169. Developing Engineered Zinc Finger Repressors of Huntingtin as a Potential Therapy for Huntington's Disease

NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY I and different amounts of the complex injected into the subretinal space of Balb/c mice. Luciferase expression was quantified up to 15 weeks post-injection. Once the optimal amount of plasmid was determined, PEGPOD was complexed with a GDNF-expressing plasmid and injected into the subretinal space of Balb/c mice. Mice were subjected to 450-nm blue light at 14, 30, and 70 days postinjection. Electroretinograms (ERG) and thickness of outer nuclear layer (ONL) were measured 7 days post-light treatment. Results: Reduced dose of the PEGPOD/DNA complex resulted in expression of the luciferase up to 10 weeks post-injection. When PEGPOD was complexed with GDNF (PEGPOD/GDNF) and injected 30 days prior to light treatment, significant functional and anatomical rescue was observed. A- and B- wave amplitudes of mice injected 30 days prior to light treatment were increased by 77% and 65%, respectively, relative to control-injected mice. While functional rescue of mice injected with PEGPOD/GDNF 70 days prior to light treatment was not significant, ONL thickness of eyes injected with PEGPOD/ GDNF was significantly increased by 25% relative to control-injected eyes. Conclusions: PEGPOD/GDNF complexes show biologically significant expression in retinas of a mouse model of light-induced degeneration up to 70 days post-injection. This study illustrates the potential of PEGPOD/GDNF to mediate long-term rescue of retinal diseases such as RP and AMD, and asserts the need for consideration of this nonviral gene transfer system as a clinically viable approach to treatment of these devastating diseases.

169. Developing Engineered Zinc Finger Repressors of Huntingtin as a Potential Therapy for Huntington’s Disease

H. Steve Zhang,1 David E. Paschon,1 Josee Laganiere,1 Bryan Zeitler,1 Matthew Mendel,1 Jennifer Cherone,1 Qi Yu,1 Jocelynn Pearl,1 Joe Vu,1 Yolanda Santiago,1 Jeffrey C. Miller,1 Edward J. Rebar,1 Lei Zhang,1 Philip D. Gregory.1 1 Sangamo BioSciences Inc., Richmond, CA. Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG-trinucleotide repeat expansion in the first exon of the Huntingtin (Htt) gene. Repeat lengths of 35 or fewer CAGs are normal and have no associated pathophysiology, while those of 40 or more CAGs lead to HD with 100% penetrance, with longer repeats lengths correlating with earlier disease onset. The degeneration process primarily affects the basal ganglia and cerebral cortex, and the disease is characterized by a progressively worsening chorea, as well as cognitive and psychiatric dysfunctions. While neither the precise function of wild-type Htt nor the mechanism by which mutant Htt (which contains an expanded polyglutamine stretch)in HD pathogenesis if fully understood, results from studying rodent models of HD demonstrate that reducing both wild-type and mutant Htt levels, via strategies such as RNAi or anti-sense oligonucleotides, can prevent or delay disease onset. Moreover, simultaneous partial down-regulation of both wild-type and the expanded Htt allele (∼50%) appears to be tolerated in rodent as well as non-human primates. On the other hand, the complete loss of wild-type Htt expression is embryonically lethal in mice and numerous studies have identified important functions of wild-type Htt in various cellular processes. Thus, strategies that selectively reduce the expression of mutant and disease causing form of Htt represent the ideal therapeutic approach. Engineered zinc-finger protein transcription factors (ZFP-TFs), which can be designed to target virtually any gene, offer an attractive approach to the regulation of Htt expression. To this end, we designed a panel of ZFP transcriptional repressors to bind the proximal promoter of Htt (which would regulate both wild type and mutant Htt alleles). We show that such pan-allele specific ZFP transcription factors were able to achieve 40-75% down-regulation of Htt mRNA in transiently transfected HEK293 and Neuro2A cells, which was confirmed at the Htt protein levels. S68

To generate reagents capable of selectively repressing the mutant Htt allele – we designed ZFP DNA binding domains to selectively bind to the expanded CAG repeat, and investigated whether alleles carrying differing numbers of CAGs could be differentially regulated. We show that ZFP repressors can be engineered such that they exhibit minimal repression of Htt alleles that contain normal CAG repeat lengths but greater than 50% repression of Ht alleles with expanded CAG repeat lengths. These results demonstrate that both non-allele-specific and allele-specific repression of Htt can be achieved with engineered ZFP repressors. In vivo testing in HD models is therefore warranted to determine as well as to compare the efficacy and safety of these two approaches to the potential treatment of HD.

170. Improved Neuroplasticity and Decreased Apoptosis by Intralesional Transplantation of Mesenchymal Stem Cells after Severe Spinal Cord Injury in Rats

Fátima M.C. Caldeira,1 Fabíola B. Fukushima,2 Mário S. L. Lavor,2 Carla M.O. Silva,2 Isabel R. Rosado,2 Paula M. Costa,2 Karen M. Oliveira,2 Jankerle N. Boleoni,2 Rogéria Serakides,2 Alfredo M. Góes,3 Eliane G. Melo.2 1 Laboratório de Tecnologias para Terapia Gênica, FAV, Universidade de Brasília, Brasília, DF, Brazil; 2Departamento de Clínica e Cirurgia Veterinárias, EV, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; 3Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. The present study aimed at evaluating the effect of mesenchymal stem cells (MSC), from bone marrow, in rats with spinal cord injury (SCI). Thus, 61 adult male Lewis rats were distributed in 3 groups: sham (CN), placebo (PLA) and MSC (GCT). All animals underwent laminectomy of T12, whereas the animals in PLA and GGT underwent SCI performed with a 70g rod for 5 minutes. After 14 days, the surgical site was exposed and for PLA and GCT, 2 μL PBS and 1.5 x 105 MSC diluted in 2 μL of PBS were administered at the injury epicenter, respectively. The experimental design was completely randomized. Neurological evaluation consisted of the Basso, Beatie, Bresnahan (BBB) test and a descriptive scale adapted method, performed every 4 days for 45 consecutive days. In the second, third and fifth weeks after inoculation, rats were euthanized, having their spinal cord collected for gene expression evaluation of BDNF, NT-3, Bcl-xL, Bax, and caspase-9 by RT-PCR. In the fifth week, the spinal cord was also assessed by immunohistochemical methods, using anti-NeuN, anti-eGFP, anti-GFAP, and anti-synaptophysin antibodies, as well as by the TUNEL technique. Neurological deficits were observed in the animals with injury, with difference between the medians of groups PLA and GCT (p <0.05). There was no difference on neurons identified by NeuN antibody (p> 0.05). The higher synaptophysin immunostaining intensity and the lower formation of apoptotic bodies for GCT was similar to CN (p> 0.05). PLA group showed a higher expression of Bax and caspase-9 (p <0.05). Thus, we conclude that the MSC preserved synaptophysin and propitiously affected apoptosis.

171. Repair of Spinal Cord Injury (SCI) Using Monocytes Transfected with Adenoviral Vector Expressing Decorin in a Rat SCI Model

Alireza Biglari,1 Reza Salarinia,1 Saeideh Mazloomzadeh,1 Iraj Jafari,1 Parisa Ranjzad,2 Paul A. Kingston.2 1 Department of Molecular Medicine & Genetics, Zanjan University of Medical Sciences, Zanjan, Islamic Republic of Iran; 2Vascular Gene Therapy Unit, Core Technology Facility, The University of Manchester, Manchester, United Kingdom. Background: Subsequent to spinal cord injury many pathological changes may occur that lead to inappropriate environment for Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy