This research proposal is an experimental project that will develop an optimal formulation method for gene therapy in the lungs using non-viral gene carriers. Cystic Fibrosis (CF) is a disease caused by a single-gene defect, which leads to impaired function of the CFTR protein. Although this gene was isolated more than 20 years ago, the development of safe and effective delivery strategies have been difficult to realize. A major limitation in the development of non-viral gene carriers has been the lack of basic understanding of the extracellular and intracellular barriers. For example, it was previously shown that the gene transfer efficiency of gene carriers exposed to mucus lining was dramatically reduced, when compared to mucus-depleted tissues. This is because mucus acts as the primary barrier in the CF lungs, and efficiently removes gene carriers prior to reaching the underlying airway epithelium. To address this, we will determine the rate limiting barriers through human CF mucus and human bronchial epithelial cells for efficient delivery of gene carriers. The approach to this range of studies will be one of systematic building from simple to more complex systems, with an emphasis on quantitative studies at each step.
The specific aims of this proposal are: (1) to formulate methods that produce gene carriers that are stable in CF mucus. (2) to determine and quantify gene carrier transport through human CF mucus;(3) to determine intracellular barriers to efficient gene carrier transport to the nucleus in human bronchial epithelial cells;and (4) to determine in vivo gene transfer efficiencies and nasal potential differences (NPD) in mice. We hypothesize that the identification of important extra- and intracellular barriers will guide the rational modification of the non-viral gene carriers for successful CF gene therapy.

Public Health Relevance

Cystic Fibrosis (CF) is a disease caused by a single-gene defect, which leads to impaired function of the CFTR protein responsible for chloride ion transport. Although this gene was isolated more than 20 years ago, the development of safe and effective delivery strategies have been difficult to realize. A major limitation in the development of effective non-viral gene therapy has been the lack of basic understanding of the extracellular and intracellular barriers. We hypothesize that the identification of important rate-limiting barriers will guide the rational modification of the non-viral gene carriers for successful CF gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32HL103137-02
Application #
8329751
Study Section
Special Emphasis Panel (ZRG1-F14-C (20))
Program Officer
Tigno, Xenia
Project Start
2011-08-29
Project End
2013-04-30
Budget Start
2012-08-29
Budget End
2013-04-30
Support Year
2
Fiscal Year
2012
Total Cost
$43,402
Indirect Cost
Name
Johns Hopkins University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Suk, Jung Soo; Kim, Anthony J; Trehan, Kanika et al. (2014) Lung gene therapy with highly compacted DNA nanoparticles that overcome the mucus barrier. J Control Release 178:8-17