Inverse Genomics as a powerful tool to identify novel targets for the treatment of neurodegenerative diseases.
Toward the prevention of neurodegeneration we have used Immusol's Inverse Genomics platform to identify gene targets involved in neuronal cell death. Inverse genomics uses a combinatorial library of unique hairpin ribozymes with randomized substrate binding sequences to cleave unique RNA transcripts, thereby decreasing translation of the encoded proteins. Using the SK-N-MC neuroblastoma cell line a cell based survival selection assay was designed with C2-ceramide or TNFalpha as an induction signal for apoptosis. SK-N-MC cells were stably transduced with a ribozyme vector library, and then exposed to 20 microM C2-ceramide or 50 ng/ml TNFalpha to induce cell death. Surviving cells were harvested, their DNA isolated, and the ribozymes rescued by PCR for re-introduction into fresh cells. After several rounds of selection and ribozyme rescue we have identified individual ribozymes that protect neuronal cells from C2-ceramide induced apoptosis. Three of the cellular targets of these ribozyme sequence tags have been validated. Microarray analysis and yeast two-hybrid screens have also been used to gain insight into the pathways involved by identifying additional players involved in these pathways. These target genes may also serve as therapeutic targets for development of drugs for Alzheimer and Parkinson's diseases.
Rhoades
Kristina
K
Immusol Incorporated, 10790 Roselle Street, San Diego, CA 92121, USA. trhoades@immusol.com <trhoades@immusol.com>
Wong-Staal
Flossie
F
D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D023281:Genomics / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D019636:Neurodegenerative Diseases / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D009474:Neurons / Q000473:pathology; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use; D020798:Two-Hybrid System Techniques
D016337:RNA, Catalytic; C418481:hairpin ribozyme