GENE THERAPEUTIC APPROACH TO PRIMARY AND METASTATIC BRAIN-TUMORS .1. CD44 VARIANT PRE-RNA ALTERNATIVE SPLICING AS A CEPT CONTROL ELEMENT

Our laboratory and others have shown alternative splicing of up to ten exons at a discrete extracellular site to be primarily responsible fo r the generation of CD44 variant (CD44v) isoforms. Based on clear diff erences in the expression of these CD44v isoforms between normal and m alignant tissues, we believe that elucidation of the mechanisms underl ying the regulation of CD44 alternative splicing may provide a new gen e therapeutic targeting approach based on CD44 pre-mRNA processing in vivo. This strategy incorporates utilization of CD44 alternative splic ing control elements into a chimeric enzyme/prodrug therapy (CEPT), a novel modification of the virus-directed enzyme/prodrug therapy (VDEPT ) approach for the treatment of brain metastases from tumors of system ic origin. As initial steps towards the development of a gene therapeu tic approach based on targeting tumor cell expression of specific CD44 v alternatively spliced isoforms, we have: (1) developed a novel in vi vo assay system that allows the rapid analyses of potentially therapeu tic CD44 alternative splicing minigene constructs; and (2) cloned the E. coli cytosine deaminase (CD) gene and fused its enzymatically activ e domain to alternatively spliced CD44 exons (CD44/CD). Deamination of cytosine by this CD44/CD chimeric fusion protein is demonstrated in E . coil cell lysates to be equal to that of wild type cytosine deaminas e.