Gene therapy techniques need substantial development to provide therapeutic possibilities for treating neurological disorders such as Parkinson's disease (PD). Based on molecular control mechanisms of noradrenergic neuron-specific gene regulation, we recently devised a gene delivery system that can efficiently target transgene expression to noradrenergic neurons in a cell-specific manner. Our long-term goal is to establish gene therapy system(s) that will drive efficient transgene expression in a dopamine (DA) neuron-specific fashion based on discovery and characterization of DA-specific genes. Toward this end, we propose to identify and isolate genes that are selectively expressed in the DA mid-brain area by analyzing gene expression profiles using the most comprehensive cDNA microarrays such as the augmented NIA 16K chip and augmented RIKEN 16 K chip. Because these chips do not cover the whole genome yet, we will also identify novel DA-specific genes by the PCR-based subtractive hybridization techniques. Expression patterns of putative DA-specific genes will be tested by semi-quantitative RT-PCR using independently isolated mRNAs, and will be confirmed by in situ hybridization. Among the isolated DA-specific genes, we will first focus on putative DNA-binding transcription factors. The consensus binding sites for these putative transcription factors will be defined and their potential promoter function will be tested by cotransfection assays using cell line systems. On the basis of the mechanism of action of the novel DA-specific transcription factor(s), synthetic promoters will be developed and optimized. The optimized synthetic promoter will be subcloned in front of the reporter lacZ gene in the context of the self-inactivated lenti viral vectors. Cell type-specific expression of the reporter gene will be examined using both in vitro mesencephalic primary neuronal cultures as well as in different rat brain areas following stereotactic injection. At the later stage of this proposal, we will plan to use our developed promoter system(s) to deliver therapeutic genes (e.g., GDNF and Bcl 2) to the DA neurons and will test whether they can efficiently ameliorate behavioral symptoms in animal models of PD. The proposed research will identify and isolate genes that are selectively expressed in the mid-brain DA area on a genome-wide scale and will characterize their transcriptional regulation. Based on these mechanisms, we will devise novel and innovative DA-specific promoter systems and test them using in vitro and in vivo systems. In combination with safe viral vectors, our developed gene delivery systems can be translated clinically into gene therapy approaches for PD and other neurological disorders, in which DA neuronal system is dysregulated.
| Mauldin, Patrick D; Guimaraes, Paulo; Albin, Roger L et al. (2008) Optimal frequency for measuring health care resource utilization in Parkinson's disease using participant recall: the FS-TOO resource utilization substudy. Clin Ther 30:1553-7 |
| Kim, Dong-Wook; Chung, Sangmi; Hwang, Mikyeong et al. (2006) Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells. Stem Cells 24:557-67 |
| Chung, S; Hedlund, E; Hwang, M et al. (2005) The homeodomain transcription factor Pitx3 facilitates differentiation of mouse embryonic stem cells into AHD2-expressing dopaminergic neurons. Mol Cell Neurosci 28:241-52 |
| Hwang, Dong-Youn; Hwang, Michelle M; Kim, Han-Soo et al. (2005) Genetically engineered dopamine beta-hydroxylase gene promoters with better PHOX2-binding sites drive significantly enhanced transgene expression in a noradrenergic cell-specific manner. Mol Ther 11:132-41 |
