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 #
5R01CA151964-04
Application #
8608428
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
2014-02-01
Budget End
2015-01-31
Support Year
4
Fiscal Year
2014
Total Cost
$276,390
Indirect Cost
$89,640
Name
University of North Carolina Chapel Hill
Department
None
Type
Schools of Pharmacy
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Juliano, R L; Ming, Xin; Carver, Kyle et al. (2014) Cellular uptake and intracellular trafficking of oligonucleotides: implications for oligonucleotide pharmacology. Nucleic Acid Ther 24:101-13
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
Ming, Xin; Carver, Kyle; Wu, Lin (2013) Albumin-based nanoconjugates for targeted delivery of therapeutic oligonucleotides. Biomaterials 34:7939-49
Juliano, Rudy (2013) Nanomedicine: is the wave cresting? Nat Rev Drug Discov 12:171-2
Ming, Xin; Carver, Kyle; Fisher, Michael et al. (2013) The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides. Nucleic Acids Res 41:3673-87
Juliano, R L; Carver, K; Cao, C et al. (2013) Receptors, endocytosis, and trafficking: the biological basis of targeted delivery of antisense and siRNA oligonucleotides. J Drug Target 21:27-43
Alam, Md Rowshon; Ming, Xin; Nakagawa, Osamu et al. (2013) Covalent conjugation of oligonucleotides with cell-targeting ligands. Bioorg Med Chem 21:6217-23
Paradzik, Tina; Ivic, Nives; Filic, Zelimira et al. (2013) Structure-function relationships of two paralogous single-stranded DNA-binding proteins from Streptomyces coelicolor: implication of SsbB in chromosome segregation during sporulation. Nucleic Acids Res 41:3659-72
Juliano, Rudolph L; Ming, Xin; Nakagawa, Osamu (2012) Cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides. Bioconjug Chem 23:147-57