The thrust of this proposal is to attain novel and fundamental insights into the cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides and the implications for pharmacological effectiveness in cancer. We have synthesized oligonucleotide conjugates bearing high affinity ligands for Integrins, G-Protein Coupled Receptors, and other receptors. We can vary the ligand type, affinity, and valency. Using these reagents we will elucidate pathways of receptor- mediated cellular uptake and trafficking. We will also manipulate those pathways utilizing both chemical inhibitors and molecular tools such as activated or dominant-negative versions of proteins involved in trafficking. We will evaluate the impact of such manipulations on the pharmacologic effectiveness of antisense and siRNA via sensitive reporter assays. We will seek to identify the uptake and trafficking pathway(s) that provide the most effective delivery to the cytosol, and nucleus, and thus allow the greatest pharmacologic effect. These studies will be performed in three different contexts. Standard two-dimensional (2-D) culture of cancer cell lines will provide a setting where molecular manipulations and quantitative confocal fluorescence microscopy can be performed with relative ease. Three-dimensional (3-D) cancer cell cultures will be used as an important intermediate that may more closely reflect the situation in tumors, but that is still amenable to manipulation. Finally, via use of intravital microscopy and other tools, we will examine the uptake, distribution, and effect of antisense and siRNA oligonucleotides in xenograft tumors. The concerted use of 2-D, 3-D and in vivo systems will provide a rich stream of correlated information that will be of importance in the design of effective strategies for receptor-mediated delivery of therapeutic oligonucleotides in cancer.

Public Health Relevance

Antisense and siRNA oligonucleotides have great potential for cancer therapeutics. However our ability to use these molecules effectively is limited by lack of detailed molecular understanding of their cellular uptake and intracellular trafficking. The current proposal will address these issues using a variety of pharmacological, molecular and imaging techniques. These studies will be pursued in single cells, multi-cellular assemblies, and tumors in vivo. This integrated approach will provide a rich stream of novel information that will enhance and expedite the development of oligonucleotides as therapeutic agents in cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA151964-02
Application #
8217099
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Arya, Suresh
Project Start
2011-04-01
Project End
2015-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
2
Fiscal Year
2012
Total Cost
$307,100
Indirect Cost
$99,600
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Pharmacy
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Yang, Bing; Ming, Xin; Abdelkafi, Hajer et al. (2016) Retro-1 Analogues Differentially Affect Oligonucleotide Delivery and Toxin Trafficking. ChemMedChem 11:2506-2510
Ming, Xin; Wu, Lin; Carver, Kyle et al. (2015) Dendritic nanoconjugates for intracellular delivery of neutral oligonucleotides. Nanoscale 7:12302-6
Yuan, Ahu; Yang, Bing; Wu, Jinhui et al. (2015) Dendritic nanoconjugates of photosensitizer for targeted photodynamic therapy. Acta Biomater 21:63-73
Hemphill, James; Liu, Qingyang; Uprety, Rajendra et al. (2015) Conditional control of alternative splicing through light-triggered splice-switching oligonucleotides. J Am Chem Soc 137:3656-62
Yang, B; Ming, X; Cao, C et al. (2015) High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides. Nucleic Acids Res 43:1987-96
Ming, Xin; Laing, Brian (2015) Bioconjugates for targeted delivery of therapeutic oligonucleotides. Adv Drug Deliv Rev 87:81-9
Yuan, Ahu; Hu, Yiqiao; Ming, Xin (2015) Dendrimer Conjugates for Light-activated Delivery of Antisense Oligonucleotides. RSC Adv 5:35195-35200
Yuan, Ahu; Laing, Brian; Hu, Yiqiao et al. (2015) Direct oligonucleotide-photosensitizer conjugates for photochemical delivery of antisense oligonucleotides. Chem Commun (Camb) 51:6678-80
Juliano, R L; Carver, K (2015) Cellular uptake and intracellular trafficking of oligonucleotides. Adv Drug Deliv Rev 87:35-45
Nakagawa, Osamu; Ming, Xin; Carver, Kyle et al. (2014) Conjugation with receptor-targeted histidine-rich peptides enhances the pharmacological effectiveness of antisense oligonucleotides. Bioconjug Chem 25:165-70

Showing the most recent 10 out of 22 publications