Final Progress Report

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

A summary of project aims. 

The project revolved around introducing small RNA (e.g. siRNA) targeting host factors that have been implicated in IBD such as TNF-alpha. 

Accomplishments towards meeting those aims. 

We were able to show the following: 

     ---siRNA uptake into mucosa—primarily epithelial lining—we did not see appreciable uptake in the subepithelial compartment. These
         studies involved a siRNA conjugated to a fluorophore. As our preparations included lipid reagents (-i.e. lipofectamine 2000), we next 
         compared different formulations including carbon nanotubules. Again, the primary location of mucosal uptake was superficial in nature. 

     ---while these studies were proceeding—we also explored the utility of microRNA—and here the very interesting findings began. Basically, 
         as a  control, we PCR amplified the precursor of a microRNA species implicated in colon cancer (that targets K-RAS) and introduces it 
         in cell culture along with bona fide miRNA expression cassettes. Very surprisingly, the PCR fragment produced mature miRNA 
         species—recall that this fragment harbors no known promoter motifs. This finding was duplicated for other miRNA.
 
POSITIVE:
 
     -  si RNA can be introduced into mucosal tissue by commercially available lipid reagents 

     -  Molecules enter RNAi machinery as in cell culture 

     -  miRNA fragments can behave as synthetic siRNA upon their creation as PCR fragments or duplex oligonucleotides 

     -  miRNA work suggests that another parallel transcriptional mechanism distinct from known RNAP I/II/III and snap-IV pathways is 
         operational in human/mouse/fly cells!!!
 
NEGATIVE
 
     -  Formulation for submucosal penetration of siRNA is a huge challenge and until this is overcome, siRNA molecules as drugs have little 
        utility in  direct mucosal instillation experiments

A lay summary of the progress report. 

Human disease revolves around the aberrant expression of host factors such as nucleic acids (RNA) or proteins. In the last decade, small RNA species have emerged as a novel means to impact the expression of RNA and protein. Specifically, these small molecules bind to the RNA that encodes proteins in a sequence specific manner and leads to their degradation or non-function. IBD is characterized by the relative over expression of a set of host proteins. We asked whether these small RNA molecules, which can be made in the lab, could serve as a potential drug. Using TNF-alpha as a model, we introduced siRNA targetingTNF-alpha into the rectal cavity and found that this treatment led to lower levels of inflammation and tissue injury in a mouse model of IBD. The problem with this therapeutic platform revolved around the efficiency of delivery. Upon rectal instillation, we found that siRNA molecules largely concentrated in the peri-rectal area and that they were largely deposited into the superficial lining of the colon. Our work identified a major challenge in that we need to improve formulation of small RNA in order for them to diffuse through out the GI tract and penetrate submucosal areas that harbor the immunoregulatory cells that are involved in IBD pathogenesis.