771. Subretinal Delivery of Recombinant AAV Serotype 8 Vector in Dogs Results in Gene Transfer to Neurons in the Brain

Development of AAV Vectors have detected a dose-response increase (up to 20-fold) in AAV1 transduction of 293 cells, that express GLUT-2, by adding STZ to the medium. Surprisingly, we found that IV injection of AAV1 vectors into a STZ-treated dog resulted in 65% of liver transduction, compared to a 5% observed in non-treated dogs. Together these results extend the range of AAV serotypes that can be efficiently used for hepatic directed gene transfer to AAV1, a serotype previously described as poorly performing in liver. This may also help decrease vector dose and vector promiscuity. Access to closed compartments of organs, like the bile duct, results in improved gene transfer and may help reducing systemic exposure to gene therapy vectors thus improving safety of hepatic gene transfer. The use of alkylating agents like STZ, a chemiotherapeutic agent used in humans, should be carefully monitored in the context of gene transfer.

771. Subretinal Delivery of Recombinant AAV Serotype 8 Vector in Dogs Results in Gene Transfer to Neurons in the Brain

Knut Stieger,1 Marie-Anne Colle,2 Laurence Dubreil,2 Alexandra Mendes-Madeira,1 Michel Weber,3 Guylene Le Meur,3 JackYves Deschamps,4 Nathalie Provost,1 Delphine Nivard,1 Philippe Moullier,1 Fabienne Rolling.1 1 INSERM U649, CHU Hotel-Dieu, Nantes, France; 2INRA UMR 703, Ecole Nationale Veterinaire de Nantes, Nantes, France; 3 Service d’Ophthalmologie, CHU Hotel-Dieu, Nantes, France; 4 Service d’Urgences, Ecole Nationale Veterinaire de Nantes, Nantes, France. Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intra-ocular delivery in rats and dogs. Subretinal delivery of a AAV2/8.gfp vector results in efficient gene transfer in the RPE, the photoreceptors, and surprisingly in the cells of the inner nuclear layer and in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because GFP was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices by PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

772. Direct Spinal Cord Injection of AAV2Mediated Insulin-Like Growth Factor 1 or Vascular Endothelial Growth Factor in a Rat Model of ALS

Colin K. Franz,1 Christina Krudy,1 Thais Federici,1 Qingshan Teng,1 Yang Jun,2 Boulis M. Nicholas.1 1 Neurosurgery, Emory University, Atlanta, GA; 2Neurosurgery, Cleveland Clinic, Cleveland, OH.

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a neurodegenerative condition characterized by a progressive loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord. As motor neurons are lost, a person with ALS increasingly loses the ability to control their muscles, which eventually leads to paralysis and death. There are presently no good treatments for ALS, and to make matters worse the causes of ALS remain poorly understood. However, it is known that approximately 2% of all ALS cases are a familial form of the disease arising from mutations in the copper-zinc superoxide dismutase-1 (SOD1) gene. Therefore, a transgenic model of ALS was originally generated in mice, and more recently became available in rats, in which the expression of an abnormal form of the human SOD1 gene leads to motor neuron loss, paralysis and death. Because of their similarities to human ALS, these animal models represent powerful tools to screen and test new therapies for this disease. For example, the delivery of neurotrophic factors to motor neurons has been shown Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

to increase their survival both in vitro and in vivo. To date, insulinlike growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) are the neurotrophic factors that have shown the greatest efficacy in preserving neuromuscular function and preventing motor neuron loss in SOD1 animals. The biggest limitation to potential clinical translation of neurotrophic factors in ALS is the challenge of achieving sustained and targeted delivery to target tissues. Gene transfer of IGF-1 and/or VEGF using adeno-associated virus serotype 2 (AAV2) may offer a means to safely provide long-term, localized delivery. Our approach has been to use direct spinal cord injections of AAV2.IGF-1 or AAV2.VEGF in order to achieve the highest rate of motor neuron transduction as well as to avoid the problematic issues surrounding remote gene delivery strategies (e.g. intramuscular) in larger animals and humans. Using this approach, we have clearly shown that AAV2 can induce robust gene expression, which is predominately confined to neurons in the spinal cord. To our surprise, when AAV2.IGF-1 was delivered by this method to 80 day old SOD1 rats we found no evidence of motor neuron protection, no improvements in motor behavior, and no delays in disease onset or death. All comparisons were made against SOD1 littermates that simultaneously received either AAV2.GFP or PBS control injections. Interestingly, when we re-examined the effects of AAV2. IGF-1 injection for only the male sub-group we found a small yet significant preservation of grip strength after disease onset, but this benefit occurred without any preservation of end-state motor neuron numbers or extension of lifespan. Currently underway are a set of therapeutic experiments where AAV2.VEGF is being injected into the spinal cords of a male only population of 70 day old SOD1 rats with the results to be presented.

773. AAV Vector Expressing Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Protects Dopaminergic Neurons Against Methamphetamine-Induced Toxicity In Vitro

Matthew J. Chiocco,1 Doug B. Howard,1 Kathleen Powers,1 Mart Saarma,2 Brandon K. Harvey.1 1 National Institute on Drug Abuse, Baltimore, MD; 2University of Helsinki, Helsinki, Finland. The dopaminergic neurons of the substantia nigra progressively degenerate in Parkinson’s disease and are the focus of many genebased therapeutic strategies. Studies on dopaminergic neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and neurturin, have led to protein and gene therapy-based clinical trials. Recently, a new family of evolutionarily conserved proteins with neurotrophic effects on dopaminergic neurons has been described and includes the mesencephalic astrocyte-derived neurotrophic factor (MANF). The MANF family of proteins exhibit protective and regenerative effects on dopaminergic neurons in vitro or in animal models of Parkinson’s disease. To further study the neuroprotective effects of MANF against dopaminergic toxicity, we cloned the human MANF cDNA into a self-complementing or double stranded (ds) AAV vector. Western blotting confirmed that the dsAAV-MANF packaging plasmid produced MANF protein, and immunostaining of neuronal cultures treated with dsAAV-MANF confirmed viral transduction and MANF protein production. To test the neurotrophic effects of dsAAVMANF on dopaminergic neurons, primary neuronal cultures derived from E15 rat embryonic ventral mesencephalon were transduced on day 6 in vitro (DIV6). The cultures were challenged with 1 mM methamphetamine on DIV9 and fixed on DIV11. Cultures were immunostained for the dopaminergic marker, tyrosine hyroxylase (TH). Methamphetamine decreased TH+ cells by 80% and dsAAVMANF significantly attenuated the loss of TH+ cells to 45%. Using this model of degeneration we may further understand the mechanisms by which MANF is neuroprotective and evaluate its full potential as a therapy for degeneration of dopaminergic neurons. S289