545 Regulation of AAV Mediated Transgene Expression in Airway Epithelium In Vivo Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S209 GENE R[.]
GENE REGULATION II AAV2/5-mediated EGFP expression to the RPE, while the inclusion of miR204Ts restricted transgene expression to PRs, as expected In addition, the use of miR204Ts efficiently restricted the AAV2/8mediated expression of AIPL1- a gene mutated in Leber congenital amaurosis, a common form of childhood inherited blindness- to porcine PRs In conclusion, we show that miRNA-based regulation of transgene expression can be applied in the retina to restrict the robust expression levels obtained using ubiquitous promoters to a specific cell type Alternatively, it can be coupled with cell-specific promoters thus providing an additional layer of fine-tuning transgene expression 543 Creation of Structural Variations in the Human Genome Using Engineered Zinc Finger Nucleases Eunji Kim,1 Hyung Joo Lee,1 Jin-Soo Kim.1 Department of Chemistry, Seoul National University, Seoul, Korea Many genetic diseases and cancer are associated with chromosomal rearrangements such as deletions, insertions, duplications, translocations, and inversions Even among healthy individuals, thousands of different structural variations (SVs) or copy number variations resulting from different chromosomal rearrangements are observed Despite the recent discoveries of and interest in numerous structural variations (SVs) in the human and other higher eukaryotic genomes, little is known about the etiology and biology of these SVs, partly due to the lack of molecular tools with which to create individual SVs in cultured cells and model organisms Here, we present a novel method of inducing targeted genome rearrangements using engineered zinc finger nucleases (ZFNs) We found that ZFNs designed to target two different sites in a human chromosome could introduce two concurrent double-strand breaks, whose repair via non-homologous end-joining (NHEJ) gives rise to targeted deletions, duplications, and inversions of the genomic segments of up to a mega base pair in length between the two sites We also found that ZFNs can induce incorporation of synthetic ODN cassettes into the targeted site and replace the cassettes with the large chromosomal DNA segments High frequencies of ZFN-induced genomic rearrangements, ranging from 0.01% to 12%, allowed us to routinely isolate clonal populations of cells whose genomes contain SVs of interest We propose that ZFNs can be employed as molecular tools to study mechanisms of chromosomal rearrangements and to create SVs in a predetermined manner so as to study their biological roles In addition, our method opens up new possibilities for correcting genetic defects caused by chromosomal rearrangements and, thus, holds promise in gene and cell therapy 544 VEGF-A Zinc Finger Activator Is Neuroprotective in Rodent Preclinical Models of Stroke S Kaye Spratt,1 Philippe M D’Onofrio,2 Mark M Magharious,2,3 Mahinthan Thayapararajah,2 Richard Surosky,1 Gary Lee,1 Martin Giedlin,1 Dale Ando,1 Michael Fehlings,3 Paulo D Koeberle.2,3 Development, Sangamo BioSciences Inc, Richmond, CA; 2Dept Surgery, University of Toronto, Toronto, ON, Canada; 3Department of Genetics and Development, Toronto Western Research Institute, and Spinal Program, Krembil Neuroscience Centre, University Health Network, Toronto, ON, Canada Vascular endothelial growth factor (VEGF) is most widely recognized for its role in angiogenesis; however recent studies have identified anti-apoptotic and neuroprotective functions for VEGF in the CNS VEGF therapy for CNS injury has been hampered by the tendency of VEGF to promote vascular permeability, edema, and immune infiltration into tissues Recently, engineered zinc finger Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy proteins that upregulate multiple isoforms of VEGF-A (VEGFZFP) in their natural biological ratios have been developed and this approach is promising because VEGF upregulation by ZFPs does not promote vascular permeability (Rebar et al, 2002) Highly reproducible models of optic nerve transection (axotomy), ophthalmic artery ligation and a pial strip traumatic injury model were used to determine if the VEGF-ZFP is neuroprotective in the adult CNS AAV2 vectors encoding VEGF ZFPs (AAV.ZFP) or control vectors encoding GFP (AAV.GFP) were delivered by intraocular injections days prior to retinal injury Retinal ganglion cells (RGCs) were retrogradely labeled with Fluorogold in order to quantify survival from fixed flatmounted retinas AAV.ZFP treatment increased RGC survival by 2-fold (n=6/group, p