Introduction Substantial progress has been made over recent years in elucidating the molecular and genomic mechanisms contributing to the development of pulmonary vascular diseases, particularly Pulmonary Arterial Hypertension (PAH). With this array of genomic and molecular targets available, therapeutic intervention using gene transfer offers exciting potential for mitigation and possibly even prevention of pulmonary vascular disease progression. In order to realize such potential, it is necessary to perform safe, long-lasting, non-immunogenic gene transfer directly to the pulmonary vasculature. Adeno- associated virus (AAV) is a leading candidate vector for such a gene transfer application since it has been shown in human clinical trials to partially or fully meet the above criteria. However, the canonical AAV vectors commonly used, which embody the Serotype 2 capsid with a cytomegalovirus (CMV) promoter, poorly infect the pulmonary vasculature when systemically administered. Transductional and Transcriptional Targeting of AAV to the Pulmonary Vasculature Michael J. Passineau, Lee Zourelias, Laurie Machen, and Raymond L. Benza Gerald McGinnis Cardiovascular Institute and Gene Therapy Program, Allegheny-Singer Research Institute Conclusions Taken together, these results indicate that AAV can be efficiently targeted to the pulmonary vasculature at both the transductional and transcriptional levels. This is the first step toward a vector delivery system capable of long-term gene expression in the pulmonary vasculature to accomplish gene repair (e.g. BMPR2) and/or therapy for PAH. Acknowledgements Support for this work was provided by the Gilead Sciences Research Scholars Program (to MJP). The authors would like to acknowledge the Gene Therapy Resource Program (GTRP) of the National Heart, Lung, and Blood Institute, National Institutes of Health for providing the gene transfer vectors used in this study. Methods As a first step toward long-term transgene expression in the pulmonary vasculature, we sought to improve AAV- mediated gene transfer with transductional and transcriptional targeting strategies. On the transductional level of targeting, we used AAV2 pseudotyped with the Serotype 9 (AAV2/9) capsid to improve transduction of the pulmonary vasculature. On the transcriptional level, we varied the promoter driving expression of our Luciferase (Luc) reporter gene. We used the canonical CMV (viral) promoter, as well as a synthetic promoter (CB7) and finally a endogenous mammalian promoter (Flt-1) previously reported to show high activity in lung. Vectors were administered to the tail vein of rats at a dosage of 2x10 12 vector genomes. Intensity of gene expression was determined by in vitro Luc assay performed on homogenized lung and liver samples, corrected for protein content, to arrive at expression metric of Relative Light Units/ug of protein. Results Our results indicated that AAV2/9 transduced the pulmonary vasculature with far greater efficiency that AAV2/2 (data not shown) but that the intensity and duration of gene expression was greatly influenced by the choice of promoter. Both the non-mammalian promoters showed very high expression at two week post-treatment and this quickly declined thereafter at 4 and 10 weeks. The Flt-1 promoter, in contrast, showed overall much lower magnitude of initial expression that was maintained >2 months. Figure 1: Design of the AAV vectors. Using a two-plasmid transfection system, a expression cassette containing GL4 Luciferase driven by the experimental promoters and flanked by AAV2 ITRs was combined with a plasmid containing Rep from AAV2 and Cap from AAV9, resulting in three different AAV2/9 vectors, driven by CMV, Flt-1, and CB7 promoters, respectively Figure 2: Reporter gene expression at various timepoints post-infusion. Promoter-modified vectors were infused via the tail vein, and animals sacrificed at the indicated timepoint and lung and liver analyzed using an in vitro Luciferase assay kit. n=1/promoter/timepoint. ITR 2 CMVLuciferase ITR 2 Flt-1Luciferase ITR 2 CMVLuciferase ITR 2 Flt-1Luciferase ITR 2 Flt-1Luciferase ITR 2 CB7Luciferase Future Studies Given the massive sequestration of our AAV2/9 vector in the liver, possibly due to a “first pass effect”, our follow-up studies will administer the vector via a jugular vein catheter, allowing the vector to pass through the cardiopulmonary circulation prior to the liver. *Note: For the CMV promoter, the week 4 animal was lost. Data from a CMV animal at week 8 was placed in the graph at the week 4 slot but may not faithfully represent gene expression at week 4. * *