887. Protection from HIV-Gp120-Mediated Protein Modifications in Neurons Using rSV40 Vectors Delivering Antioxidant Enzymes

Neurologic - Genetic and Peripheral Nerve after injection showed less than a two fold difference in vector DNA level whereas the RNA level decreased by ~10 fold. These data suggest that the reduction in FVIII expression was primarily due to transcriptional silencing. Recent work by our group suggests this effect may be transgene specific (see abstract by Wang et al.). The use of a different promoter, delivery system, or increasing the number of readministrations could prolong the period of correction. We are presently testing PTMs in hemophilia A mice using lentiviral delivery. Liver toxicity was assessed by analyzing ALT, AST and AP levels at several time points (2 days to 12 weeks). All three proteins in prebleed plasma were in the normal range. At 2 days after injection there was a transient increase in ALT and AST but levels returned to normal by week 1. AP levels remained in the normal range at all time points. These studies demonstrate that 1) SMaRT™ can be used to correct the coagulation deficit in hemophilia A mice by trans-splicing to highly expressed albumin pre-mRNA, 2) readministration of minicircle PTMs is possible to sustain expression, in the absence of FVIII inhibitor formation, and 3) the delivery procedure, minicircles and trans-splicing to endogenous albumin do not cause long term liver toxicity in mice.

Neurologic – Genetic and Peripheral Nerve 886. AAV-Mediated Aipl1 Gene Replacement in a Mouse Model of Leber Congenital Amaurosis

Claudio Mussolino,1 Simona Neglia,1 Chiara Abrescia,1 Enrico M. Surace.1 1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy.

Leber Congenital Amaurosis (LCA) is a severe retinal degenerative disease associated with mutations in genes preferentially expressed in photoreceptors. LCA is genetically heterogeneous and it is characterized by an early poor vision or complete blindness from birth. Twelve different genes have been associated to LCA. The Aipl1 gene accounts for ~ 8% of all cases and 25 different mutations have been described to date. This study is aimed at correcting the retinal genetic defect of the Aipl1 knock-out (Aipl1-/-) mouse model of LCA with a gene replacement approach. To this end, we generated Adenoassociated viral vectors (AAV) serotype 5 containing the human or murine Aipl1 gene driven by human Rhodopsin (hRho) promoter. The 2 vectors were administrated in two separate groups of Aipl1-/- mice (n=9/group) with a single subretinal injection at post-natal day 4 (P4) before the onset of the retinal degeneration. Histological analysis of treated Aipl1-/- retinae revealed a significant preservation of the outer nuclear layer in the area transduced by the AAV2/5 containing the human AIPL1 transgene and the AIPL1 protein was correctly expressed in photoreceptors as assessed by immunofluorescence analysis. By contrast, with the murine AIPL1 transgene morphological rescue was not observed. Despite the protection of photoreceptors from death and the presence of the protein in the correct location, electroretinograms (ERG) performed one month after injection (P30) showed no improvement in retinal functions in all treated animals. Given the severity of the LCA photoreceptor disease and the difficulties of an early intervention, to delay the photoreceptor loss we co-injected AAV2/1 vector containing the Glial cell-line Derived Neurotrophic Factor (GDNF) with AAV2/5 harboring the human AIPL1 transgene. No functional retinal improvement was measured by ERG analysis despite a significant recovery of retinal structure. We recently demonstrated that AAV serotype 8 with the human Rhodopsin promoter provides the highest levels of photoreceptor transduction and transgene expression in vitro and in vivo. Since severe photoreceptor diseases may require faster onset and higher levels of gene expression, we have generated AAV2/8-hRho-hAipl1 vectors to deliver the therapeutic gene to mutant retinae. We are Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

currently evaluating morphologically and functionally the impact of this novel vector serotype to treat Aipl1-/- mice retinae.

887. Protection from HIV-Gp120-Mediated Protein Modifications in Neurons Using rSV40 Vectors Delivering Antioxidant Enzymes

Lokesh Agrawal,1 Jean-Pierre Louboutin,1 Beverly A. S. Reyes,2 Elisabeth Van Bockstaele,2 David S. Strayer.1 1 Department of Pathology, Thomas Jefferson University, Philadelphia, PA; 2Neurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA. HIV-1 infection in brain is associated with cognitive impairment and with neuronal apoptosis in the hippocampus, basal ganglia and cerebral cortex. Neuron loss leads to varying degrees of clinical impairment, ranging from mild motor and sensory neurological deficits to severe dementia. HIV-1-associated dementia (HAD) is thought to involve neuron damage mediated in part by HIV-1 gene products, which cause oxidation, nitration of neuronal proteins and cytotoxic levels of lipid peroxidation. Among the key HIV-1 gene products implicated in HIV encephalopathy is the envelope (Env) glycoprotein, gp120. We have created a model for HIV-1 Ba-L gp120-induced apoptosis both in vitro and in vivo. We studied the parameters of that system with regard to neuron apoptosis, protein oxidation, nitration and lipid peroxidation. We compared primary neuron cultures or rat brains exposed to recombinant gp120 alone, and studied the effectiveness of anti-oxidant gene delivery using rSV40-delivered antioxidant enzymes Cu/Zn superoxide dismutase SV(SOD1) or glutathione peroxidase SV(GPx1). In vitro studies were performed with cultured primary human neurons. These cells were transduced with SV(SOD1) or SV(GPx1), or mock-transduced, then exposed to HIV-1 Ba-L gp120. In vivo studies were performed using Sprague-Dawley rats, given vectors and gp120 in sequence by stereotaxic injection into the caudate-putamen. Treatment with gp120 both in vitro and in vivo led to increased protein oxidation and nitration when compared to control cultures or to control rats injected with saline. Unlike nitration, which was limited to a single protein, mol size of 65 kDa, oxidation was found to be associated with several neuronal proteins with prominent bands observed between 25-40 kDa including neuromodulin, alpha-internexin and neurofilament protein. Gp120-treated neurons also showed 2-3 fold greater malonaldehyde (MDA, 3-4 µM) levels, a marker for lipid peroxidation when compared with control cultures or saline injected rats. Treatment with SV(SOD1) or SV(GPx1) prior to challenge with gp120 led to significant reduction in oxidation and nitration of neuronal proteins. Interestingly, transduction with SV(GPx1), but not SV(SOD1), led to significantly less MDA release as compared to controls after challenge with gp120. Therefore, antioxidant gene delivery strongly protects from the types of oxidative modifications to proteins and lipids that occur as a consequence of exposure to HIV-1 gp120. Understanding the role of chronic production of gp120 in mediating apoptosis, protein oxidation and lipid peroxidation with subsequent protection using SV(SOD1) or SV(GPx1) may help develop new therapeutic strategies for dementia associated with HIV.

888. IGF-1 Rescues Motor Neuron Toxicity in an In Vitro Stem Cell-Based Model of ALS

Amanda M. Haidet,1 Mark Hester,2 Brian K. Kaspar.1,2,3 Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, OH; 2Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 3Department of Pediatrics, The Ohio State University, Columbus, OH.

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Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder mediated by the death of motor neurons leading to paralysis S331