimental result can be found out by accompanying drawing 1 that shown in accompanying drawing 1-4 novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention has significant reticent effect to epo protein; As seen from Figure 2, Epo siRNA and negative control have significant difference.Can be found out that by accompanying drawing 3 novel choroidal neovascularization gene therapy medicament Epo-siRNA of the present invention has significant inhibitory action to the choroid/retinal neovascularization of induced with laser; Can be found out that by accompanying drawing 4 Epo siRNA and negative control have significant difference.

Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment the present invention has been done detailed description; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.

Claims (8)

1. novel choroidal neovascularization gene therapy medicament Epo-siRNA, it is characterized in that, described medicine Epo-siRNA is as the basis take the RNA perturbation technique, the basic principle that from GenBank, obtains the cDNA sequence of Epo gene, selects according to the siRNA target sequence, be the siRNA of 21 nucleotide for the length of Epo gene design, it is the single catenary suspension state that the 5 ' end of described medicine Epo-siRNA has two deoxyribonucleotides, and described medicine Epo-siRNA has carried out the modification that methylates.

2. novel choroidal neovascularization gene therapy medicament Epo-siRNA as claimed in claim 1, it is characterized in that, 5 ' end of the antisense strand of described medicine Epo-siRNA is A/U, have 5 A/U at least in 7 bases of 5 ' end of described antisense strand, 5 ' end of the positive-sense strand of described medicine Epo-siRNA does not have continuous GC fragment more than 9 for G/C in the described siRNA sequence.

3. novel choroidal neovascularization gene therapy medicament Epo-siRNA as claimed in claim 2 is characterized in that the oligonucleotide sequence of described medicine Epo siRNA is:

Positive-sense strand: 5'GACCCUUCAGCUUCAUAUATT3';

Antisense strand: 5'UAUAUGAAGCUGAAGGGUCTT3'.

4. novel choroidal neovascularization gene therapy medicament Epo-siRNA as claimed in claim 1 is characterized in that, described medicine Epo-siRNA can play the silence effect that Epo is expressed.

5. novel choroidal neovascularization gene therapy medicament Epo-siRNA as claimed in claim 1 is characterized in that described medicine Epo-siRNA is inhibited to the mrna expression of Epo gene.

6. novel choroidal neovascularization gene therapy medicament Epo-siRNA as claimed in claim 1 is characterized in that described medicine Epo-siRNA is inhibited to the Epo protein level.

7. novel choroidal neovascularization gene therapy medicament Epo-siRNA as claimed in claim 1 is for the preparation of the purposes in the medicine for the treatment of choroidal neovascularization disease.

8. purposes as claimed in claim 7 is characterized in that, described choroidal neovascularization disease comprises degeneration of macula, idiopathic choroidal neovascularization, pathologic myopia and angioid streak.

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