The papillomaviruses are epitheliotropic small DNA tumor viruses that cause both benign and malignant lesions in humans and animals. These viruses productively infect only squamous epithelia and progression of the viral life cycle is dependent on differentiation of the host cell, the keratinocyte. An oncogenic subset of the human papillomaviruses (including HPV-16, 18, 31, and 33) is associated with the vast majority of cervical cancers as well as squamous cell carcinomas in other locations. The E6 and E7 viral oncogenes interfere with the functions of p53 and pRb, respectively, and thus disrupt regulation of the cell cycle. The role of a third viral oncogene, E5, in the viral life cycle is less well understood. The L1 and L2 ORFs encode the major and minor capsid proteins, respectively. Expression of papillomavirus genes is regulated at both transcriptional and posttranscriptional levels. We are currently exploiting HPV alternative splicing to develop novel RNA-based antiviral and anticancer therapies. In addition, in the process of studying the post-transcriptional regulation of papillomavirus gene expression, we have developed considerable expertise in RNA processing and in the design and execution of isoform-specific real time QRT-PCR assays. We are continuing to apply this expertise to the analysis of viral and cellular mRNA levels and alternative splicing in collaborations with other investigators. Some of these collaborations are described below. Targeting HPV infected cells using specific trans-splicing: The ideal cancer therapy would kill cancer cells and have no effect on normal cells. The expression of spliced human papillomavirus E6/E7 pre-mRNAs by the vast majority of cervical cancers provides an absolute difference between cancer cells and normal cells that can be exploited for the specific targeting of these cells. Through a CRADA between NCI and Intronn Inc., we are investigating the use of Intronn's Spliceosome Mediated RNA Trans-splicing (SMaRT[TM]) Technology to develop a novel RNA-based suicide gene therapy for cervical cancer. SMaRT involves the trans-splicing of a pre-trans-splicing molecule (PTM) to a target pre-mRNA, effectively reprogramming genetic information at the mRNA level. SMaRT can be used to replace or repair 5' exon(s), internal exon(s), and 3' terminal exon(s) and to make chimeric mRNAs. Target specificity is obtained by tethering the PTM to the target pre-mRNA through base-pairing of the PTM binding domain (BD) to the pre-mRNA target. We have used HPV-16 as a model system to study the factors that affect SMaRT efficacy. HPV-PTMs were designed to target 5' splice sites of HPV-16. Cotransfection of PTM and HPV-16 pre-mRNA expression vectors showed up to 80 percent trans-splicing. The PTM binding domain (BD) was shown to enhance trans-splicing efficiency and confer splice site selectivity in a sequence dependent manner. Trans-splicing efficiency was influenced by PTM level and by PTM cis-splicing due to cryptic 5' splice sites within the BD. Target titration experiments demonstrated that trans-spliced mRNA levels were directly proportional to target pre-mRNA levels over a 300-fold dynamic range.
