Final Progress Report

Proposal No. IBD-0078
Principal Investigator:  Takeshi Sano, Ph.D.
Applicant Organization:  Beth Israel Deaconess Medical Center (Boston, Massachusetts, U.S.A.)
Project Title:  A colon-directed gene transfer technology for gene therapy of inflammatory bowel disease
Period of Award:  November 1, 2003 - October 31, 2005

A.  Summary of project aims

The long-term goal of this project is to develop a safe, efficient gene transfer technology for the colorectal system toward the realization of gene therapy of inflammatory bowel disease (IBD). This project aims to develop strategies for safe, focused virus-mediated delivery of therapeutic transgenes to inflamed lesions in the colon. The development of such strategies is based on our novel gene transfer technology, in which viral vectors [adenoviral vectors (Ad5) derived from adenovirus serotype 5, and adeno-associated viral vectors (AAV) derived from adeno-associate virus serotype 2] are attached stably to the surfaces of microbeads and delivered to target cells in the form of virus-microbead conjugates. Such virus-microbead conjugates can infect cells at efficiencies equivalent to or even greater than free viral vectors in solution. In particular, the transduction of target cells, which are poorly permissive to infection by free viral vectors, can be considerably enhanced, providing the attached viral vectors with broader tropism. Our original hypothesis was that this gene transfer technology allows for efficient transduction of the colon upon intra-colonical administration of virus-microbead conjugates. If this hypothesis is proven to be correct, this gene transfer technology can be used to develop an effective gene therapy strategy for IBD.

This project consisted of two aims. The first aim was to investigate, by using a mouse colitis model, if virus-microbead conjugates containing Ad5 or AAV could be used for efficient delivery of transgenes to inflamed lesions in the colon. The second aim was to test, by using a mouse colitis model, the efficacy of the focused delivery of the interleukin-10 (IL-10) gene to the colon using virus-microbead conjugates for the amelioration of colitis.

B.  Accomplishments toward meeting those aims and a list of significant results

In this project, we made several key achievements. The major experimental findings are summarized below:

•  Ad5 can transduce the colon with chemically induced colitis in mice efficiently when Ad5 is administered intra-colonically by enema in the form of Ad5-microbead conjugates containing a lectin, concanavalin A (Con A). Con A serves both as an anchoring agent and as an infectivity enhancer for Ad5-microbead conjugates. The co-attachment of Con A was found to be essential for enhanced transduction of the colon with colitis.
• The transduction of the colon in mice by Ad5-microbead conjugates containing Con A was dependent on the status of the mucosa. The transduction of the colon was poor when mucosal layers were intact (normal colon), but it was enhanced considerably following the destruction of the mucosal layer, caused by the development of colitis. This means that Ad5-microbead conjugates containing Con A could serve as efficient gene transfer agents with selective transduction of inflammation sites where the destruction of the mucosa has occurred.
• Ad5 carrying the gene for mouse IL-10, a potent anti-inflammatory factor, showed a potential to provide enhanced amelioration of colitis in a mouse colitis model when administered intra-colonically by enema in the form of Ad5-microbead conjugates containing Con A. This enhanced therapeutic effectiveness may be derived from the fact that intra-colonical administration of Ad5 carrying the IL-10 gene in the form of Ad5-microbead conjugates containing Con A can considerably raise local IL-10 levels in the colon by efficient transduction of inflammation sites where the destruction of the mucosa has occurred.
• Virus-microbead conjugates, prepared by using AAV (AAV-microbead conjugates), efficiently transduced, in vitro, colonic cell lines that are poorly permissive to infection by free AAV, when Con A was co-attached to the conjugates. This infectivity enhancement by the co-attachment of Con A is derived, presumably, from the ability of AAV-microbead conjugates containing Con A to associate stably with target cells.
• AAV showed the potential ability to efficiently transduce the colon with chemically induced colitis when administered intra-colonically by enema in the form of AAV-microbead conjugates containing Con A.

C.  Lay summary

Inflammatory bowel disease (IBD), which consists of Crohn's disease and ulcerative colitis, involves chronic intestinal inflammation, which causes severe destruction of the mucosa. An intense, local immune response is likely to be responsible for the initiation and progression of the inflammatory processes. Thus, one potential therapeutic strategy for IBD is to repress such inflammatory processes by using anti-inflammatory cytokines. Although daily administration of certain anti-inflammatory cytokines has shown some therapeutic effect for Crohn's disease, a few critical problems with this strategy make its application to the therapy of IBD unrealistic. Gene therapy approaches are very attractive as therapeutic strategies for IBD, since localized, long-term expression of anti-inflammatory cytokines at inflamed lesions in the colon is potentially achievable. In fact, a few recent studies have demonstrated the potential efficacy of the local delivery of the genes for anti-inflammatory cytokines to the colon as a therapeutic strategy for IBD. One genuine concern in an effort to bring this and other gene therapy approaches to the clinical stage is with the safety in using viral vectors in human subjects. The need for a much greater attention to safety than previously considered has now become apparent for any gene therapy protocol prior to its implementation in human subjects. This project aimed to develop strategies for safe, focused virus-mediated delivery of therapeutic transgenes to inflamed lesions in the colon toward the realization of gene therapy of IBD. This effort was based on our novel gene transfer technology, which can offer enormous enhancements of both safety and efficiency to virus-mediated gene transfer strategies. By using two viral vector species, we have demonstrated in a mouse colitis model that viral vectors can efficiently be delivered to inflamed lesions in the colon by using our gene transfer technology. When viral vectors carrying the gene for a potent anti-inflammatory cytokine, interleukin-10, were delivered to the colon in a mouse colitis model by using our gene transfer technology, the local IL-10 level in the colon was raised considerably, resulting in enhanced amelioration of colitis. These experimental results suggest great potential that our gene transfer technology can be used to develop an effective therapeutic modality for IBD, which could used either alone as a gene therapy strategy or in combination with other therapeutic modalities, such as drug therapy and surgery.