464. Development of a Novel Gene Regulation System Downstream of Promoter

Abstract

Current regulation systems for vector delivered transgene has focused primarily on controlling promoter activity. In this study, we assayed the ability to regulate expression of transgene cassettes after vector delivery downstream of promoter activity through use of alternative splicing. A unique human beta globin intron carrying a point mutation causing aberrant splicing was engineered into the open reading frame of vector expressing reporter or therapeutic transgene cassettes under constitutively expressing CBA promoter. After introduction into target cells, vector derived mRNAs are aberrantly spliced and no functional proteins are produced. Administration of target antisense oligonucleotides (AON) specific for alternative splice site restores wild type splicing and functional protein expression. Using this approach, we transfected luciferase reporter transgene cassettes carrying IVS2-654 intron plasmids into 293 cells. 24 hr post transfection cell lysates were obtained and luciferase assays preformed. We observed 6-8 folds increase in luciferase activity after AON treatment equivalent to levels obtained with wile type intron. This restoration of correct splicing and protein expression was not dependent on location of IVS2-654 intron. Regulated expression in vivo was tested using AAV vector carry regulated transgene cassette in mouse model. One week after portal vein injection of 1010 genomes, 25mg/kg AON was administered IP. We observed an 8-10 folds increase over control that persisted for over 1 month before returning to base-line level. Long-term study shows AON regulation 1yr. post vector administration. To further optimize this regulation system, we constructed a series of intron mutants that removed 250-400nt of 5' sequences as well as engineered point mutations that would strengthen use of alternative splice sites. Results of these efforts resulted in mutant intron cassettes that reduced background expression dramatically and resulted in 160-230 folds increase in protein expression after oligo treatment.