The potential of the human osteopontin promoter and single-nucleotide polymorphisms for targeted cancer gene therapy.

Abstract

Regulatory elements of the osteopontin (opn) gene are attractive candidates for expressiontargeted gene therapy because numerous malignant cancers are marked by opn overexpression. The maximum opn promoter ((P)opn)-driven reporter intensity obtained for tested cancer cell lines was as strong (102.69%) as positive-control transfections. At the same time, (P)opn-driven reporter expression was reduced by ~90% in non-cancer cell lineages. Deletion analysis of the -922 bp region opn promoter did not confirm published reports of a repressor area within 922 bases upstream of the transcriptional start site. Further enhancements to targeting and expression were obtained through incorporation of single-nucleotide polymorphisms (SNPs) into the promoter sequence. It was found that the SNPs -443C, -155GG, -66T led to increased (P)opn-driven transfection in cancer cells (fold increase of 1.23 ~ 3.48), with a concomitant decrease in reporter expression in normal controls (fold change of 0.69). Further investigations to confirm a correlation between endogenous opn mRNA levels and (P)opn-driven reporter expression produced a surprising lack of correlation (R(2)=0.24). However, taking into account opn mRNA splicing variants showed a strong negative correlation between mRNA levels of the variant opn-a and (P) opn-driven transgene activity (R(2)=0.95). These data have implications on how future searches for expression-targeting promoters should be conducted.