Over the past few years, there has been increasing exploration of the potential for gene silencing or knockdown therapies in the treatment of neurodegenerative disorders. The technology has progressed to a point in which a phase 1 human therapeutic trial for familial amyotrophic lateral sclerosis has been initiated. Current knock-down therapies under investigation include viral vector delivery of shRNAi or microRNA mimics, delivery of naked RNAi and RNAi complexed with various reagents to facilitate uptake, and delivery of modified antisense DNA oligonucleotides. The mechanisms of action for these approaches include modulation of mRNA translation, modulation of pre-mRNA splicing, and degradation of mRNA and pre-mRNA. These various approaches have been tested in pre-clinical animal models to varying extents with varying levels of efficacy. A glaring limitation of these studies that have been conducted thus far is that it has generally been impossible to monitor the efficacy of knock-down in real time. To overcome this limitation, we propose two Aims that are designed to build capability to track the efficacy of knock-down in real time and provide proof of concept studies in mouse models of two neurodegenerative diseases that are potential targets for gene silencing efforts. Taking advantage of the expertise of the investigators involved, we plan to focus on models for Huntington s disease and fronto-temporal dementia. These disorders are prime candidates for gene-silencing therapeutics and previous work in modeling these disorders in mice has produced models that recapitulate aspects of each disorder. In the approach described here, we seek to generate models in which we will produce assayable and observable behavioral phenotypes while simultaneously being able to monitor the efficacy of gene silencing reagents in real-time.
In Aim 1, we will generate mice that express mutant forms of human tau fused in-frame to luciferase.
In Aim 2, we will similarly generate mice that express mutant Nterminal fragments of huntingtin fused in-frame to luciferase. In both constructs we will employ a technique that facilitates post-translational processing of the poly-protein to liberate the luciferase so it can be assayed independently of pathologic accumulations of mutant tau or huntingtin. We propose to use new in vivo imaging techniques to detect and measure bioluminescence catalyzed by the expressed luciferase. In the tau model we propose to generate, we expect the animals to develop measurable memory deficits with neuropathological abnormalities that include neuronal loss and neurofibrillary tangle pathology. In the Huntington s model, we similarly expect to induce assayable phenotypes, which include motor function deficits, reduction in the transcription of a subset of genes in striatum, hypoactivity, and premature death. Thus, one could ultimately have models with dual readout capability in which reductions in expression could be monitored in real-time by monitoring luciferase activity levels while simultaneously having disease-relevant phenotypes to assay.

Public Health Relevance

The studies proposed in this application are designed to produce and characterize new animal models for pre-clinical testing of gene silencing approaches for intractable human neurodegenerative disease. The innovative models we propose will allow real-time, in vivo, assessments of the efficacy of gene silencing or gene knockdown therapies, allowing investigators to move to Phase I clinical trials with the most optimized and specific therapeutic available. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of th individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
Project #
Application #
Study Section
National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
Program Officer
Sutherland, Margaret L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Florida
Schools of Medicine
United States
Zip Code