[50] Cross-species comparison of in vivo reporter gene expression after recombinant adeno-associated virus-mediated retinal transduction

Publisher Summary This chapter describes several methods to perform subretinal recombinant adeno-associated virus (rAAV)-mediated gene transfer in eyes of several different species of mammals. In preparing and evaluating purified recombinant virus, the investigator should assure that all manipulations are performed in accordance with institutional and national biosafety guidelines. The transgene cassette used in the study is a cDNA encoding green fluorescent protein (GFP) driven by a cytomegalovirus (CMV) promoter. Although any gene that fits into the rAAV vector could be used, the advantage of the GFP transgene is that the presence of its protein product can be evaluated noninvasively as a function of time after injection. A comparative study reveals a similar cellular specificity of transgene expression, stability of transgene expression, and immunological response to the rAAV across species. However, differences in delivery methods and time of onset of rAAV-mediated transgene expression are noted. The approach for generating rAAV-GFP is to package the recombinant DNA into AAV particles by complementation with a system expressing AAV rep and cap genes.