293. Optimizing the Efficacy of Spliceosome-Mediated RNA Trans-Splicing (SMaRT™) for Suicide Gene Therapy of Cervical Cancer

Abstract

SMaRT|[trade]| is a platform technology that has the ability to reprogram genetic information at the mRNA level. SMaRT involves the trans-splicing of a pre-trans-splicing molecule (PTM) to a target pre-mRNA. PTMs are RNAs that are designed to contain a binding domain (BD) complementary to a target pre-mRNA, splice elements necessary for efficient and specific trans-splicing and the coding domain. Target specificity is obtained by tethering the PTM to the target pre-mRNA through the BD in the PTM. SMaRT can be used to replace or repair 5' exon(s), internal exon(s), and 3' terminal exon(s) and to make chimeric mRNAs. We are investigating the use of SMaRT to specifically target the expression of a therapeutic molecule to cervical cancer, which is associated with oncogenic human papillomaviruses (HPVs). We have designed HPV-specific PTMs that target the pre-mRNAs encoding E6 and E7 oncoproteins and have shown efficient trans-splicing to HPV pre-mRNAs. We are using this system to study the factors that affect trans-splicing efficiency and specificity. Previously, we have shown that the BD confers both target specificity as well as splice site specificity. Trans-splicing efficiency is very high (80%) in cotransfection experiments and the amount of trans-spliced product is directly proportional to the amount of target over at least a 300-fold range. The efficiency of trans-splicing to endogenous targets is generally 10-fold lower. We investigated SMaRT by cotransfecting GFP and PTM expression vectors into cells stably expressing a mini-gene HPV pre-mRNA target and sorting cells for high and low GFP expression using flow cytometry. Those cells that expressed the most GFP also expressed the highest levels of PTM and had higher levels of trans-splicing (|[sim]|17%). In contrast, trans-splicing efficiency in low GFP-expressing cells was only 4%. We conclude from these experiments that trans-splicing to endogenous targets can be improved significantly with efficient delivery and high expression of PTMs. Cervical cancer cell lines such as CaSki and SiHa express endogenous HPV-16 pre-mRNAs, but have been refractory to efficient transfection. We have now generated recombinant adenoviral vectors expressing HPV PTMs and are using them to efficiently deliver PTMs to cervical cancer cells. Experiments are in progress to assess trans-splicing efficiency and potential therapeutic effects as a function of multiplicity of infection.