Neural progenitor cell transduction with AAV serotypes 1 and 4

CENTRAL NERVOUS SYSTEM I 229. AAV4 Mediated Gene Transfer in Mucopolysaccharidosis Type VII Mice Gumei Liu,1,2 John A. Chiorini,5 Beverly L. Davidson.1,2,3,4 1 Program in Gene Therapy; 2Departments of Internal Medicine; 3 Neurology; 4Physiology & Biophysics, University of Iowa, Iowa City, IA; 5National Institutes of Health, Bethesda, MD. Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease (LSD) caused by a deficiency of beta-glucuronidase. The central nervous system (CNS) manifestations of MPS VII include enlarged ventricles, progressive loss of neurological function and mental retardation. Due to the nature of the blood brain barrier (BBB) in humans, systematic treatments may have little effect on the progressive CNS deficits. However, the epithelia lining the ventricular system, the ependyma, is an attractive target for gene transfer. Ependymal cell-expression of beta-glucuronidase would lead to enzyme activity in the cerebrospinal fluid (CSF), which permits global distribution of enzyme. Previous data in our lab showed that adeno-associated virus type 4 (AVV4) specifically transduced ependyma following intraventricular infusion. In this study, we investigated the feasibility of AAV4 mediated betaglucuronidase gene transfer in the CNS of MPS VII mice. AAV4betagluc vectors were injected unilaterally into the lateral ventricle of MPS VII and C57BL/6 wildtype mice. Mice were killed 10 days, 4 weeks and 6 weeks after injection, and assayed in situ for enzyme activity. Beta-Glucuronidase expression patterns were similar in all groups. Enzyme activities were detected as early as 10 days postinjection and persisted through all time points. Beta-glucuronidase expression was most extensive along the ventricular system. In addition, scattered positive cells were observed in the hippocampus, striatum and cortex. Positive cells in these regions were most often associated with small blood vessels, which indicated that betaglucuronidase enzymes or the AAV4 traveled through the VirchowRobin spaces lining the penetrating blood vessels to reach the brain parenchyma. These results suggest that AAV4 vectors may be a useful vehicle for gene transfer in the LSDs.

230. Neural Progenitor Cell Transduction with AAV Serotypes 1 and 4 Gumei Liu,1,2 Robert M. Kotin,5 John A. Chiorini,5 Beverly L. Davidson.1,2,3,4 1 Program in Gene Therapy; 2Departments of Internal Medicine; 3 Neurology; 4Physiology & Biophysics, University of Iowa, Iowa City, IA; 5National Institutes of Health, Bethesda, MD. The mucopolysaccharidoses (MPS) and the ceroid lipofuscinoses (NCLs) are devastating childhood onset diseases. Some forms of MPS and all NCLs have a severe CNS component. MPS VII, or beta-glucuronidase deficiency, and late-infantile NCL, or tripeptidyl protease deficiency are due to mutations in mRNAs encoding soluble lysosomal enzymes. Prior work in our laboratory and others’ demonstrated that overexpression of beta-glucurondiase or TPP-1 from transduced cells in brain can lead to enzyme activity in nontransduced cells remote from the injection site, and improvements in cellular pathology and behavioral deficits. This occurs through a process called cross-correction. We hypothesized that crosscorrection could also occur from gene-corrected endogenous neural progenitor cells (NPCs). Transduction of endogenous NPCs with virus expressing beta-glucuronidase or TPP-1 may allow enzyme secretion at several sites: 1) in the SVZ (with enzyme secreted into the ventricular system); 2) during migration through the corpus callosum (with enzyme secreted along the extensive white matter tracts of the corpus callosum); and 3) from differentiated neurons in the olfactory bulb (OB). We tested AAV1 and AAV4 for their ability to target endogenous neural progenitors. AAV4 vector injected unilateral into the lateral ventricle of adult or P0 C57BL/6 mice Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

resulted in highly specific transduction of ependymal cells along the ventricular wall. Only scattered transgene positive cells were observed in the rostral migratory stream (RMS) and olfactory bulb in tissues harvested from several days to 8 weeks after gene transfer, regardless of injection time. Transgene positive cells did not express neuronal cell marker, indicating that NPCs were not a major target of AAV4 transduction in vivo. In contrast to AAV4, transgene positive migrating neuroblasts and olfactory bulb neurons were obvious after unilateral intraventricular injection of AAV1. However, numbers remained too few to consider AAV1-transduced NPCs as a significant source of recombinant lysosomal enzymes. Thus, transduction of endogenous NPCs with AAV4 does not occur, and with AAV1 is limited, preclude the use of these vectors for therapeutic application via NPCs.

231. Supplementation of Peripherin/rds Rescues Mutation-Associated Rod and Cone Photoreceptor Defects in Transgenic Mice May Nour,1 Muna I. Naash,1 Steven J. Fliesler.2 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Deptartments of Ophthalmology and Pharmacological & Physiological Sciences, Saint Louis University School of Medicine, St. Louis, MO. 1

Blindness due to genetic abnormalities in photoreceptor-specific proteins represents a significant and largely intractable health problem worldwide. In the present study, we generated transgenic mice carrying point mutations in the photoreceptor-specific protein, peripherin/rds (P/rds), to model human hereditary diseases that involve either rod- or cone-dominant retinal degeneration. We tested the ability of supplemental expression of normal mouse P/rds (NMP) to over-ride the retinal degenerative phenotype and to promote normal visual function in these transgenic lines. Electroretinography, histology at the light and electron microscopy levels, immuno-gold cytochemistry (using anti-rod and -cone opsin antibodies), Western blot analysis, and limited tryptic digestion were used to assess rescue of the retinal disease phenotypes. Herein, we provide the first evidence for P/rds supplementation-mediated rescue of both rod and cone-dominant disease-causing mutations. This efficacious strategy may have broader implications for clinical intervention in P/rds-associated retinal diseases as well as for the treatment of other hereditary blinding disorders.

232. Transplantation of Human Embryonic Stem Cell Derived Oligodendroglial Progenitors for the Treatment of Spinal Cord Injury Jane Lebkowski,1 Gabriel Nistor,2 Giovanna Bernal,2 Minodora Totoiu,2 Catherine Priest,1 Scott Thies,1 Jerrod Denham,1 Hans Keirstead.2 1 Geron Coporation, Menlo Park, CA; 2Reeve-Irvine Research Center UC Irvine, Irvine, CA. Human embryonic stem cells (hESCs) can replicate indefinitely in culture yet upon appropriate cues differentiate into elements of all three embryonic germ layers. Because of these proliferative and differentiative capabilities, hESCs could serve as as the source material for transplantable cells for tissue restorative therapies. To this end, we have developed protocols to direct the differentiation of hESCs into candidate target cell populations for therapeutic activity. Using combinations of extracellular matrix factors, growth factors, and select media components, we have produced cardiomyocytes, neural progenitors, neurons, oligodendroglial progenitors, hematopoietic cells, osteoblasts, and hepatocytes from hES cells. In all cases, the differentiated cells display both a panel of markers and appropriate

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