680. Optimization of rAAV-Mediated Expression for Large Transgenes Using a Synthetic Promoter and Tandem Array Enhancers
Authors: Ziying Yan, Xingshen Sun, Zehua Feng, Guiying Li, John T. Fisher, Zoe A. Stewart, John F. Engelhardt
Journal: Molecular Therapy (2015)

rAAV is a widely used viral vector for the human gene therapy, but its inherently small 4.679kb genome is a significant challenge for delivering effective expression of a large gene. Our main goal is to develop rAAV vectors for delivering the cystic fibrosis transmembrane conductance regulator (CFTR) for cystic fibrosis lung disease gene therapy. To express the CFTR gene of 4.43kb coding sequence from a rAAV vector, a short promoter well functioned in airway epithelium is required. In the present study, we screened a set of 100-mer synthetic enhancer elements for their ability to augment CFTR expression from a short 83-bp synthetic promoter. With an empirical approach, screening for their effectiveness in the delivery of reporter gene expression were conducted in step-wise fashion – in plasmids, proviral vectors, and rAAV vectors. Our initial studies in monolayer (non-polarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI) in the context of AAV vector infection found that the combination of F5tg83 (183bp) is the most efficient promoter in both ALI cultures, leading to 19.6-fold and 57.5-fold increases reporter expression, respectively, over the enhancer-less counterpart. We also demonstrated that the F5tg83 promoter produced the highest level of transgene expression in the ferret airway in vivo. Since rAAV-CFTR vectors greater than 5.0kb in size are dramatically impaired with respect to vector efficacy, we utilized a shortened ferret CFTR minigene with a 159 bp deletion in the R-domain to construct a rAAV vector (AV2/2. F5tg83-fCFTRDR). This vector yielded an ≈17-fold increase in vector derived CFTR mRNA transcription and significantly improved Cl- currents in human CF ALI cultures when the vectors were administrated through basolateral infection. We also tested the F5tg83 promoter activity in the context of rAAV/HBoV1 vector, which packages an oversized rAAV genome up to 5.5kb in human bocavirus virus 1 (HBoV1) caspid and efficiently transduces HAE-ALI from apical membrane. We found that the F5tg83 promoter delivered robust and persistent expression of different reporter genes (firefly luciferase or the secreted guassia luciferase) from the HAE-ALI following apical infections of the AV2/HBoV1 chimeric vectors. The expanded package capacity of rAAV2/HBoV1 vector makes it possible to further optimize the F5tg83 promoter directed CFTR gene expression with the full-length CFTR gene. In summary, our study has identified a small enhancer/promoter combination that may have broad utility for rAAV-mediated CF gene therapy to the airway. Furthermore, the enhancer screening approach may be useful for rAAV applications in other organs that seek to deliver large transgenes that approach the packing capacity of rAAV.