Experimental Study On The Gene Therapy Of Bladder Cancer With RAAV-TK-IRES-Endostatin

Bladder transitional cell carcinoma(BTCC) is one of the most commonurological malignant diseases. Its recurrent rate is almost 60% to 90% after operationalthough as a major method to prevent this tumor. Local radiotherapy and systematicchemotherapy are rarely used due to their severe side effects and drug resistance.Nowadays, intravesical biotherapy and immunotherapy instillation are regarded as aneffective adjuvant method, but almost 30% to 45% patients had no response to thistherapy, furthermore, the clinical effect was limited by its severe side effects,drugresistance and fewer drug taking.Numerous scholars are paying close attentions to suicide gene treatment thesedays and gene engineering is used to transduce suicide gene into tumor cells. Suicidegene included in virus or bacterial genome, encodes special enzyme. Nontoxicprodrug can be metabolized by the latter, then become venenous production and leadto tumor cell's death. Therefore, the treatment is called "virus oriented enzymolysisprodrug medication" or "molecular chemotherapy". Thymidine kinase gene treatmentis one of most used suicide gene treatments. The enzyme metabolizes nuclide analoginto diphosphorylation and then becomes triphosphorylation which against tumor andselectively metabolizes GCV. GCV becomes triphosphorylation and then interferesand intercepts DNA normal synthesis and cellular proliferation. In the treatment fortumor, herpes simplex virus-thymidine kinase gene is considered to be the mosteffective and also licensed into clinical trial only. It can play a part in preventing DNA chain elongating, so that selectively kill and wound suicide gene cells inmultiplicative division period.Vascular formation is regulated by stimulating factors and inhibiting factors, andmany stimulators and inhibitors have been identified by now. Among all the inhibitors,endostatin has been testified as the strongest one. It could inhibit the proliferation ofendothelial cells, induce apoptosis of tumor cells, inhibit tumor growth and metastasiseffectively, and has no side effect. So endostatin maybe the most preferred agent fortumor anti-angiogenic therapy. In 1999, it has been used to tumor patients as thefirst stage clinical experiment in US, after two years' observation, it was proved thatendostatin therapy can inhibit tumor growth and progression with little side effects.Adeno-associated virus (AAV) offers many desirable features of retrovirusestherapy. The recombinant AAV vectors can transducer both dividing andnon-dividing cells in vitro and in vivo. Efficient and long-term transduction in vivoand the lack of both cytotoxicity and cellular immune responses are the hallmarkfeatures of AAV mediated gene transfer. AAV was regarded as one of the extremevectors in gene therapy in future. Although AAV vectors have been extensively usedin gene therapy for genetic and metabolic diseases, few studies have used this vectorsystem for bladder cancer gene therapy.With the development of molecular biology, gene therapy are now regarded as awholly new and promising method to conquer human carcinoma. But, we have tosolve two key cruxes: a) vector choose, to select a kind of high expressed, low levelantigen and safety vector is the major aim toward this. b) therapeutic gene select, aswe all known, the occurrence of tumor is an multi-controlled pathway progress. So,the gene therapeutic effect was limited by only one gene selection.Therefore, in our study, we aims to combined the endostatin and suicide genetogether by pIRES vector via recombined AAV, which was regarded as one of the extreme vectors in gene therapy in future. Enhanced the effect of TK on the basis oftumor anti-angiogenic gene therapy of endostatin by double targeted at these twogenes and then established the express way of the therapeutic gene with little sideeffects.Part 1 Construction of the non-melted plasmid AAV contain TK and endostatin geneObjective: To construct the non-melted plasmid AAV contain TK and endostatingene via pIRES vector.Methods:1. PCR TK segment from pAdTrack-CMV-TK and then cloned it tovector pMD-19T simple. Digested pMD-19Tsimple-TK and pAAV-ES by ClaI,XbaIand then construct pAAV-TK and verified by sequence indentification.2. PCR IRES,TK,ES framents from pIRES-MCS, pAAV-TK, pAAV-ES and thencloned these genes into vector pMD-19T simple to construct pCMV-TK-IRES-ES.verified by sequence indentification and enzyme digestion.3. Double digested pCMV-TK-IRES-ES and pAAV-MCS, connection fragmentsto construct pAAV-TK-IRES-ES and then digested by ClaI and XbaI to verification.4. To avoid the lost of ITR, we identified pAAV-TK and pAAV-TK-IRES-ESthisby PCR and enzyme digestion.Results: 1. Extract pAdTrack-CMV-TK, PCR TK fragment and verified byelectrophoresis. Digested by ClaI and XbaI, the fragment is about 1.1kb, whichestablished the successfully construction of pAAV-TK.2. Extract pAAV-ES and pIRES-MCS and then PCR them, we got two framentsabout 700bp and 650bp which are in accordance with correct sequences. By digeseting pMD-19Tsimple-TK and pCMV and connectded them by T4 ligase, wesucessfully constructed pCMV-TK. Also, By digeseting pMD-19Tsimple-IRES andpCMV-TK and connectded them by T4 ligase, we sucessfully constructedpCMV-TK-IRES. Digested pCMV-TK-IRES-ES by XbaI and BamHI, we got fragmentabout 700bp, which is the correct size. So, we successfully constructedpCMV-TK-IRES-ES.3. Digested pCMV-TK-IRES-Es and pAAV-MCS, and then connected themtogether by T4 ligase, and then digested its clone by ClaI and XbaI, the fragment isabout 2.3kb. We successfully constructed pAAV-TK-IRES-ES.4. When pAAV-Tk and pAAV-TK-IRES-ES were digested by PstI and NotI, wegot 137bp fragment by electrophoresis. The PCR product were about 3kb, which wereas expected.Part 2 The package, purification, concentration and idenfication of recombination AAVObjective: To package, purify, concentrate and idenfy of the recombinationAAV.Methods: 1. AAV-293 cells were co-transfected by electroporationusing pAAV -TK(or Paav-TIE), pRC and pHelper to package rAAV-TK and rAAVTIE. the cells were under Chloroform-NaCI sediment-Chloroform and ice alcoholto dissociate, purify and concentrate rAAV.2. Viral particle of purified rAAV were assayed by AVSachTM ELISA. Use.SDS-PAGE Coomassie brilliant blue staining to observe capsid protein of rAAV. And use electric microscope to observe the size and the procedure of rAAV.Results: We obtained high quality of rAAV after dissociating and purifying. Theviral particlesof rAAV were 2×10^11-12. Under SDS-PAGE and Coomassie brilliantblue staining, we could find three notable viral capsid protein strap of AAV Weobserved the viral particle under electron microscope and found that rAAV werelocated uniformly, the diameters were about 20 to25 nm and the figure werepolyhedron. The successful packaging of rAAV established the foundation for laterexperiment using this vector in vivo and in vitro.Part 3 Study on rAAV-TIE gene therapy of bladder cancer in vitroObjective: To observe the effect on apoptosis and growth of cell bladder cancercell by transfecting with rAAV-ES, rAAV-TK-IRES-ES, rAAV-TK; To indentify theHUVEC cell and the influence of bladder tumor cell by tranfected by rAAV-TK.Methods: 1.To indenfy the HUVEC cell by HE and AgNo₃ staining, IHC andelecric microscope.2. To value the MOI by transfect the T24 cell by rAAV-EGFP; We identify thebiological effect of rAAV by extracting the gene of ES, TK and TK-IRES-ES(TIE)transfected by rAAV-ES, rAAV-MCS, rAAV-TK and rAAV-TK-IRES-ES; Also, weuse microscope and etc. to study the influence of T24 transfected by rAAV-TK.3. T24 cell was transfected by rAAV-ES and rAAV-TIE, collected the serum andidentified the concentration of Endostatin by ELISA. A t the same time, we use FCMto value the apoptosis extent cultured by the serum which contain the Endostatin.4. We study the dose and time effect of rAAV-TK and rAAV-TIE/GCV system by MTT test. And also we use JC-1 and FCM to observe the apoptosis effect of thissystem.Results: 1. After the HUVEC cell was stained by AgNO₃, we saw the silverparticles just like the stone road. When scaned by the electric microscope, we saw theW-P body, whichi was the charicteristic of HUVEC cell.2. After the bladder tumor cell was transfected by rAAV-EGFP about 72 hours,lots of GFP was expressed in the core of cell. The MOI was about 1×10⁶v.p./cell.The T24 cell was little affected after transfecting by rAAV-tk by FCM test.3. The concentration of endostatin was tested by ELISA and just as followings indifferent groups: rAAV-ES group(60.08ng/mL),rAAV-TIE group(58.14ng/mL),rAAV-MCS group(0 ng/mL). The cell in the rAAV-ES group and rAAV-TIE groupwere more easy to apoptosis than control group.(rAAV-ES37.02%, rAAV-TIE32.14%)4. After 72h of transfecting by rAAV-TK and rAAV-TIE/GCV system, theapoptosis were 34.12%(rAAV-TK) and 36.91%(rAAV-TIE).Part 4 The study of rAAV-TIE gene therapy of bladder cancer of nude mice in vivoObjective: To construct bladder cancer animal model using nude mice and studythe therapeutical effect of rAAV-TIE model in vivo.Methods: 1. Cell suspension of T24 cells were injected into thesubcutaneously of right scapular region of nude mice, The nude mice were raised under SPF condition and observed the xenograft tumor growth.2. Bearing tumor nude mice were randomly divided into 5 groups: rAAV-MCSgroup, rAAV-Tk group,rAAV-ES group,rAAV-TIE group. 4 weeks later of treatment,the nude mice were killed by dislocate vertebrae cevicales. The xenografts tumorswere fixed for HE stain. The liver tissue and nephridial tissue were also fixed for HEstain.3. Four weeks later, nude mice were sacrificed for blood sample and endostatinconcentration was assayed by ELISA.Results: 1. After 3 weeks of injected with T24 cells on nude mice, 25 showvisible tumor on the injected location. The rate of tumor formation is 93%.2. After 9 days injected by rAAV-ES, rAAV-TK, rAAV-TIE, the tumor volumewere: rAAV-ES group(0.75±0.08)cm³, rAAV-TK group (0.71±0.11)cm³, rAAV-TIEgroup (0.52±0.09)cm³, rAAV-MCS group(1.27±0.13) cm³ and control group1.24±0.17)cm³.3. The MVD in the different groups were as followings: rAAV-ES group(18.72±2.53)/HP, rAAV-TK group (21.74±4.62)/HP, rAAV-TIE group(12.73±1.78)/HP, rAAV-MCS group (52.38±6.46)/HP and control group(49.94±7.17)个/HP4. The endostatin concentration in the different groups were as followings:rAAV-ES group (38.52±6.53)μg/L and rAAV-TIE group(40.33±7.48)μg/L.5. HE statin confirmed the tumor. The liver tissue and kidney tissue of eachgroup has no obviously cell degeneration and necrosis.Conclusions: We successfully constructed rAAV-TK and rAAV-TIE. Experiment in vitro and in vivo indicated that interested gene could express in hostcell mediated by rAAV. In vitro study shows that rAAV-TIE could inhibit tumorinduced angiogenesis and suppress both the initiation and the subsequent growth ofhuman bladder cancer. Gene therapy with rAAV-TIE may be an effective adjuvantmethod.