907. Gene Therapy for Genetic Lipoprotein Lipase (LPL) Deficiency; an Update

INBORN ERRORS OF METABOLISM: LIVER AND PANCREATIC DISEASES relatively high intrahepatic pressures achieved, there was minimal evidence of liver injury; no histological abnormalities were observed and only minimal and transient elevation of AST and ALT were noted. The procedure itself was uneventful and very well tolerated with no clinical manifestations of acute or chronic toxicities. This novel method of delivering HDAd is simple, minimally invasive, and clinically relevant since it increases the therapeutic index of HDAd for liver-directed gene therapy by allowing high efficiency hepatic transduction with low vector doses and with negligible acute or chronic toxicities.

907. Gene Therapy for Genetic Lipoprotein Lipase (LPL) Deficiency; an Update Jaap Rip,1 Melchior C. Nierman,1 Jeroen A. Sierts,1 Colin J. Ross,2 Michael R. Hayden,2 Albertine de Jong,3 Paul Dijkhuizen,3 Wim Hermens,3 Jaap Twisk,3 John J. Kastelein,1 Jan Albert Kuivenhoven,1 Janneke M. Meulenberg.3 1 Department of Vascular Medicine, Academic Medical Centre, Amsterdam, Netherlands; 2Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC, Canada; 3Amsterdam Molecular Therapeutics, Amsterdam, Netherlands. Lipoprotein lipase (LPL) deficiency cannot be effectively treated and causes severe hypertriglyceridemia and pancreatitis. We have reported that adeno-associated virus serotype 1 (AAV1) based gene therapy normalizes triglyceride levels in LPL deficient mice and cats. The current study evaluates safety and biodistribution of the same vector, AAV1-LPLS447X, and addresses clinical application. Toxicological analysis after intramuscular administration of AAV1LPLS447X (1E11-1E13 gc/kg) in murine dose-escalation studies did not reveal clinical or hematological changes. Shortly after administration (7 days), vector DNA was found in injected muscle, draining lymph nodes, and filtering organs while spread to reproductive organs was limited. 28 and 90 days post injection, vector DNA levels dissipated in all tissues except for injected muscle and lymph nodes. Furthermore, we studied 6 LPL deficient patients to assess eligibility for a first clinical trial. All presented with absence of LPL catalytic activity but LPL protein levels were 19-103% of normal. Infection of patient’s myoblasts with AAV1-LPLS447X resulted in the secretion of catalytically active LPL in all cases. The efficacy and safety data obtained so far support the initiation of a clinical trial to test this vector in LPL deficient patients.

908. Evaluation of the Sleeping Beauty Transposon System in OTC Deficient Spf-Ash Mice: A Non-Viral Approach to a Cell Autonomous Metabolic Disease Andy Wilber,1 Joel Frandsen,1 Hiroki Morizono,2 Mendel Tuchman,2 R. Scott McIvor.1 1 GCD, University of Minnesota, Minneapolis, MN; 2Children’s National Medical Center, George Washington University, Washington, DC. In humans, ornithine transcarbamylase (OTC) deficiency is the most common error of the urea cycle. The limited potential of medical procedures for life-long correction suggests liver-directed gene therapy as an alternative. Some 15 years of research using viral vectors in OTC deficient mice generated 1 report of long-term correction using helper-dependent adenovirus (Ad) to express a stabilized human OTC gene. However, the failure of a recombinant Ad clinical trial argues that non-viral vectors may be the more appropriate choice for molecular therapy of this disorder. The Sleeping Beauty (SB) transposon has been demonstrated to mediate insertion and

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

expression of secreted or advantageous gene products in hepatocytes. Here, we present the first application of a plasmid system for therapy of a cell autonomous disorder. Localization of OTC enzyme to the mitochondria suggests a potential limitation with respect to the gene transfer efficiency that can be achieved by non-viral vectors. To address this issue, we studied the delivery of cis-acting transposons by evaluating the effect of DNA dose on stable gene expression using in vivo bioluminescent imaging (IVI) to monitor luciferase (luc) activity after infusion of a CAGS-regulated luc transposon with a ubiquitinregulated SB transposase (pT2/CL//UbSB) into C57BL/6 mice. Dose response experiments comparing 2, 20, and 200 µg of pT2/CL// UbSB showed the highest levels of persistent luc activity resulting from the 2 µg dose. We conducted studies using transposons encoding fluorescent proteins and luc on the same transcript, in cis with a ubiquitinregulated transposase (pKT2/CFIL//UbSb) to correlate the number of fluorescent cells in liver sections or hepatocyte suspensions with transient and stable luc activity measured by IVI. Stable in vivo luc activity of 7x107 photons/sec was observed following a single injection of (pKT2/CFIL//UbSB) and defined transient expression to 1 in 10 cells and stable expression to 1 in 200 cells. Finally, OTC deficient mice were infused with bifunctional transposons with the mouse OTC cDNA (pKT2/COIL//UbSB), monitoring in vivo luc activity and urinary orotic acid levels as a measure of OTC expression. OTC deficient mice infused with pKT2/ COIL//UbSB mimicked luc levels achieved in mice administered fluorescent protein/luc transposons, indicating similar levels of transcription. OTC activity assayed in liver extracts from deficient mice infused with OTC/luc transposons was on average 100% higher after 2 days compared to uninjected controls, verifying presence of the OTC enzyme. Additional mice (n=5) infused with OTC/luc transposon +/- transposase exhibited maintenance of in vivo luc activity in animals receiving the transposase. These results suggest a correlation between in vivo luc activity and the number of genetically modified hepatocytes. In addition, these results are encouraging with respect to anticipated use of the SB transposon for treatment of diseases that do not benefit from a secreted or selective gene product, thus encompassing a wide range of genetic disorders. R. Scott McIvor has a financial interest in Discovery Genomics, Incorporated.

909. Sustained Correction of Glycogen Storage Disease Type II by rAAV1 Vector-Mediated Gene Therapy Kerry O. Cresawn,1 Cathryn S. Mah,1,2 Thomas J. Fraites, Jr.,1 Melissa A. Lewis,1 Irene Zolotukhin,2 Barry J. Byrne.1,2 1 Pediatrics and Powell Gene Therapy Center, University of Florida, Gainesville, FL; 2Cellular and Molecular Therapy, University of Florida, Gainesville, FL. Glycogen storage disease type II (GSDII), is a lysosomal storage disease caused by a partial to complete deficiency in the lysosomal hydrolase, acid α-glucosidase (GAA). From gestation, affected individuals store glycogen in their lysosomal compartments, resulting in severe hypertrophic cardiomyopathy and respiratory insufficiency. Previous studies with intrahepatic and intramuscular delivery of recombinant adeno-associated viral (rAAV) vectors to GSDII mice resulted in a variable anti-GAA immune response and insufficient levels of circulating protein in immune-responsive mice. In this study, we evaluated the potential for intravenously delivered rAAV vectors to correct the biochemical, histological and functional phenotype in GSDII mice over a sustained period of time. Neonatal GSDII mice were intravenously administered 5 x 1010 particles of rAAV serotype 1 vector encoding human GAA under control of the S351