826 AAV mediated gene transfer for the treatment of the alport syndrome

826 AAV Mediated Gene Transfer for the Treatment of the Alport Syndrome Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S316 PRECLINICAL AND CLINIC[.]

PRECLINICAL AND CLINICAL APPLICATIONS OF AAV 824 Pharmacokinetics of GDNF Protein Infused into the Rat Brain and Striatal Levels of GDNF after Transduction with AAV2-GDNF Piotr Hadaczek,1 Louisa Johnston,1 John Forsayeth,1 Krystof Bankiewicz.1 Neurological Surgery, University of California San Francisco, San Francisco, CA Over the past few years, glial cell derived neurotrophic factor (GDNF) has attracted a lot of attention as a mean to control and manipulate restoration of degenerating dopaminergic (DA) neurons in Parkinsonian patients Delivery of GDNF into the brain (either by gene therapy vectors or direct GDNF protein infusion) has been used in animal models and clinical trials These studies remain inconclusive to date Part of the problem lies in the uncertainty of the optimal therapeutic dose and treatment regimen Little is known about the exact half-life of the infused GDNF protein within the brain as well as the relation between the exact amounts of GDNF produced from AAV2-GDNF vector Our studies were designed to understand pharmacokinetics of GDNF as a therapeutic agent for Parkinsonian patients and help defining its optimal therapeutic dose level We examined the dynamics of GDNF expression from AAV2-GDNF vector infused into the rat brain The level of GDNF production from AAV2-GDNF transduction showed a dose-dependant correlation The three doses of vector used: 1.65 x 1011, 9.07 x 1010, and 1.65 x 1010 vg resulted in GDNF tissue levels of almost 12, 8, and ng/mg protein, respectively GDNF expression reached a plateau at about week at all doses and remained stable for at least months To examine the distribution, half-life, and the response of the dopaminergic system to a single GDNF protein infusion, we infused two different doses of GDNF (15 and µg) into the rat striatum We euthanized animals (n=17) after 3, 7, 14, 21, and 28 days The brains were processed for ELISA, HPLC, and immunohistochemical staining (IHC) against GDNF The striatal infusion of 15 or µg GDNF protein resulted in about 60 or 13 ng /mg tissue protein on day respectively At both doses, there was a rapid decrease in GDNF tissue level observed between day and The high-dose group still showed elevated levels of GDNF even after one month after infusion, but the low-dose group (3 µg) reached baseline one week earlier Immunohistochemical (IHC) staining against GDNF showed similar results, but was less sensitive and there was little signal detectable beyond day 14 after injection Interestingly, IHC revealed GDNF presence in the septum and the base of the brain The dopamine (DA) turnover (measured as a ratio of DA metabolites, DOPAC and HVA, to DA) was significantly increased up to at least days after a single GDNF infusion 825 Pre-Existing Immunity to Different AdenoAssociated Virus Serotypes in Serum and Synovial Fluid of RA Patients: Implications for Vector Efficacy Margriet J Vervoordeldonk,1,2 Federico Mingozzi,3 Shyrie Edmonson,3 Rogier Thurlings,2 Katherine High,3,4 Paul P Tak.1,2 Div of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands; 2Arthrogen BV, Amsterdam, Netherlands; 3Center of Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia; 4Howard Hughes Medical Institute, Philadelphia Objective: AAV is considered as one of the most potent vectors for gene therapy for rheumatoid arthritis (RA) Humoral immunity against AAV vector represents an important barrier to gene transfer Recent reports indicate that synovial fluid has the potential to inhibit AAV-mediated transduction of chondrocytes and fibroblastlike synoviocytes, however little is known about the presence and relationship between serum and synovial fluid (SF) neutralizing S316 antibody (Nab) titers to AAV We looked at NAb of matched serum and SF samples against a range of AAV serotypes In addition, the effect of anti-CD20 administration on anti-AAV2 antibodies was also evaluated Methods: Anti-AAV-2, -5, -6, and -8 Nab titers were determined in 11 matched serum and SF samples of RA patients Anti-AAV1 capsid Ig subclasses were measured with an ELISA In addition, anti-AAV2 Nab titers in serum of 16 subjects receiving a single course of infusions rituximab were measured Serum samples were collected at baseline and 4, 16, 24 weeks thereafter Results: NAb to AAV-2 were the most prevalent, with a titer >1:316 detectable in 6/11 subjects, compared to 0/11, 1/11, and 1/11 for AAV-5, AAV-6, and AAV-8, respectively Conversely, titers 0.5 µM in the IM injection group and >2 µM with RI delivery group We have demonstrated that rAAV1-hAAT vector produced by the HSV-based method can safely be administered by different delivery methods into nonhuman primates and the serum hAAT levels of more than µM were observed with the vascular delivery routes Detailed analyses of vector copy number and distribution in the NHPs will be presented 829 AAV9-Mediated Targeted Overexpression of Catalase in Mitochondria Enhances Running Performance of BL6 Mice Dejia Li,1 Yongping Yue,1 Yi Lai,1 Dongsheng Duan.1 Department of Molecular Microbiology and Immunology, Univeristy of Missouri-Columbia, Columbia, MO Mitochondria are the major source of cellular free radicals Recently it was reported that ecotopic overexpression of catalase in mitochondria reduces oxidative damage, delays age-associated pathology and extends murine lifespan We hypothesized that catalase overexpression in mitochondria could also improve the muscle contractility To test this hypothesis, we generated AAV9 vector carrying the mitochondrial leader sequence tagged calatase expression cassette At three months after systemic delivery in neonatal BL6 mice, we evaluated catalase expression by western blot, immunofluorescence staining, catalase activity assay and zymography These studies confirmed mitochondrial expression of AAV derived catalase Quantitatively, catalase activity was increased by to10-fold in skeletal muscle and heart Interestingly, ectopic catalase expression did not influence the in vitro performance of the extensor digitorum longus muscle Twitch force, titanic force, the fatigue profile and the eccentric contraction profile were not altered However, we did observe a significant improvement in the treadmill performance in AAV infected mice Taken together, our data suggest that targeting catalase to mitochondria may represent an novel approach to improve exercise performance Alpha-1 antitrypsin (AAT) deficiency is one of the most common genetic disorders and is estimated that as many as in every 2500 individuals are affected by this disease Gene transfer is an appealing Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S317 ... medulla region of the kidney In conclusion, our study represents a first step forward to AAV- based therapy of chronic kidney disease 827 AAV- Based Gene Therapy Approaches for the Treatment of Giant... Replacement of the GAN gene is a potential therapeutic option for GAN Gene therapy approaches for GAN are similar to those for ALS, SMA, and other motor degenerative diseases of the CNS Namely,... The current progress of these comparisons will be presented, including novel strategies for efficient and widespread delivery of rAAV to the CNS via systemic administration The relevance of these