Deficiency of the gas nitric oxide (NO) has been implicated in a number of gastrointestinal motility disorders including achalasia, diabetic gastroparesis, pyloric stenosis, chronic constipation, and Hirschsprung's Disease. However, the therapeutic potential of NO has been hindered by its short biological half-life in seconds, rapid diffusion, and multiple targets of action. Ideally, local and chronic delivery of NO to these deficient areas is necessary for treatment. Our broad objective is to study how embryonic and somatic stem cells may be manipulated, not simply for cell replacement therapy as has been the initial focus for their use, but as a novel means of chronic and local drug delivery or gene therapy in the adult gastrointestinal tract. Our preliminary work has revealed that embryonic stem (ES) cells can be engineered for stable production of NO. Moreover, after injection in the adult pylorus, these cells remained in the area, reconstituted the native tissues, respected the local architecture, and continued to express NO. These exciting findings support the feasibility for a novel approach to directed gene therapy in the gut. This proposal entails three specific goals using mice as the cell and animal model for this stem cell technology: (1) the establishment of ES cell lines for optimal delivery of nitric oxide using (a) fluorescent probes to help distinguish injected cells from its host and (b) a bicistronic vector which allows for selection of cells expressing high amounts of NO;(2) assessment of the histological and physiological impact of NO delivery in wild type and knockout mice lacking neuronal NO synthase to establish the pluripotency of the ES cells in adult gut and to assess the ability to complement a specific genetic defect;and (3) harvesting non-embryonic stem cells, namely from intestinal epithelium and bone marrow, for gene delivery and potential cell replacement therapy in the gut. We hypothesize that stem cells can be optimized for directed gene therapy in the adult gut. This approach appears particularly well suited for such diffusible, unstable and transiently bioactive compound such as nitric oxide. Using stem cells for delivery of gene products provides a novel and widely applicable technique for future treatment of a number of inflammatory and degenerative conditions.
PROJECT NARRATIVE The ability of stem cells to be expanded and differentiated into various tissue types holds great promise in the replacement therapy of degenerative diseases. However, the potential of stem cells to be engineered for delivery of specific products locally and chronically at sites of implantation has not been largely explored. This proposal raises a novel cell-based therapy using a gastroparetic mice model for complementing a specific deficiency in neuronal nitric oxide. This approach is uniquely suited for local and chronic delivery of such an important gaseous neurotransmitter with otherwise fleeting and diverse actions throughout the body, where traditional pharmacological approaches would not be possible. This would have great implications for the treatment of neurodegenerative diseases not only in the gut, but in a wide range pathologies prevalent in our aging Veteran population such as spinal cord injuries, male impotence, urinary incontinence in which nitric oxide play a major role, and in a wide range of neuropathies resulting from diabetes and aging.