which are greater than 70% NG2 positive and express Olig 1,. PDGFR, and several other markers indicative of the oligodendrocyte lineage. These cells have ...
CENTRAL NERVOUS SYSTEM I function characteristic of the target cell. For all of these differentiated cell populations, a thorough cell compositional analysis has been completed or is underway. The hESCs have been differentiated into several different populations of neural cells. Such includes oligodendroglial progenitors which are greater than 70% NG2 positive and express Olig 1, PDGFR, and several other markers indicative of the oligodendrocyte lineage. These cells have been transplanted into the spinal cord of myelin basic protein deficient shiverer mice where they were found to induce the myelination of axons with the production of myelin basic protein. These cells upon transplantation into the injury site of immunosuppressed rats 7 days after a moderate 200 kilodyne contusion injury migrated up to 12 mm and induced either directly or indirectly the remyelination of denuded axons. No evidence of tumor formation was observed in animals 2 months post transplant. Rats treated with the hESC derived oligodendroglial progenitors demonstrated a statistically significant 2-4 point improvement in their BBB locomotor scores compared to untreated or human fibroblast treated controls. Improvements included superior recovery of weight bearing capacity, paw placement, toe clearance, and tail elevation. Advanced studies are underway to determine the therapeutic window for such transplantation and the requirements for immunosuppression. Further preclinical studies are underway to test the safety and efficacy of these hES cell derived oligodendroglial cells for the treatment of spinal cord injury. In preparation for clinical use, banks of the H1 and H7 cell lines have passed a series of tests for pathogens including amphotropic, xenotropic and ecotropic retroviruses as well as adventitious agents of human, bovine, murine and porcine origin. The hESCs have been grown exclusively feeder-free culture conditions using conditioned medium from mouse embryonic fibroblasts over the last three years. More recently, we have developed defined conditions in the absence of conditioned medium or serum for the propagation of the undifferentiated cells. Production of the differentiated cells is being scaled for advanced preclinical studies.
233. Gene Control as a Therapeutic Intervention: Zinc-Finger Protein Transcription Factors as Regulators of the Molecular Determinants of Neuropathic Pain Yann Jouvenot,1 John R. Forsayeth,2 Siyuan Tan,1 Andrew McNamara,1 Raymond A. Chavez,2 Philip Gregory.1 1 Sangamo Biosciences, Inc., Richmond, CA; 2Avigen, Inc., Alameda, CA. Neuropathic (or chronic) pain is a complex disorder resulting from injury to the nerve, spinal chord or brain. The American Pain Society estimates that nearly 50 million Americans are totally or partially disabled by pain, however, few effective yet well-tolerated treatments are available. Indeed, existing therapeutics cause a range of undesirable side effects primarily due to the difficulty in developing small-molecule drugs capable of specifically targeting the receptor/ channel of choice. Treatment of chronic pain is a therefore a promising target for gene therapy strategies. The study of the molecular mechanisms triggering neuropathic pain has identified several genes to be abnormally over-expressed in sensory neurons of the Dorsal Root Ganglion (DRG) in models of neuropathic pain. We have chosen to focus on three of these targets; (i) the Vanilloid Receptor 1 (VR1), a non-selective cationic channel responding to thermal, pH and capsacin stimulation, was found to be up-regulated after partial nerve injury (Hudson et a., 2001). The VR1 antagonist capsazepine was also found to reverse mechanical hyperalgesia in models of inflammatory and neuropathic pain (Walker et al., 2003); (ii) TRKA (Tyrosine kinase A receptor or high-affinity NGF receptor), shown to up-regulated in DRG neurons after chronic spinal chord injury (Qiao et al., 2002) and; (iii) the sodium channel S90
Nav1.8 (a.k.a PN3 or SCN10A), expression of which was found to be increased in patients with neuropathic pain symptoms (Coward et al., 2000). Moreover, reduced levels of Nav1.8 correlate with inhibition of neuropathic pain in the rat spinal nerve injury model of chronic pain (Lai et al., 2002). Thus, VR1, TRKA and Nav1.8 are potential targets for molecular therapies aimed at down-regulating their expression. To this end, we have developed zinc-finger protein transcription factors (ZFP TFs) for the repression of gene expression (Snowden et al. 2003; Tan et al. 2003). ZFP TFs hold therapeutic promise as they have been shown to be highly efficacious and yet function with singular specificity within the mammalian genome. Here we report the development of ZFP TF repressors targeted to each of the neuropathic pain target genes VR1, TRK-A and Nav1.8. In transient transfection experiments these gene-specific ZFP TFs are shown to be capable of potent target gene repression at the mRNA level. Furthermore, by using immunochemistry and cell sorting techniques we show that these ZFP TF repressors are capable of reducing the levels of the targeted protein. These reagents are currently being tested for efficacy in combination with Adenoviral Associated Vector (AAV) delivery. Efficacy in these cellular systems will form the basis for pre-clinical evaluation of ZFP TF as therapeutics for neuropathic pain.
234. Correction of Pathology in Galactosialidosis Mice Crossed into Transgenic Mice Expressing PPCA in Neural Cells Huimin Hu,1,2 Linda Mann,1 Erik Bonton,1 Taylor Walker,1 Alessanrdra d’Azzo.1 1 1Department of Genetics and Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, TN; 2International Outreach Programs, St. Jude Children’s Research Hospital. Protective protein/cathepsin A (PPCA) is a lysosomal carboxypeptidase that is deficient in Galactosialidosis (GS), an autosomal recessive, neurodegenerative lysosomal disorder affecting most of the systemic organs and the central and peripheral nervous systems. The GS mouse model resembles closely the severe human condition and develops a progressive neurological phenotype including loss of hearing and motor coordination. To determine whether different neural cell populations respond differently to therapeutic modalities meant to correct the CNS phenotype in the GS model, we have generated a transgenic mouse line that overexpress a human PPCA minigene under the control of the rat neuron specific enolase promoter (NSE-PPCA). While expression of the transgene in this transgenic line was strictly neuronspecific the distribution of PPCA positive neurons reflected only in part that of the endogenous NSE protein. In particular cerebellar Purkinje cells did not express the transgene. These transgenic mice were therefore a useful model to assess whether regional neuronal expression of the transgene cross-corrected the CNS phenotype in GS mice crossed into the transgenic background. Crossed mice were analyzed at different time points and examined by using different methods: in situ hybridization, immunohistochemistry, enzyme assays and histological staining. The overall brain morphology was completely restored in the crossed mice. The extensive vacuolation of perivascular macrophages and endothelial cells characteristic of the GS brain phenotype was reverted. In addition, we observed long-term preservation of Purkinje cells and prevention of the hearing loss. The NSE-PPCA/GS mice also had a longer lifespan (1.5 years), although their visceral organs progressively deteriorated. These findings clearly underscore the occurrence of cross correction between neural cells in the CNS. (Supported in part by NIH grant DK 52025).
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
