Methods of identifying modulators of TDP-43 mediated cellular toxicity
Inventors
Gitler, Aaron D. • Elden, Andrew
Assignees
University of Pennsylvania Penn • National Institutes of Health NIH • US Department of Health and Human Services
Publication Number
US-8865411-B2
Publication Date
2014-10-21
Expiration Date
2030-03-26
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Abstract
Compositions and methods are disclosed for identifying agents useful for the treatment of proteinopathies.
Core Innovation
The invention relates to compositions and methods for identifying agents useful for the treatment of proteinopathies, particularly those associated with aberrant protein aggregation and cellular toxicity mediated by the protein TDP-43 or its functional C-terminal variants. The core innovation includes identification of genetic modulators that augment or suppress TDP-43 mediated toxicity and aberrant protein aggregation. These modulators serve as therapeutic targets for treating neurological diseases such as amyotrophic lateral sclerosis (ALS), fronto-temporal lobar degeneration (FTLD), Parkinson's disease, Huntington's disease, and Alzheimer's disease (AD).
The problem addressed arises from the increasing incidence of neurodegenerative diseases associated with aging populations, characterized by accumulation of insoluble protein aggregates in the brain and nervous system. TDP-43 has been identified as a major protein in pathological inclusions in ALS and FTLD-U. Mutations in TDP-43 gene have been found in sporadic and familial ALS patients, implicating TDP-43 as central to disease pathogenesis. Understanding the mechanisms of TDP-43 toxicity and aggregation is crucial for developing new therapeutic approaches to these devastating proteinopathies.
Claims Coverage
The claims present seven main inventive features covering methods of inhibiting TDP-43 toxicity, methods of identifying agents that modulate TDP-43 toxicity, and engineered transgenic yeast cells for studying TDP-43 activity.
Method of inhibiting TDP-43 mediated cellular toxicity using agents targeting specific genes
A method involving contacting a cell expressing a toxicity-inducing C-terminal variant of TDP-43 (amino acids 188-414) with an agent that inhibits expression or activity of at least one gene selected from a defined group of genes (including DBR1, PCL6, SAK1, etc.) or increases expression or activity of another defined group of genes (including FMP48, VTS1, XRS2, etc.), where inhibition agents are antisense, ribozyme, or siRNA molecules complementary to the target gene mRNA, and increased expression is achieved by introducing a vector encoding the respective polypeptide.
Use of siRNA targeting DBR1 to inhibit TDP-43 toxicity
A method wherein the gene targeted is specifically DBR1, using an siRNA comprising a guide strand complementary to DBR1 mRNA to inhibit its expression, resulting in reduction of TDP-43 mediated cellular toxicity.
Method for identifying agents which inhibit TDP-43 mediated cellular toxicity
A screening method comprising providing a cell expressing the C-terminal TDP-43 variant (amino acids 188-414) associated with increased protein aggregation and toxicity, contacting the cell with an agent that either inhibits expression or activity of a specified group of genes or increases expression or activity of another group of genes (overlapping with that in other claims), and measuring cellular toxicity relative to a control to identify agents that reduce toxicity.
Assessment of effects of candidate agents on TDP-43 mediated protein aggregation
A method of further evaluating candidate agents identified by screening for their effects on TDP-43 mediated protein aggregation, to ascertain their ability to reduce aggregation alongside toxicity.
Transgenic yeast cells expressing TDP-43 variant and TDP-43 activity modulators
Engineered yeast cells comprising a genetic construct expressing the TDP-43 C-terminal variant (amino acids 188-414) along with a heterologous nucleic acid encoding a modifier of TDP-43 activity selected from a defined set of modifiers (including genes such as DBR1, PCL6, SAK1, etc.), where these modifiers alter TDP-43 toxicity when overexpressed.
Modifiers that reduce TDP-43 cytotoxicity upon over-expression
Over-expression of certain identified genetic modifiers in the transgenic yeast cells reduces TDP-43 induced cytotoxicity, indicating their potential as therapeutic targets.
Modifiers that enhance TDP-43 mediated cytotoxicity upon over-expression
Over-expression of certain other identified genetic modifiers enhances TDP-43 mediated cytotoxicity, identifying genes whose inhibition may be therapeutic.
The claims comprehensively cover methods of modulating cellular toxicity induced by TDP-43 via specific genetic targets, methods of identifying agents modulating this toxicity, and transgenic yeast models expressing TDP-43 and modifiers, enabling screening and therapeutic development focused on the modulation of gene expression or activity to treat proteinopathies.
Stated Advantages
Identification of TDP-43 activity modulators provides new beneficial therapeutic targets for treatment of disorders associated with aberrant protein folding and deposition.
The yeast model system enables screening for agents that modulate TDP-43 mediated toxicity and aggregation, thus facilitating the development of novel therapeutics.
Agents inhibiting DBR1 debranching activity can effectively reverse toxic effects of TDP-43, providing specific molecular targets for treatment.
Documented Applications
Treatment and prevention of proteinopathies such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD), fronto-temporal lobar degeneration (FTLD), Parkinson's disease (PD), Huntington's disease (HD), and Alzheimer's disease (AD).
Screening assays to identify agents useful for modulating TDP-43 activity to treat neurological diseases associated with aberrant protein aggregation.
Use of transgenic yeast strains expressing TDP-43 and TDP-43 activity modulators for drug screening and mechanistic studies of proteinopathies.
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