which are responsible for significant morbidity and mortality, remain refractory to treatment. Conjugation of bacterlal Bcgalactosidase to an anti-transferrin receptor antibody has resulted in transport ofthe complex across the blood-brain barrier and as such may be useful for delivering missing enzyme for the correction ofthe CNS manifestations. However, the antibody conjugation procedure can be labor intensive and clinical treatment would require repeated injections ofthe complex. We sought to determine whether we could employ a DNA-based fusion gene strategy to target IDUA to the transferrin receptor, which could be delivered in vivo for continuous enzyme production. In vitro we showed enzymatic activity, proper targeting to the lysosome, and transferrin receptor mediated uptake ofthe fusion protein. In vivo, using the MPS I murine model, we compared the bio-distribution ofenzyme from an IDUA-based plasmid (MonoIDUA) to a transferrin-IDUA fusion gene-based plasmid (Tf-IDUA). The Tf-IDUA fusion protein showed a broader distribution to many organs than that of native enzyme. Specifically, the heart and brain ofMPS I animals showed enhanced uptake ofTf-IDUA (p<.001 for brain and p$.O I for heart), After short-term (3 week) treatment of adult MPS I mice with Tf-IDUA we noted pathology reduction in peripheral tissues (liver, spleen , kidney, etc) and substantial clearing ofglycosaminoglycans at refractory sites such as the meninges and choroid plexus. Some Purkinje cells of the cerebellum were also cleared of storage material in Tf-IDUA treated mice while MonoIDUA treated animals showed no correction ofCNS pathology. Our results arc the first to provide evidence that a transferrin receptor targeted IDUA fusion protein can access refractory sites and may represent an alternative to conventional therapies.
96. Insulin-Like Growth Factor-1 Rescues Galactocerebrosidase-Mediated Toxicity in Hematopoietic Stem Cells: Relevance for Gene Therapy of Globoid Cell Leukodystrophy Ilaria Vlsigalll,' ? Martina Cesani.l -' Margherita Neri ,l.2 Laura Tononi,' Silvia Ungari,' Sergio Marchcsini.t Angela Gritti,' Luigi Naldini.P [San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; lSan Raffaele University, Vita-Salute San Raffaele University, Milan, Italy; J Experimental Neurology Institute , San Raffaele Scientific Institute, Milan, Italy; ~ Biomedical Science and Biotechnology, University ofBrescia, Brescia, Italy. Globoid cell leukodystrophy (GLD) is a lysosomal storage disorder due to the deficiency of the enzyme Galactocerebrosidase (GALC). The defect results in intracellular storage of undegraded metabolites in central and peripheral nervous systems, leading to progressive demyelination. We are testing the efficacy of a gene therapy strategy based on hematopoietic stem and progenitor cells (HSPC) and lentiviral vectors (LV) in the murine models ofthe disease. We recently demonstrated the occurrence of an acute toxicity upon LV-mediated GALC over-expression in murine (m) and human HSPC. GALC.LV transduced HSPC showed a significant impairment in proliferation and dilTerentiation and, most importantly, failed to rescue transplanted recipients from lethal conditioning. This functional impairment was associated to apoptosis of GALC over-expressing HSPC. Interestingly, more differentiated cells, such as microglia, showed only minor signs oftoxicity upon transduction , indicating a lower sensitivity to GALC over-expression. In order to overcome this major limitation and improve the efficacy of HSPC gene therapy for GLD, we are evaluating whether the anti-apoptotic molecule Insulin-like Growth Factor I (IGFI) could exert a protective elTecton GALC-transduced HSPC. Several studies have implicated IGFI in preventing cell death due to the activation of the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway in a number of cell types, including hematopoietic cells. Further, the S38
primary product of GALC-dependent metabolism, ceramide, acts as strong pro-apoptotic stimulus and inhibits the phosphorylation of Akt. Intriguingly, we have evidence of ceram ide accumulation in GALC.LV transduced mHSPC short term after gene transfer. Remarkably, short-term in vitro treatment with IGFI rescued GALC.LV transduced mHSPC from apoptosis, and from the proliferation and dilTerentiation impairment due to GALC over-expression, without affecting enzymatic activity. Importantly, in preliminary in vivo studies IGFI -treated, GALC -transduced mHSPC rescued transplanted mice from lethal conditioning. Evaluation ofthe therapeutic efficacy of this strategy in murine models ofGLD is currently on going.
97. Long-Term Somatic Correction in MPS II Mice Using a Self-Complimentary AAV2 Vector Joseph Mucnzcr,' Lu Kang,' Haiyan Fu,2 Douglas M. Mcf.arty.! [Department ofPediatric, Division ofGenetics and Metabolism, University ofNorth Carolina at Chapel Hill, Chapel Hill, NC; 'Pediatrics. Centerfor Gene Therapy, Columbus Children s Research Institute, Ohio State University, Columbus, OH. Mucopolysaccharidosis II (MPS II) is an X-linked lysosomal storage disorder due to the deficiency of the enzyme, iduronate sulfatase (Id-S). In the severe form ofMPS II, the deficiency of IdS leads to the intralysosomal accumulation ofglycosaminoglycans in the central nervous system (CNS) and somatic tissues/organs. No treatment is available for the CNS disease in MPS II patients , but recently recombinant enzyme replacement therapy has become available to treat the somatic disease. AAV gene therapy is a promising treatment for MPS disorders. Traditional AAV vectors deliver a single-stranded DNA genome (ssAAV), which must be converted by host-cell-mediated DNA synthesis to double-stranded DNA for active expression. The self-complementary AAV (scAAV) vector described by McCarty et al (Gene Therapy (2003) 10: 2112-2118) allows more efficient expression by delivering a duplex genome and bypassing second strand DNA synthesis which may be the rate limiting step in AAV transduction. In this study, scAAV2 vector expressing human Id-S (U Ia promoter) was compared to traditional ssAAV expressing human Id-S (CMV promoter) in MPS II mice. The scAAV2 and ssAAV2 vectors were administered to the adult MPS II mice intravenously (Sx l O!' viral particles) and intracisternally (5x IOIU viral particles) after pretreatment with mannitol (1-2mglgm body weight). Four scAAV treated MPS II mice were sacrificed at 20-22 months ofage when they developed neurological symptoms, such as bladder distension, weight loss and gait abnormalities. Our result demonstrated that complete correction ofglycosaminoglycan (GAG) storage in multiple tissues were observed in the scAAV treatment group , including liver, spleen , kidney, heart, lung, intestine, and muscle, compared to the nontreated mice (p<0.05). The ssAAV2 treated MPS II mice group shows complete GAG correction in liver, partial GAG correction in spleen , heart, lung, intestine and muscle and no correction in the kidney. Id-S enzyme activity was higher than normal levels in liver and spleen in the scAAV2treated group, and about 10-100% of the normal activity in the kidney. In contrast , Id-S enzyme activity can only be detected in the livers, but not in the spleens or kidneys ofthe ssAAV2 treatment group . These results suggest scAAV2 mediated gene delivery results in significant wider distribution ofId-S expression compared to ssAAV. These results suggest that scAAV2 vector is a promising candidate for treating the somatic disease in MPS II.
Molecular Therapy Volume 15.Supplement I, ~b y 2007 Co pyr ight © "1111: American S ociety o f Gen e Th erapy