Specific and Efficient Transduction of the Cochlear Inner Hair Cells with Recombinant Adeno-Associated Virus Type 3 Vector

NEURO GENE THERAPY II: NEW APPROACHES FOR DOMINANT, RECESSIVE, DEGENERATIVE AND AUTOIMMUNE DISEASES robust and persistent TPP-I expression, thus paving the way for a clinical study to assess safety and efficacy in human subjects with LINCL.

1061. Specific and Efficient Transduction of the Cochlear Inner Hair Cells with Recombinant Adeno-Associated Virus Type 3 Vector Yuhe Liu,1,2,7 Takashi Okada,2 Tatsuya Nomoto,2 Kuniko Shimazaki,3 Kianoush Sheykholeslami,4 Shin-Ichi Muramatsu,5 Rahim Ajalli,2 Koichi Takeuchi,6 Hiroaki Mizukami,2 Akihiro Kume,2 Shuifang Xiao,7 Keiichi Ichimura,1 Keiya Ozawa.2 1 Department of Otolaryngology, Jichi Medical School, Tochigi, Japan; 2Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan; 3Department of Physiology, Jichi Medical School, Tochigi, Japan; 4Department of Otolaryngology, University of Tokyo, Tokyo, Japan; 5Division of Neurology, Department of Medicine, Jichi Medical School, Tochigi, Japan; 6Department of Anatomy, Jichi Medical School, Tochigi, Japan; 7Department of Otolaryngology, Peking University First Hospital, Beijing, China. Background: Virally mediated gene transfer to the adult mammalian cochlea appears to be a powerful strategy to investigate gene function in the auditory system and to develop new therapeutic treatment for hearing impairment. Recombinant adeno-associated virus (AAV) vectors have been of interest in the context of cochlear gene therapy because of their ability to mediate efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. Because of the controversy on AAV2-mediated transduction of hair cells, further progress would be awaited through the development of vectors based on other AAV serotypes. Methods: Seven serotypes of AAV vectors (AAV1-5, 7 and 8) containing chicken beta-actin promoter associated with cytomegalovirus immediate-early enhancer (CAG)-driven enhanced green fluorescent protein (EGFP) and Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) were directly microinjected into the scala tympani to transduce the murine cochlear cells. The cochlear function was monitored with evoked auditory brain stem responses (ABR). The transgene expression patterns in the cochlear cells were determined by visualizing EGFP expression. Results: Expression patterns of EGFP showed that each of these serotypes successfully targets distinct cell types within the cochlea. In contrast to other serotypes, AAV3 vector specifically and most efficiently transduced the cochlear inner hair cells in vivo, while AAV1, 2, 5, 7, and 8 could also transduce inner hair cells as well as other cell types such as spiral ganglion cells or spiral ligament cells. Additionally, the inner Sulcus cells were also transduced with AAV1, 5, 7 and 8 vectors; as well as outer supporting cells with AAV5 and 7 vectors. According to the ABR monitoring, there was no loss of the cochlear function over the frequencies tested after the injection of AAV vectors. Conclusion: There was a distinct difference between AAV2 and AAV3 vectors for transduction in the cochlear cells. While AAV2 vectors provide broad expression property in the cochlea, AAV3 vectors are suitable for specific gene transfer into the cochlear inner hair cells. Development of a viral vector that infects the cochlear hair cells without virus-induced ototoxic effects is crucial for further molecular studies of the cochlear hair cells. These include investigation of the role of genes in influencing the development of inner hair cells, gene replacement strategies to treat certain forms of inherited deafness due to monogenic mutations, and otoprotective strategies to prevent acquired hearing disorders.

Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

1062. Functional Rescue of Parkinsonian NonHuman Primates by a Dopamine Producing Multicistronic Lentiviral Vector Bechir Jarraya,1 Mimoun Azzouz,2 James Miskin,2 Scott G. Ralph,2 Fraser Wilkes,2 Jonathan Rohll,2 Lucy E. Walmsley,2 Fiona Ellard,2 Susan M. Kingsman,2 Kyriacos A. Mitrophanous,2 Philippe Hantraye,1 Stephane Palphi,1 Nicholas D. Mazarakis.2 1 1URA CEA CNRS 2210, Service Hospitalier Frédéric Joliot, 4 Place du Général Leclerc, Orsay Cedex, France; 2Oxford BioMedica (Ltd), The Oxford Science Park, Oxford, United Kingdom. Parkinson’s disease (PD) is a neurodegenerative disease affecting 1% of the population over 55 years, whose cardinal symptoms include rigidity, resting tremor, bradykinesia and impaired postural reflexes. PD is characterised by a selective loss of dopaminergic neurons in the substantia nigra, creating dopamine deficiency in the target projection area: the striatum. We and others have demonstrated that expression of the dopamine biosynthetic enzymes tyrosine hydroxyalse (TH), aromatic L-amino acid decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1), in the striatum, results in a significant improvement in motor function in animal models of the disease (Azzouz et al., J Neurosci. 2002 22:10302-12; Shen et al., Hum Gene Ther 2000 11:1509-19; Muramatsu et. al., Hum Gene Ther 2002 13:345-354). We have generated an optimised EIAV tricistronic vector LentiVector® (EIAV-TRIC), with an improved expression and safety profile, that can express all the three enzymes in transduced striatal neurons, both in vitro and in vivo. Stereotactic delivery of these vectors in the sensorimotor putamen of MPTP treated macaques with severe parkinsonism, led to significant motor recovery and improvement in clinical rating scales. This was specific to the EIAV-TRIC treated macaques, for similar treatment with a vector expressing a marker gene failed to yield any such improvement. Our results indicate that intraputamenal delivery of dopamine synthesising genes by LentiVector®, can achieve functional rescue in a primate PD model, thus opening the potential for its use in gene therapy of late stage PD patients.

1063. In Utero β-Glucuronidase Gene Transfer with AAV Vectors Results in Widespread Transduction, Enzyme Activity, and Disease Correction in the MPS VII Mouse Brain Brian A. Karolewski,1,2 John H. Wolfe.1,2 1 Department of Pathobiology and Center for Comaparative Medical Genetics, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA; 2Pediatric Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA. Mucopolysaccharidosis (MPS) VII is a heritable lysosomal storage disease caused by the deficiency of b-glucuronidase (GUSB) enzyme. MPS VII is a chronic and progressive multiorgan disorder with signs of pathology already present during fetal life. Most therapeutic approaches for MPS VII have focused on postnatal treatments. Although these approaches can reverse storage lesions, the central nervous system (CNS) is more difficult to treat. We investigated in utero gene transfer in the MPS VII mouse CNS with AAV 1 and AAV2. We injected 1 µl of AAV1 (3.3X1013 genomes/ ml) or AAV2 (1.4X1012 genomes/ml) into the lateral ventricle of the fetal brain on day 15.5 of gestation. AAV1 injected brains demonstrated extensive and widespread GUSB activity and mRNA expression the brain. There was retrograde transport of GUSB to the ganglion cells of the retina. GUSB activity was detected in the gray matter throughout most of the spinal cord segments. Storage lesions were reversed in the brain and spinal cord. Ubiquitin immunoreactivity was significantly reduced compared to the affected control. This suggested that in utero gene transfer with AAV1 S407