143. Ribozyme Gene Delivery for Gene Target Discovery and Functional Validation 

Xinqiang Li, Peter J. Welch, Mark C. Leavitt, Flossie Wong-Staal, and Jack R. Barber 
Immusol, Inc., 3050 Science Park Road, Second Floor, San Diego, CA 92121  
barber@immusol.com 

Ribozymes (Rzs) are RNA molecules that can be engineered to cleave and inactivate other RNA molecules in a sequence-specific fashion. Thus, Rzs can be designed to selectively inactivate the expression of any target gene ("gene knockdown") and its corresponding protein. We have used Rz genes, delivered with viral vectors, as a tool for gene functional validation and discovery. We have used hairpin ribozyme gene delivery to rapidly and effectively inhibit expression of a number of viral and cellular genes.  

To expedite the process of associating genes with cellular function, we have developed Rz gene vector libraries. Retroviral and Adenoassociated viral vectors have been generated that efficiently deliver and express Rz genes whose target recognition sequences have been randomized, generating a library of Rz genes capable of recognizing a total of more than 1x107 possible gene target sequences. The library of Rz genes is delivered into large numbers of tissue culture cells (one Rz gene per cell for each Rz gene in the library), followed by selection for individual cells that have lost a particular function. The sequence of the Rz target recognition domain thus identified allows the identification and cloning of genes that are necessary for a given cellular function. As an example of the power of the technology, we will present data demonstrating its use to identify a novel tumor suppressor gene, without prior sequence information.