Adeno-associated viral vectors for tissue-specific gene delivery in vivo
Qureshi, Wajeeha Rashid
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OBJECTIVE: Clinical gene therapy is increasingly becoming a favorable method for the targeting and treatment of various human diseases. One such gene-transfer method is the use of adeno-associated viruses (AAVs) as a tool for tissue-specific delivery of genes. AAV vectors are attractive vector candidates due to their low pathogenicity, biological safety, and ability to infect both dividing and quiescent cells for sustained gene expression. We sought to develop a protocol for the production, propagation, purification, and characterization of various AAV serotypes to determine their tropism and to observe their integrative and expressive abilities using different reporter genes in vitro and in vivo. METHODS: We used subcloning techniques to introduce our genes of interest into an AAV expression plasmid and recombined with two other AAV vectors required for packaging, cell-specific targeting, and integration capabilities. Serotypes AAV1/8/9 and 5 which are cross-packaged with the prototype AAV2 were used to individually target cardiac cells/skeletal muscle and neuronal cells respectively in eC57BL/6J mice. Viral particles were propagated in vitro and purified using an iodixanol gradient. Purified AAV vectors were introduced into cells in culture and injected into live mice via tail vein injection or using robotic stereotaxic surgery directly into brain opposite saline controls to determine expression capabilities in vitro and in vivo, respectively. Live mice were imaged using the IVIS bioluminescent imaging system to measure luciferase luminescent readout. Separately, sections were acquired from the brains of the mice injected with serotype AAV2/5 and imaged using live cell imaging fluorescence microscopy to demonstrate integrative ability of the vectors into neurons based on GFP reporter signal. RESULTS: A protocol for the generation of neuron-specific AAVs was developed for the successful integration of the reporter gene GFP. The reporter gene was cloned into the AAV expression cassette. Efficient transfection methods were determined along with optimal culture conditions for the propagation of the viral particles in vitro. Promoter-driven reporter gene expression was observed in serotype-targeted cell types in vitro and in vivo. Expression was limited to neuronal cells based on AAV serotype specificity and SYN1 promoter. Integration and expression of the luciferase gene was not observed in the IVIS system. CONCLUSION: Here we demonstrate expression of a GFP reporter in specific neuronal cell types which indicates successful integration and expression abilities of AAV vectors. This specific tissue-targeting technique using the AAV vector highlights the potential for the further development of these vectors as a promising gene transfer system.