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 #
5R01EB005028-08
Application #
8473070
Study Section
Biomaterials and Biointerfaces Study Section (BMBI)
Program Officer
Tucker, Jessica
Project Start
2005-04-01
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
8
Fiscal Year
2013
Total Cost
$372,867
Indirect Cost
$125,767
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
Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M (2014) Detergent induction of HEK 293A cell membrane permeability measured under quiescent and superfusion conditions using whole cell patch clamp. J Phys Chem B 118:2112-23
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
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
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
Shen, Wenwen; van Dongen, Mallory A; Han, Yingchun et al. (2014) The role of caveolin-1 and syndecan-4 in the internalization of PEGylated PAMAM dendrimer polyplexes into myoblast and hepatic cells. Eur J Pharm Biopharm 88:658-63
van Dongen, Mallory A; Dougherty, Casey A; Banaszak Holl, Mark M (2014) Multivalent polymers for drug delivery and imaging: the challenges of conjugation. Biomacromolecules 15:3215-34
Matz, Rebecca L; Erickson, Blake; Vaidyanathan, Sriram et al. (2013) Polyplex exposure inhibits cell cycle, increases inflammatory response, and can cause protein expression without cell division. Mol Pharm 10:1306-17
van Dongen, Mallory A; Desai, Ankur; Orr, Bradford G et al. (2013) Quantitative analysis of generation and branch defects in G5 poly(amidoamine) dendrimer. Polymer (Guildf) 54:4126-4133
Mills, Maria; Orr, Bradford G; Banaszak Holl, Mark M et al. (2013) Attractive hydration forces in DNA-dendrimer interactions on the nanometer scale. J Phys Chem B 117:973-81
van Dongen, Mallory A; Vaidyanathan, S; Banaszak Holl, Mark M (2013) PAMAM Dendrimers as Quantized Building Blocks for Novel Nanostructures. Soft Matter 9:

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