117. Long-Term Somatic and CNS Correction in MPS II Mice after Combined Intravenous and Intrathecal Administration of a Self-Complementary AAV2 Vector

117. Long-Term Somatic and CNS Correction in MPS II Mice after Combined Intravenous and Intrathecal Administration of a Self-Complementary AAV2 Vector

Inborn Errors of Metabolism I vector-treated mice. These studies show that rAAV-mediated LCAD delivery ameliorated local and systemic pathologies obse...

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Inborn Errors of Metabolism I vector-treated mice. These studies show that rAAV-mediated LCAD delivery ameliorated local and systemic pathologies observed in partially-deficient LCAD mice.

117. Long-Term Somatic and CNS Correction in MPS II Mice after Combined Intravenous and Intrathecal Administration of a SelfComplementary AAV2 Vector

Joseph Muenzer,1 Lu Kang,1 Douglas D. McCarty,2,3 Haiyan Fu.2,3 Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Center for Gene Therapy, Columbus Children’s Research Institute, Columbus, OH; 3Department of Pediatrics, College of Medicine, Ohio State University, Columbus, OH. 1

Mucopolysaccharidosis II (MPS II) is an X-linked lysosomal storage disorder due to the deficiency of the enzyme, iduronate sulfatase (Id-S). No definite treatment is available for MPS II patients. AAV gene therapy is a promising treatment for MPS disorders. Traditional AAV vectors contain a single-stranded DNA genome (ssAAV), which must be converted by host-cell-mediated DNA synthesis to double-stranded DNA for active expression. The newer self-complementary AAV (scAAV) vector allows more efficient expression by delivering a duplex genome and bypasses second strand DNA synthesis which may be the rate limiting step in AAV transduction. In this study, both the ssAAV2 and the scAAV2 vector expressing human Id-S were administered to MPS II mice by two different delivery methods. First, a group of adult MPS II mice (4-6 weeks of age) were injected with scAAV2 vector intravenously (2-4 x 1011 viral particles) (IV) after pretreatment with mannitol (1-2 mg/gm body weight). Secondly, both scAAV2 and ssAAV2 were administered to adult MPS II mice intravenously (5 x 1011 viral particles) and intrathecally (5 x 1010 viral particles) (IV+IT) after pretreatment with mannitol. Treated MPS II mice were sacrificed at 16 to 24 months of age when they developed severe neurological symptoms. Our result demonstrated complete correction of glycosaminoglycan storage in multiple tissues in the scAAV treatment group (IV+IT), including liver, spleen, kidney, heart, lung, intestine, and muscle, compared to the non-treated mice (p<0.05). The ssAAV2 treated MPS II mice group had complete correction in liver, partial correction in spleen, heart, lung, intestine and muscle and no correction in the kidney in both the IV injected animals and the IV+IT injected animals. Id-S enzyme activity was > normal level in liver and spleen of the scAAV2 treated group, and about 10-100% of the normal activity in the kidney. In contrast, Id-S enzyme activity could only be detected in liver, but not in the spleen or kidney of the ssAAV2 treatment group. Decreased CNS lysosomal storage was shown by histopathology in the brain of the IV+IT ssAAV2 and scAAV2 treated animals, but not in the IV ssAAV2 group. We also demonstrated that the lifespan of MPS II mice are significantly improved in all three groups of animals after AAV2 treatment compared with non-treatment group. These results suggested that IV combined with IT injections of AAV2 vector following mannitol pretreatment is a promising approach for treating both somatic and CNS disease in lysosomal storage disorders and scAAV2 mediated gene delivery results in significant wider distribution of Id-S expression compared to ssAAV. These results suggest that scAAV vector is a promising candidate for the treatment of MPS II.

Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

118. Incorporation of a Human Replication Signal and a Superactive Factor IX Variant Stablizes and Enhances Long-Term Functional Activities Following Nonviral Gene Transfer in Hemophilia B Mice

Peiqing Ye,1 Alicia Buechler,1 Dominika Jirovska,1 Chia-Ni Lin,2 Shuwha Lin,2 Steven Chen,1 Carol H. Miao.1 1 Department of Pediatrics, Seattle Childlren’s Hospital Research Institute and University of Washington, Seattle, WA; 2Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan. Improving the efficiency of gene expression cassette in a gene transfer vector can enhance transgene expression levels per vector genome, therefore can facilitate gene transfer protocols to achieve a therapeutic effect for treating genetic diseases, or in the case of highlevel expression, can reduce vector dosages thus reducing toxicity of or immune responses to the vectors. We have previously established a high expressing, liver-specific gene transfer vector, pBS-HCRHPFIXIA, which produced high-levels of factor IX (FIX) in hemophilia B mice following hydrodynamics-based nonviral gene transfer. The vectors were shown to remain in predominantly episomal forms, leading to gradual decrease of transgene expression levels over time due to slow turnover of hepatocytes and degradation of plasmids. We found that incorporation of one or two copies of a human consensus replication sequence into the vector enhanced and stabilized gene expression levels of FIX over long term. However little plasmid replication was detected by DpnI restriction analysis of DNA extracted from the treated mouse livers. At 8 weeks post plasmid treatment, partial hepatectomy was performed and both groups of mice lost >60% of the gene expression levels due to the loss of episomal vectors. Gene expression did not increase however remained stable and persistent for the next 80 days, indicating efficient retention of plasmid vectors but lack of plasmid replication. Next, we inserted a 690 bp Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) into the vector. No significant enhancement in hFIX gene expression or activity was obtained. WPRE sequence did not function additively or synergistically with the intron element to stabilize RNA in our construct. To further improve the efficacy of gene transfer for hemophilia B, we have incorporated a series of hFIX variants for increased functional activity. A FIX variant (FIX-Triple), containing alanine replacements at positions 86, 277, and 338 of FIX-WT, was found to bind factor VIIIa with much higher affinity than FIX-WT. The liver-specific construct containing the FIX-Triple variant (pBS-HCRHPI-FIXTMA) produced ~3 fold higher gene expression and ~10 fold higher total clotting activity than the construct containing the FIX-WT post-naked DNA transfer. Combining the replication signal with the superactive FIX variant in our vector (pBS-HCRHPI-FIXTMA-RepA), persistent, high-level gene expression and complete phenotypic correction in hemophilia B mice was achieved over long periods of time.

119. Long-Term Correction of Hyperphenylalaninemia in a Mouse Model for PKU by Intramuscular Delivery of AAV Expressing PAH with Serotypes 1, 2 or 8

Alex Rebuffat,1 Cary O. Harding,2 Zhaobing Ding,3 Thony Beat.1 1 Department of Pediatrics, Universitiy of Zurich, Zurich, Switzerland; 2Department of Molecular and Medical Genetics and Department of Pediatrics, Oregon Health and Science Universtiy, Portland, OR; 3Current address, Institute of Bioengineering and Nanotechnology, Singapore, Singapore. Phenylketonuria (PKU) is the most frequent inherited disorder of amino acid metabolism. PKU is caused by a severe deficiency of hepatic phenylalanine hydroxylase resulting in accumulation of S45