670. Intracellular Shigatoxin 1A1 Delivered by Adenovirus-Mediated Segmental Trans-Splicing Causes Apototic Cell Death In Vitro and In Vivo
Authors: Katsutoshi Nakayama, Robert G. Pergolizzi, Ronald G. Crystal
Journal: Molecular Therapy (2004)

The concept of virus-mediated gene transfer of intracellular toxins, that kill the cell in which they are expressed, holds promise for cancer therapy and ablation of excess non-malignant tissues. However, it has been difficult to produce the virus vectors coding for toxin genes due to the intrinsic dilemma that even low level expression of toxin molecules kills the virus producing cells. To overcome this problem and to develop a virus vector system for delivering intracellular toxins to target cells, we employed a strategy we call segmental trans-splicing (STS), in which two engineered DNA fragments coding for 5' (“donor”) and 3' (“acceptor”) segments of a toxin gene, respectively, are constructed in virus vectors. Neither of these fragments alone generates a toxic product, but when co-delivered to target cells, the two vectors generate two toxin pre-mRNA fragments which are spliced by the target cell spliceosomes in the nucleus to produce mature, functional mRNA coding for the toxin. As an example of using STS delivery of an intracellular toxin to treat established tumors, we designed STS vectors to deliver shigatoxin, a specific N-glycosidase that cleaves a single adenine residue from 28S ribosomal RNA and provokes the inhibition of protein synthesis and apoptotic cell death. As a model, we used the shigatoxin 1A1 (Stx1A1) gene, an enzymatic fragment of shigatoxin 1 derived from a pathogenic E. coli strain. The Stx1A1 coding sequences were split into two adenovirus (Ad) vectors, the 5' sequences were inserted into the “Shigtoxin1A1 donor” (AdStxDo) and the 3' sequences into the “Shigatoxin1A1 acceptor” (AdStxAc), respectively. RT-PCR analysis of RNA from HeLa cells infected with AdStxDo and AdStxAc showed expression of correctly trans-spliced mature mRNA for Stx1A1, confirmed by sequencing analysis. HeLa, HEp2, and A549 cells infected with AdStxDo and AdStxAc (104 particle units/cell) demonstrated a dose-dependant inhibition of protein synthesis and reduction in cell viability. HeLa and HEp2 cells showed apoptotic DNA fragmentation. To demonstrate the ability of the combination of AdStxDo and AdStxAc to suppress tumor growth in vivo, 5×1010 particle units of AdStxDo and AdStxAc injected into pre-existing HeLa, HEp2, and A549 flank tumors established in immunodeficient mice (HeLa and HEp2 in BALB/c scid mice, A549 in BALB/c nu/nu mice). Over time, there was significant inhibition of tumor growth in all 3 tumor types (% reduction in tumor volume compared to PBS control on day 24: HeLa- 29%, HEp2- 49%, A549- 43%; all p<0.05). In contrast, no inhibition of tumor growth was observed after administration of the controls, including PBS, AdNull, AdStxDo alone, AdStxAc alone, or either Stx vector in combination with AdNull. Despite the tropism of Ad vectors for the liver, intratumoral injection of AdStxDo and AdStxAc did not result in expression of Stx1A1 in liver, as assessed by RT-PCR and serum liver enzyme analysis, indicating that no significant leakage of vector or toxin occurred from the injection site. These result suggest that STS may provide a novel paradigm for the delivery of intracellular toxins such as Stx1A1 by gene transfer, and that this strategy is an effective method for local cancer therapy.