Towards Gene Therapy of Inflammatory Bowel Disease: Questions and answers in murine models

Abstract

Inflammatory bowel disease (IBD) is an increasingly prevalent disorder for which a suitable treatment is still lacking. IBD is thought to be caused by a loss of tolerance to resident enteric bacteria in genetically prone individuals resulting in T cell activation, leukocyte infiltration, and production of pro-inflammatory mediators. In this scenario the most obvious approach of the -so called- biological therapies should be either to increase the expression of immunoregulatory genes, such as IL-10, or to block the effects of proinflammatory mediators, such as TNFα. Both approaches should help overcome the spreading, chronification and worsening of the inflammation. A number of anti-TNFα-based strategies are currently in use for IBD treatment, but any of them is entirely devoid of adverse reactions and refractoriness. The introduction of nucleic acid into target cells in order to increase or decrease the expression of a specific gene during a sustained period of time, as well as the development of efficacious and safe gene therapy vectors are sought as primary objectives to reach therapeutic efficacy in such diseases. The aim of this work was to assess the therapeutic potential of different gene therapy strategies: in one hand, employing a defective adenovirus (Ad) or CpG-free plasmids, both encoding the murine IL-10 cytokine and given by the intravenous route (chapter 1); in the other hand, employing chemically modified or not modified siRNAs to silence the proinflammatory TNFα cytokine and given by the intrarectal route (chapter 4). Both interventions were designed to treat the murine DSS colitis in C57BL/6 mice, independently. Also, the cellular and humoral immune responses generated after repetitive administrations of different Ad encoding immunoregulatory (IL-10) or not immunoregulatory (GFP) transgenes were studied in mice (chapter 2). In order to find a vector with specific colonic biodistribution to selectively alter the gene expression in this tissue, we analyzed the biodistribution and biosafety of several chimeric Ad obtained from combinations of Ad5 -with hepatic tropism- and Ad40 -with enteric tropism- elements (chapter 3). These studies allowed us to compare the therapeutic efficacy of systemic IL-10 up-regulation obtained with two totally different gene therapy vectors, and to evidence the potential of local TNFα-silencing strategies as an alternative to the systemic blockade of this cytokine after administration of monoclonal antibodies or soluble receptors. The CpG-free plasmid encoding the IL-10 gene was able to produce an important therapeutic efficacy without adverse effects at short- and mid-term time points. On the contrary, late toxicity generated after i.v. Ad5IL-10 administration caused a significant increase in the mortality rate, being therefore a short-coming for its clinical application. The siRNA TNF78 (siTNF78) with the double O-Methyl and propanediol modifications was able to induce an important phenotypic amelioration in colitic mice, higher than that of the rest of siRNAs assayed, accordingly with its increased silencing potential both in vitro and in vivo and with the absence of unwanted unspecific off-target effects. The change of the Ad5 fiber with the Ad40 short fiber has allowed our consolidated group and other members of IBDnet to generate a chimeric Ad5/40S with selective tropism for the colon. After the characterization of the potent immune response generated after repetitive administration of Ad5 in vivo as well as the confirmation of the immunomodulatory effects exerted by the IL-10 transgene in these responses, we suggest the use of IL-10 as transgene in a gene therapy vector with local biodistribution (i.e., Ad5/40S) as a promising alternative for the treatment of chronic intestinal diseases such as IBD.

Date of Award	8 Jul 2011
Original language	Undefined/Unknown
Supervisor	Ester Fernandez Gimeno (Director)