129. Does Transcription Influence AAV-Mediated Homologous Recombination?

Abstract

Recombinant adeno-associated viral (AAV) vectors constitute one of the most promising tools for gene transfer. While the majority of AAV transduction events are episomal, our laboratory recently exploited the vector's ability to induce homologous recombination (HR) to design promoterless vectors that utilize chromosomal homology arms flanking a ribosomal skipping P2A-therapeutic coding sequence construct to integrate sequences just upstream of the stop codon of an endogenous gene. When targeting the albumin gene in the liver, a chimeric mRNA transcript capable of producing both albumin and a second therapeutic protein is consequently created. Not only do these vectors offer the permanence of gene transfer associated with integration, but a vector lacking a promoter reduces the chance for oncogene activation from off-target vector integration. AAV-mediated HR appears to be more efficient when targeting transcriptionally active loci, yet it is unclear if transcription itself or other factors that secondarily influence transcription, such as chromatin state, are directly linked to AAV-mediated HR. We therefore set out to establish if the transcriptional rate in particular is a major determinant for this type of HR.To do this, we developed a strategy to quantify precision AAV-mediated transgene integration by exploiting an engineered locus whose transcriptional rates could be controlled by drug administration. To this end, we used lentiviral vectors to generate a series of clonal HeLa cell populations each harboring a single-copy, doxycycline-inducible genomic site expressing the human alpha 1-antitrypsin (hAAT) cDNA. Upon induction with doxycycline, transgene expression from this site increases by greater than 1,000-fold. Each clonal cell population was infected with an AAV serotype DJ vector containing homology arms to the integrated hAAT cDNA and a P2A- codon-optimized human coagulation factor IX (hFIX) cDNA or P2A-eGFP construct that would allow for multicistronic genomic expression of both peptides from a single mRNA only if HR occurred. Transgene hFIX or eGFP expression as a measure of HR is established by quantitative RT-PCR and by transgene protein measurements. By modulating the rate of transcription just prior to rAAV vector administration, we can establish if transcription from a given genomic locus influences the frequency of AAV-mediated HR. Our preliminary results suggest that transcription per se might not be the predominant factor influencing the rate of HR. Further studies will provide more insight into the mechanism of gene targeting by AAV, optimal target site selection, and potentially expand the use of AAV-mediated gene targeting for treating various genetic and acquired diseases.