381. Retrograde Transport of AAV8 Vector to Central Nervous System Via Intramuscular Delivery

NEUROLOGIC & OPHTHALMIC I this phenomenon remain poorly understood. In order to study this phenomenon, we monitored the fate of HSPC upon transplantation using Magnetic Resonance Imaging (MRI) and MR contrast media for cell labeling, and lentiviral vectors (LV) encoding for GFP. By the means of MRI, we observed short-term CNS-specific homing of a fraction of the transplanted HSPC in both irradiated and non-irradiated MLD and GLD mice. These cells preferentially migrated within areas of adult neurogenesis, such as the subventricular zone and the dentate gyrus of the hippocampus, and of tissue damage, like the corpus callosum and the cerebellum. FACS analysis on brain leukocytes confirmed these findings and allowed precise quantification of these CNS migrating cells. Long-term monitoring of GFP-expressing cells by FACS analysis and immunohistochemistry revealed the persistence of the transduced cells (either the transplanted HSPC or their progeny) in the same regions in which we reported their short term homing, at comparable or even increased rate, in non-irradiated animals. These cells proliferate to a minor extent, and express the hematopoietic marker CD45 and the microglia marker Iba1, even if retaining an undifferentiated morphology. When comparing long-term the non-irradiated versus the conditioned and transplanted mice, we found abundant donor cells and reconstitution of mature microglia in the latter, suggesting that in the absence of myeloablation and peripheral engraftment, CNS migrating HSPC are not able to provide full and extensive reconstitution of brain microglia, but remain in an undifferentiated state and within specific brain regions. We will investigate deeply this phenomenon, in particular which are the critical factors afffecting microglia reconstitution.

379. CNS Delivery of Human SMN1 by AdenoAssociated Virus Is Highly Efficacious in a Mouse Model of Spinal Muscular Atrophy Type I

Marco A. Passini,1 Jie Bu,1 Eric M. Roskelley,1 Amy M. Richards,1 S. Pablo Sardi,1 Catherine R. O’Riordan,1 Katherine W. Klinger,1 Lamya S. Shihabuddin,1 Seng H. Cheng.1 1 Genzyme Corporation, Framingham, MA.

Spinal muscular atrophy (SMA) is a neuromuscular disease and the most common autosomal recessive disorder that affects children. SMA is caused by a deficiency of functional SMN protein due to mutations in the survival motor neuron (SMN1) gene. Loss of SMN activity results in motor neuron cell death in the spinal cord leading to weakness of the proximal muscles responsible for crawling, walking, head/neck control and swallowing, and the involuntary muscles that control breathing and coughing. Consequently, patients present with increased tendencies for pneumonia and other pulmonary problems such as restrictive lung disease. An approach to treating this monogenic disorder is to utilize a recombinant adeno-associated virus (AAV) vector to deliver SMN to the CNS. To this end, an AAV vector containing the human SMN1 cDNA (AAV2/8-CBA-hSMN1) was injected at birth into the CNS of an aggressive mouse model of SMA (SMN-/-, hSMN2+/+, SMNdelta7+/+). These injections resulted in hSMN expression throughout the spinal cord. Treated SMA mice contained a higher number of motor neurons compared to untreated, age-matched mutants. The size of myofibers from multiple muscle groups in treated SMA mice approximated those observed in wild type. Furthermore, the neuromuscular junction (NMJ) in treated SMA mice did not contain pathological accumulation of neurofilament protein at the pre-synaptic termini. Treated SMA mice also displayed significant improvements on a battery of behavioral tests suggesting that the NMJ was functional. Importantly, AAV2/8-CBA-hSMN1 significantly increased the median lifespan of SMA mice to 50 days compared to 15 days in untreated mutants. These data demonstrate that CNS-directed, AAV-mediated SMN augmentation is highly efficacious in addressing both the neuronal and muscular pathologies of a mouse model of SMA Type I. S148

380. AAV Gene Therapy with Cholesterol 24-Hydroxylase Reverses Alzheimer Phenotype of APP23 Mouse Model

Eloise Hudry,1 Debby Van Dam,2 Wim Kulik,3 Peter P. De Deyn,2,4 Femke F. Stet,3 Ornella Ahouansou,1 Abdellatif Benraiss,1 Andre Delacourte,5 Pierre Bougneres,6 Patrick Aubourg,1 Nathalie Cartier.1 1 INSERM UMR745/University Rene descartes, Paris, France; 2 Laboratory of Neurochemistry & Behaviour, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium; 3Laboratory of Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands; 4Department of Neurology/ Memory Clinic, Middelheim General Hospital, Antwerp, Belgium; 5 Inserm U837, Lille, France; 6Inserm U561 - University Paris Descartes, Paris, France. The development of late onset Alzheimer disease (AD) is closely connected with cholesterol metabolism. Cholesterol increases the production and deposition of amyloid-β (Aβ) peptides, a hallmark of the pathology, and is abnormally retained in AD neurons. In the brain, cholesterol is synthesized in situ but cannot be degraded nor cross the blood-brain-barrier. The major exportable form of brain cholesterol is 24S-hydroxycholesterol, an oxysterol generated by the neuronal cholesterol 24-hydroxylase encoded by the CYP46A1 gene. To determine if CYP46A1 gene overexpression in the brain of APP23 mice affects the processing of APP and the production of amyloidogenic Aß peptides, we have injected stereotactically AAV vector expressing the human CYP46A1 gene in the cortex and hippocampus of 3-month old APP23 mice. Behavioral studies were performed at 6 months and mice were sacrified at 12 months to study the load of amyloid plaques, the production of Aß peptides, the levels of Aß oligomers and C-terminal fragments (CTFs), neuronal connectivity, microgliosis and astrocytosis. We demonstrate that increasing brain CYP46A1 gene expression through AAV-mediated gene transfer markedly reduces amyloid deposits, Aß40/42 peptides, Aß oligomers, reverses microgliosis and astrocytosis and improves spatial memory defects in APP23 mice before the onset of amyloid deposits. In vitro and in vivo studies converge to demonstrate that these effects are not mediated through the activation of ABCA1 and ApoE gene expression by 24S-hydroxycholesterol acting as ligand on liver-X receptors (LXR). Rather, in vitro and in vivo studies consistently suggest that the decrease of Aβ40/42 peptides induced by CYP46A1 gene expression is caused by a decreased γ-secretase cleavage of amyloid precursor protein. Overall, these data demonstrate that selective overexpression of CYP46A1 in neurons can reduce Aß peptides and amyloid deposits in vivo and indicate that viral vector delivery of CYP46A1 could find therapeutic applications in Alzheimer disease.

381. Retrograde Transport of AAV8 Vector to Central Nervous System Via Intramuscular Delivery

Hui Zheng,1,2 Chunping Qiao,2 Juan Li,2 Chi-Hsien Wang,2 Jianbin Li,2 Cheng Zhang,1 Xiao Xiao.2 1 Department of Neurology, The First Affiliated Hospital of Sen Yet-sun University, Guangzhou City, Guangdong Province, China; 2 Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina, Chape Hill, NC. Introduction: Blood brain barrier (BBB) is a regulated interface between peripheral circulation and the central nervous system (CNS). Its high selectivity limits nearly all molecule and drugs to reach the brain and spinal cord for many disorders of the CNS. So far, seeking for an approach to deliver the therapeutic particles into the central nervous system has proved challenging. Previously, we have revealed that AAV8 can efficiently transducer whole-body skeletal Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

NEUROLOGIC & OPHTHALMIC I and cardiac muscles after simply systemic injection. In this study, we investigated the ability of AAV8 vector to retrograde transport to CNS after intramuscular injection. Methods: AAV8 encoding LacZ gene driven by a CMV promoter was injected into the tibialis anterior and gastrocnemius of three weeks old ICR mice (2x 1012 v.p.). The injection was performed on only one leg and the other was used as negative control. They were sacrificed at either one, two or three months after injection and both injected and uninjected muscles, sciatic nerve, spinal cord and brain were collected for LacZ expression analysis and histology examination. Results: Our results revealed that beta-gal positive cells were found in the injected muscles, uninjected ipsilateral, but not contralateral muscles, ipsilateral peripheral nerves, dorsal root ganglion (DRG), spinal cord and brain. The LacZ positive cells traveled to the higher level of the spinal cord, even the brain, three months after vector delivery. Colabeling of neurofilament (NeuF), myelin basic protein (MBP) and beta-gal (LacZ) expression revealed that many neuronal axons showed AAV8-LacZ gene expression. In addition, we noticed some myelin expression cells were LacZ positive, suggesting Schwann cells were transduced. We also observed that there were more LacZ positive cells in the lower spine portion than the higher spine portion, indicating the entrance of AAV to the nervous system was through retrograde transport. Conclusions: AAV8 vector can be efficiently retrograded to the central nervous system via intramuscular delivery. This method can be potentially useful for the treatment of many neuromuscular diseases such as amyotrophic lateral sclerosis (ALS), chronic pain, multiple sclerosis (MS), MDC1A and Charcot-Marie-Tooth (CMT) disease, etc.

382. A Regulatable Gene Therapy Approach Using the Human Glycine Receptor To Treat Pain

James R. Goss,1 Michael Cascio,1 Shaohua Huang,1 David M. Krisky,1 Richard J. Clarke,2 Jon W. Johnson,2 Michael Gold,3 Joseph C. Glorioso.1 1 Dept. of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Dept. of Neuroscience, University of Pittsburgh, Pittsburgh, PA; 3Dept. of Anesthesiology and the Pittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, PA.

Over the past several years there have been significant advances in the use of viral vector mediated gene transfer to address a variety of medical conditions. One concern has been the potential need to develop regulatable vectors, especially for the treatment of chronic conditions such as pain. The most common approach used thus far employs a systemically active substance such as tetracycline to control vector transgene expression. However, this may not be the best long term solution to the problem due to potential adverse effects from the systemic activator and the less than one hundred percent control of gene expression demonstrated in these systems. An alternative approach is to design a system in which a constitutively produced gene product only functions in the presence of an exogenously applied activator that can be targeted to the vector infected cells. For the treatment of pain, neurotransmitter-gated receptors are attractive candidates for use in such a system. In the adult CNS, the ionotropic glycine receptors (GlyR) are typically inhibitory and act to silence neurons by transiently permitting inward chloride currents upon activation. The distributions of these receptors are regulated temporally and spatially, and are expressed predominantly in spinal cord and the lower brain. We hypothesized that we could reduce pain by directly expressing the alpha1 subunit of the GlyR in primary sensory neurons using a non-replicating herpes simplex virus (HSV)-based vector and then activating this receptor via a local application of exogenously applied glycine. We tested this approach using two rodent models of pain, the formalin footpad test in the rat and the osteolytic sarcoma model of bone cancer-induced pain in the Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

mouse. Immunohistochemical analysis of the hindpaw inoculated with GlyR-expressing vector (vHGlyRa1), but not control vectors, confirmed the restricted expression of GlyR in peripheral nerve fibers. Electrophysiological analysis confirmed functional GlyR expression was restricted to afferents innervating the site of inoculation. Finally, rats inoculated with vHGlyRa1 exhibited significantly reduced nociceptive behavior during the second phase of formalin-induced pain following subcutaneous injection of glycine into the footpad, whereas expression of the receptor alone had no effect on pain. The anti-nociceptive effect of the glycine was reversed by a subsequent injection of strychnine, a GlyR antagonist. Similarly, tumorbearing mice treated with vHGlyRa1showed a significant decrease in nociceptive behavior and improvement in mechanical allodynia following an injection of glycine. These studies suggest that the selective activation of HSV-mediated alpha1 GlyRs expressed in peripheral neurons by exogenously applied glycine could function as a regulatable gene therapy system and could find use for the treatment of pain.

383. Long-Term Delivery of a HuntingtinSpecific shRNA Causes Toxicity in the Brain

Jodi L. McBride,1,3 Ryan L. Boudreau,1 Ines Martins,1 Patrick D. Staber,1 Barrie J. Carter,2 Haim Burstein,2 Sergio Ojeda,3 Beverly L. Davidson.1 1 Internal Medicine, University of Iowa, Iowa City, IA; 2Targeted Genetics, Seattle, WA; 3Neuroscience, Oregon National Primate Research Center, Beaverton, OR. Huntington’s disease (HD) is a neurodegenerative disorder resulting from an expanded glutamine tract in exon 1 of huntingtin (htt). The polyglutamine expansion confers a toxic gain of function on htt, causing degeneration of striatal and cortical neurons in the brain. HD is characterized by devastating motor, cognitive, and psychiatric deficits that usually emerge in the fourth or fifth decade of life and always causes death. RNA interference is a viable approach to reduce expression of disease genes by degrading the encoding mRNA and viral delivery allows for stable and long-term expression of these molecules in the brain. We have previously identified a shRNA sequence (sh8.2) that decreases both mutant and wildtype htt expression for up to 4 months post-injection without inducing striatal toxicity. Here, we expand this finding and investigate the long-term behavioral and neuropathological consequences of delivering sh8.2 to the striatum of CAG140 HD mice. We confirm the ability of recombinant adeno-associated virus (AAV) to express sh8.2 in striatal neurons for over one year (14 months) and decrease htt expression by ∼50% (p<0.001 for both low titer [5e11] and high titer [5e12] viral preparations compared to controls). Interestingly, while sh8.2 was efficacious in decreasing htt expression, we observed significant loss of DARPP-32 expression, a marker for medium spiny projection neurons, within striata of AAV-sh8.2 treated mice compared to both buffer-injected (p<0.01) and sh8.2mismatch-injected controls (p<0.01). Additionally, we noted a concomitant increase in striatal GFAP expression ,a marker of reactive astrocytes, in mice treated with AAV-sh8.2 compared to buffer-injected (p<0.01) and sh8.2 mismatchinjected (p<0.01) controls. Mice treated with AAV-sh8.2 showed no improvement over controls on behavioral measures including the accelerating rotarod and staircase tests (p>0.05 for each assay). Furthermore, mice treated with the high titer viral preparation showed a significant decrease in rotarod performance to buffer-injected controls (p<0.05). Taken together, these data demonstrate that, while short-term expression of sh8.2 in the striatum is well-tolerated, longterm delivery causes toxicity. Ongoing experiments will determine if this effect is specific to the long-term decrease in htt expression or results from the long-term expression of this particular shRNA sequence (either by inhibiting the RNAi machinery or by eliciting off-sequence silencing). Because the mice did not demonstrate S149