Scientific Abstract

Proposal No. IBD-0194R
Principal Investigator:  Bharat Ramratnam, M.D.
Applicant Organization:  Rhode Island Hospital (Providence, U.S.A.)
Project Title:  Transient gene therapy for inflammatory bowel disease
Period of Award:  March 15, 2007  – March 14, 2009

This proposal focuses on the therapeutic potential of RNA interference (RNAi) to prevent and treat inflammatory bowel disease (IBD). RNAi refers to the sequence-specific degradation or translational inhibition of RNA mediated by short single and double stranded RNA. In human cells, the cellular introduction of short interfering (si) RNA leads to sequence specific degradation of mRNA. RNAi has emerged recently as a powerful tool to probe the function of genes of known sequence in vitro and in vivo. Investigators have proven the principle that RNAi can be used to inhibit the expression of a wide range of disease-specific genes in a variety of animal models.  We hypothesize that the power of RNAi can be harnessed to create new therapies for IBD.  Increased understanding of the molecular basis of IBD now affords opportunity for highly targeted therapy using siRNA against genes critical for maintaining the chronically active inflammatory state characteristic of IBD. Effectively targeting these siRNAs directly to the colonic mucosa in vivo to knock down corresponding mRNA expression should thereby prevent or decrease the severity of colitis in IBD without incurring the side-effects of systemically administered therapies.  Our preliminary data in mouse models demonstrate that siRNA targeting somatic and hematopoietic genes can be specifically targeted to mucosal surfaces using liposomal formulations. The siRNAs are efficiently taken up by mucosal cells along the full thickness of the mucosa and mediate durable and specific gene silencing. In preliminary experiments using the dextran sodium sulfate (DSS) model of IBD in mice, we found that mucosal application of siRNA targeting TNF-α led to relative protection from colitis.  Although severe colonic inflammation (grade 4) was evident in all mice treated with DSS alone, those treated with DSS and TNF-α siRNA had a mean grade of 1.9 in the left colon (p<0.003). Colonic TNF-α mRNA levels were 58-fold increased in the DSS alone arm but only 5.5-fold increased in mice who received liposomal TNF-α siRNA intrarectally (p <0.01). siRNA treatment was highly specific as it had no appreciable effect on levels of >100 interferon-related genes in mucosal tissue compared to liposome-only treated animals. These results provide proof of concept for the development of mucosally-delivered siRNA preparations to interrupt/inhibit the inflammatory cascade responsible for IBD.

The experiments outlined in this proposal involve the use of rectally-administered liposomal siRNAs targeting TNF-α and/or the pro-inflammatory transcription factor NFκ-B first in the DSS murine colitis model in order to dissect (1) the time-course, (2) the dose-response relationship, (3) effects on the expression of downstream pro-inflammatory molecular targets, and (4) degree and extent of colitis following RNAi-mediated gene knockdown. Determining the optimal conditions and targets for luminally-administered siRNA therapy in DSS-induced colitis will shape the design of further therapeutic trials in more complex and diverse rodent models of spontaneous colitis, and will provide a bridge to translate the potential of this powerful, highly-specific and novel therapy to clinical trials in patients with chronic colitis.  RNAi based therapies are underway in Phase I/II clinical trials in several disease areas including age-related macular degeneration and respiratory viral infections. Support from the Broad Medical Research Program would allow us to rapidly expand upon the promising results we have already obtained. New data would permit NIH grant applications and serve as a foundation for planning human studies involving RNAi mediated treatment of IBD.