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. Expression of papillomavirus genes is regulated at both transcriptional and posttranscriptional levels. One focus of this laboratory is the regulation of HPV-16 and HPV-31 alternative splicing in both acute infections and during cancer progression. We have developed very sensitive assays for HPV alternative splicing using real time quantitative RT-PCR (QRT-PCR). These assays are currently being used to follow alternative splicing in the E6 region which has the potential to regulate the levels of functional full length E6 protein. These studies currently use serially passaged HPV-16 expressing keratinocytes as models for cancer progression but will eventually focus on precancer and cancer cells obtained from cervical tissue using Laser Capture Microdissection (LCM). Essentially all cervical cancers express human papillomavirus E6/E7 pre-mRNAs and these pre-mRNAs undergo significant alternative splicing. We are exploiting this fact to develop a novel RNA-based suicide gene therapy for cervical cancer and HPV infections based on Spliceosome Mediated RNA Trans-splicing (SMaRTTM) Technology developed at Intronn Inc. This work is being done through a CRADA between NCI and Intronn. Trans-splicing refers to the joining together of exons from two different pre-mRNAs to form a single chimeric mRNA. Trans-splicing is thought to be very rare in mammalian cells. However, pre-trans-splicing molecules (PTMs) can efficiently and specifically trans-splice to a target pre-mRNA if they contain an antisense binding domain that tethers the PTM to the target pre-mRNA. Thus a therapeutic molecule encoded by a PTM exon will be expressed only when the specific target pre-mRNA is present. We have designed PTMs that target both the nt 226 and nt 880 5' splice sites in the HPV-16 early region. We have also designed very specific and sensitive real time QRT-PCR assays to analyze trans-splicing efficiency and specificity. Cotransfection of 293 cells with PTM and target expression plasmids converted up to 77% of the HPV pre-mRNA into chimeric trans-spliced mRNA, indicating that SMaRT can be very efficient. These assays were used to identify the factors that affect trans-splicing efficiency and specificity. The binding domain controls both the splice site specificity within a pre-mRNA as well as target pre-mRNA specificity and also has affects on efficiency. Cryptic splice sites within the binding domain promote cis-splicing of the PTM which competes with trans-splicing. Elimination of these cryptic splice sites significantly improves PTM efficiency. The absolute amount of trans-spliced mRNA depends both on the abundance of the target pre-mRNA as well as the ratio of PTM to target. In addition, pre-mRNAs expressed from endogenous genes are targeted less efficiently than those expressed from transiently transfected expression plasmids. We are currently using 5' RACE (rapid amplification of cDNA ends) to assess the specificity of SMaRT and will use this information to design PTMs with improved specificity. Future work will involve the design of PTMs containing toxins or pro-drug activating enzymes.
