A novel approach for gene therapy

DESCRIPTION (provided by applicant): Many human diseases have been traced back to single genetic mutations in the genome that are causative for the disorder, such as Lesch-Nyhan Syndrome, Cystic Fibrosis, Tay-Sachs disease and Duchenne muscular dystrophy. The goal of this proposal is to proof the feasibility of a newly developed technology for gene- therapy that targets RNA transcripts of mutated genes. The targeting of RNA molecules has several competitive advantages over strategies to correct disease-caus ing mutations with DNA gene-therapy, which to date have had very limited success in the clinic. There is a high need for the development of new gene- therapeutic technologies since for many genetic diseases there is currently either no effective treatment available or limited to alleviating disease symptoms. RNA directed gene-therapy corrects mutations in short- lived, transient messenger molecules and therefore avoids potential side-effects caused by unwanted changes in genomic DNA sequences that are due t o nonspecific activity of DNA targeting drugs. Treating mutated messenger molecules also conserves the natural expression level of the target gene, whereas gene-replacement strategies can lead to artificial levels of expression. To proof feasibility of thi s new, proprietary approach we have chosen to design a RNA targeting molecule that corrects a disease-causing mutation in hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene transcripts known to cause Lesch-Nyhan syndrome. The specific aims of the p roposed studies are to 1) design, test and optimize a constructed protein that recognizes a specific, previously characterized ssRNA sequence and selectively repairs a single nucleotide mutation within transcripts of a luciferase reporter gene; and 2) usin g expression cloning to develop targeting molecules that specifically bind to HPRT gene transcripts and correct a known mutation that causes Lesch-Nyhan syndrome on the RNA level. Cellular transfection assays of targeting molecules and reporters will be us ed to assess the selectivity and efficiency of the targeting molecules. Selection of functional proteins will be achieved through reporter gene activity measurements of transfected cells and gene modification activity and specificity will be assessed throu gh RT-PCR and sequence analysis.