Comparison of Recombinant Adeno-Associated Virus Gene Therapy Vector Packaging Methods.
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Adeno-associated virus is being developed as a human gene therapy vector. Recombinant AAV vectors are constructed by replacing the wild type viral genome with a transgene of interest. This recombinant genome is then packaged into infectious virions. Virion production requires a vector plasmid containing the transgene of interest, wtAAV rep and cap genes, and helper virus functions. Standard protocols provide rep and cap either through stable rep/cap cell lines, or through co-transfection of a rep/cap plasmid. Helper virus functions are provided by adenovirus-5 or herpes simplex virus-1. Viral infection results in loss of some recombinant virions due to cell death as well as potential contamination of the final stock. To overcome these problems, several helper-virus-free protocols have been developed using plasmids containing essential Ad-5 genes. Standard purification protocols involve CsCl gradient centrifugation followed by dialysis.The centrifugation takes several days, and product is lost in the dialysis buffer.Several protocols have been developed utilizing nonionic and nontoxic iodixanol gradients which require much shorter centrifugation times and use ion-exchange heparin affinity columns.To directly compare standard protocols to newer ones, plasmids containing helper virus genes will be tested against HSV-1 infection, and iodixanol/heparin purification will be tested against CsCl gradient/dialysis purification.The relative efficiencies of the protocols will be visualized using a blue and a yellow fluorescent protein cloned into a unique AAV vector plasmid, CWRSP.