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Optimization of the Key Technology for Production and Quality Control of rAAV

Author: ChenZuo
Tutor: DiaoYong
School: Huaqiao University
Course: Polymer Chemistry and Physics
Keywords: Adeno-associated virus Plasmid DNA Cetyltrimethylammonium bromide genome titer qPCR
CLC: TQ464.6
Type: Master's thesis
Year: 2013
Downloads: 1
Quote: 0
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Abstract


Adeno-associated virus (AAV) is small (25nm) nonenveloped virusese thatpackage a linear,single-sttanded DNA.AAV viral vectors is the most promising toolsin human gene therapy.This is due mainly to its nonpathogenicity in humans, lowinflammatory response, high transduction efficiency, and broadhost cell tropism.Recombinant AAV(rAAV) gained enormous interest in experimental and clinicalgene therapy applications. Concordant with that progress has been an increased focuson the preparation of a large number of rAAV and development of rigorous qualitycontrol assays to support regulatory applications for new product licensure.Plasmid DNA is raw materials of AAV preparation. In order to preparation of alarge number of rAAV.We have to preparation of a large number plasmid DNA.Todevelop a simple and economic procedure for large-scale purification of plasmidDNA. Purify plasmid DNA by a procedure consisting of alkaline lysis,Cetyltrimethylammonium bromide (CTAB) technology and inorganic saltprecitate.Perform overall control tests on the purified plasmid DNA by gelelectrophoresis, BCA protein assay kit, endotoxin assay kit and Real time-PCR.Thebiological activity of the purified plasmid DNA was detected by cell and animaltransfection experiments. The purified supercoiled plasmid DNA contained was morethan90%and the yield of procebure was more than80%. The purified plasmid DNAbiological activity was high. The purified plasmid DNA met the requirements forplasmid DNA for therapeutic use.One of the most important quality control assays for gene therapy vectors is thatof vector genome titration, as the vector genome is the key mediator of therapeuticeffect for any genetic therapy, and vector genome quantification is almost alwaysused for dose determination. In this papper, we used a wide variety of methods todetermine the AAV vector genome titer, including ultraviolet (UV)spectrophotometry, native gel, PCR and qPCR. The genome titer determined by UVspectrophotometry and native gel is higher than determined by PCR and qPCR. Thesize of the amplified fragment will not effect determined of rAAV genome titer by PCR and qPCR. but the location of primer design will affect the determined of rAAVgenome titer.The inverted terminal repeats (ITR) sequences are the only cis-regulatedviral elements required for rAAV vector generation. We describes the qPCR systemfacilitating the detection and quantification of rAAV ITR sequences. Because thismethod can be used universally for all rAAV genome-based vectors, it willsignificantly simplify rAAV vector titrations in the future.

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