At present, there are no methods to selectively transfer genes to alveolar epithelial type II cells without also transfering them to type I cells. This is a major problem in the development of gene therapy approaches to treat diseases with type II cell involvement, including surfactant deficiencies, and acute lung injury. We have developed a way to overcomethis problem. While many aspects of vector design are being addressed, one critical area that needs more research is the nuclear import of vector DMA. Our goal is to design more effective gene therapy vectors for use in the lung by understanding the molecular mechanisms by which DMA and DNA-protein complexes are actively transported into the nucleus. We have identified a DNA sequence that increases nuclear localization and subsequent gene expression uniquely in alveolar epithelial cells. The DNA is the proximal portion of the surfactant protein C (SP-C) promoter which contains binding sites for several cell-specific transcription factors, including TTF-1, GATA-6, and TAZ. These transcription factors mediate the cell-specific transcription of this promoter, and we hypothesize that they also mediate the nuclear import of the DNA. Our working hypothesis is that transcription factors, containing nuclear localization signals (NLSs) for their nuclear import, bind to specific SP-C DNAsequences thereby 'coating' the DNA with NLSs, and allowing the DNA to utilize the NLS-mediated import machinery for nuclear entry. Further, we have developed a new technique for in vivo gene delivery using electric fields that can be used to target these nonviral type II cell specific vectors to the alveolar epithelium. Using this in vivo electroporation, we are in a unique position to test the effects of this cell-selective nuclear import sequence on type II cell transfection in animal models for acute lung injury. We hypothesize that the SP-C DNA nuclear targeting sequence will lead to gene transfer and expression only in type II cells, and not in other lung cells of living animals. This proposal is designed to test this hypothesis and will lead to the creation of new alveolar gene therapy vectors that are both cell-specific and capable of greater gene transfer efficiencies. The proposed experiments will molecularly characterize the mechanisms of alveolar epithelial cell DNA nuclear import and will extend the findings to an in vivo model system to transfer the genes for the Na+,K+-ATPase to increase alveolar fluid clearance in injured lungs.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Lung Injury, Repair, and Remodeling Study Section (LIRR)
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Harabin, Andrea L
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University of Rochester
Schools of Dentistry
United States
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Lin, X; Barravecchia, M; Kothari, P et al. (2016) ?1-Na(+),K(+)-ATPase gene therapy upregulates tight junctions to rescue lipopolysaccharide-induced acute lung injury. Gene Ther 23:489-99
Dean, David A (2013) Cell-specific targeting strategies for electroporation-mediated gene delivery in cells and animals. J Membr Biol 246:737-44
O'Reilly, Michael A; Yee, Min; Buczynski, Bradley W et al. (2012) Neonatal oxygen increases sensitivity to influenza A virus infection in adult mice by suppressing epithelial expression of Ear1. Am J Pathol 181:441-51
Yao, Hongwei; Chung, Sangwoon; Hwang, Jae-woong et al. (2012) SIRT1 protects against emphysema via FOXO3-mediated reduction of premature senescence in mice. J Clin Invest 122:2032-45
Lin, Xin; Dean, David A (2011) Gene therapy for ALI/ARDS. Crit Care Clin 27:705-18
Dean, David A; Gasiorowski, Joshua Z (2011) Microinjecting cells using a constant-flow microinjection system. Cold Spring Harb Protoc 2011:prot5590
Dean, David A; Gasiorowski, Joshua Z (2011) Nonviral gene delivery. Cold Spring Harb Protoc 2011:top101
Staversky, Rhonda J; Vitiello, Peter F; Yee, Min et al. (2010) Epithelial ablation of Bcl-XL increases sensitivity to oxygen without disrupting lung development. Am J Respir Cell Mol Biol 43:376-85
Lam, A P; Dean, D A (2010) Progress and prospects: nuclear import of nonviral vectors. Gene Ther 17:439-47
Kaufman, C D; Geiger, R C; Dean, D A (2010) Electroporation- and mechanical ventilation-mediated gene transfer to the lung. Gene Ther 17:1098-104

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