Millions of individuals in the US are estimated to suffer from drug addiction, associated with aggravated health problems and thousands of deaths annually. Cocaine, which binds to the dopamine receptor 2 (D2R), is the most widely abused drug. The observation that greater cocaine sensitivity is associated with reduced D2R or MAO A expression seems counterintuitive. We hypothesize that external positron emission tomographic (PET) imaging and quantitation of neuronal mRNAs that encode D2R or MAO A will enable realtime analysis of cocaine sensitivity in preclinical models and human subjects. We have designed and demonstrated a novel technology to visualize intracellular mRNAs from outside the body. The mRNA targeting agents are peptide nucleic acids (PNAs). When radiolabeled and administered i.v., these sequences hybridize specifically to mRNA copies of activated genes. We added a small peptide analog to allow the mRNA imaging agents to be taken up by target cells that express a characteristic cell surface receptor. Finally, we added a chelator to bind a positron-emitting radionuclide to the mRNA imaging agents to permit external PET imaging. Tail vein administration of mRNA imaging agents for CCND1, IRS1, MYCC, and KRAS mRNAs have enabled us to visualize gene expression in live mice bearing breast cancer, pancreas cancer, and prostate cancer xenografts. Mismatch probes and control cells yielded background signals. Other laboratories have demonstrated that PNA conjugates can cross the blood-brain barrier. For endocytosis of mRNA imaging agents specifically into neuronal cells, we need to use a characteristic neuronal receptor. High levels of mu opioid receptor 2 (MOPR) are highly expressed by neuronal cells that also express D2R and MAOA proteins. An enkephalin derivative, N-Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO), binds tightly to MOPR and induces internalization. Based on the above observations, we propose a pilot study to design and test novel mRNA PET imaging agents for D2R and MAOA mRNAs expressed in neuronal cells, through two specific aims.
Specific Aim 1 : We will design, synthesize, purify, and characterize D2R and MAOA mRNA imaging agents with DAMGO specific for MOPR for neuronal cell endocytosis. We will also prepare fluorescent probes for confocal microscopy studies. For both gene targets, we will also synthesize and evaluate PNA and peptide mismatch controls.
Specific Aim 2 : We will determine whether or not the D2R and MAOA mRNA imaging agents show greater than or equal to 3-fold greater accumulation in CHO-HArMOR cells in culture, vs. mismatch controls, and vs. control cells that do not express MOPR. Fluorescent probes will be used to study endocytosis and intracellular trafficking. If DAMGO fails to promote cytoplasmic localization, alternate enkephalin derivatives will be selected and tested. Once we have identified a ligand that induces MOPR-mediated endocytosis, Radiolabeled mRNA imaging agents will be used to quantitate uptake. Results consistent with our hypothesis would permit testing of mRNA imaging agents for neuronal mRNAs in animal models.
We propose to develop a genetic nuclear medicine procedure to reveal and measure the activity of brain cell genes from outside the body. We will study connections between cocaine sensitivity and brain cell gene activity with our new method. In the future, beyond the scope of this proposal, nuclear medicine imaging of brain cell gene activity in humans might explain why some individuals become addicted more easily than others.
|Wickstrom, Eric (2015) DNA and RNA derivatives to optimize distribution and delivery. Adv Drug Deliv Rev 87:25-34|
|Sethi, Dalip; Chen, Chang-Po; Jing, Rui-Yan et al. (2012) Fluorescent peptide-PNA chimeras for imaging monoamine oxidase A mRNA in neuronal cells. Bioconjug Chem 23:158-63|