Using Induced Pluripotent Stem Cells to Characterize Cystic Fibrosis

Project Summary/Abstract This proposal details a 5-yr plan to prepare the candidate, Ruobing Wang, MD, for a career as an independent physician-scientist positioned to impact our understanding of pediatric lung diseases, particularly Cystic Fibrosis (CF).

As a clinician taking care of CF patients, she has identified rare individuals with homozygous F508del CFTR mutations who met clinical criteria for inclusion as: 1) CF Long-term non-progressors (LTNP), with preserved lung function 2) CF Rapid progressors (RP), whose lung function declines rapidly.

Whole exome sequencing uncovered rare missense polymorphisms in i) SCNN1 (which encodes epithelial sodium channel ENaC) in LTNPs, and ii) two genes in epithelial alternative chloride channels (ANO1 and SCL26A9) in RPs.

The central hypothesis of the proposal is that the extreme phenotypes of CF are due to the alterations in epithelial ion and fluid transport driven by these gene defects, and that the extreme-phenotype disease severity and mechanism can be modeled and studied in vitro with a reprogrammed cell-based platform.

Using a novel protocol, Dr.

Wang generated airway basal-like cells from induced pluripotent stem cells (iPSCs) which can then differentiate into basal, multi-ciliated, and secretory lineages on air-liquid interface, forming a functioning airway epithelium with intact barrier function and aberrant trans-epithelial chloride transport.

Normalization of chloride transport in these cells by CFTR gene editing confirms the reliability of her model to recapitulate CF phenotype.

The central goals of the project are to establish this novel iPSC-platform for CF disease modeling and study the epithelial function of the extreme-phenotype patients.

She is now uniquely poised to complete the aims to 1) To test whether iPSC-derived airway epithelia can serve as a platform to model airway epithelial ion and fluid transport and muco-ciliary transport in CF, 2) to model extreme-phenotype CF patient and interrogate the role of candidate modifier genes and their impact on ion and fluid transport, and 3) To establish the iPSC-platform for personalized drug response, and test the therapeutic role of pharmacologic targeting of alternative ion channel candidates.

Dr.

Wang has 80% protected time from Boston Children's Hospital (BCH) Division of Respiratory Diseases and the Department of Medicine. Her co-sponsors are 1) Dr.

Darrell Kotton at the Center for Regenerative Medicine (CReM) at Boston University (BU) with whom she has trained for the past 1.5-years, and 2) Dr. Benjamin Raby, the chief of BCH Division of Respiratory Diseases. Furthermore, Dr.

Wang has assembled a team of extraordinary scientific advisory members, each bringing their specific expertise, to assist her career development and scientific research.

A detailed training plan is presented that includes mentored research, didactic coursework, presentations at meetings, and a timeline for completion of the research aims, preparation of manuscripts, and future R01 application.

The proposed research, training plan, mentorship committee, and scientific-clinical environment at BCH and BU will position the candidate to transition to independence by the end of the award.