CN110423281A - For treating the fusion protein, viral vectors and drug of senile macular degeneration - Google Patents
For treating the fusion protein, viral vectors and drug of senile macular degeneration Download PDFInfo
- Publication number
- CN110423281A CN110423281A CN201910705207.1A CN201910705207A CN110423281A CN 110423281 A CN110423281 A CN 110423281A CN 201910705207 A CN201910705207 A CN 201910705207A CN 110423281 A CN110423281 A CN 110423281A
- Authority
- CN
- China
- Prior art keywords
- structural domain
- ser
- leu
- thr
- lys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The invention discloses the fusion proteins, viral vectors and drug for treating senile macular degeneration, are related to gene therapy technology field.The fusion protein disclosed by the invention for being used to treat senile macular degeneration comprising the structural domain 1 and structural domain 2 selected from VEGFR1, the structural domain 3 selected from VEGFR2 and the Fc segment selected from immunoglobulin.The fusion protein can treat senile macular degeneration, have the characteristics that few dosage, effect lasts, low immune risk.
Description
Technical field
The present invention relates to gene therapy technology fields, in particular to the fusion for treating senile macular degeneration
Albumen, viral vectors and drug.
Background technique
Senile macular degeneration (AMD) is increasingly prominent a kind of seriousness eye disease after the mankind enter aging society
Disease is the common eye disease of 50 years old or more crowd, and the main reason for lead to visual loss.According to whether having abnormal new
The presence of angiogenic is clinically divided into atrophic or stemness AMD and two type of exudative or moist AMD (wAMD)
Type.Stemness AMD is gradual visual loss, accounts for 90%, and moist AMD disease is rapid, is cause visual loss main
One of factor only accounts for 10%, but the blindness caused by it accounted for it is all because of 90% or more of blindness caused by AMD.Data are aobvious
Show, arrive the year two thousand twenty, global about 200,000,000 people of patients with macular degeneration number, it is contemplated that the year two thousand forty, will increase to 300,000,000 people, become old
The main arch-criminal of people's blindness.AMD illness rate in 45 years old Chinese or more crowd is 2.44%-18.98%, and in city
Brain worker's incidence is higher, with the quickening of population aging process, to the year two thousand fifty, Chinese macular degeneration patient populations
About fifty-five million people.On the other hand, the high energy blue light of electronic world penetrates the cornea of eyes and crystalline lens reaches retina, most
First make brain excitement without feeling fatigue, the decaying of long-time eyesight can induce eye fatigue, dry eyes sign, circadian rhythm disturbances, directly
Penetrating crystalline lens arrival retina can accelerate to damage macular area, result in the acceleration of the fine macular area degree of injury of eye, from
And increase the younger of the AMD course of disease and extension.AMD has become the third-largest blinding eye disease, is classified as whole world effort exploration and grinds
One of disease areas studied carefully.
Moist AMD is mainly that abnormal new vessels are grown under the retina of macula area, referred to as choroidal neovascularization
(CNV), macular area local edema or bleeding is caused to cause the protuberance and limitation retinal pigment epithelium detachment of macular area.It eventually leads to
Scar is formed, and damages retinal photoreceptor cells so as to cause visual loss.Therefore by inhibiting vascular endothelial growth factor
(VEGF), the growth of blocking lesion new vessels can effectively treat retinopathy.
It is intravitreal injection protide VEGF inhibitor that a generally acknowledged line of moist AMD, which treats mode,.Currently, for controlling
The protide VEGF inhibitor major product for treating moist AMD has the bevacizumab (bevacizumab) of Roche, Roche/Novartis
Fab antibody segment Lucentis, VEGFR-Fc (VEGF-trap) the fusion protein aflibercept of regeneration member and Sichuan
The VEGFR-Fc fusion protein Conbercept of Kang Hong medicine company.
The VEGF target drug listed
Although the appearance of VEGF inhibitor has brought the moist revolutionary progress in the field AMD, problem is still remained.
Firstly, leakage can be reduced in intraocular injection VEGF with albumen, eyesight is improved, is continued slowly however, the generation of VEGF is one
Process, therefore in VEGF and when concentration of the albumen in vitreum is lower than treatment level, leakage and neovascularization growth are multiple
Hair；Secondly, the frequently intracorporal VEGF inhibitor injection of glass cavity, is all great burden for doctor and patient；Furthermore albumen
Class drug price is expensive, by taking VEGF Trap as an example, need to inject every year 8 times, price reaches 60,000 yuan/person/year；In addition, clinically
The study found that the long-term prognosis that anti-vegf drug carries out treatment 4 years or more is not good enough, VEGF inhibitor drug resistance is also one
Huge problem.How to reduce dosage rate, develop the occurrence of new administration route and reduction drug resistance, is wet at present
Property AMD treat the main bottleneck that faces.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of for treating the fusion protein of senile macular degeneration.The fusion protein
It can treat senile macular degeneration, have the characteristics that few dosage, effect lasts, low immune risk.
Another object of the present invention is to provide a kind of nucleic acid molecules.The nucleic acid molecules can express above-mentioned fusion egg
It is white, have the characteristics that expression efficiency is high.
Another object of the present invention is to provide a kind of expression cassettes.The expression cassette can give expression to above-mentioned fusion protein, tool
Have the characteristics that expression efficiency is high.
Another object of the present invention is to provide a kind of carriers.The carrier can give expression to above-mentioned fusion protein, have table
Up to feature high-efficient, that expression continues, dosage is few.
Another object of the present invention is to provide a kind of drugs for treating senile macular degeneration.The drug can treat
Senile macular degeneration has the characteristics that few dosage, effect lasts, low immune risk.
The present invention is implemented as follows:
In a first aspect, the present invention provides a kind of fusion proteins for senile treatment macular degeneration comprising be selected from
The structural domain 1 and structural domain 2 of VEGFR1 (vegf receptor 1), the structural domain 3 selected from VEGFR2 (vegf receptor 2) and selected from exempting from
The Fc segment of epidemic disease globulin.
Fusion protein provided by the invention can be realized by the structure optimization to functional protein to Age related macular
The treatment of denaturation has the characteristics that few dosage, effect lasts, low immune risk.
Further, in some embodiments of the present invention, the amino acid sequence of the structural domain 1 selected from VEGFR1
Column are as shown in SEQ ID NO.9；
Preferably, the structural domain 1 is located at the N-terminal of the fusion protein；
Preferably, the N-terminal of the fusion protein to C-terminal is followed successively by the structural domain 1, the structural domain 2, the structure
Domain 3 and Fc sections described；
Preferably, the amino acid sequence of the structural domain 2 selected from VEGFR1 is as shown in SEQ ID NO.10；
Preferably, the amino acid sequence of the structural domain 3 selected from VEGFR2 is as shown in SEQ ID NO.11.
Preferably, the immunoglobulin is human IgG1；
Preferably, the amino acid sequence of the Fc segment is as shown in SEQ ID NO.12；
Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.8.
Second aspect, the present invention provides a kind of nucleic acid molecules, encode fusion protein as described above.
Preferably, shown in the base sequence SEQ ID NO.1 of the nucleic acid molecules.
Nucleic acid molecules provided by the invention can expeditiously express above-mentioned melt by the optimization to base sequence
Hop protein, improve therapeutic effect, and advantageously reduce deliver the nucleic acid molecules make to intracorporal carrier (such as viral vectors)
With dosage, help to reduce immune risk.
The third aspect, the present invention provides a kind of expression cassettes, containing described in nucleic acid molecules as described above and driving
The promoter of nucleic acid molecules expression.
Further, in some embodiments of the present invention, the promoter is CB7 promoter；
Preferably, the base sequence of the CB7 promoter is as shown in SEQ ID NO.2；
Preferably, the expression cassette also contains Kozak sequence, the Kozak sequence be located at the downstream of the promoter and
Positioned at the upstream of the nucleic acid molecules；
Preferably, the expression cassette also contains intron sequences；The intron sequences are located at the downstream of the promoter
And it is located at the upstream of the Kozak sequence；
Preferably, the expression cassette contains also terminator；The terminator is located at the downstream of the nucleic acid molecules；
Preferably, the terminator is SV40polyA.
Expression cassette provided by the invention can expeditiously express above-mentioned melt by the optimization to each Expression element
Hop protein improves therapeutic effect, and advantageously reduces and deliver the use of the expression cassette to intracorporal carrier (such as viral vectors)
Dosage helps to reduce immune risk.
Fourth aspect, the present invention provides a kind of carriers, contain expression cassette as described above.
Further, in some embodiments of the present invention, the carrier is plasmid vector or viral vectors.
Preferably, the viral vectors is recombined glandulae correlation viral vectors.
Further, in some embodiments of the present invention, the recombined glandulae correlation viral vectors be selected from AAV1,
Any one in AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh10；
Preferably, the recombined glandulae correlation viral vectors are AAV2, AAV5, AAV8 or AAV9.
Adeno-associated virus (AAV) is the simplest single stranded DNA defective virus of a presently found class formation, is needed auxiliary
Viral (usually adenovirus) is helped to participate in duplication.Recombined glandulae correlation viral vectors (rAAV) are by cap the and rep gene in AAV
It is substituted for target gene, target gene is not inserted into the chromosome of cell, in the form of independent exchromosomal DNA is episomal
It is retained in nucleus.Since its safety is good, host cell range is wide, division and non-dividing cell, immunogenicity can be infected
Low, the features such as expression alien gene time is long in vivo, it is considered as most promising gene therapy vector, worldwide
Gene therapy and vaccine research in be used widely.Also an attractive tool is provided for intraocular gene delivery.
The clinic of the principle and AAV carrier of moist AMD in single-gene disorder is treated in view of VEGF-trap antagonism VEGF
Using above-mentioned nucleic acid molecules are delivered to layer of retina cell using AAV carrier and realize high efficient expression by the present invention, and are used
Dosage is lower, reduces immune risk, can achieve the purpose of effectively treatment AMD.
5th aspect, the present invention provides a kind of drugs for treating senile macular degeneration, contain and melt as described above
Hop protein or carrier as described above；
Preferably, the drug further includes pharmaceutically acceptable auxiliary material.
Further, in some embodiments of the present invention, the dosage form of the drug is injection；
Preferably, the dosage form of the drug is suitable for the injection using vitreous chamber or subretinal injection.
The drug can treat senile macular degeneration, have the characteristics that few dosage, effect lasts, low immune risk.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment
Attached drawing is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not to be seen as
It is the restriction to range, it for those of ordinary skill in the art, without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the structural schematic diagram of the expression vector of the embodiment of the present invention 1.
Fig. 2 is the structural schematic diagram of the control vector 1 of the embodiment of the present invention 1.
Fig. 3 is the structural schematic diagram of the control vector 2 of the embodiment of the present invention 1.
Fig. 4 is the structural schematic diagram of the control vector 3 of the embodiment of the present invention 1.
Fig. 5 is the structural schematic diagram of the control vector 4 of the embodiment of the present invention 1.
Fig. 6 is the testing result of the protein expression efficiency of each carrier in the embodiment of the present invention 1.In figure: Untreated
It represents: untransfected plasmid group.
Fig. 7 is in expression vector vivoexpression VEGF in the embodiment of the present invention 2 and the influence that is proliferated to HUVEC of albumen；
In figure: negative control represents not plus VEGF-165 stimulates HUVEC proliferation group, and positive control, which represents, only adds VEGF-165 stimulation
HUVEC proliferation group adds untransfected HEK293 cell after Untreated represents addition VEGF-165 stimulation HUVEC proliferation
Supernatant processing.Untreated group in each embodiment represent be untransfected HEK293 cell supernatant.
Fig. 8 be the embodiment of the present invention 3 in expression vector vivoexpression VEGF in and albumen to HUVEC at the influence of pipe.
In figure: EGM2 is represented: the positive controls of EGM2 medium treatment.
Fig. 9 is in expression vector vivoexpression VEGF in the embodiment of the present invention 4 and the influence that migrates to HUVEC of albumen.
In figure: oh control is represented: 0 hour control group of scratch；Non- sugaring represents: the negative control of non-sugaring induction HUVEC migration
Group, high sugar represent: the positive controls of high glucose induction HUVEC migration are added；Untreated is represented: untransfected is added
The supernatant of HEK293 cell is handled.
Figure 10 is the affinity inspection in the expression vector vivoexpression VEGF in the embodiment of the present invention 5 with albumen to VEGF
Survey result.
Figure 11 is the inspection in the internal VEGF expression of the recombined glandulae correlation viral vectors in the embodiment of the present invention 7 with albumen
Survey result.
Figure 12 be the embodiment of the present invention 8 in give recombined glandulae correlation viral vectors after detect monkey under different time
In VEGF in right eye and the result of albumen.
Figure 13 is to give recombinant adenoviral vector treatment to choroidal neovascularization model monkey in the embodiment of the present invention 8
The fluorescein fundus angiography inspection result of front and back.
Figure 14 is the structure chart in the VEGF expression in embodiment 1 with the AAV packaging plasmid of protein gene.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer
It is recommended that condition carry out.Reagents or instruments used without specified manufacturer is the routine that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
(1) structure of expression vector
Expression vector provided in this embodiment contains the nucleic acid expressed for treating the fusion protein of senile macular degeneration
Molecule, structure as shown in Figure 1, its from 5 ' end to 3 ' end contain: CB7 promoter, introne, Kozak sequence (gccacc),
The structural domain 1 of VEGFR1, the structural domain 2 of VEGFR1, the structural domain 3 of VEGFR2, immunoglobulin human IgG1 Fc segment with
And SV40polyA.Wherein, the base sequence of CB7 promoter is as shown in SEQ ID NO.2, the base sequence of introne such as SEQ
Shown in ID NO.3, the coded sequence of the structural domain 1 of VEGFR1 is as shown in SEQ ID NO.4, the volume of the structural domain 2 of VEGFR1
Code sequence as shown in SEQ ID NO.5, the coded sequence of the structural domain 3 of VEGFR2 is as shown in SEQ ID NO.6, immunoglobulin
The coded sequence of the Fc segment of IgG1 is as shown in SEQ ID NO.7.
Treated expressed by above-mentioned expression vector the structure of the fusion protein of senile macular degeneration from N-terminal to C-terminal according to
It is secondary are as follows: the structural domain 1 and structural domain 2 of VEGFR1, the structural domain 3 of VEGFR2 and the Fc piece selected from immunoglobulin human IgG1
Section；The amino acid sequence of the fusion protein (in hereinafter referred to as VEGF and albumen) is as shown in SEQ ID NO.8；The knot of VEGFR1
The amino acid sequence in structure domain 1 is as shown in SEQ ID NO.9, the amino acid sequence of the structural domain 2 of VEGFR1 such as SEQ ID NO.10
The amino acid sequence of shown, VEGFR2 structural domain 3 is as shown in SEQ ID NO.11, the Fc segment of immunoglobulin human IgG1
As shown in SEQ ID NO.12.The coded sequence of the fusion protein is encoded as shown in SEQ ID NO.1.(2) structure of expression vector
It builds
Expression cassette is constructed, the conventional molecular biologicals operation building such as digestion, connection, conversion and colony screening identification is passed through
In VEGF expression and the AAV packaging plasmid (see Figure 14) of protein gene, successively include: CB7 promoter, introne,
Kozak sequence, the structural domain 2 selected from VEGFR1, the structural domain 3 selected from VEGFR2, is selected from the structural domain 1 selected from VEGFR1
The Fc segment and SV40 polyA of Immunoglobulin IgG1, expression cassette two sides are inverted terminal repeat (ITR), knot
Composition schematic diagram is shown in that Fig. 1, SphI and AgeI are restriction enzyme site.
(3) the vivoexpression efficiency of expression vector
By the above-mentioned expression vector of 120ng in 96 orifice plates transfected HEK 293, collect cell conditioned medium after 48h, use
In ELISA method detection VEGF and protein content, and control vector (control vector 1-4) is set, compares the expression egg of these carriers
White efficiency；
The structure difference of control vector 1-4 is as shown in Figure 2-5:
Control vector 1 lacks the structural domain 1 and VEGFR2 of VEGF expression R1 compared to the expression vector of the present embodiment 1
The nucleic acid molecules of structural domain 3；Albumen expressed by control vector 1 is named as fusion protein A, by 2 He of structural domain of VEGFR1
The Fc segment of immunoglobulin human IgG1 is constituted.
Control vector 2 lacks the nucleic acid molecules of the structural domain 1 of VEGF expression R1 compared to the expression vector of the present embodiment 1；
Albumen expressed by control vector 2 is named as fusion protein B (i.e. VEGF Trap albumen), by the structural domain 2 of VEGFR1,
The structural domain 3 of VEGFR2 and the Fc segment of immunoglobulin human IgG1 are constituted.
Control vector 3 increases WPRE sequence and bGH tune in Fc segment downstream compared to the expression vector of the present embodiment 1
Control sequence.
WPRE sequence is as shown in SEQ ID NO.13, and bGH regulating and controlling sequence is as shown in SEQ ID NO.14.
Control vector 4 compared to the present embodiment expression vector CMV promoter instead of CB7 promoter；Control vector
Albumen expressed by 3 and 4 is consistent with fusion protein structure expressed by 1 expression vector of the present embodiment.
As a result as shown in fig. 6, being as the result is shown pair with the content of albumen in VEGF expressed by the expression vector of the present embodiment
According in carrier 1,2,3 and 4 VEGF expressions and 2-2.4 times of protein content, which absolutely proves that the embodiment of the present invention 1 is mentioned
The expression vector of confession can effectively improve in VEGF expression and the efficiency of albumen.
Embodiment 2
The influence that HUVEC is proliferated with albumen in expression vector vivoexpression VEGF
Cell suspension is diluted to 1.5 × 10 using EGM2 complete medium4A/ml, 200 holes μ l/ spread 96 orifice plates, carefully
After the adherent 4h of born of the same parents, culture medium in hole is changed to 100 μ l M199+5%FBS culture mediums；37 DEG C of incubator cultures for 24 hours, change liquid for 24 hours
Afterwards, it inhales and abandons M199+5%FBS culture medium, 100ul VEGF165 containing 10ng/ml is added, and (VEGF165 plays promotion to HUVECS
The effect of proliferation, be purchased from the general glad biology in Shanghai) and by 1 expression vector of embodiment in the institute of 6 orifice plates transfected HEK 293 48h
The cell conditioned medium (in VEGF and concentration of the albumen in the cell conditioned medium of 100ul be 22000pM) of collection contains the load in supernatant
In the VEGF that body surface reaches and albumen.37 DEG C of incubator culture 72h detect cell proliferative conditions, study the expression vector of embodiment 1
Influence in expressed VEGF with albumen to the HUVECs proliferation of the VEGF-165 stimulation of 10ng/ml, with 2 table of control vector
Cell conditioned medium and the VEGF Trap (specification: 40mg/ml, 4mg, 0.1ml of the B containing fusion protein reached；It is purchased from: Bayer) conduct pair
According to.
As a result as shown in fig. 7, melting in the VEGF that the expression vector of embodiment 1 is expressed with what albumen, control vector 2 were expressed
HUVECs under hop protein B and VEGF Trap processing group can effectively inhibit VEGF-165 to stimulate is proliferated, and is inhibited between each processing group
Effect is without significant difference.Illustrate, 1 expression vector of embodiment expression VEGF in and albumen can achieve it is identical as VEGF Trap
Effect.
Embodiment 3
In expression vector vivoexpression VEGF and albumen to HUVEC at the influence of pipe
Matrigel matrigel (50 hole μ l/) is taped against in 96 orifice plates, after 37 DEG C of incubation 1h, 500ng/ml will be used respectively
The cell conditioned medium of the B containing fusion protein that expresses of control vector 2, the expression of embodiment 1 expression vector containing in VEGF and albumen
In 96 orifice plates that each group HUVECs cell inoculation of transfection supernatant VEGF Trap processing is covered with Matrigel to bottom, it is placed in
37 DEG C, 5%CO2It is incubated for 8h in incubator, observes the formational situation of the HUVECs cell tubule of each processing group, and in microscope
Under take pictures, evaluation respectively to by the post-stimulatory HUVECs cell of VEGF-165 at pipe ability influence.As a result, it has been found that waiting real
It is equal to apply fusion protein B and VEGF Trap processing group that in the VEGF of the expression vector expression of example 1 and albumen, control vector 2 are expressed
The formation of HUVECs lumen can effectively be inhibited (see Fig. 8).
Embodiment 4
The influence that HUVEC is migrated with albumen in expression vector vivoexpression VEGF
Using scratch experiment research, by the transfection supernatant of control vector 2,1 expression vector of embodiment transfection supernatant Ah
Bai Xipu (protein concentration of three processing is 500ng/ml) is respectively to the shadow of the HUVECs cell migration ability of high glucose induction
It rings.Scratch for 24 hours after, as a result, it has been found that control vector 2 transfection supernatant, 1 expression vector of embodiment transfection supernatant VEGF Trap
Processing group can effectively inhibit the HUVECs of high glucose induction to migrate (see Fig. 9) under 500ng/ml protein concentration.
Embodiment 5
With albumen to the affinity of VEGF in expression vector vivoexpression VEGF
By transfecting control vector 2 and 1 expression vector of embodiment in HEK293 cell, cell conditioned medium, Yi Abai are collected
It is western it is general be positive control, react respectively with the VEGF of equivalent in identical VEGF with the supernatant of albumen molar concentration, detect react
Residue VEGF content in liquid, draws and corresponds to remaining VEGF content curve under each protein concentration, to evaluate in VEGF and egg
White and VEGF binding affinity.As the result is shown in the fusion protein B of control vector 2 and the VEGF of 1 expression vector of embodiment expression
With albumen from VEGF Trap under different protein concentrations in conjunction with VEGF165 active consistent (see Figure 10).
Embodiment 6
AAV virus preparation and purification
The method packed with reference to reports such as Martin Lock and purify recombination AAV virus, using PEI by the Rep of AAV and
Cap protein expression plasmid (pAAV2/2), helper plasmid (pAd Δ F6) and 1 expression vector of embodiment, cotransfection HEK293 cell
Packaging prepares recombinant adeno-associated virus, after transfecting 48h, harvests cell and culture supernatant, uses Iodixanol hypervelocity gradient
Centrifugal purification AAV virus [Martin Lock, Mauricio Alvira, Luk H. Vandenberghe.Rapid, Simple,
and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at
Scale.HUMAN GENE THERAPY, 2010.21:1259-1271], recombined glandulae correlation viral vectors are obtained, sonde method is used
Measure virus titer.
Embodiment 7
The internal detection of expression of recombined glandulae correlation viral vectors
Respectively in 6 week old mouse glass intracavitary administrations 9 × 109GC/eye、3×109GC/eye and 1 × 109GC/eye titre
Embodiment 6 recombined glandulae correlation viral vectors, 6 weeks execution mouse after injection detect in intraocular VEGF and the table of albumen
It reaches, shows as the result is shown down to 1 × 109Under the dosage of GC/eye, reach in intraocular VEGF with the expression quantity of albumen
3.53ng/ml；3×109It is 21.77ng/ml under the dosage of GC/eye, 9 × 109It is under the dosage of GC/eye
42.71ng/ml.Prior art report is injected with identical injection dosage same way, and in intraocular VEGF and expressing quantity is only
0.65ng/ml(P Pechan1,H Rubin. Novel anti-VEGF chimeric molecules delivered by
AAV vectors for inhibition of retinal neovascularization.Gene Therapy(2009)
16, 10–16).Thus illustrate, the recombined glandulae correlation viral vectors of the embodiment of the present invention 6 can be expressed expeditiously in vivo
In VEGF and albumen (see Figure 11).
Embodiment 8
The cylinder therapeutic effect of recombined glandulae correlation viral vectors detects
To 2 3-6 years old rhesus macaquies (being respectively designated as monkey 1 and monkey 2), the left eye intravitreal injection injection of every rhesus macaque
Express control viral vectors (difference of the control viral vectors compared to the recombined glandulae correlation viral vectors of embodiment 6 of EGFP
It is to be played control with albumen, the viral vectors for expressing EGFP instead of in VEGF with EGFP, eliminate unrelated sequences and virus
Influence to therapeutic effect.), the recombined glandulae correlation viral vectors of right eye intravitreal injection embodiment 6, give 4 respectively ×
1010GC/eye dosage takes aqueous humor and serum on the 2nd, 4,9 week before administration, after administration, uses VEGF neutralizing
Protein-ELISA method detects in each time point aqueous humor of every monkey in VEGF and protein content, as the result is shown every monkey note
It penetrates in the equal successful expression VEGF of right eye of the recombined glandulae correlation viral vectors of embodiment 6 and albumen, in VEGF and protein expression
Amount is shown in Table 1, and at the 9th week, expression quantity rose to 1781.9ng/mL, and left eye is not detected in VEGF and protein expression is (see figure
12)。
After table 1 is administered in aqueous humor under different time in VEGF and expressing quantity
| 2nd week | 4th week | 9th week | |
| Monkey 1 | 179.67ng/mL | 857.88ng/mL | 1781.9ng/mL |
| Monkey 2 | 128ng/mL | 308.9ng/mL | 1372.18ng/mL |
Laser photocoagulation choroidal neovascularization (CNV) modeling was carried out to each eye in the 6th week after administration, 2 days after modeling
Fluoroscopic visualization inspection models situation, checks Fluorescein Leakage area improvement rate and Fluorescein Leakage in fluoroscopic visualization assessment in the 9th week
Area reduction is to assess therapeutic effect of the recombined glandulae correlation viral vectors to monkey CNV model of embodiment 6.As the result is shown
The monkey left eye fluorescence leakage of (see Figure 13), injection control viral vectors are serious, and percolation ratio injects embodiment 6 up to 33%
The monkey right eye fluorescence leakages of recombined glandulae correlation viral vectors just fade away, percolation ratio 0%.Illustrate the weight of embodiment 6
Group gland relevant viral vector can effectively prevent vascular leakage, achieve the purpose that treat CNV.
VEGF Trap coded sequence is used in existing scheme, is initially designed as produced in vitro VEGF Trap albumen note
Agent is penetrated, is directly delivered to the encoding gene of VEGF Trap albumen intraocularly using rAAV gene delivery technique.Usually all it is at present
The expression of VEGF Trap albumen within the eye is promoted by the optimization of each Expression element, however passes through the excellent of Expression element merely
It is all very limited to change promotion protein expression.Ruslan Grishanin etc. is up to 2 × 10 by monkey intraocular injection12GC/ eye agent
The AAV recombinant virus of amount realizes VEGF Trap albumen, expression quantity within the eye up to 5800ng/mL (mean value 3400ng/mL, but
Its injection dosage is 50 times of injection dosage of the present invention), however, injection high dose virus has increase body to be immunoreacted
Risk；Michael Lukason etc. reports monkey injection 2 × 1010The AAV2-sFLT01 of GC/eye dosage, as a result monkey eye
Interior sFLT01 expressing quantity is extremely low.This explanation delivers VEGF Trap expressed sequence or sFLT01 table by AAV within the eye at present
There is a problem of that destination protein expression efficiency is low up to sequence.
We are in VEGF and protein gene optimizes transformation in an embodiment of the present invention, in conjunction with to gene expression
The optimization of box, using rAAV as gene delivery vector, in retina or optic ganglion cell, realize VEGF in and albumen
High-efficiency continuous expression achievees the purpose that treat wAMD to persistently inhibit new vessels.By improving in target gene VEGF
With the expression of albumen, and in continuous expression VEGF and albumen (after injection the 2nd week later expression and expression quantity is at any time
Passage increases, and sees Figure 12), rAAV carrier dosage is reduced, and then reduce immune risk.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Chengdu Jin Weike Biotechnology Co., Ltd
<120>for treating the fusion protein, viral vectors and drug of senile macular degeneration
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 1683
<212> DNA
<213>artificial sequence
<400> 1
atggtttctt actgggatac cggcgtgctg ctgtgtgccc tgctgtcttg tctgctgctg 60
accggctcta gcagcggcag caagctgaag gatcccgagc tgagcctgaa gggcacccag 120
catattatgc aggccggcca gacactgcac ctccagtgta gaggcgaagc cgctcacaaa 180
tggtccctgc ctgagatggt gtccaaagag agcgagcggc tgagcatcac caagagcgcc 240
tgtggcagaa acggcaagca gttctgcagc accctgacac tgaatactgc ccaggccaac 300
cacaccggct tctacagctg caagtatctg gccgtgccta ccagcaagaa gaaagaaacc 360
gagagcgcca tctacatctt catctctgat accggcagac ccttcgtgga aatgtacagc 420
gagatccccg agatcatcca catgaccgag ggcagagagc tggtcatccc ctgcagagtg 480
acaagcccca acatcaccgt gactctgaag aagttccctc tggacacact gatccccgac 540
ggcaagagaa tcatctggga cagccggaag ggcttcatca tcagcaacgc cacctacaaa 600
gagatcggcc tgctgacctg tgaagccacc gtgaatggcc acctgtacaa gaccaactac 660
ctgacacaca gacagaccaa caccatcatc gacgtggtgc tgagccctag ccacggcatt 720
gaactgtctg tgggcgagaa gctggtgctg aactgtaccg ccagaaccga gctgaacgtg 780
ggcatcgact tcaactggga gtaccccagc agcaagcacc agcacaagaa actggtcaac 840
cgggacctga aaacccagag cggcagcgag atgaagaaat tcctgagcac cctgaccatc 900
gacggcgtga ccagatctga ccagggcctg tatacttgtg ccgccagctc tggcctgatg 960
accaagaaaa acagcacctt cgtgcgggtg cacgagaagg acaagaccca cacctgtcct 1020
ccatgtcctg ctccagaact gctcggcgga ccttccgtgt tcctgtttcc tccaaagcct 1080
aaggacaccc tgatgatcag cagaacccct gaagtgacct gcgtggtggt ggatgtgtcc 1140
cacgaggatc ccgaagtgaa gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 1200
aagaccaagc ctagagagga acagtacaat agcacctaca gagtggtgtc cgtgctgacc 1260
gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaaggcc 1320
ctgcctgctc ctatcgagaa aaccatctcc aaggccaagg gccagcctag ggaaccccag 1380
gtttacacac tgcctccaag cagggacgag ctgacaaaga accaggtgtc cctgacctgc 1440
ctggtcaagg gcttctaccc ttccgatatc gccgtggaat gggagagcaa tggccagcct 1500
gagaacaact acaagacaac ccctcctgtg ctggacagcg acggctcatt cttcctgtac 1560
agcaagctga cagtggacaa gagcagatgg cagcagggaa acgtgttcag ctgcagcgtg 1620
atgcacgagg ccctgcacaa ccactacacc cagaagtccc tgagcctgtc tcctggcaaa 1680
taa 1683
<210> 2
<211> 666
<212> DNA
<213>artificial sequence
<400> 2
ctagtcgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60
atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120
cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180
tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300
ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360
acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc 420
ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg 480
cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg 540
ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa 600
agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc 660
gggcgg 666
<210> 3
<211> 1065
<212> DNA
<213>artificial sequence
<400> 3
gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 60
cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 120
cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 180
gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg 240
tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 300
gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg 360
gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt 420
gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc 480
cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg 540
gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg 600
ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg 660
gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc 720
agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc 780
cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg 840
gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc 900
ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga 960
ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc 1020
tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaag 1065
<210> 4
<211> 384
<212> DNA
<213>artificial sequence
<400> 4
atggtttctt actgggatac cggcgtgctg ctgtgtgccc tgctgtcttg tctgctgctg 60
accggctcta gcagcggcag caagctgaag gatcccgagc tgagcctgaa gggcacccag 120
catattatgc aggccggcca gacactgcac ctccagtgta gaggcgaagc cgctcacaaa 180
tggtccctgc ctgagatggt gtccaaagag agcgagcggc tgagcatcac caagagcgcc 240
tgtggcagaa acggcaagca gttctgcagc accctgacac tgaatactgc ccaggccaac 300
cacaccggct tctacagctg caagtatctg gccgtgccta ccagcaagaa gaaagaaacc 360
gagagcgcca tctacatctt catc 384
<210> 5
<211> 300
<212> DNA
<213>artificial sequence
<400> 5
tctgataccg gcagaccctt cgtggaaatg tacagcgaga tccccgagat catccacatg 60
accgagggca gagagctggt catcccctgc agagtgacaa gccccaacat caccgtgact 120
ctgaagaagt tccctctgga cacactgatc cccgacggca agagaatcat ctgggacagc 180
cggaagggct tcatcatcag caacgccacc tacaaagaga tcggcctgct gacctgtgaa 240
gccaccgtga atggccacct gtacaagacc aactacctga cacacagaca gaccaacacc 300
<210> 6
<211> 315
<212> DNA
<213>artificial sequence
<400> 6
atcatcgacg tggtgctgag ccctagccac ggcattgaac tgtctgtggg cgagaagctg 60
gtgctgaact gtaccgccag aaccgagctg aacgtgggca tcgacttcaa ctgggagtac 120
cccagcagca agcaccagca caagaaactg gtcaaccggg acctgaaaac ccagagcggc 180
agcgagatga agaaattcct gagcaccctg accatcgacg gcgtgaccag atctgaccag 240
ggcctgtata cttgtgccgc cagctctggc ctgatgacca agaaaaacag caccttcgtg 300
cgggtgcacg agaag 315
<210> 7
<211> 684
<212> DNA
<213>artificial sequence
<400> 7
gacaagaccc acacctgtcc tccatgtcct gctccagaac tgctcggcgg accttccgtg 60
ttcctgtttc ctccaaagcc taaggacacc ctgatgatca gcagaacccc tgaagtgacc 120
tgcgtggtgg tggatgtgtc ccacgaggat cccgaagtga agttcaattg gtacgtggac 180
ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagtacaa tagcacctac 240
agagtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa agagtacaag 300
tgcaaggtgt ccaacaaggc cctgcctgct cctatcgaga aaaccatctc caaggccaag 360
ggccagccta gggaacccca ggtttacaca ctgcctccaa gcagggacga gctgacaaag 420
aaccaggtgt ccctgacctg cctggtcaag ggcttctacc cttccgatat cgccgtggaa 480
tgggagagca atggccagcc tgagaacaac tacaagacaa cccctcctgt gctggacagc 540
gacggctcat tcttcctgta cagcaagctg acagtggaca agagcagatg gcagcaggga 600
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660
ctgagcctgt ctcctggcaa ataa 684
<210> 8
<211> 560
<212> PRT
<213>artificial sequence
<400> 8
Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser
1 5 10 15
Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro
20 25 30
Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr
35 40 45
Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro
50 55 60
Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala
65 70 75 80
Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr
85 90 95
Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val
100 105 110
Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile
115 120 125
Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
130 135 140
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
145 150 155 160
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
165 170 175
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
180 185 190
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
195 200 205
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
210 215 220
Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
225 230 235 240
Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr
245 250 255
Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys
260 265 270
His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly
275 280 285
Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
290 295 300
Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
305 310 315 320
Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr
325 330 335
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
340 345 350
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
355 360 365
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
370 375 380
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
385 390 395 400
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
405 410 415
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
420 425 430
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
435 440 445
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
450 455 460
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
465 470 475 480
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
485 490 495
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
500 505 510
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
515 520 525
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
530 535 540
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545 550 555 560
<210> 9
<211> 128
<212> PRT
<213>artificial sequence
<400> 9
Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser
1 5 10 15
Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro
20 25 30
Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr
35 40 45
Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro
50 55 60
Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala
65 70 75 80
Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr
85 90 95
Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val
100 105 110
Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile
115 120 125
<210> 10
<211> 100
<212> PRT
<213>artificial sequence
<400> 10
Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
1 5 10 15
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
35 40 45
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
50 55 60
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
65 70 75 80
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95
Gln Thr Asn Thr
100
<210> 11
<211> 105
<212> PRT
<213>artificial sequence
<400> 11
Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val
1 5 10 15
Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val
20 25 30
Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys
35 40 45
Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys
50 55 60
Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln
65 70 75 80
Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn
85 90 95
Ser Thr Phe Val Arg Val His Glu Lys
100 105
<210> 12
<211> 227
<212> PRT
<213>artificial sequence
<400> 12
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 13
<211> 595
<212> DNA
<213>artificial sequence
<400> 13
cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 60
tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 120
ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 180
gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 240
cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 300
ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 360
gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct 420
gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc 480
cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 540
tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcat 595
<210> 14
<211> 232
<212> DNA
<213>artificial sequence
<400> 14
cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 60
tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 120
attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 180
aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gc 232
Claims (10)
1. a kind of for treating the fusion protein of senile macular degeneration, which is characterized in that it includes the structure selected from VEGFR1
Domain 1 and structural domain 2, the structural domain 3 selected from VEGFR2 and the Fc segment selected from immunoglobulin.
2. fusion protein according to claim 1, which is characterized in that the amino acid of the structural domain 1 selected from VEGFR1
Sequence is as shown in SEQ ID NO.9；
Preferably, the structural domain 1 is located at the N-terminal of the fusion protein；
Preferably, the N-terminal of the fusion protein to C-terminal be followed successively by the structural domain 1, the structural domain 2, the structural domain 3 with
And the Fc segment；
Preferably, the amino acid sequence of the structural domain 2 selected from VEGFR1 is as shown in SEQ ID NO.10；
Preferably, the amino acid sequence of the structural domain 3 selected from VEGFR2 is as shown in SEQ ID NO.11；
Preferably, the immunoglobulin is human IgG1；
Preferably, the amino acid sequence of the Fc segment is as shown in SEQ ID NO.12；
Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.8.
3. a kind of nucleic acid molecules, which is characterized in that it encodes fusion protein as claimed in claim 1 or 2；
Preferably, shown in the base sequence SEQ ID NO.1 of the nucleic acid molecules.
4. a kind of expression cassette, which is characterized in that it contains nucleic acid molecules as claimed in claim 3 and the driving nucleic acid molecules
The promoter of expression.
5. expression cassette according to claim 4, which is characterized in that the promoter is CB7 promoter；
Preferably, the base sequence of the CB7 promoter is as shown in SEQ ID NO.2；
Preferably, the expression cassette also contains Kozak sequence, and the Kozak sequence is located at the downstream of the promoter and is located at
The upstream of the nucleic acid molecules；
Preferably, the expression cassette also contains intron sequences；The intron sequences are located at the downstream of the promoter and position
In the upstream of the Kozak sequence；
Preferably, the expression cassette contains also terminator；The terminator is located at the downstream of the nucleic acid molecules；
Preferably, the terminator is SV40 polyA.
6. a kind of carrier, which is characterized in that it contains expression cassette described in claim 4 or 5.
7. carrier according to claim 6, which is characterized in that the carrier is plasmid vector or viral vectors；
Preferably, the viral vectors is recombined glandulae correlation viral vectors.
8. carrier according to claim 7, which is characterized in that the recombined glandulae correlation viral vectors be selected from AAV1, AAV2,
Any one in AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh10；
Preferably, the recombined glandulae correlation viral vectors are AAV2, AAV5, AAV8 or AAV9.
9. a kind of drug for treating senile macular degeneration, which is characterized in that it contains fusion egg of any of claims 1 or 2
The white or described in any item carriers of claim 6-8；
Preferably, the drug further includes pharmaceutically acceptable auxiliary material.
10. drug according to claim 9, which is characterized in that the dosage form of the drug is injection；
Preferably, the dosage form of the drug is suitable for the injection using vitreous chamber or subretinal injection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910705207.1A CN110423281B (en) | 2019-07-31 | 2019-07-31 | Fusion protein, viral vector and drug for treating age-related macular degeneration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910705207.1A CN110423281B (en) | 2019-07-31 | 2019-07-31 | Fusion protein, viral vector and drug for treating age-related macular degeneration |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110423281A true CN110423281A (en) | 2019-11-08 |
| CN110423281B CN110423281B (en) | 2021-04-30 |
Family
ID=68413642
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910705207.1A Active CN110423281B (en) | 2019-07-31 | 2019-07-31 | Fusion protein, viral vector and drug for treating age-related macular degeneration |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110423281B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322281A (en) * | 2021-05-12 | 2021-08-31 | 成都金唯科生物科技有限公司 | Recombinant adeno-associated virus for efficiently expressing RS1 protein in tissue specificity and application thereof |
| WO2023155918A1 (en) * | 2022-02-21 | 2023-08-24 | 上海瑞宏迪医药有限公司 | Vegf-binding molecule and pharmaceutical use thereof |
| WO2024002076A1 (en) * | 2022-06-27 | 2024-01-04 | 上海鼎新基因科技有限公司 | Aav drug for treating angiogenesis-related fundus diseases |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102219859A (en) * | 2011-05-20 | 2011-10-19 | 烟台荣昌生物工程有限公司 | Fusion protein for antagonizing angiogenesis inducible factor and application thereof |
| CN102869779A (en) * | 2010-03-29 | 2013-01-09 | 宾夕法尼亚大学托管会 | Pharmacologically induced transgene ablation system |
| CN107001457A (en) * | 2014-11-25 | 2017-08-01 | 瑞泽恩制药公司 | Method and formulation for treating vascular eye |
| WO2019067540A1 (en) * | 2017-09-27 | 2019-04-04 | Regenxbio Inc. | Treatment of ocular diseases with fully-human post-translationally modified anti-vegf fab |
| CN109661470A (en) * | 2016-04-15 | 2019-04-19 | 宾夕法尼亚州大学信托人 | New A AV8 mutant capsids and composition containing it |
-
2019
- 2019-07-31 CN CN201910705207.1A patent/CN110423281B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102869779A (en) * | 2010-03-29 | 2013-01-09 | 宾夕法尼亚大学托管会 | Pharmacologically induced transgene ablation system |
| CN102219859A (en) * | 2011-05-20 | 2011-10-19 | 烟台荣昌生物工程有限公司 | Fusion protein for antagonizing angiogenesis inducible factor and application thereof |
| CN107001457A (en) * | 2014-11-25 | 2017-08-01 | 瑞泽恩制药公司 | Method and formulation for treating vascular eye |
| CN109661470A (en) * | 2016-04-15 | 2019-04-19 | 宾夕法尼亚州大学信托人 | New A AV8 mutant capsids and composition containing it |
| WO2019067540A1 (en) * | 2017-09-27 | 2019-04-04 | Regenxbio Inc. | Treatment of ocular diseases with fully-human post-translationally modified anti-vegf fab |
Non-Patent Citations (1)
| Title |
|---|
| 刘彦尧 等: "血管内皮生长因子受体-2在湿性年龄相关性黄斑变性中的研究进展", 《重庆医科大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322281A (en) * | 2021-05-12 | 2021-08-31 | 成都金唯科生物科技有限公司 | Recombinant adeno-associated virus for efficiently expressing RS1 protein in tissue specificity and application thereof |
| CN113322281B (en) * | 2021-05-12 | 2024-01-05 | 成都金唯科生物科技有限公司 | Recombinant adeno-associated virus for high-efficiency tissue-specific expression of RS1 protein and application thereof |
| WO2023155918A1 (en) * | 2022-02-21 | 2023-08-24 | 上海瑞宏迪医药有限公司 | Vegf-binding molecule and pharmaceutical use thereof |
| WO2024002076A1 (en) * | 2022-06-27 | 2024-01-04 | 上海鼎新基因科技有限公司 | Aav drug for treating angiogenesis-related fundus diseases |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110423281B (en) | 2021-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102574810B1 (en) | Compositions for the treatment of wet age-related macular degeneration | |
| AU2013257450B2 (en) | Restoration of visual responses by in vivo delivery of rhodopsin nucleic acids | |
| AU2013263159B2 (en) | Treatment of amd using AAV sFlt-1 | |
| US20180353614A1 (en) | VE-PTP Extracellular Domain Antibodies Delivered by a Gene Therapy Vector | |
| JP6449175B2 (en) | Methods and compositions for the treatment of ocular gene related disorders | |
| KR20210022524A (en) | Composition for the treatment of wet age-related macular degeneration | |
| US11510950B2 (en) | Variant AAV capsids for intravitreal delivery | |
| CN110423281A (en) | For treating the fusion protein, viral vectors and drug of senile macular degeneration | |
| KR102545070B1 (en) | Gene Therapy for Eye Disorders | |
| KR20210149803A (en) | Factor H vectors and their uses | |
| CN110023327A (en) | The identification and its application method being mutated in there is the channel opsin variant for improving photosensitivity | |
| US20230340529A1 (en) | Adeno-associated virus for delivery of kh902 (conbercept) and uses thereof | |
| WO2023227043A1 (en) | Rpgr-encoding nucleic acid and use thereof | |
| US20230048017A1 (en) | Adeno-associated virus for delivery of kh902 (conbercept) and uses thereof | |
| TW202235618A (en) | Treatments for intraocular pressure related disorders | |
| Luo et al. | A novel capsid-XL32-derived adeno-associated virus serotype prompts retinal tropism and ameliorates choroidal neovascularization | |
| KR20240010489A (en) | Compositions and methods for improving vision function | |
| CN117337331A (en) | Compositions and methods for enhancing visual function | |
| CN115003820A (en) | Compositions and methods for ocular treatment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |