Alternative splicing as a novel of means of regulating the expression of therapeutic genes.
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.
Hayes
Gregory M
GM
Department of Radiation Oncology, UCLA Center for Health Sciences, Los Angeles, California 90095-1724, USA.
Carpenito
Carmine
C
Davis
Peter D
PD
Dougherty
Shona T
ST
Dirks
Julie F
JF
Dougherty
Graeme J
GJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D017398:Alternative Splicing; D000970:Antineoplastic Agents / Q000627:therapeutic use; D015153:Blotting, Western; D002295:Carcinoma, Transitional Cell / Q000628:therapy*; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers / Q000737:chemistry; D005091:Exons; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D015316:Genetic Therapy; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics* / Q000378:metabolism; D007438:Introns; D008297:Male; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011471:Prostatic Neoplasms / Q000235:genetics / Q000628:therapy*; D014162:Transfection; D014410:Tumor Stem Cell Assay; D001749:Urinary Bladder Neoplasms / Q000628:therapy*
D000970:Antineoplastic Agents; D017931:DNA Primers; D018960:Hyaluronan Receptors; D000469:Alkaline Phosphatase