Basic Biology III
Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasion population affecting approximately 1 in 3000 births. The ΔF508 mutation in the cystic fibrosis transmembrane receptor CFTR is the predominant cause of CF. The mutant CFTR channel dysregulates ionic homeostasis of several essential ions, and causes the loss of cell surface conductance, elevated mucus levels, bronchial obstruction, bacterial infections, inflammation, premature lung failure and a shortened lifespan. Despite recent advancements in CF research, current treatments, such as antibiotics, mucolytics, dietary supplements, and asthma therapy slow disease progression by alleviating the symptoms, while not addressing the cause. Due to the difficulty of obtaining primary pulmonary cells from patients, information regarding mutant ΔF508 CFTR behavior has been generated from immortalized cell lines containing artificial reporter constructs. Although these reporter constructs provide an opportunity to approach misfolding diseases like CF, artificial systems can dramatically differ from primary cell sources, which are very limiting. In addition to cell culture models, there are several CF mouse models available. However, many of these models only display the gastrointestinal symptoms associated with CF, but do not recapitulate the bronchial pathology responsible for premature lung failure. By using potent and selective HDACi and selectively silencing expression of histone deacetylases, we have found that histone deacetylase 7 (HDAC7) plays a critical role in regulating the ΔF508 CFTR mutant. This application is aimed at understanding the molecular basis of how HDAC7 silencing impacts the cellular environment in manner that functionally trafficks the CFTR to the surface instead of being degraded. CF induced pluripotent stem cells (iPSCs) provide us with an opportunity to explore HDAC7 as a master regulator of ΔF508 CFTR function in an endogenous setting. By introducing a fluorescent protein reporter sensitive to select ions, we can monitor CFTR activity levels during propagation of iPSCs and differentiation to primary lung cells. We have shown that silencing HDAC7 changes the levels of several chaperone proteins that participate in CFTR regulation. We now would like to pursue what their roles are and how they relate to HDAC7 mediated pathways. By looking at genes directly associated HDAC7, transcriptional changes that occur in its absence, and proteins interacting with CFTR, we can distill a set of critical proteins/pathways responsible for rescuing ΔF508 CFTR function. In addition, iPSCs provide a unique opportunity to discern if environmental changes are carried from progenitor to terminally differentiated cells and to what extent. Ultimately, these studies will contribute to our ongoing understanding of pliable cellular systems and how subtle changes in pathways associated with pathogenic genes can have tremendous impact on disease severity.
Statement of Benefit to California:
The availability of primary lung cells from patients has been a limiting reagent in the efforts to approach diseases like Cystic Fibrosis. With the recent developments in induced pluripotent cells derived from patient skin cells, we have the opportunity to move the field into a more relevant endogenous setting, by generating primary lung cells from a renewable resource. The studies proposed in this application will yield information on how we can modulate key elements in the cellular environment to restore functionality to misfolded pathogenic proteins. Not only will be of benefit to the State of California, but to therapeutic approaches in general.
Project Synopsis: The goal of the proposed research is to use disease-in-a-dish modeling to elucidate molecular mechanisms relevant to cystic fibrosis (CF) and its potential treatment. The applicant will employ induced pluripotent stem cells (iPSC) derived from a patient harboring deltaF508, a common disease-causing mutation in the cystic fibrosis transmembrane regulator protein (CFTR) that prevents this ion channel from being properly trafficked to the cell surface. The goal of Aim 1 is to explore the role of histone deacetylase 7 (HDAC7) in restoring deltaF508 CFTR function at the cell surface. In Aim 2, the applicant will investigate the molecular mechanisms responsible for this HDAC7 mediated rescue of the CF phenotype. Significance and Innovation: - The rationale for employing iPSC in these studies, as opposed to using immortalized lung cells, was not convincing; the applicant did not provide compelling arguments for their use. - The proposed research is innovative, particularly in its use of small molecule inhibition to modulate CFTR activity. - The significance of this work is potentially high, as a better understanding of the role of HDAC7 on mutant CFTR processing could lead to improved treatments for CF. Feasibility and Experimental Design: - A major weakness is the lack of detailed and defined plans for the stem cell component of this project and, particularly, the differentiation studies. - The preliminary data provided no evidence that the applicant has experience or proficiency with any of the required techniques. Of greatest concern, there is no evidence that the applicant can derive lung cells from iPSC. - Reviewers found the quality of the iPSC shown in the preliminary data to be questionable. It is not clear that the necessary expertise for deriving and characterizing iPSC cells is established in the applicant’s laboratory. - The experimental design lacked essential information about data interpretation, anticipated results, potential pitfalls, or alternative approaches. - A list of candidate genes has already been generated and described in the preliminary data; the extent to which Aim 2 would improve on those results is unclear. - The preliminary data convincingly demonstrate the ability of HDAC inhibitors to improve mutant CFTR folding and trafficking. Principal Investigator (PI) and Research Team: - The principal investigator (PI) is well suited to investigate the action of HDAC and CFTR in lung cells. However, he/she lacks substantial experience with pluripotent stem cells and has not demonstrated any ability to derive lung cells from iPSC. - The actual status and independence of the PI within the applicant institution is unclear, and there are no letters of support or collaboration accompanying the application. - There is no evidence that the PI has previously directed a research project, and he/she has no publications as senior author. - The proposed experiments will require a large research infrastructure, and it is not clear that this will be available to the PI. - The research team comprises only two personnel, the PI and an unnamed postdoctoral scientist. Both are committing 100% effort. Responsiveness to RFA: - The proposed research exploits human iPSC to study the molecular basis of disease. As such, the project adequately and appropriately addresses the goals and objectives of the RFA.