(582ap) Use of the Osteopontin Promoter for Expression-Targeted Cancer Gene Therapy

Compared to chemotherapy, expression-targeted cancer gene therapy is a promising treatment that could deliver therapeutic levels of tumor cell death without bystander effects.  To aid in reducing such side effects, it is critical to find DNA regulatory elements that are associated with strong specificity and high efficacy in cancer cells.  

Osteopontin (OPN) plays an important role in cancer invasion and metastasis and is over-expressed in various types of malignant cancers.  The regulatory elements of the opn gene are therefore attractive candidates for expression-targeted gene therapy.  The ^Popn promoter (^Popn) was extracted from the human genome via the polymerase chain reaction, and deletion analysis was used to assess important control regions within the promoter.  The engineered plasmids were used to demonstrate that ^Popn yields excellent selectivity for the cancer cells tested. The maximum ^Popn driven green fluorescent protein expression intensity in the cancer cells tested was as strong as in transfections where the cytomegalovirus promoter (^Pcmv) was used: the average value was 102.69% of the average value obtained using ^Pcmv.  However, the maximum intensity of ^Popn-driven expression in normal cells was only 11.57% of the average value obtained using ^Pcmv to drive transcription.  This combination of strength and selectivity suggests that ^Popn has excellent therapeutic potential in expression-targeted gene therapy.  For instance, the promoter could be used to safely drive the expression of pro-apoptotic genes in cancer cells.

The correlation between endogenous opn mRNA levels and ^Popn-driven transgene expression was also investigated.  As has been noted in the literature with several other promoters used for expression-targeting, there was no apparent correlation between endogenous opn mRNA levels and ^Popn-driven transgene expression.  However, when different isoforms of the OPN protein were taken into account, an obvious inverse relation was obviated with OPN-A (Pearson correlation coefficient r=-1, p=0.0003, n=4).  A possible mechanism to explain the inverse relation involves mRNA splicing in generating the various endogenous isoforms of osteopontin, where OPN-A levels may be decreased for the cell to produce the other two isoforms (OPN-B and OPN-C).  When transgenes under the control of a single exon are delivered into cells, the splicing of opn mRNA is not a factor for transgene expression.  The data observed herein suggest that, in (cancer) cells strongly utilizing ^Popn, OPN-A levels will be reduced but ^Popn-driven transgene expression will be increased.

Single-nucleotide polymorphisms (SNPs) within ^Popn were also examined.  SNPs are often overlooked in expression-targeted gene delivery, but may hold an important key to customized medicine.  We examined six different SNP combinations and found that different cell types respond differently to distinct SNP constructs.  The implications of this are that, for expression-targeted gene therapy to be successful in the clinic, plasmids should be tailored for individual patients.  A one-size-fits-all approach should no longer be expected to yield a successful genetic treatment for cancer.