Juan Sanchez-Ramos, M.D. Ph.D. - US grants

The candidate is a pharmacologist and neurologist with research experience in behavioral pharmacology and clinical research training in movement disorders. His-special interests include extrapyramidal syndromes and neurotoxic consequences of drug use. Research proposed here concerns MPTP (l-methyl-4-phenyl-1,2,3,6- tetrahydropyridine), a meperidine analogue which produces changes that mimic the lesion of Parkinson's disease in primates and other species. Despite advances in understanding some aspects of the mechanism by which the drug causes neuronal degeneration, many basic questions remain unanswered due primarily to the complexity of in vivo research on the central nervous system. One approach to this problem is to elucidate the mechanism of action of MPTP in vitro by studying the effects of the toxin on cell cultures of dopaminergic neurons and upon striatal (or midbrain) slices. Specific aims of this research are: 1) to characterize the selectivity and irreversibility of MPTP and MPP+ toxicity towards dopamine neurons in culture, 2) to test the hypothesis that MPP+ acts primarily on neuronal fibers, with subsequent retrograde degeneration of the neuron, 3) to test the hypothesis that oxyradicals mediate MPP+ induced destruction of dopamine neurons, 4) to test the hypothesis that mitochondrial electron transport is inhibited by MPP+ in rat (or mouse) striatal and midbrain slices as measured with scanning spectrophotometer, and 5) to test the hypothesis that target tissue (striatal cells) influences MPP+ neurotoxicity and facilitates recovery of dopamine neurons from sub-lethal damage. The University of Miami Dept of Neurology provides outstanding resources for development of a clinical investigator: fully equipped laboratories, basic research staff, out-patient clinics, hospital neurology ward, students and housestaff.

? DESCRIPTION (provided by applicant): Gene therapy of brain diseases is hampered by the requirement for invasive stereotaxic injection into brain, or infusion of therapeutic agents into te intrathecal space. The surgical approach is not optimal for neurodegenerative diseases that require treatments throughout life. This obstacle has given impetus to the design of a new nose-to-brain delivery system for nucleic acids or drugs that cannot pass the blood-brain-barrier. The overall goal of this project is to refine and test a nanocarrier system consisting of chitosan-based, manganese-containing nanoparticles (mNPs) loaded with therapeutic nucleic acids: small interfering RNA (siRNA) and dsDNA. The experiments are designed to assess the mechanisms and extent to which the nanocarriers transport their payload from the olfactory mucosa to olfactory bulb and other brain regions to suppress marker genes in mouse models of rapid onset Huntington's Disease. Specific Aim 1: Testing optimized nanocarriers loaded with siRNA against a marker gene expressed in transgenic green mice that constitutively express green fluorescent protein (GFP) in brain neurons. mNPs will be packaged with siRNA directed against GFP. Primary end-points will be a) the extent and time-course of dissemination of mNPs from olfactory bulb to other regions of brain assessed by MRI T1-weighted imaging; b) GFP mRNA and GFP protein expression by comparative quantitiative PCR and Western blot analysis, respectively. Specific Aim 2: Study of gene-silencing in a mouse model of HD. siRNA directed against htt will be loaded into NPs and administered intra-nasally or microinjected directly into striatum in rapid-onset mouse models of Huntington's Disease. Primary-end points will be a) extent and time-course of dissemination of mNPs from olfactory bulb (or from striatum) to other regions of brain assessed by microbore MRI, b) quantitative analysis of htt mRNA expression by quantitative PCR and htt protein expression by Western blot in various brain regions, c) effects on behavior and locomotor activity. Specific Aim 3 a) Iterative optimization of the nanocarrier system in cell cultures expressing a marker gene guided by results from concurrent in vivo experiments in Aim 1 and b) studies of cellular extrusion of exosomes containing mNPs as an hypothesized mechanism for cell-to-cell dissemination. Specific Aim 4: Testing capacity of mNPs to deliver DNA (gene encoding a red fluorescent protein) in vivo. mNPs will be loaded with plasmid dsDNA encoding red fluorescent protein (RFP) and intranasally instilled into normal C57BL6 mice. End-points will be the same as those in Aim 2. Clinical Significance: The ability to dose patients chronically and non-invasively via intra-nasal administration of nanocarriers of gene-silencing agents or other large therapeutic molecules will have a dramatic impact in the therapeutics of brain disorders.