The ability to restrict gene delivery and expression to particular cell types is of paramount importance for gene therapy, since ectopic expression of a transgene could lead to deleterious host inflammatory responses or dysregulation of normal cellular functions. The same is true for delivery of drugs and chemotherapeutic agents, as well as imaging agents. At present there are only three methods to limit delivery/expression of drugs, probes, and/or genes to specific cell types and tissues: physical delivery to a desired target organ, use of ligands or antibodies for cell surface receptors, and cell-specific promoters to drive transcription in desired cell types. We have developed a fourth approach for cell-specific delivery of nonviral DNA-based vectors based on our elucidation of the mechanisms of plasmid DNA nuclear import. We have shown that the nuclear localization of plasmids in the absence of cell division is sequence-specific and requires transcription factors that bind to these sequences in the cytoplasm and facilitate DNA nuclear import. We have identified a number of DNA sequences that show cell-specificity of nuclear import because they bind to cell-specific transcription factors. To date, we have identified cell-specific DNA nuclear targeting sequences that act in smooth muscle cells, osteoblasts, endothelial cells, and alveolar type 2 epithelial cells. These sequences support DNA nuclear import and subsequent gene expression from the plasmids only in their respective cells in vitro and in living animals. Our goal now is to screen a large number of potential DNA sequences for cell-specific DNA nuclear targeting activity using a panel of cell types and develop a library for cell-specific gene and drug delivery. In this application, we propose to construct and screen a library of 1000 cell-specific mammalian promoters for nuclear import activity in an array of 100 cell types from tissues throughout the body. Quantum dot-labeled plasmids will be electroporated into cells and assayed by quantitative image analysis on an ImageStream flow cytometer. Potential DNA nuclear targeting sequences will be validated using a traditional microinjection approach and the mechanisms of their cell-specificity and nuclear import will be characterized. Finally, validated hits from the previous aims will be delivered by electroporation to individual organs in vivo to test for cell- and tissue-specific gene delivery.

Public Health Relevance

Gene therapy is an exciting and potentially very useful approach to treat a number of diseases at the molecular level. Unfortunately, when targeting genes or drugs to any tissue, one would like the delivery to be targeted and specific for the desired cell type;delivery of agents to the wrong cells could be disasterous. We have developed a new approach to limit gene delivery to desired cell types by focusing on how genes move within cells. In this proposal, we will screen a large number of DNA sequences to identify a panel of elements that can direct cell-specific delivery of genes and drugs to a set of 100 different cell types that represent tissues throughout the body. This study will create a new group of agents that can promote gene and drug delivery to specific cells while avoiding others.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM094228-03
Application #
8327223
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Ainsztein, Alexandra M
Project Start
2010-09-30
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$425,189
Indirect Cost
$149,986
Name
University of Rochester
Department
Pediatrics
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Dean, David A (2013) Cell-specific targeting strategies for electroporation-mediated gene delivery in cells and animals. J Membr Biol 246:737-44
Lin, Xin; Dean, David A (2011) Gene therapy for ALI/ARDS. Crit Care Clin 27:705-18