This project will develop and explore Magnetic Resonance (MR) visible probes to image intracellular gene transcription activator proteins. Drug addiction is a major health problem that severely hampers the productivity of many members of our society. While studies indicate that drug addiction results from combined influences of genes and environment, recent studies suggest that epigenetic modifications of gene transcription factors may play an important role in the development of addictions in humans. Advances in cellular and molecular biology in the past century have led to identification of novel gene markers, the signaling pathways they influence and gene activities they regulate. Two such activities that they have shown modification in in studies of drug addiction are intracellular activator protein-1 (AP-1) and nuclear factor kappa- beta (NF-k-b). The inability to produce a functional AP-1 protein to bind at the AP-1 site in a transgenic mouse blocks sensitization of drugs of abuse. With biopsy the only source of tissue for conventional assays, such studies are not permitted in humans.
We aim to develop and investigate a targeted MR imaging technique and to apply it for studies of drug addiction in a series of live brains. With a long-term goal of enabling specific manipulation of epigenetic process to non-human primates (NHP), we formulate a hypothesis: short double- stranded (ds) DNA aptamers with the sequence domain for AP-1 protein will function as decoys for endogenous AP-1 protein binding. The aptamers will be linked to an MR-visible contrast agent (superparamagnetic iron oxide nanoparticles-NeutrAvidin, or SPION-NA, 49 nm in diameter), which permits imaging AP-1 proteins in live animal subjects. We have outlined the following steps to validate our hypothesis: Step 1. Design a nuclease-resistant dsAP-1 aptamer with high affinity for AP-1 protein binding Step 2. Quantify rhodamine (Rhd)-labeled dsAP-1 aptamer binding in vitro Step 3. Demonstrate SPION-dsAP1 delivery for MRI and compare in vivo MR signal in C57black6 mice and a mutant strain known to produce no or less AP-1 protein (A-FOS/NSE or FosB knockout mutants) Step 4. Demonstrate Rhd-dsAP1 uptake specificity in cells that express green fluorescence protein (GFP) directed by AP-1 protein [B6;DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J] Step 5. Demonstrate dsAP-1 at high dose blocks AP-1 protein-induced activity in mice after amphetamine Step 6. Demonstrate that SPION-dsAP1 delivery allows a window for MRI in live non-human primates and detects elevation of AP-1 protein after amphetamine exposure This application is designed to enable highly innovative and conceptually creative research of molecular targeting of gene products using an unconventional, novel targeted MRI technique for in vivo systems. Because transcription factor binding domains are conserved from rodents to humans, our innovative technique, once validated, has potential for theranostic application, from rodents to primates.
(Aptamer Imaging in Non-human Primates: A Theranostic Strategy for Substance Abuse) This project will develop and explore magnetic resonance (MR) visible probes to image intracellular gene transcription activator proteins. The underlying mechanism that is associated with gene activities regulated by activator proteins after exposure to drugs of abuse is not totally understood, but it occurs across the general population, affecting all ages, genders and races. There are few assays to assess intracellular proteins in the brain except in biopsy or post mortem samples. We will examine if this novel technique offers the promise of imaging important, but previously unmeasurable, aspects of live cerebral gene activities in the awake rhesus monkey (Macaca mulatta) before a crucial leap to future clinical applications in neural activity of humans.
