Edgardo Rodriguez, Ph.D. - US grants

Huntington's disease (HD) is a progressive devastating neurodegenerative disorder that results in motor dysfunction, cognitive impairment and development of psychiatric symptoms. HD is characterized by a loss of srtiatal neurons. It is caused by a mutant form of a protein termed huntingtin (htt). The mutation is an expansion of CAG repeats in exon 1 of the HD gene. This expansion, which is translated into a polyglutamine tract, causes mutant htt to acquire a gain of toxic function. Various hypotheses have been brought forward as to what this toxic function may be. One hypothesis points to a disruption, caused by mutant htt, in the expression of other genes required for neuronal survival. It has been shown that htt is required during embryogenesis but the function of both mutant and normal htt in adults remains unknown. By using a well established HD-like mouse model (R6/2) this proposal will study the effects of reducing expression of mutant htt in striatal neurons. This reduction will be achieved by delivering RNA cleaving enzymes (ribozymes) that specifically recognize and cleave the htt mRNA. These ribozymes will be delivered by stereotaxic injections using a recombinant adeno-associated viral vector (rAAV) into the striatum of R6/2 mice. Reducing striatal mutant htt expression will help us elucidate the function of this protein in adult mouse striatum and identify which genes, if any, are directly inhibited by mutant htt. This proposal will also address the therapeutic potential of delivering trophic factors into the brain of R6/2 mice. Results gained from this study will enhance our understanding of the molecular pathogenesis of HD and offer new therapeutic alternatives to a fatal disease with no known cure.

DESCRIPTION (provided by applicant): Spinocerebellar Ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a dominantly inherited, progressive ataxia for which there is no cure. It is one of at least nine neurodegenerative disorders caused by CAG repeats encoding polyglutamine expansions in the respective disease genes. The expansion in SCA3 confers a toxic property on the ATXN3 disease gene product, a nonessential protein known as ataxin-3. Accordingly, suppressing the expression of ATXN3 is a potentially powerful therapeutic strategy for SCA3. Using a YAC transgenic mouse model expressing the full length human ATXN3 disease gene, we propose to test the in vivo efficacy and safety of viral-mediated RNA interference (RNAi) as therapy for SCA3. Preliminary studies have optimized microRNA-like short hairpin RNAs (shRNAs) that effectively target human ATXN3. Aim 1 will test and compare the ability of recombinant adeno-associated viruses (AAV) expressing these shRNAs to suppress expression of mutant human ATXN3 in injected cerebellum of adult mice. Parallel studies in aim 1 will also assess the safety of RNAi reagents targeting ATXN3. In Aim 2, the RNAi AAVs tested and confirmed in aim 1 will be injected bilaterally in the cerebellum, the primary neuropathological target in SCA3. Long term efficacy of RNAi to suppress both ATXN3 expression and disease-related phenotypes will be assessed. Because our AAV reagents employ elements already used in human gene therapy trials, a successful anti- ATXN3 RNAi reagent that fulfills safety and efficacy measures will represent an immediate candidate for preclinical studies leading toward a human clinical trial. PUBLIC HEALTH RELEVANCE: The proposed research will test viral-mediated RNA interference as a therapeutic strategy for the neurodegenerative disease, spinocerebellar ataxia type 3, a currently untreatable, fatal disorder. Studies carried out in a mouse model of disease are anticipate to establish that RNAi may be an effective strategy in humans with the disease.

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type-6 (SCA6) is a dominantly inherited ataxia caused by a polyglutamine (polyQ)- encoding CAG repeat expansion near the 3'-end of the Cav2.1 calcium channel gene, CACNA1a. Selective loss of cerebellar Purkinje neurons is the pathological hallmark of SCA6, a slowly progressive and debilitating disease for which there is no treatment. The preclinical studies proposed here will test a novel splice-isoform- specific RNA interference (SIS-RNAi) therapy approach for SCA6, using a genetically accurate mouse model of the disease. Alternative splicing at the 3'-end of the Cav2.1 transcript produces mRNA isoforms that encode two classes of Cav2.1 variants, one lacking and one containing the polyQ domain near the c-terminal end of the protein. In SCA6, recent studies have shown an aberrant increase in the levels of splice isoforms encoding for mutant (expanded) polyQ-containing Cav2.1 variants. Levels of toxic polyQ-containing Cav2.1 variants appear to increase only in cerebellar Purkinje neurons, potentially explaining their selective degeneration in SCA6. The overall hypothesis is that a novel SIS-RNAi approach developed by our research group will selectively suppress expression of polyQ-containing Cav2.1 variants in Purkinje neurons and thus will prove to be of significant therapeutic benefit in SCA6. Aim 1 will establish the capacity of recombinant adeno-associated (rAAV)-delivered SIS-RNAi molecules to selectively suppress the expression of human, polyQ-encoding Cav2.1 splice-isoforms in Purkinje neurons of an SCA6 knock-in mouse model. SCA6 knock-in mice express expanded polyQ-encoding Cav2.1 transcripts under the control of the endogenous mouse Cav2.1 promoter. Importantly, these mice faithfully recapitulate motor and histo-pathological features observed in human SCA6. Aim 2 will examine the molecular and behavioral effects of long-term rAAV-SIS-RNAi expression on the progressive neurological dysfunction observed in SCA6 knockin mice. The primary impact of the proposed studies is that they are expected to provide preclinical data supporting the development of SIS-RNAi as therapy for humans with SCA6. Moreover, they will highlight the potential of SIS-RNAi as a therapeutic strategy in other hereditary diseases and forge novel reagents useful to our understanding of Cav2.1 channel function and regulation. PUBLIC HEALTH RELEVANCE: The proposed research will test splice-isoform-specific RNA interference (SIS-RNAi) as a therapeutic strategy for Spinocerebellar Ataxia Type 6, a currently untreatable, severely debilitating neurodegenerative disorder. These studies will assess the activity, specificity and safety profiles of SIS-RNAi in vivo using a genetically accurate mouse model of the disease. Results are anticipated to support continued pre-clinical development of SIS-RNAi as a therapy for humans with SCA6.

Abstract A number of neurological disorders are characterized by the preferential dysfunction and/or loss of specific neuronal cell populations and the mechanisms underlying this cell type specific vulnerability remain poorly understood. It has been proposed that the differential neuronal vulnerability observed in these disorders arises from disruptions in cell type specific gene expression regulatory programs responsible for maintaining the physiological identity and function of the affected neurons. MicroRNAs (miRNAs) are small, non-coding RNAs involved in the regulation of gene expression networks at the posttranscriptional level. Studies, including ours, have implicated altered miRNA expression in the pathogenicity of several neurological disorders. Here, we propose the development of a novel molecular approach to investigate the role that neural cell type specific changes in miRNA function play in the pathogenicity of neurological diseases. The approach relies on a recombinant adeno-associated virus (rAAV)-based, Cre recombinase-dependent genetic Flp-excision switch (FLEX switch) that restricts the expression of an ectopically delivered miRNA-binding protein Argonaute-2 to Cre expressing cells. We call this approach miFLAGO (miRNA-associated, FLEX switch regulated, AGO2). Experiments in Aim-1 will validate the functionality and reproducibility of miFLEX in the cerebellum of adult transgenic mice engineered to selectively express Cre recombinase in Purkinje cells. Purkinje cell-specific miFAGO-miRNA complexes will be isolated and used to build miRNA libraries for high-throughput RNA sequencing analysis. In Aim-2, we will use miFLAGO to investigate the role that cell type specific changes in miRNA function play in the pathogenicity of a mouse model of Spinocerebellar ataxia type-1, a polyglutamine disorder caused by the expansion of a CAG repeat in the ATXN1 gene. The short-term goal of this proposal is to provide the research community with a validated novel molecular tool for the study of cell type specific miRNA function. Long-term, we aim to fine-tune our understanding of neural cell type specific gene expression regulatory programs and how they might mediate differential vulnerability in neurological disease, with the goal of identifying novel therapeutic routes.

? DESCRIPTION (provided by applicant): Parkinson's disease is the second most prevalent neurodegenerative disorder in the United States. A continued lack of curative treatments poses a burden not only to PD patients but also to our society as a whole. Thus, the development of disease modifying therapies is imperative. This will require paradigm-shifting strategies designed to modulate, not the cause of symptoms, but the molecular underpinnings driving neuronal dysfunction in the PD brain. Numerous genetic and biochemical studies firmly place the SNCA gene, which encodes for a-synuclein, at the center of the patho-molecular events driving onset and progression of genetic and sporadic forms of PD. Aberrantly polymerized forms of a-synuclein are the major component of Lewy bodies and Lewy neurites that are classically observed in PD. Also, a-synuclein inclusion pathology tracts with the neuronal degeneration that is observed in PD. Moreover, recent studies have suggested that a-synuclein fibrils could act in a prion-like mechanism to seed and spread a-synucleinopathy throughout, perhaps underlying the toxicity observed in PD and other forms of dementia. Because a-synuclein-induced pathology requires sustained a-synuclein expression, it is hypothesized that curtailing SNCA expression could impede the onset or progression of disease. Here, we propose to investigate the feasibility of a novel, paradigm- shifting gene targeting approach to inactivate the SNCA gene in somatic brain tissue. Experiments in aim-1 of this proposal will develop, test and determine whether adeno-associated virus (AAV)-delivery of a new CRISPR/Cas9 system into adult mammalian brain results in safe and effective inactivation of the SNCA locus. Results from this first set of experiments are expected to validate AAV-CRISPR/Cas9 as a novel, powerful tool that can be used by the entire neurodegenerative disease research community to query gene function in the brain. Proof-of-principle studies in aim-2 of this proposal will determine if AAV-CRISPR/Cas9-mediated inactivation of SNCA in a novel mouse model of a-synucleinopathy is efficient enough to modify the onset or progression of a-synuclein pathology in disease relevant areas of the brain. Results from these experiments are anticipated to provide the basis for further development of this novel molecular therapy for PD and other incurable neurodegenerative diseases.