The objective of this proposal is to generate a regulatable target gene which can be expressed specifically in a selected site at a desired time. We plan to use this system in animals to test the ability of the gene of interest to rescue neurodegenerative disorders. To generate a regulatable system, we will take advantage of our recent finding that a carboxyl (C)-terminal mutant of the progesterone receptor fails to respond to progesterone but is capable of binding RU486 and other antagonists. Importantly, this truncated form of the receptor can activate a target gene in the presence of antagonists. To eliminate possible activation of endogenous progesterone-responsive genes we will replace the DNA binding domain of this mutated form of the progesterone receptor with the yeast transcription factor GAL4 DNA binding domain, allowing recognition of specific GAL4 binding sites. In this way, only regulatable target genes containing specific GAL4 DNA binding sites will be activated by progestin antagonists. Thus, this modified form of the progesterone receptor would be a suitable regulator of the expression of target genes in the presence of progesterone antagonists. To test the validity of this regulatable system, we will use Rat models which stimulate aspect of Parkinson's disease and Alzheimer's disease. Parkinson's disease involves the extensive loss of dopaminergic neurons in the substantia nigra. Parkinsonian phenotype can be generated in rats using 6-hydroxydopamine treatment and used to analyze the effectiveness of the regulatable gene system. For this purpose, the modified steroid receptor regulator will be co-expressed with a regulatable target capable of expressing tyrosine hydroxylase (TH), an enzyme which converts tyrosine to l-dopa and thereby stimulates the level of intrinsic dopaminergic input. Regulatable expression of TH in the striata of rats should serve as a good model for assessing the therapeutic gene treatments for Parkinson's disease. Alzheimer's disease is a complex neurodegenerative disorder of heterogeneous origins. The most common defect is the gradual loss of cholinergic function. Therefore, we would like to use the regulatory system to express neurotrophic factors, such as NGF, in the fimbria-fornix lesioned rats and to assess the ability of neurotrophins in the reduction of lesion-induced cholinergic neuronal degeneration. This approach will be useful for rescuing central cholinergic defects associated with Alzheimer's patients. Similarly, the expression of neurotrophin may enhance the survival and maintenance of dopaminergic neurons. The success of this novel approach could have an impact ont he development of gene therapies for other inheritable and acquired genetic defects.
Showing the most recent 10 out of 48 publications
