Our research group at [REDACTED] has had a long-standing interest in understanding the cause of several disorders that result in severe, and often times fatal forms of diarrhea in children. These diarrheal disorders are inherited, and somehow lead to poor absorption of nearly all forms of nutrients, including protein, sugars and fats. Why children with these disorders have impaired absorption of nutrients is one of the main unsolved mysteries, but they generally require life-long the daily infusion of intravenous nutrients or an intestinal transplantation to sustain proper growth and nutrition.
The goal of this grant application is to develop personalized disease-in-a-dish models that can be used to solve these and other gastrointestinal disorders that are poorly understood. Specifically, we propose to develop custom-made "diarrhea-in-a-dish" models that will use pluripotent stem (iPS) cells derived from skin biopsies of individuals with various forms of diarrhea. These iPS cells will be induced to form gut epithelium that we believe will resemble various characteristics of the subjects native intestine. We will also develop methods that are already established in mice, to isolate and expand human intestinal (somatic) stem cells in cell incubators and in fat compartments of immunodeficient mice. We believe that the resulting intestinal units can be manipulated using various commonly used tools to introduce and/or suppress genes that might control the histology and function of the gut.
We are also using newly developed genetic tools where the entire important (coding) region of the human genome is sequenced to identify genes that are defective, and thereby may account for the diarrheal disorder under investigation. This new approach generally identifies several genes that are defective, and we propose to introduce the normal forms of these various genes into the stem cell derived gut tissue to see which gene might reverse the abnormality. We believe that the combination of these various approaches will likely assist us in defining the cause of various forms of diarrhea.
While short-term bouts (acute) of diarrhea are very common, approximately 5% individuals experience chronic (>2 weeks) diarrheal symptoms, and some may be life-long. Unfortunately, Physicians and Scientist alike have a very poor understanding of why so many patients experience chronic diarrhea. While the congenital diarrheal disorders under investigation in this grant are rare conditions, improving our understanding of these types of genetic disorders will undoubtedly provide new insight into how nutrients are absorbed, and may enhance our understanding of several common but poorly understood disorders, including IBD, IBS, drug-induced and other idiopathic forms of chronic diarrhea. Developing, refining and expanding the tools described here may also set the foundation to study other common disorders, and to screen for novel drug discovery.
Chronic diarrheal disorders are common aliments among Californians, and result in frequent and prolonged hospitalizations, outpatient doctor visits and lost wages from sick leave. A subset of young children develop diarrhea shortly after birth, and some will require either daily life-long intravenous nutrition and/or intestinal transplantation. While the medical cost incurred from all forms of chronic diarrhea is daunting, we have a very poor understanding of the basis of most diarrheal conditions, and currently available drug therapies have limited efficacy.
In this grant application, we propose to use pluripotent and somatic stem cells to assist us in solving the cause of several forms of congenital diarrheal disorders. We believe that these "diarrhea-in-a-dish models" can be used in the future to understand the cause of other common forms of diarrhea, and to screen for potential drug targets. Furthermore, the approaches taken here may provide alternative advances to small bowel transplantation in these more difficult cases of chronic diarrhea.
Public and private insurers spend hundreds of millions of dollars in the diagnosing and management of children and adults with various forms of chronic diarrhea. More importantly, patients and their families are frequently immersed in the consequences of suffering from chronic aliments that are debilitating, hamper quality of life, and require frequent medical attention. Given our incomplete understanding of these conditions, the limited therapeutic options available, and the direct and indirect cost to the state of California and its citizens, novel approaches only recently available with the use of stem cell technology may provide new insight, and possible solutions and hope for those affected by these conditions.
This proposal is focused on the development of in vitro models of inherited diarrheal disorders. The applicant identifies the absence of relevant “diarrhea-in-a-dish” models as a translational bottleneck. These models could be used to understand basic disease mechanisms as well as for drug screening. The applicant proposes two specific aims: (1) to generate induced pluripotent stem cells (iPSCs) from patients with various inherited diarrheal disorders, differentiate them into intestinal epithelial cells and perform screens for potential disease-causing genes; and (2) to develop methods to grow human small bowel epithelium in culture from tissue-specific stem cells obtained from patients with inherited diarrheal disorders.
Reviewers agreed that this proposal addresses a significant bottleneck in the translation of therapies for intestinal disorders. They noted that diarrheal disorders are poorly understood, understudied and associated with significant mortality and health care costs. Thus any improvement in treatment options could have a large and meaningful impact. Reviewers found the rationale for the proposal to be logical and scientifically sound. They noted that while the methods and tools proposed are not particularly innovative, their application to the field of gastrointestinal diseases is novel and laudable.
Reviewers’ main criticism of this proposal was that there is insufficient preliminary data to predict that the specific aims will be successfully achieved. They noted that that the research plan relies heavily on techniques that are as yet unpublished or have not been established in the applicant’s laboratory. For example, there is no preliminary data suggesting that they will be able to obtain a sufficient number of crypts from small human biopsy samples to grow gut epithelium in culture. Even if this tissue is grown, there is little acknowledgement of the difficulties associated with using these primary cultures to perform the detailed and technologically complex experiments proposed. The applicant plans to transduce cultured gut organoids with lentiviral vectors, which the reviewers suggested could be extremely difficult. Given that these experiments comprise a significant portion of both aims, reviewers would have appreciated data demonstrating the feasibility of this technique. The applicant proposes to use functional assays to measure the uptake of several nutrients, experiments which are also unsupported by preliminary data. Finally, the reviewers noted that potential pitfalls and alternative approaches are only superficially discussed and quantitative success criteria are not clearly described.
The reviewers described the Principal Investigator (PI) as one of the world’s experts in rare inherited diarrheal disorders and highly qualified to oversee the project. They noted that an enormous strength of the proposal is the PI’s access to a large cohort of identified patients from which to make iPSCs and obtain biopsies. The PI is well published in the field, including articles in high impact journals that are relevant to the proposed studies. The reviewers appreciated that the PI has assembled a talented group of collaborators and generally found the research team well-qualified to carry out the proposed research.
Overall, while reviewers praised several aspects of this proposal, including the potential impact on inherited diarrheal disorders and the strong research team, they ultimately did not find the research plan feasible, due to a lack of preliminary data.
- This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was suggested, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.
- Peter Zandstra