543. Efficient Transduction of Canine Muscle Using rAAV6

AAV VECTORS III (I) improve gene transfer to murine hepatocytes for the phenotypic correction of hemophilia B; (II) achieve high efficiency transduction of mouse retina for the potential treatment of ocular diseases; and (III) high efficiency transduction of monocyte-derived dendritic cells (moDCs) for their potential use as anti-cancer vaccines. However, since ubiquitination occurs on lysine residues, we reasoned that sitedirected mutagenesis of surface-exposed lysine residues might prevent ubiquitination of AAV2 capsids, which in turn, might circumvent subsequent vector degradation by the cellular proteasomal machinery. We replaced each of the 7 surface-exposed lysine (K) residues (K258, K490, K527, K532, K544, 549, and K556) on the AAV2 capsid with glutamic acid (E). The transduction efficiency of K490E, K544E, K549E, and K556E scAAV2 vectors expressing the EGFP reporter gene was increased up to 5-fold compared with WT AAV2 vectors, with the K556E mutant being the most efficient, in HeLa cells in vitro at 2,000 vgs/cell. Intravenous delivery of 1x10e10 vgs/animal of WT and K-mutant ssAAV2 vectors expressing the firefly luciferase (Fluc) reporter gene and bioluminescence imaging two weeks post injection further corroborated these results. Next, the two most efficient mutants were combined to generate a double-mutant (K544+556E). The transduction efficiency of this double-mutant, packaging Fluc (ssAAV2-Fluc), increased by approximately 2-fold compared with each of the single mutants, and about 10-fold compared with WT ssAAV2 vectors in murine hepatocytes in vivo. Since AAV8 vectors have previously been shown to transduce murine hepatocytes exceedingly well, and since some of the surface-exposed K residues are also conserved in this serotype, we generated K530E-, K547E-, and K569E-mutant ssAAV8-Fluc vectors. The transduction efficiency of the K547E and K569E vectors was increased by approximately 3-fold and 2-fold, respectively, compared with WT ssAAV8 vectors, in murine hepatocytes in vivo. These studies suggest that targeting the surface-exposed lysine residues is a promising strategy for the development of more efficient AAV serotype vectors for their potential use in human gene therapy.

542. Transduction of Murine, Nonhuman Primate, and Human Hematopoietic Stem Cells (HSCs) by Recombinant AAV Serotype Vectors: Identification of AAV6 as the Most Efficient Serotype for Human HSCs, and Further Augmentation in Transduction Efficiency In Vitro and In Vivo with Point-Mutations of Tyrosine Residues in the Viral Capsid

Liujiang Song,1 M. Ariel Kauss,2 Etana Kopin,2 Manasa Chandra,2 Tahira Ul-Hasan,2 Erin Miller,2 Giridhara R. Jayandharan,3 Angela E. Rivers,4 George V. Aslanidi,5 Chen Ling,5 Baozheng Li,5 Wenqin Ma,5 Xiaomiao Li,5 Lourdes M. Andino,6 Li Zhong,7 Alice F. Tarantal,8 Mervin C. Yoder,9 Kamehameha K. Wong, Jr,2 Menqun Tan,1 Saswati Chatterjee,2 Arun Srivastava.5 1 Physiology, Central South University and Shenzhen Institute of Xiangya Biomedicine, Shenzhen, Guongdong, China; 2Virology, Beckman Research Institute of the City of Hope Medical Center, Duarte, CA; 3Haematology, Christian Medical College, Vellore, Tamil Nadu, India; 4Pediatrics, University of Illinois at Chicago, Chicago, IL; 5Pediatrics, University of Florida, Gainesville, FL; 6 Cranofacial Biology, Medical University of South Carolina, Chaleston, SC; 7Pediatrics, University of Massachusetts Medical School, Worcester, MA; 8Pediatrics and Cell Biology and Human Anatomy, University of California at Davis, Davis, CA; 9 Pediatrics, Indiana University, Indianapolis, IN. Transplantation of genetically-modified autologous hematopoietic stem cells (HSCs) is the most promising alternative to allogeneic transplantation to potentially cure genetic diseases such as -thalassemia and sickle cell disease. In a recent clinical trial, a Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

recombinant lentiviral vector-mediated -globin gene transfer in an adult patient with severe -thalassaemia led to transfusionindependence. However, the observed therapeutic benefit was also accompanied by transcriptional activation of a cellular proto-oncogene, HMGA2, leading to clonal expansion of myeloid cells. Thus, alternatives to lentiviral vectors are needed. Although recombinant AAV2 vectors have gained attention, owing to their safety and efficacy in a number of Phase I/II clinical trials, their transduction efficiency in HSCs has been reported to be low. Only a handful of additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. In our present studies, we evaluated the transduction efficiency of the 10 available AAV serotype vectors in primary HSCs from mice, cynomolgus monkeys, and humans, respectively. Transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. We report here that: (i) AAV1 vectors transduce primary murine HSCs most efficiently both in vitro and in vivo; (ii) None of the 10 AAV serotype vectors transduce cynomolgus monkey HSCs well in vitro; (iii) AAV6 vectors are the most efficient in transducing primary human HSCs, both in vitro, and in a mouse xenograft model in vivo; and (iv) The transduction efficiency of AAV6 vectors is further augmented, both in vitro and in vivo, following site-directed mutagenesis of specific surface-exposed tyrosine residues. These studies suggest that the optimized AAV6 vectors may be a safe and effective alternative for their potential use in HSC-based gene therapy in humans.

543. Efficient Transduction of Canine Muscle Using rAAV6

Jane T. Seto,1 Julian N. Ramos,1 James M. Allen,1 Eric Finn,1 Guy L. Odom,1 Jeffrey S. Chamberlain.1 1 Department of Neurology, University of Washington, Seattle, WA. There are recent reports that galectin-3-binding protein (G3BP), which is present in canine and human sera, aggregates strongly with rAAV-6 and affects vector transduction efficiency. We have screened over 30 canine sera samples (obtained from Marshall Bioresources) by inhibition assays in HT1080 cells using rAAV6-CMV-hPlAP. Of those, only 1 serum sample showed low levels of inhibition. Since rAAV9 does not bind G3BP, we further compared the transduction efficiency of rAAV6 and 9 by jugular vein administration. Beagles were injected with rAAV6-CK8-hPlAP or rAAV9-CK8-hPlAP. AP staining was present in small regions of the heart of the rAAV6injected dog, while minimal staining was observed in the heart of the rAAV9-injected dog. Interestingly, intramuscular injection of the same vectors in a 6 month old beagles showed similar results. At 1e12 vg, rAAV9 injected muscles did not show any positive AP staining, while rAAV6 injected muscles showed large patches of positive AP staining. In sum, our preliminary data suggest that rAAV6 may have higher transduction efficiency than rAAV9. Further studies using dogs from various sources may be required to determine if the circulating levels of G3BP present in canine sera is sufficiently high to indeed inhibit the transduction efficiency of rAAV6.

544. Integrated Transcriptional and Post Transcriptional Targeting Results in Cardiac Biased Transgene Expression

Jinhui Wang,1 Joshua Gorsky,1 Jonathan Peterson,1 Joseph Rabinowitz.1 1 Center for Translational Medicine, Department of Pharmocology, Temple University, Philadelphia, PA. We have previously reported that promoters of myocardial specific genes including alpha cardiac actin (a-CARD), Troponin I (Tni), and Troponin T (Tnt) lead to bias AAV mediated gene expression S209