Novel Research Tool to Study TDP-43 Dysfunction

University of Pittsburgh researchers have developed a novel RNA biosensor named CUTS-1 (CFTR-UNC13A TDP-43 Loss of Function Sensor), capable of detecting TDP-43 dysfunction in cells. TDP-43 loss of function is a hallmark in the neuropathology of several neurodegenerative conditions including amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), and Alzheimer’s disease (AD). CUTS-1 is designed to identify cells with TDP-43 dysfunction, providing a direct measure of disease severity. Additionally, CUTS-1 could be used to deliver targeted therapy to these cells leading to new treatment strategies for ALS, FTD, and AD. 

A novel RNA biosensor has been developed to selectively identify cells with TDP-43 dysfunction which are linked to several neurodegenerative conditions. In cells with TDP-43 expression, the biosensor is spliced and no EGFP will be expressed; however, in the absence of TDP-43 EGFP expression will occur, identifying dysfunctional cells. The amount of EGFP expressed is a direct marker of TDP-43 dysfunction and could monitor disease progression or regression. 

Description

ALS, FTD, and AD are debilitating neurodegenerative diseases. Common to all three diseases is the loss of function of TDP-43, a regulator of RNA splicing. This dysfunction leads to aberrant RNA processing resulting in the inclusion of cryptic exons (non-conserved sequences) in RNA. CUTS-1 has been developed to harness TDP-43 dysfunction and is deactivated by TDP-43-induced splicing. It is only in cells with loss of TDP-1 function that the reporter gene EFGP is expressed allowing researchers to identify dysfunctional cells using fluorescence. CUTS-1 has a variety of applications in understanding the progression and regression of these diseases in response to treatment and could be used to selectively target dysfunctional cells with therapies.

Applications

- Amyotrophic lateral sclerosis research
- Frontotemporal degeneration research
- Alzheimer’s disease research

Advantages

The loss of TDP-43 function and the resulting inclusion of cryptic exons in the RNA is a biological feature of ALS, FTD, and AD. This novel biosensor, CUTS-1, can selectively detect and quantify cells with TDP-43 dysfunction using the presence of these cryptic exons resulting in the expression of EGFP, a reporter that fluoresces under UV light allowing for real-time, direct observation of disease severity by researchers. The unique design of this biosensor, where complete expression of genes encoded on CUTS-1 is controlled by the presence or absence of TDP-43, allows this approach to selectively deliver treatments to dysfunctional cells reducing the risk of off-target effects, improving patient safety.

Invention Readiness

CUTS-1 has been developed and produced. Key components of the biosensor are: mCherry coding sequence to act as a control reporter, a sequence incorporating a cryptic exon sequence that is a fusion of two well-described cryptic exons alongside TDP-43 binding sites, a stop codon for mCherry, and a sequence for the reporter gene, EGFP. In normally functioning cells, TDP-43 will bind to CUTS-1, leading to splicing and removal of the cryptic exons with only mCherry expressed. However, In the absence of TDP-43, the cryptic exon is retained leading to EGFP expression. In vitro studies have validated CUTS-1 as a biosensor to detect TDP-43 dysfunction and EGFP expression is proportional to loss of function.

IP Status

https://patents.google.com/patent/WO2025262636A1

Related Publication(s)

Xie, L., Merjane, J., Bergmann, C. A., Xu, J., Hurtle, B., & Donnelly, C. J. (2024). CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function. Cold Spring Harbor Laboratory. https://doi.org/10.1101/2024.07.12.603231