Establishment Of Adeno-associated Virus Vector Scalable Mass Production System And Mechanism Study

Adeno-associated virus (AAV) is a nonpathogenic gene therapy viral vector.Epidemiological studies revealed that80%population has been infected by AAV butwithout any AAV-concerned diseases. Recombinant AAV (rAAV) deleted all theviral genome, only preserved two terminal regulatory elements termed as invertedterminal repeats (ITR), which improved its safety. Additionally, AAVs areParvoviridae with small particle and stable physical character, making them suitablecandidates of biological medicine. AAV has been widely adopted in many diseasestherapy demonstrating outstanding results, and was selected as one of the Top TenTechnical Advances in2009by Science due to its excellent success in Leber'sCongenital Amaurosis. New Engl J Med reported AAV was used in Leber'sCongenital Amaurosis gene therapy, the recipients restored the vision and could dosports as normal without any severe adverse in the years follow-up. Journals such asNature, Nat Med et al. have reported several times that AAV could be adopted toexpress HIV antibodies, resulting in completed deletion of HIV in mouse and monkeymodels. Besides, AAV also has outstanding performances in nervous system diseases,metabolic diseases, as well as tumors. In conclusion, AAV being as gene transfervector has attracted world-wide attentions.Presently, there are39clinical trials utilizing AAV in progress in the world,among which28trials are being carried out in America,7trials in Europe,2trials inMiddle East, and1trial in India and South Africa respectively. However, in SouthAmerica, Australia and other technologically advanced regions such as Canada,Singapore, Japan and so on, there are no AAV concerned clinical trials. A criticalfactor that restricts AAV clinical trials in the other areas is the difficulty of AAVpackage, especially when employed in mass production. For the difficultiesencountered in the trials on large animals and clinical trials, even in the US, there isnot a standard design for AAV mass production. Thus, the establishment of large-scale AAV production technology has a very important significance to accelerate theapplied research of AAV vector and to occupy the high ground of the related fields.Traditional AAV producing methodology used three plasmids to co-transfectHEK293cells, if you need1014vg virus, you should repeatedly transfected a totalamount to over5000175cm2culture flasks cells, which is a great limit to thetechnology operation, costs and human resources. To this end, the international community has been looking for ways to mass production of AAV, established avariety of production systems including Ad/AAV, HSV/AAV, production cell lines,as well as insect cell-baculovirus expression vector system. Among them, using theinsect cell-baculovirus expression vector system (IC-BEVS) to product AAV is apotent production system which has aroused widespread concerns in recent years.Although, it has been extensively studied in mammalian cells, the AAV packagingcomponents's localization and interaction in insect cells are almost unknown, andthese localization and ineraction are important for AAV genome duplication, capsidassembly and genome packaging.This research focused on establishing the scalableproduction and purification technology of AAVbased on the insect cell-baculovirusexpression vector system, and on this basis, firstly carried out a systematic dynamiccomparative study of1,2,8,9four serotypes AAVs'recombinant processes in insectcells.Part I: Establishment of adeno-associated virus production system based on thethe IC-BEV systemObjective: To construct recombinant baculovirus vectors based on pFBDbackbone vector which were required for packaging AAV, and produce1,2,8,9fourserotypes AAVs.Methods:(1) Use pFBD vector as a skeleton, construct recombinant baculovirustransfer vectors pABP1,2,8or9anyone of which carry the AAV2Rep gene and theserotype specific Cap gene, introduce Intron-ph promoter in defined sites of the Repgene and Cap gene to initiate the expression of Rep and Cap gene in insect cells;(2)Use pFBD vector as a skeleton, construct recombinant baculovirus transfer vectorspABVN-hCMV-EGFP and pABVN-hCMV-H794carrying AAV2ITR expressioncassette;(3) Transpositional recombine the six vectors pABP1,2,8,9and pABVN-hCMV-EGFP and pABVN-hCMV-H794respectively with DH10BacTMbacteria, andblue-white clones picking up of the corresponding recombinant baculovirus vectorpABPM1,2,8,9as well as pABVNM-hCMV-EGFP, and pABVNM-hCMV-H794;(4)Package the recombinant baculovirus ABPM1,8,9as well as ABVNM-hCMV-EGFP, and ABVNM-hCMV-H794;(5) Produce rAAV1-hCMV-EGFPor H794,rAAV2-hCMV-EGFP or H794,rAAV8-hCMV-EGFP or H794,rAAV9-hCMV-EGFP or H794by recombinating baculovirus ABPM1,2,8,9, with ABVNM-hCMV-EGFP or ABVNM-hCMV-H794respectively;(6) Titercorrespondingvirus by RT- PCR, and detect the Rep and Cap protein expression by Western Blot, verify theviability of rAAV by cell experiment.Results: Successfully constructed pABP1,2,8,9and pABVN-hCMV-EGFPpABVN-hCMV-H794vectors, and obtained corresponding recombinant baculovirusby transpositional recombination and virus packaging; successfully recombined1,2,8and9serotypes AAV viruses; RT-PCR, Western blot and cell infection experimentssuccessfully determined the virus titer, protein expression, and verified the viability ofthe virus.Conclusion: Successfully established adeno-associated virus production systembased on the insect cell-baculovirus expression vector system (IC-BEVS).Part II: The large-scale production, purification and identification of adeno-associated virus vectorsObjective: Based on the adeno-associated virus production system which hasbeen established on IC-BEV system, amplify AAV production scale, exploitcompatible scalable production and purificationprocess, and establish thecorresponding virus identification and assessment system.Methods: Use the500ml Falcon culture shake flasks of BD Company torecombine and produce rAAV1,2,8and9in a thermostatic culture oscillator,comparatively study a variety of cell lysis methods, buffer systems, ion concentrations,and chromatographic buffer pH values and optimize the process, purify rAAV byusing AVB affinity chromatography in the AKTA Purifier10protein purificationdevice, and determine titer by RT-PCR, measure purity by Silver Staining, as well asobserve and calculate the ratio of full virus particlesto empty by transmission electronmicroscopy, verify the functionality of the purified virus by cell test.Results: We established scalable AAV production and purification technologybased on IC-BEVs, the current optimized conditions are suitable for the isolation andpurification of rAAV1,2and8,100ml of the culture system can produce up to1013VG virus, a single batch can obtain1014VG virus with a great producingefficiency improvement compared with traditional method that needs to transfectmore than5000175cm2flasks culture. The purity of obtained virus is over95%, fullvirus particles accounted for about85%, and the virus has good ability of infection. Conclusion: Based on the IC-BEVS, We established large-scale production andpurification technology which has reached world advanced level from the view ofyield and purity, and laid the foundation for the production of large-scaleindustrialization and related researches of AAV. The conditions of serotype9AAVpurification need to be explored further.PartIII: the dynamic mechanism of AAV production in IC-BEV systemObjective: the production of AAV in mammalian cells has been extensivestudied, but the AAV packaging process in insect cells is almost unknown. We firstlysystematically comparatively study the dynamic mechanism of rAAV1,2,8,9fourserotypes AAVs assembly in insect cells, providing clues for further understandingand optimization of large-scale production and purification technology of AAV basedon the IC-BEVS.Methods: With antibodies respectively reacted with Rep protein, all types of Capprotein, intact rAAV1and rAAV2virus particles and baculovirus surfaceglycoprotein protein gp64, study dynamic mechanism of assembly of eachcomponents during AAV production in insect cells by immunofluorescence at12,24,48and72and96hours past infection. At the same time of48hpi, observe the cellstructural changes when AAV was recombined and produced in insect cells bytransmission electron microscopy.Results: In insect cell the Rep protein soon entered into nucleus after beingtranslated, and gradually increases to a high concentration in clusters distribution overtime. The Cap proteins entered into the nucleus slower than Rep proteins, buteventually wholly deposited in nucleus. The distribution of Cap proteins were closelyrelated with Rep proteins, especially serotype2and9Cap proteins. All serotypesAAVs were packaged in nucleus, and the packaging position was closely related tothe baculovirus matrix and the fibrillar structures. Packaged AAVs were mainlydistributed in the perinulcear ring zone, which place also was where huge numberbaculovirus accumulation. Immunofluorescence showed that Rep and Cap proteinshad interactionat that place.Conclusion: Rep and Cap proteins located in the ring zone surroundingbaculovirus matrix and recombined AAV particles. Rep proteins may haveinteractions with Cap proteins, and the distribution of baculovirus, fibrillar structures and AAV particles were closely related. The position AAV packaged in Sf9cellsapparently was not in the nucleolus region reported in mammalian cells. However, thefine interaction mechanisms of Rep, Cap, AAV particles and baculovirus in Sf9cellsneed further research.In summary, we have successfully established a large-scale production andpurification technology platform of AAV based on IC-BEVs. The productioncapability (1013VG per100ml cell culture) and purity(95%) all have reached the goldstandard developed by the worldwide AAV cutting-edge laboratories; The first timein the world as we know we explored the dynamic mechanism on rAAV1,2,8and9package in Sf9cells, which laid the foundation for further understanding andoptimization of this production system; The technology platform we have establishedovercomes the bottleneck of AAV mass production in clinical trials, that is alsoimportant in theorectical research and practical applications.