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. Our previous studies have focused on the regulation of papillomavirus alternative splicing and polyadenylation using the bovine papillomavirus BPV-1 as a model. These studies identified multiple positive and negative regulatory elements that control splicing and polyadenylation. We have now shifted our focus to HPV-16 and HPV-31 and are examining the regulation of splicing in both acute infections and during cancer progression. Much of our effort has been focused on the development and testing of very sensitive splice-specific assays for viral mRNAs. These assays use real time quantitative RT-PCR (QRT-PCR) and are being used to monitor viral transcription and splicing in acute and productive HPV infection and during cancer progression. Of particular interest to us is splicing in the E6 region since it 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). We are also exploiting the fact that the vast majority of cervical cancers express oncogenic HPV mRNAs encoding the E6 and E7 oncoproteins to develop a novel RNA-based therapy for cervical cancer and HPV infections based on Spliceosome Mediated RNA Trans-splicing (SMaRTTM) Technology developed at Intronn LLC. 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, Intronn has developed novel RNAs known as pre-therapeutic molecules (PTMs) that efficiently trans-splice with target pre-mRNA. The PTMs are designed to specifically interact with the intron of the target pre-mRNA via base pairing, inhibiting cis-splicing and enhancing trans-splicing. SMaRTTM technology restricts the expression of a therapeutic molecule (TM) to only those cells that express the target pre-mRNA. Therefore our goal is to use this technology to express a disruptive or toxic protein only in cancer cells expressing HPV pre-mRNAs. We have designed and tested PTMs which are targeted against the E6 and E7 regions of HPV-16 and trans-splice the TM exon to HPV E6 and E7 sequences using the 5' splice sites at nt 226 and nt 880. Trans-splicing efficiency is assayed using real time QRT-PCR. Cotransfection of 293 cells with a PTM expression plasmid and a plasmid in which the HPV-16 early region is transcribed from the SV40 early promoter gave trans-splicing efficiencies up to 70%. Trans-splicing can also be detected in cervical carcinoma cell lines transfected with PTM expression plasmids, but efficiency is considerably less due at least partially to poor transfection efficiency with these cells. Future work will focus on improving delivery and trans-splicing efficiency in cervical cancers and on the development of PTMs containing toxins.
