Alternative splicing as a novel of means of regulating the expression of therapeutic genes

Abstract

In order to determine the potential of alternative splicing as a means of targeting the expression of therapeutic genes to tumor cells in vivo, a series of episomal plasmid -based ''splice -activated gene expression'' ( pSAGE ) vectors was generated, which contain minigene cassettes composed of various combinations of the three alternatively spliced exons present in the differentially expressed adhesion protein CD44R1 ( v8, v9, and v10 ) with or without their corresponding intronic sequences, positioned in -frame between the CD44 leader sequence and a ''leaderless'' human liver / bone / kidney alkaline phosphatase ( ALP ) cDNA. Because both the v8 -v9 and v9 -v10 introns contain multiple in -frame stop codons, the expression and enzymatic activity of ALP are dependent upon the accurate removal of intronic sequences from the pre -mRNA transcripts encoded by these constructs. The various pSAGE constructs were introduced into CD44H -positive ( T24 ) and CD44R1 -positive ( PC3 ) target cells by electroporation and transfectants selected in hygromycin B. ALP expression was determined by staining with the ALP substrate, BCIP / INT, and the transfected cells tested for their sensitivity to the inactive prodrug, etoposide phosphate. ALP -mediated dephosphorylation of etoposide phosphate generates the potent topoisomerase II inhibitor etoposide. The data obtained indicate that whereas the v8 -v9 intron is spliced in both CD44H -and CD44R1 -positive cells, the v9 -v10 intron is efficiently and accurately removed only in CD44R1 -positive cells. Furthermore, only CD44R1 -positive cells were sensitized to etoposide phosphate when transfected with the v9 -v10.ALP construct. These data emphasize the potential usefulness of alternative splicing as a novel means of targeting gene expression to tumor cells in vivo. Cancer Gene Therapy ( 2002 ) 9, 133 -141 DOI: 10.1038 / sj / cgt / 7700427 Keywords: CD44; alternative splicing; cancer gene therapy G ene therapy shows great promise as a modality in the treatment of cancer.1 As the result of progress made in recent years, there is now no shortage of candidate genes which, if expressed at a high -enough level, can either directly kill tumor cells or sensitize them to the cytotoxic effects of ionizing radiation and/ or chemotherapeutic agents.2 A major outstanding challenge is to develop a safe, effective, and practical means of targeting such genes to tumor cells in vivo while minimizing as much as possible the damage inflicted on normal tissues.3 In this regard, promising results have been obtained using engineered viral vectors that exhibit restricted cellular tropism. 4 Similarly, a number of promoter /enhancer elements have been identified, which can be used to control gene expression in particular cell types or tissues. 5,6 Although little explored, there are additional ways in which gene expression could potentially be targeted to particular cell types in vivo. Specifically, because tumor cells and normal cells differ in their ability to alternatively splice certain pre -mRNA t...

Molecular mechanisms regulating the tumor-targeting potential of splice-activated gene expression

“…4 A similar procedure was used to create the v8-v9-v10.ALP construct. Briefly, a fragment containing the CD44 v8-v9-v10 region, including the intron sequences located between v8-v9 and between v9-v10, was amplified from genomic DNA by PCR using the following primer pair: 5 0 -CCATCGATGGCAGTCA-TAGTACAACGC-3 0 (v8-5 0 ) and 5 0 -CCGCTCGAGG CGATTGACATTAGAGTTGG-3 0 (v10-3 0 ).…”

“…A PCR product of approximately 3.3 kb was isolated, digested with ClaI and XhoI and cloned into the ClaI-XhoI site of plasmid pBS.CD44L. 4 A leaderless ALP fragment was then added to the 3 0 end of this construct exactly as previously described. 4 A slightly modified procedure was used to generate v8/v9-v10.ALP, a construct in which the v8 and v9 exons are already joined together and only the intron between v9 and v10 remains.…”

“…4 A leaderless ALP fragment was then added to the 3 0 end of this construct exactly as previously described. 4 A slightly modified procedure was used to generate v8/v9-v10.ALP, a construct in which the v8 and v9 exons are already joined together and only the intron between v9 and v10 remains. Briefly, the v8/v9 (no intron) and the v9-v10 (intron containing) PCR products obtained as previously described 4 were ligated together and used as a template for a PCR reaction employing the v8-5 0 and v10-3 0 primers indicated above.…”

“…3 Among a number of recently developed approaches that have shown particular promise, we have demonstrated that alternative splicing constitutes a potentially powerful means of targeting therapeutic genes to cells that possess certain differentially expressed phenotypic characteristics. 4 Splicing is the process by which intronic sequences are removed from an initial pre-mRNA transcript generated by RNA polymerase II and the exons assembled to give a contiguous message that then undergoes translation to produce the corresponding protein product. 5 Splicing can be either constitutive, where all potential exons are included in the final transcript, or alternative, if particular exons are included or excluded in a developmentally regulated, activation-dependent and/or cell-specific manner.…”

“…11 Recently, we have extended these studies and demonstrated that differential alternative splicing of CD44 can also be exploited in the context of tumor-targeted gene therapy. 4 Specifically, we have developed a series of ''splice-activated gene expression'' (pSAGE) vectors that contain minigene constructs in which a putative therapeutic gene is linked to two or more CD44 exons along with their corresponding introns such that expression of a chimeric protein incorporating the gene of interest can only occur if the intronic sequences are completely and accurately removed from the transcript that is produced. We have evaluated this approach using alkaline phosphatase (ALP) as a model therapeutic gene.…”