Ret Signaling and Ameliorates Axonal Degeneration in Diabetic Rats

Neurologic - Novel Approaches 7.5 months. Gene therapy results in mice and cats strongly support the continued development of AAV vectors for human gangliosidosis clinical trials.

994. 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 Joseph C. Glorioso.1 1 Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA; 2Neuroscience, 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 not found in sensory neurons. We hypothesized that we could reduce pain by directly expressing the alpha1 subunit of the GlyR in primary sensory neurons using a herpes simplex virus (HSV)-based vector and then activating this receptor via a local application of exogenously applied glycine. We previously tested this approach using the formalin footpad test of acute inflammatory pain in the rat and demonstrated a glycine dependent reduction in nociceptive behavior during the second phase of formalin-induced pain. Because the neuronal mechanisms underlying acute inflammatory pain and chronic neuropathic pain are different we have expanded on this result by examining the effectiveness of our vector in treating nociception in the osteolytic sarcoma model of bone cancer induced pain in the mouse. NTCT 2472 osteolytic sarcoma cells were implanted into the medullary space of the right femur of C3H/HeJ mice. One week later the ipsilateral lumbar DRG were transduced by subcutaneous inoculation into the plantar surface of the hind foot with 1 x 107 pfu of vector vHGlyRa1or a control vector vHG. Nociceptive behavior was evaluated 2 weeks after vector inoculation by assessing spontaneous ambulatory pain (SAP) and measuring mechanical allodynia (MA). There was no difference in SAP or MA in tumor bearing mice treated with either vector. However, 20 minutes after injecting 100 uM glycine into the right rear footpad, tumor-bearing mice treated with vHGlyRa1 demonstrated significant improvements in both SAP and MA whereas the vHG group showed no effect. This study suggests 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 for the treatment on chronic pain.

995. NT-3 Gene Transfer Improves Peripheral Nerve Pathology and Function in Trembler Mice

Zarife Sahenk,1,2,3 Chris Edwards,3 K. Reed Clark,1,3 Jerry R. Mendell,1,2,3 Brian K. Kaspar,1,3 Jiangchao Zhang,3 Janet Oblinger.3 1 The Ohio State University, Pediatrics, Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2The Ohio State University, Neurology, Research Institute at Nationwide Children’s Hospital, Columbus, OH; 3Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital, Columbus, OH. Charcot-Marie-Tooth (CMT) neuropathies are one of the most common inherited neurological conditions affecting 1 in 2500 people in the United States. Both children and adults are affected causing sensory and motor dysfunction, pain, and a need for ambulatory aids. A primary Schwann cell (SC) disorder represents the single most common form of CMT neuropathy. Previous studies from this laboratory have shown that in animal models for CMT there is an impairment of nerve regeneration (Sahenk et al., 2003, 2005). These observations are best explained by the crucial role of SCs in the support of axonal regeneration and associated myelination. TremblerJ (TrJ) mice and patients with CMT1A, both harboring mutations of the PMP22 gene, demonstrated augmentation of nerve regeneration following exogenous delivery of NT-3 (Sahenk et al., 2005). The latter is a component of the autocrine regulatory system promoting survival and differentiation of mature SCs in the absence of axons. For translational therapy, however, the short serum halflife of NT-3 makes it impractical for clinical application. A gene therapy approach offers the potential for sustained NT-3 delivery through secretion by muscle cells. In normal mice, following rAAV. NT-3 delivery to gastrocnemius muscle, we established that NT-3 serum levels are easily detectable as early as 3 weeks and remain elevated on repeated tests for up to 9.9 months. In TrJ mice (n=16), rAAV.NT-3 or PBS was injected into the gastrocnemius muscle. At 3 weeks post-injection, sciatic nerves were crushed unilaterally and grip strength data was collected weekly. Mice were killed at 20 weeks post-crush and both sciatic nerves were removed. Grip strength improved in limbs harboring the regenerating sciatic nerves by 16 weeks compared to the PBS treated group. Furthermore, the hind limb strength in an additional group with bilateral intact sciatic nerves were significantly better than capsid injected TrJ mice (n=3). In agreement with these functional data, quantitative morphometric studies showed significant increases in the myelinated fiber densities and increases in myelin thickness, in both regenerating and intact sciatic nerves from the rAAV.NT-3 treated TrJ mice compared to PBS or capsid treated group. This is the first study to illustrate that rAAV.NT-3 gene transfer into muscle tissue will result in therapeutic blood levels sufficient to provide functional and histopathological improvements in trembler nerve regeneration and hypomyelination. This study provides an impetus for considering gene transfer of rAAV.NT-3 in CMT neuropathies, as well as other nerve diseases with impaired nerve regeneration.

996. Peripheral Gene Transfer of Glial CellDerived Neurotrophic Factor Restores GDNF/Ret Signaling and Ameliorates Axonal Degeneration in Diabetic Rats

Guei-Sheung Liu,1,3 Jhih-Yin Shi,2 Ming-Hong Tai.1,2 1 Department of Medical Education & Research, Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan, Taiwan; 2 Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung City, Taiwan, Taiwan; 3Departments of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Taiwan.

Deprivation of neurotrophic factors may contribute to the pathogenesis of diabetic neuropathy. The role of glial cell-derived Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

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Muscle Gene Therapy: Systemic Delivery neurotrophic factor (GDNF) in the pathogenesis and therapeutics of diabetic neuropathy is not well defined. In streptozotocin (STZ)induced diabetic rats, the expression of GDNF signaling complex in the peripheral nerves was examined when the abnormalities in electrophysiological parameters were initially detected. After STZ injection for 14 days, diabetic rats began to exhibit significant neuropathic deficits, which were followed with evident alteration in the structure of axons and myelination in the sciatic nerve. Expression analysis revealed that GDNF and its receptors, Ret and GFRa1, were downregulated in the sciatic nerve during onset of neuropathic symptoms. After detection of neuropathy, intramuscular GDNF gene transfer evoked a sustained rise of circulating GDNF concentration and restored the GDNF level in the sciatic nerve to normal threshold, which significantly improved the neurological functions of diabetic rats. Such improvement was correlated with significant reduction in axon demyelination and loss of schawnn cells in the sciatic nerve. In summary, peripheral GDNF gene delivery alleviates the neuropathic deficits by attenuating the hyperglycemia-induced deficiency in GDNF signaling pathway and axonal degeneration in the sciatic nerve, thereby holding potential for treatment of diabetic neuropathy.

Muscle Gene Therapy: Systemic Delivery 997. Systemic AAV-9 Delivery in Normal Dog Leads to High-Level Persistent Transduction in Whole Body Skeletal Muscle

Yongping Yue, Arkasubhra Ghosh, Chun Long, Brian Bostick, Bruce F. Smith, Joe N. Kornegay, Dongsheng Duan. 1 Dept of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO; 2Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO; 3Scott-Ritchey Research Center, Auburn University, Auburn, AL; 4Dept of Pathology and Laboratory Medicine, University of North Carolina - Chapel Hill, Chapel Hill, NC. Adeno-associated virus (AAV) is a promising vector for muscle disease gene therapy. The dog model represents an ideal intermediate system prior to human trials. Previous attempts to deliver AAV directly to canine muscle have largely failed to achieve efficient transduction because of a strong immune response. In this study, we evaluated systemic AAV-9 gene delivery in newborn dogs by local and systemic delivery. Transgene expression was examined at different time points after AAV infection by biopsy or whole body necropsy. In contrast to the previous reports of low expression and strong immune reaction in dog muscle, we observed efficient transduction at 4 weeks following intramuscular gene delivery. Importantly, systemic gene delivery resulted in impressive whole body skeletal muscles transduction for up to 6 months. In more than 20 different muscle groups (including head, neck, chest, abdominal, thoracic and pelvic limbs), we observed ≥ 80% transduction in the majority of muscles throughout the entire muscle length. Taken together, our results provide the first evidence that systemic AAV delivery can reach multiple muscles in a large animal and that body size is not a barrier to intravascular AAV gene transfer. Our results raise the hope of whole body correction for many muscle diseases such as Duchenne muscular dystrophy. (Supported by NIH and MDA).

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998. Long-Term Mini-Dystrophin Expression without Immunosuppression in GRMD Dogs after AAV8-Mediated Gene Delivery by Hydrodynamic Limb Vein Injection

Joseph Kornegay,1 Chunlian Chen,2 Juan Li,2 Janet Bogan,1 Dan Bogan,1 JIwei Chen,2 Bing Wang,4 Tong Zhou,4 Richard J. Samulski,3 Xiao Xiao.2 1 Department of Pathology School of Medicine, School of Medicine, Univerity of North Carolina at Chapel Hill, Chapel Hill, NC; 2 Division of Molecular Pharmaceutics, School of Pharmacy, Univerity of North Carolina at Chapel Hill, Chape Hill, NC; 3Gene Therapy Center, School of Medicine, Univerity of North Carolina at Chapel Hill, Chapel Hill, NC; 4Dept. of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA. Golden retriever muscular dystrophy (GRMD) dogs are employed as a large animal model of Duchenne muscular dystrophy (DMD) for AAV vector mediated gene therapy studies. Specifically, AAV serotype 8 (AAV8) is selected as the vector of choice for the delivery of a canine mini-dystrophin gene, which is under the transcriptional control of CMV promoter. The AAV8-CMV-cMinidys vector was injected into the hind legs of multiple young adult GRMD dogs (5 kg to 10 kg in weight) by the hydrodynamic limb vein injection method (isolated retrograde limb vein perfusion). The vector dose used in each dog was at 1 x 10e13 v.g/kg body weight. The injection volumes ranged from 10 ml/kg to 50ml/kg with a consistent injection rate of 1 ml/second. During and immediate after injection, blood circulation in the hindlimbs was block by a tourniquet for a total of 10 minutes. No overt vector-related adverse effect was observed during and after the procedure. At various time points, muscle biopsy samples were taken and analyzed for mini-dystrophin expression and immune responses. Canine minidystrophin gene expression was detected at both short term and long term (6 months) time points post vector injection. There was no discernable CTL responses against the canine minidystrophin gene. Immunofluorescent staining of CD4+ and CD8+ cells on muscles from vector-injected legs and the contralateral salineinjected control legs revealed no statistic differences. Furthermore, a normal dog similarly perfused with an AAV8-CMV-GFP vector also showed strong GFP expression at 2 week and 10 week time points without CTL immune responses. By contrast, a normal dog similarly perfused with an AAV2-CMV-GFP vector triggered robust CTL responses. These results suggest that the AAV8 vectors triggers minimal or no CTL responses against the canine minidystrophin in GRMD dogs and GFP in normal dogs when delivered by the limb perfusion method.

999. Effective Transduction of Dystrophic Dogs with rAAV Serotype 8 Sachiko Ohshima, Jin-Hong Shin, Akiyo Nishiyama, Katsutoshi Yuasa, Yuko Kasahara, Takashi Okada, Shin’ichi Takeda. 1 Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan.

Background: Duchenne muscular dystrophy (DMD) is an X-linked, lethal disorder of the striated muscle caused by mutations in the dystrophin gene, which encodes a large sub-sarcolemmal cytoskeletal protein dystrophin. The absence of dystrophin associated with the loss of dystrophin-glycoprotein complex from the sarcolemma results in progressive muscle weakness, cardiomyopathy, and early mortality. Several treatment modalities have been attempted to correct the dystrophic phenotypes, but more effective therapy still needs to be developed. A recombinant adeno-associated virus (rAAV) has been utilized in the various preclinical and clinical studies. However, many questions associated with the host immune reaction have been raised and innate immune response against the rAAV has not been studied. Here we investigated the transduction efficiency and immune response Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy