The development of non-viral vectors for oligonucleotide delivery of plasmid DNA (pDNA), antisense DNA (asDNA), and small interfering RNA (siRNA) remains a substantial scientific challenge. Current methods suffer from low transgene expression levels (pDNA) and problems with transport inside the cell and degradation in the cytoplasm (pDNA, asDNA, siRNA). This research program will test the role of cell plasma membrane disruption in triggering cellular responses that inhibit transfection and/or expression. The structure and dynamics of the nanoscale pores induced in the membrane will be explored and their relationship to the triggering of cellular defense mechanisms determined. The creation of nanoscale pores in the cell membrane will be examined to see if they cause an increase in cytoplasmic nuclease activity. Understanding the cellular responses induced by the non-viral vectors is critical to rational development of these oligonucleotide delivery agents.
The specific aims of this research are: 1) Assessment of polymer and polyplex cell membrane disruption 2) Assessment and quantification of the role of plasma membrane permeability in triggering cellular defense mechanisms that inhibit transfection and expression. 3) Design and quantification of polyplex structure.

Public Health Relevance

This work is important to public health because it will uncover the details of cellular mechanism that inhibit efficient use of gene therapies. Gaining understanding of how non-viral vectors activate cell-based defenses against the introduction of foreign oligonucleotides will allow rational optimization of vector design.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
2R01EB005028-05
Application #
7985421
Study Section
Biomaterials and Biointerfaces Study Section (BMBI)
Program Officer
Zullo, Steven J
Project Start
2005-04-01
Project End
2014-05-31
Budget Start
2010-08-15
Budget End
2011-05-31
Support Year
5
Fiscal Year
2010
Total Cost
$448,589
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Merzel, Rachel L; Orr, Bradford G; Banaszak Holl, Mark M (2018) Distributions: The Importance of the Chemist's Molecular View for Biological Materials. Biomacromolecules 19:1469-1484
Shakya, Anisha; Dougherty, Casey A; Xue, Yi et al. (2016) Rapid Exchange Between Free and Bound States in RNA-Dendrimer Polyplexes: Implications on the Mechanism of Delivery and Release. Biomacromolecules 17:154-64
Vaidyanathan, Sriram; Chen, Junjie; Orr, Bradford G et al. (2016) Cationic Polymer Intercalation into the Lipid Membrane Enables Intact Polyplex DNA Escape from Endosomes for Gene Delivery. Mol Pharm 13:1967-78
Dougherty, Casey A; Vaidyanathan, Sriram; Orr, Bradford G et al. (2015) Fluorophore:dendrimer ratio impacts cellular uptake and intracellular fluorescence lifetime. Bioconjug Chem 26:304-15
Ma, Qian; Han, Yingchun; Chen, Cong et al. (2015) Oral absorption enhancement of probucol by PEGylated G5 PAMAM dendrimer modified nanoliposomes. Mol Pharm 12:665-74
Manono, Janet; Dougherty, Casey A; Jones, Kirsten et al. (2015) Generation 3 PAMAM dendrimer TAMRA conjugates containing precise dye/dendrimer ratios. Mater Today (Kidlington) 4:86-92
Vaidyanathan, Sriram; Anderson, Kevin B; Merzel, Rachel L et al. (2015) Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane. ACS Nano 9:6097-109
van Dongen, Mallory A; Orr, Bradford G; Banaszak Holl, Mark M (2014) Diffusion NMR study of generation-five PAMAM dendrimer materials. J Phys Chem B 118:7195-202
Rattan, Rahul; Bielinska, Anna U; Banaszak Holl, Mark M (2014) Quantification of cytosolic plasmid DNA degradation using high-throughput sequencing: implications for gene delivery. J Gene Med 16:75-83
Dougherty, Casey A; Furgal, Joseph C; van Dongen, Mallory A et al. (2014) Isolation and characterization of precise dye/dendrimer ratios. Chemistry 20:4638-45

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