The Establishment Of Recombinant Adeno-associated Virus Preparation Method

Gene therapy,as a new method for treatment a variety of human genetic diseases has aroused great attention,which is a process correcting genetic defects by introducing functional gene into the target cells by the vector.Vector is the key factor in gene therapy,which is mainly divided into two types,viral vector and non-viral vector.At present,most of the vectors used in gene therapy are viral vectors.The most common used viral vector includes adenovirus vector,retrovirus vector,lentivirus vector and adeno-associated virus vector.These viral vectors have their own advantages and disadvantages.Adeno-associated virus vector has an ability for its site-specific integration in the genome and does not cause any detectable side-effect,which is one of the most attractive viral vectors for gene therapy in clinic.High titers of viral vector were required for in vivo gene therapy;however,the most commonly used method in laboratory for AAV production was currently carried out by transfecting three plasmid to HEK 293 cells,which has some problems such as low transfection and low packing efficiency.Hence,to establish new method for preparation of recombinant AAV has a significant importance.In this study,two methods were used for rAAV preparation,(1)rAAV production by co-transfecting two plasmids into HEK293 cells.AAV2 rep-cap gene was inserted into E3 region of the adenovirus backbone vector by homology recombination,then the universal vector was constructed.By using the vector,the adenovirus backbone vector E3 region carrying AAV2 rep with the cap gene of different serotypes were obtained.The adenovirus backbone plasmid carrying AAV rep-cap and the shuttle plasmid pAAV carrying AAV2 ITR and exogenous gene were co-transfected into HEK293 cells to prepare rAAV.(2)rAAV production by stable cell line.A cell line with the potential to produce rAAV was firstly established by integrating AAV rep-cap and Cre gene in the genome of HEK 293 cells by using lentivirus vector carrying these genes followed by introducing the shuttle plasmid pAAV carrying AAV2 ITR and exogenous gene into the cells,which was named potential rAAV producer cell line.Then adenovirus with packing signal flanked by loxp site was used to infect the potential rAAV producer cell line to generate rAAV viral vector.The research project includes the following aspects.1.rAAV production by co-transfecting two plasmids into HEK293 cells.(1)Cap gene amplification of different AAV serotypes.Through the analysis of different transduction ability of different AAV serotypes,AAV1,AAV5,AAV8 DJ and AAV9 were selected for this project.The full-length cap gene encoding AAV1,AAV5,AAV8 DJ and AAV9 was amplified,respectively by designed primers based their sequences,which included BglII and PmeI enzyme sites at the 5’ and 3’ of the gene,respectively.(2)Construction of adenovirus backbone plasmid pAd5-backbone-E3-AAV2 rep-cap containing AAV2 rep-cap.AAV2 rep-cap was cloned into the adenovirus E3 shuttle vector,then the resultant plasmid pAd5-backbone-AAV2 rep-cap was obtained by homologous recombination between E3 shuttle plasmid carrying AAV2 rep-cap with the adenovirus backbone vector.(3)Construction of the adenovirus backbone plasmid containing AAV2 rep with cap gene of different serotypes.The lacZ gene fragment was obtained by PCR,then LacZ was cloned into pAd5-backbone-AAV2 rep-cap by replacing AAV2 cap gene.The resultant plasmid was named pAd5-backbone-AAV2 rep-lacZ.Then AAV1,5,8 DJ and 9 cap gene were cloned into pAd5-backbone-AAV2 rep-lacZ by replacing lacZ,respectively.The obtained plasmids were named the pAd5-backbone-AAV2 rep-AAV1 cap,pAd5-backbone-AAV2 rep-AAV5 cap,pAd5-backbone-AAV2 rep-AAV8 DJ cap and pAd5-backbone-AAV2 rep-AAV9 cap respectively.(4)Preparation of rAAV.The plasmid pAAV-ITR-CMV-puromycin-IRES-GFP-Sv40pA-ITR was co-transfected into HEK293 cells with the adenovirus vector carrying AAV2 rep and cap of different serotypes into the HEK293 cell,then rAAV was prepared.2.rAAV production by stable cell lines.(1)Construction of lentivirus vectors containing AAV rep-cap and Cre gene.The Cre gene fragment was obtained by PCR,then Cre and AAV rep-cap were sequentially cloned into lentivirus vector by means of molecular cloning.The resultant plasmid was named pCDH-CMV-Cre-AAV2 rep-cap-EF 1 a-Neomycin.Using the similar strategy,lentivirus vector carrying AAV2 rep,Cre and four different serotypes cap,respectively was obtained,which was named as pCDH-CMV-Cre-AAV2 rep-AAV1 cap-EF1a-Neomycin,pCDH-CMV-Cre-AAV2 rep-AAV5 cap-EFla-Neomycin,pCDH-CMV-Cre-AAV2 rep-AAV8 DJ cap-EF 1 a-Neomycin and pCDH-CMV-Cre-AAV2 rep-AAV 9 cap-EF1a-Neomycin,respectively.(2)Construction of the cell line stably expressing the AAV2 rep-cap and Cre gene.pCDH-CMV-Cre-AAV2 rep-cap-EF1a-Neomycin,the lentivirus packing plasmid pPackage and pVSVGE were co-transfected into HEK293 cells,then supernatants containing lentivirus were collected 72h post transfection and used to infect HEK293 cell.Finally,HEK293/Cre-AAV2 rep-cap cell line stably expressing the AAV2 rep-cap and Cre was obtained through G418 selection.(3)Construction of the potential rAAV producer cell line.The plasmid pAAV-ITR-CMV-puromycin-IRES-GFP-Sv40pA-ITR was transfected into HEK293/Cre-AAV2 rep-cap,then the potential rAAV producer cell line named as HEK293/pAAV-Cre-AAV2 rep-cap was obtained through puromycinscreening.(4)Construction of the potential rAAV producer cell line expressing cap of different AAV serotypes.The plasmid pAAV-ITR-CMV-puromycin-IRES-GFP-Sv40pA-ITR was transfected into HEK293 cell,and then HEK293/pAAVcell line was obtained after puromycin screening.Based on this cell line,fourpotential rAAV producer cell lines expressing cap of different AAV serotypes were obtained after G418 selection by infecting four lentivirus carrying four cap genes of different AAV serotypes.(5)Preparation of recombinant helper adenovirus.Adenovirus vector pAd5-loxp-Ψ-loxp-PGK-mCherry-Sv40pA was obtained by homologous recombination between backbone vector pAd5-backbone digested by ClaI and the adenovirus El shuttle vector pAd5-E 1-loxp-Ψ-loxp-PGK-mCherry-Sv40pA digested by ScaI in E.coli BJ5183.The PacI linearized plasmid was transfected into HEK293 cells,then high titer adenovirus Ad5-backbone-loxp-Ψ-loxp-PGK-mCherry-Sv40pA which packing signal was flanked by loxp site was prepared by propagation and purified by CsCl density gradient centrifugation.(6)Preparation of rAAV.Recombinant helper adenovirus was used to infect the potential rAAV producer cell line,then rAAV was prepared.This project achieved the following results.1.Cap gene of AAV1,AAV5,AAV8 DJ and AAV9 cap gene were obtained by PCR,respectively.2.By means of molecular cloning,the adenovirus backbone vector pAd5-backbone-AAV2 rep-cap and the universal backbone vector pAd5-backbone-AAV2 rep-Lacz were obtained.Then pAd5-backbone-AAV2 rep-AAV 1 cap,pAd5-backbone-AAV2 rep-AAV5 cap,pAd5-backbone-AAV2 rep-AAV8 DJ cap and pAd5-backbone-AAV2 rep-AAV9 cap were constructed by replacing LacZ gene on the basis of the universal backbone vector.3.The plasmid pAAV-ITR-CMV-puromycin-IRES-GFP-Sv40pA-ITR was co-transfected into HEK 293 cells with adenovirus vector carrying AAV2 rep,cap and four different serotypes cap,respectively,then the rAAV was prepared successfully.4.By means of molecular cloning,the lentivirus vector pCDH-CMV-Cre-AAV2 cap-rep-EFla-Neomycin was obtained,then HEK293/Cre-AAV2 rep-cap cell line stably carrying Cre and AAV2 rep-cap was obtained by lentivirus infection of HEK293 cells.Finally,the potential rAAV producer cell line HEK293/pAAV-Cre-AAV2 rep-cap was obtained by transfecting pAAV vector pAAV-ITR-CMV-puromycin-IRES-GFP-Sv40pA-ITR to HEK293/Cre-AAV2 rep-cap cell line followed by puromycin screening.5.Four lentivirus vectors,pCDH-CMV-Cre-AAV2 rep-AAV 1 cap-EF1a-Neomycin,pCDH-CMV-Cre-AAV2 rep-AAV5 cap-EF1a-Neomycin,pCDH-CMV-Cre-AAV2 rep-AAV8DJ cap-EFla-Neomycin and pCDH-CMV-Cre-AAV2 rep-AAV9 cap-EF1a-Neomycin,were constructed by using molecular cloning technique.Then potential rAAV producer cell line expressing cap of AAV1,AAV5 AAV8 DJ and AAV9,respectively was established by lentivirus infection of HEK293/pAAV cell line.6.Recombinant helper adenovirus Ad5-loxp-Ψ-loxp-PGK-mCherry-Sv40pA containing mCherry cassette and package signal flanked by loxp sites was obtained by propagation and purified by CsCl density gradient centrifugation.