CN1167801C - Constitution of recombinant human VEGF adenovirus carrier - Google Patents

Abstract

The present invention relates to the constitution of recombinant adenovirus carriers with human vascular endothelial growth factors, which can be used for treating serious coronary diseases and circumferential serious ischemic diseases. The method comprises: human VEGF. cDNA is inserted under a CMV promoter of a shuttle plasmid pHCMVP1A in a positive direction for constituting a shuttle plasmid pAd-hVEGF; the latter and pJM17 cotransfects 293 cells by a liposome; homologous recombination is used for obtaining recombinant adenoviruses Ad-hVEGF with human VEGF genes; a PCR coamplification method is used for identifying whether Ad-hVEGF is correct or not; the vascular titer is calculated according to the ultraviolet absorbance (A260) of 260 nm; a result VEGF. CDNA is successfully inserted into pHCMVSP1A carriers in the positive direction; genome DNA of the recombinant adenoviruses is used as a template; simultaneously, a VEGF genic fragment of 576 bp and an 860 bp adenovirus skeleton genic fragment are enlarged; the accuracy of the Ad-hVEGF is proved; measured A260 is 0.097 and the vascular titer is 1.94*10<12>pfu/ml.

Description

Recombinant human VEGF adenovirus carrier and application thereof

Technical field

The present invention relates to a kind of adenovirus carrier of the VEGF165 of containing gene, this construction of recombinant adenovirus containing and they are as the application of the therapeutical agent of coronary heart disease and peripheral arterial ischemic angiopathy etc.

Background technology

Coronary heart disease (coronary artery disease, control CAD) has had considerable progress, but the treatment of CAD still has many stubborn problems.Percutaneous tranluminal coronary angioplasty (PTCA) postoperative 3-6 month restenosis rate is up to 30-50%.The study on prevention of restenosis is many, confirms effective means in the experimentation on animals, and is then invalid or result of treatment is not obvious when applying to human body; Control PTCA postoperative restenosis method was once placed hope on the application of intracoronary stent in the clinical position, but the restenosis rate behind the Stent is still up to 15-54% (referring to listed document 1); In experimentation on animals in recent years and the clinical trial proof coronary artery ³²The P radiotherapy is effective to control PTCA postoperative restenosis, but Verin etc. reported with the β ray ( ⁹⁰Y) carry out the coronary artery internal radiotherapy, the incidence of restenosis is 40% after 6 months.Coronary artery bypass graft surgery (CABG) postoperative bridge vascular lesion makes the people puzzled.For serious diffusivity coronary artery pathological changes, the routine medication weak effect, can not be vascular interventional treatment and CABG, adopt the therapeutic vasculogenesis of pHVEGF165 gene or bFGF, promote new vascularization, set up side Zhi Xunhuan, myocardial ischemia obtains medical treatment, even cure (referring to listed document 3,4), allow people see the hope of gene therapy.Equally, (Peripheral artery disease PAD), has also obtained challenging effect to ischemic limb muscle treatment peripheral arterial ischemic angiopathy with the phVEGF165 gene injection.In the application cell somatomedin is done at present experimentation on animals and clinical study, VEGF studies to have entered the cell growth factor (referring to listed document 3-7) of clinical trial the most, and the specific endotheliocyte that acts on promotes side Zhi Xunhuan to form.

In recent years, the foreign scholar adopts injection for curing PAD in exposed plasmid phVEGF 165 lower limb muscles of carrier for expression of eukaryon of VEGF165, has obtained challenging effect, and part patient has avoided amputation (referring to listed document 5 and 6).The existing in recent years report of the gene therapy of CAD, can be when CABG, injection or adopt left side wall of the chest micro-incision will expose plasmid phVEGF165 or bFGF is expelled to (referring to listed document 3 and 4) in the ischemic myocardium in lesion vessels blood supply district cardiac muscle, intractable CAD patient's angina pectoris attacks number of times is obviously reduced, and the pannonit daily dosage portion obviously reduces, and exercise tolerance obviously increases, the 1-2 month after the gene therapy, coronarography shows that side Zhi Xunhuan is open-minded in the myocardial ischemia district, and blood supply of cardiac muscle improves.Rosengart etc. adopt adenovirus mediated VEGF121 injection for curing CAD in the wall of the chest micro-incision ischemic myocardium of left side, and treatment back angina pectoris attacks number of times obviously improves ventricular wall motion improvement (referring to document 7).Illustrated that CAD and PAD clinical application of gene therapy are worth.Injection DNA or method in directly myocardium by transduceing in the liposome coronary artery, the gene transfer rate is low, less than 1%, and having confirmed in pig chronic myocardial ischemia model has higher genetic expression in the scope of 0-15mm around the injection point, from injection point cardiac muscle far away, genetic expression is lower.Barr report by import in the conduit underwent coronary contain bacterium lacZ gene duplicate deficient adenovirus (AdCMV.lacZ) 10 ¹⁰Pfu/ml, myocardium transfection efficiency is up to 32% (referring to listed document 13).Quiding gene is expressed few the peripheral organ, the Giordano report only has 1.3% adenovirus to arrive (referring to listed document 14) beyond the coronary circulation.In addition, inject inflammation antisense and the myocardial fibrosis (referring to listed document 15) that DNA often causes cell in the cardiac muscle.Relative open chest surgery, quiding gene is AT in the coronary artery, and imports recombinant adenoviral vector in the underwent coronary, can not detect inflammatory reaction and the myocardial fibrosis followed.Not seeing the report of the adenovirus mediated above-mentioned disease of VEGF165 gene therapy at present both at home and abroad as yet, by importing adenovirus mediated gene therapy in the heart catheter underwent coronary, may be a kind of safe and effective procedure, has great clinical value.

Summary of the invention

The present invention has made up the recombinant adenoviral vector of expressing human VEGF165 gene, lays a good foundation for adopting VEGF165 gene therapy CAD and PAD.

VEGF is the cytokine of most important short vasculogenesis, is the strongest mitogen of endothelial cell specific, and VEGF has four kinds of monomer existence forms, is called VEGF121, VEGF165, VEGF189 and VEGF206.Wherein the biological activity of VEGF165 accounts for 60% of VEGF, and the signal conduction by its specific receptors KDR/flt-1 on the endotheliocyte promotes endothelial cell proliferation, migration.In ischemic tissue, vascular endothelial cell raises VEGF mRNA and receptor expression thereof, grows neovascularity (referring to listed document 11) by division in the gemma mode from female blood vessel, sets up side Zhi Xunhuan.VEGF is the Survival Factor (referring to listed document 12) of new life's immature blood vessel, can impel the ripe and differentiation of new vessel.

One of gene therapy the key link is exactly how to import foreign gene in the target cell effectively and make its stably express.The gene transfer vector that adenovirus carrier is considered to efficiently express (referring to document 10), because the adenovirus host range is wide, but infection duplication splitted cell not only also can infect cell stationary phase; Bale capacity is big, can insert the allogenic gene fragment of 7.5kb; Can not be incorporated in the karyomit(e) of host cell, not have danger such as activating oncogene or insertion sudden change; Virus can reach very high titre, and reach 100% infection rate through breeding, purifying; Character is more stable, to plurality of advantages such as the mankind are comparatively safe, makes it have greater advantage in transgenosis, becomes topmost carrier in the present gene therapy.

The Ad-hVEGF165 that the present invention makes up is the replication-defective adenoviral of E1 district disappearance, E1 district gene is the necessary gene of adenoviral replication, this just requires duplicating of this complete adenovirus to carry out in the area gene transfected cell of E1, and 293 cells are just for adapting to the packing cell that these needs have transformed E1 district gene, this has just determined replication-defective adenoviral infection chance and do not have replication for once in target cell, thereby finish the function of adenovirus carrier, avoid the infringement of adenovirus itself to target cell, reach the purpose of transgenosis, but the Ad-hVEGF165 of the expressing human VEGF that we make up is inserted into people VEGF165.cDNA forward under the CMV promotor of shuttle plasmid pHCMVSP1A, make up shuttle plasmid pAd-hVEGF165, the latter and pJM17 obtain to contain the recombinant adenovirus Ad-hVEGF165 of people VEGF165 gene by liposome cotransfection 293 cells through homologous recombination.Cut evaluation and the evaluation of pcr amplification method through EcoRI, NcoI and XhoI enzyme, prove that the shuttle plasmid pAd-hVEGF165 and the recombinant adenovirus Ad-hVEGF165 that make up are correct, the latter is VEGF expression in target cell.

Description of drawings

Fig. 1 is that the enzyme of agarose gel electrophoresis evaluation pAd-hVEGF 165 is cut the result:

1、PCR?marker：237、377、515、697、994、1543bp；

2, pAd-hVEGF165 (Nco I+Xho I): the 511bp fragment is arranged;

3, pAd-ahVEGF165 (Nco I+Xho I): the 101bp fragment is arranged;

4、pAd-hVEGF165(EcoR?I)：576、6797bp；

5、pAd-ahVEGF165(EcoR?I)：576、6797bp。

Fig. 2 is the PCR coamplification result that agarose gel electrophoresis is identified Ad-hVEGF165:

1、PCRmarker：237、377、515、697、994、1543bp；

2, pAd-hVEGF165 is a template, amplifies the VEGF165 fragment of 576bp;

3, pHCVSP1A is a template, amplifies 860bp adenovirus skeleton gene fragment;

4, the Ad-hVEGF165 virus genom DNA is a template, amplifies two fragments of 576bp and 860bp simultaneously.

Fig. 3 is in the clinical trial before example 2 patient treatments and the arteria coronaria dextra radiography after the treatment, and side Zhi Xunhuan increases between diagram treatment back arteria coronaria dextra tip and the arteria coroaria sinistra.

In the following embodiments, we have selected VEGF165 is goal gene, and with the effective carrier of replication-defective adenoviral conduct of E1 district disappearance, the recombinant adenovirus Ad-hVEGF165 of structure, virus titer are 1.94 * 10 ¹²Pfu/ml, purity is 1.5, illustrates that virus titer and purity are all higher, can satisfy the needs in the body gene therapy, lays a good foundation for treating CAD and PAD.

Describe the present invention by the following examples, should be noted that, cited embodiment should not be construed as limitation of the present invention.

Embodiment

Embodiment 1 carries the structure of the adenovirus shuttle plasmid of VEGF165

1, material and method

1.1 reagent

EcoRI, T ₄DNAligase is available from Huamei Bio-Engrg Co.; Calf intestinal alkaline phosphatase (CIAP), low melting-point agarose are available from promega company; NcoI, XhoI are available from BioLabs company; Superfine new-born calf serum is available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.; DMEM, Lipofectamine are available from GIBCO/BRL company; CsCl is available from Sigma company.

1.2 plasmid and bacterial strain

PHCMVSP1A (having adenoviral gene) and pJM17 are provided by Military Medical Science Institute two; DH5 α is provided by intracardiac section of No.1 Hospital Affiliated to Beijing Medical Univ.; PUCCAGGS/hVEGF165 is provided by pathology system of medical college of Kyushu University.

1.3 clone

293 cells are the human embryonic kidney cell line of adenovirus E 1 district gene transformation, and culture condition is the high sugared DMEM nutrient solution of 20% new-born calf serum, at 37 ℃, 5%CO ₂Cultivate in the incubator.

1.4 instrument

CO2gas incubator (Sanyo, Japan), high speed freezing centrifuge (Jouan, Italy), superspeed refrigerated centrifuge (Hitachi, Japan), inverted phase contrast microscope (Olympus IX-70, Japan), ultraviolet spectrophotometer (752 types, Shanghai), airbath, shaking bath.

1.5 carry the structure of the adenovirus shuttle plasmid of VEGF165

PHCMVSP1A after the EcoRI enzyme is cut, 70 ℃ of deactivations 15 minutes, make carrier 5 ' end dephosphorylation with CIAP after, reclaim the 6797bp fragment.With the carrier segments of dephosphorylized 6797bp and the VEGF165 cDNA fragment T of 576bp ₄4 ℃ of connections of DNA Ligase are spent the night, obtain recombinant shuttle plasmid, latter's transformed competence colibacillus bacillus coli DH 5 alpha, the choosing colony amplification, cut with the EcoRI enzyme after extracting plasmid in a small amount, if obtained 576bp and two fragments of 6797bp behind the electrophoresis, proved that then VEGF165 cDNA has been inserted into adenovirus shuttle vector.To insert the shuttle plasmid of VEGF165 cDNA with NcoI, XhoI double digestion, the recombinant adenovirus shuttle plasmid pAd-hVEGF165 of 511bp person for just VEGF165 arranged behind the electrophoresis, the recombinant adenovirus shuttle plasmid pAd-ahVEGF165 of 101bp person for antisense VEGF 165 arranged.

1.6 the pAd-hVEGF165 and pJM17 cotransfection 293 cells of Lipofectamine mediation

(1) with 293 cell inoculations in the plate of 9cm, 37 ℃, 5%CO ₂Cultivate in the incubator, carry out transfection when making it reach 80% fusion;

(2) in the Eppendorf pipe, press liposome complex such as DNA/ such as configuration such as mole number such as grade: dilution plasmid pAd-hVEGF165 and pJM17 in 1ml serum-free DMEM nutrient solution, rotation 1sec adds the Lipofectamine suspension, mixing again, place 30min under the room temperature, DNA is combined on the liposome;

(3) inhale the DMEM substratum that removes to contain in the culture dish serum, add 1ml serum-free DMEM nutrient solution and wash cell 1 time, the DMEM of sucking-off serum-free adds the DNA/ liposome complex, cultivates 4h for 37 ℃;

(4) in culture dish, add the DMEM that contains serum of capacity again, continue to cultivate 8h;

(5) inhale and to remove DMEM/DNA/ liposome mixture, add 0.5% agarose and cover and (contain 1 * DMEM in 0.5% agarose, 10%FCS), treat that it solidifies to be placed on 37 ℃, 5%CO ₂Cultivate observation of cell pathology situation in the incubator.

1.7 the PCR of the adenovirus carrier of recombinant human VEGF 165 identifies

Behind cotransfection, occur getting culture supernatant 100ul cytopathic 293 cells, centrifugal removal cell debris, collect supernatant and add lysate (0.5%SDS, 20mmol/L EDTA, 50ug/ml ProteinaseK) 400ul, behind 55 ℃ of effect 1h, add equal amounts of phenolic/chloroform/primary isoamyl alcohol extracting 1 time, on reset and add 3mol/L NaAc (pH5.2) the deposit D NA of 2 times of volume dehydrated alcohols and 1/10 volume.Wash 1 time with 70% ethanol, precipitation is dissolved in the 20ul sterilization tri-distilled water, and is that template is carried out pcr amplification with this DNA.Primer 1 and primer 2 are the primer of amplification VEGF165 gene, primer 3 and 4 primers for amplification adenovirus skeleton gene fragment.The PCR reaction system is for getting template DNA 10ul, each 5ul of 20umol/L primer, 10 * PCRBuffer 5ul, 2mmo/L dNTP 5ul, Taq enzyme 1ul, add water to PCR reaction final volume 50ul, cover with mineral oil, 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 60s circulate 30 times, last 72 ℃ are extended 10min, carry out electrophoresis at 1.2% agarose and identify pcr amplification product, amplify the VEGF165 gene fragment of 576bp and the adenovirus skeleton gene fragment person of 860bp simultaneously, then for containing the recombinant adenovirus Ad-ahVEGF165 of people VEGF165.Primer sequence is as follows:

Primer 1:5 ' atg aac ttt ctg ctg tct tgg 3 '

Primer 2: 5 ' tca ccg cct cgg ctt gtc aca 3 '

Primer 3:5 ' teg ttt ctc agc agc tgt tg 3 '

Primer 4:5 ' cat ctg aac tca aag cgt gg 3 '

1.8 amplification, purifying and the titer determination of recombinant human VEGF 165 adenovirus Ad-hVEGF165

Get recombinant adenovirus supernatant 500ul, be added in 90% 293 cells that merge 37 ℃, 5%CO ₂Cultivate in the incubator, after observing 95%～100% cell under the mirror and pathology occurring, centrifugal collection supernatant, and with cell multigelation 3 times between-80 ℃ and 37 ℃, with the centrifugal collection supernatant of 3000r/min, method (referring to listed document 9) by Graham is carried out CsCl density gradient centrifugation purifying to the adenovirus supernatant, with the adenovirus supernatant behind the purifying to dialyzate (10mmol/L Tris-HCl, 1mmol/L MgCl ₂, 10% glycerine) and 4 ℃ of dialysis 2 times, each 24h.Viral liquid after the dialysis is carried out titer determination.Get the viral liquid 100ul behind the purifying, 10%SDS 20ul, PBS 880ul measures the A of virus genom DNA ₂₆₀And A ₂₈₀Absorbance value, calculate the particle and the purity of virus, 1A in view of the above ₂₆₀=10 ¹²Pfu/ml, A ₂₆₀/ A ₂₈₀＞1.3 show that purity is higher, virus titer pfu/ml=A260 * extension rate * 10 ¹²

2 results

2.1 the enzyme of recombinant plasmid pAd-ahVEGF165 is cut qualification result

Obtained 576bp and two fragments of 6767bp after pAd-hVEGF165 cuts with the EcoRI enzyme, confirmed that people VEGF165 has inserted under the promotor of adenovirus shuttle plasmid.In order to differentiate the adenovirus shuttle plasmid of forward and the reverse VEGF165 of insertion, with NcoI and XhoI double digestion, obtain the recombinant adenovirus plasmid pAd-hVEGF165 of the segmental just VEGF165 of 51Ibp and the recombinant adenovirus plasmid pAd-ahVEGF165 of the segmental antisense VEGF 165 of 101bp.See Fig. 1.

2.2 the pcr amplification method is identified the result of Ad-hVEGF165

Virus genom DNA with extraction is a template, adds the primer and the adenovirus skeleton gene fragment primer of VEGF165 gene simultaneously, has obtained the VEGF gene fragment of 576bp and the adenoviral gene fragment of 860bp simultaneously after amplification.With pHCMVSP1A is that template amplification goes out the 860bp fragment, is that template amplification goes out the 576bp gene fragment with pAd-hVEGF165.After having confirmed pAd-hVEGF165 and pJM17 cotransfection 293 cells, produced the recombinant adenovirus that contains people VEGF, seen Fig. 2 through homologous recombination.

2.3 titre and the purity of reorganization Ad-hVEGF165

Behind the breeding of recombinant adenovirus poisons, amplification, the purifying, record A ₂₆₀Be 0.097 and A ₂₈₀Be 0.066, calculating virus titer is 1.94 * 10 ¹²Pfu/ml, purity is 1.5, illustrates that virus titer and purity are all higher, can satisfy the needs in the body gene therapy.

Embodiment 2 clinical trials

1, data and method

1.1 case data

Example 1, the man, 50 years old, stenocardia 5 years increased the weight of 1 year, and extensive anterior myocardial infarction took place January calendar year 2001, capable at that time reperfusion as treatment, after this frequently show effect severe cardiac angina and left heart insufficiency, coronary angiography show to circle round (LCX) in left anterior descending branch (LAD), a left side and arteria coronaria dextra (RCA) all entirely shuts near section, only depend on the branch vessel blood supply.

Example 2, the man, 59 years old, extensive anterior myocardial infarction took place in February, 2000 in pectoralgia history 2 years, and blood pressure reduces at that time, obviously has difficulty in breathing, and lung's bubble can not be put down for sleeping in.Coronary angiography shows the narrow 50-90% of section far away in the left trunk, and LAD, LCX are all from the section start obturation, and arteria coronaria dextra is the narrow 50-80% of section closely.

1.2 adenovirus carrier imports in the skin coronary artery

Agreement through patient and family members, adenovirus carrier imported in passed through the skin coronary artery on April 3rd, 2000, untoward reaction does not appear in the injection process, shiver with cold, heating all appearred in 1 hour after surgery in 2 routine patients, 39 ℃ of body temperature, intravenous injection dexamethasone 20mg is behind promethazine 25mg and the infusion treatment, body temperature is normal after 3 hours, does not after this occur heating again.Leave hospital after 2 weeks.Every 1-2 week of postoperative follows up a case by regular visits to once.

2, result

2.1 clinical symptom

Two routine patients week beginning angina pectoris attacks after treatment alleviates, number of times reduces.Example 1 time was withdrawn medicines such as the glad health of southern Shandong, Diltiazem after surgery in 2 weeks voluntarily, but stenocardia continues to alleviate, and every day, only night-time attack 2 times, the time length was short, and stenocardia is classified as the II level.Example 22 whens week pectoralgia outbreak after surgery obviously alleviates, and mobility increases, the medicines such as sorbitrate, Diltiazem, Asprin of stopping using, but 6 week of postoperative level walking 5km only have and slightly breathe hard, no stenocardia is shown effect, stenocardia is classified as the II level.

2.2 coronary angiography result

Example 1, postoperative 6 during week left side coronary angiography branch and side Zhi Xianying degree the same, and during the arteria coronaria dextra radiography circular cone prop up, sharp-edged prop up between and with left anterior descending branch, septal branch between visible abundant collateral circulation, make section development far away among LAD, the LCX, reach side Zhi Xunhuan 3+ level.

Example 2, postoperative 6 during week left side trunk section far away entirely shut, rarely seen two tiny branches develop, the nearly section of right hat pathology is the same, and side Zhi Xunhuan was abundant between far-end and LAD, LCX and septal branch, diagonal angle propped up, and made LAD, LCX and diagonal angle Zhi Xianying more abundant, reach side Zhi Xunhuan 3+ level, see Fig. 3.

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Claims (4)

1, a kind of plasmid-type adenovirus carrier is characterized in that it contains VEGF 165 genes.

2, the plasmid-type adenovirus carrier of claim 1 is characterized in that VEGF 165 genes are connected under the CMV promotor of adenovirus shuttle plasmid.

3, claim 1 or 2 plasmid-type adenovirus carrier are in the application that is used for preparing the therapeutical agent for the treatment of coronary heart disease.

4, claim 1 or 2 the plasmid-type adenovirus carrier application in the therapeutical agent of preparation treatment peripheral arterial ischemic angiopathy.

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