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Proof of Concept

The goal of this project is to design and test RNA-based strategies that prevent TDP-43 aggregation, a pathological hallmark of amyotrophic lateral sclerosis (ALS). Our dual approach uses antisense oligonucleotides (ASOs) to suppress stress granule (SG) formation and RNA aptamers to block TDP-43 recruitment into SGs. Together, these tools aim to intervene in the earliest steps of protein aggregation, creating a layered therapeutic framework for ALS.

ASO Design Results

Successfully reduced DAZAP1 (23%) and FAM98A (38-49%) expression using antisense oligonucleotides

Aptamer Development

Computational modeling and molecular dynamics simulations to target TDP-43 C-terminal region

Validated Measurements

RT-qPCR with validated primer efficiency curves confirmed reproducible gene expression changes

ASO Designs

We identified three SG-associated proteins (DAZAP1, FAM98A, and SND1) whose knockdown reduces SG assembly. For each, a panel of ten ASOs was designed. Transfections were performed in SH-SY5Y cells, and knockdown efficiency was quantified by RT-qPCR. Cells were subsequently stressed with sodium arsenite to induce oxidative stress, and SG formation was assessed by immunofluorescence. ASOs were ranked based on transcript reduction and functional suppression of SG formation.

Methodology

ASO Design

Panel of 10 ASOs designed for each target protein (DAZAP1, FAM98A, SND1) to maximize knockdown efficiency while minimizing off-target effects.

Cell Transfection

SH-SY5Y neuronal cells transfected with selected ASOs. Transfection efficiency monitored to ensure adequate cellular uptake.

Oxidative Stress

Cells stressed with sodium arsenite to induce stress granule formation, mimicking ALS cellular stress conditions.

RT-qPCR Analysis

Gene expression quantified using validated primer efficiency curves. Immunofluorescence assessed stress granule formation.

Knockdown Efficacy Results

DAZAP1
0%
Knockdown

ASO-A and ASO-B

Reduced DAZAP1 expression

FAM98A
0%
Knockdown

ASO-C and ASO-D

Reduced FAM98A expression by 38-49%

SND1
⚠
Inconclusive
Initial Trials

SND1 Knockdown

Initial trials showed inconsistent results (377% Primer efficiency - excluded from analysis)

Primer Efficiency Standard Curves
Primer efficiency standard curves for RT-qPCR validation.
ASO Knockdown Efficiency
Relative expression fold change of DAZAP1 and FAM98A after ASO knockdown.

These findings confirmed that ASOs can feasibly reduce SG-related transcripts in neuronal-like cells, laying the foundation for testing their effect on TDP-43 aggregation.

Aptamer Designs

In parallel, RNA aptamers were computationally modeled to bind the C-terminal region of TDP-43, where aggregation initiates. Using HADDOCK and GROMACS molecular dynamics simulations, candidate aptamers were docked and ranked with machine learning. A top candidate was synthesized and tested for cellular binding.

Aptamer vs Antibody Labeling
Aptamer vs antibody labeling of TDP-43, showing reduced but detectable aptamer signal.

Results

Immunocytochemistry revealed that the aptamer localized to protein structures in SH-SY5Y cells, but consistent co-localization with TDP-43 was not confirmed. This outcome suggests that while aptamers are structurally capable of binding cellular proteins, further refinement and staining optimization are needed to confirm specific TDP-43 interaction.

Measurements

To ensure reproducibility, gene expression changes were measured by RT-qPCR with validated primer efficiency curves. Aptamer interactions were evaluated through fluorescence microscopy, binding assays, and protein localization analysis. Together, these measurements provided quantitative and qualitative data to benchmark early intervention in TDP-43 biology.

Primer Efficiency Results

0%

DAZAP1
Primer Efficiency

0%

FAM98A
Primer Efficiency

0%

GAPDH
Primer Efficiency

Analysis and Next Steps

This proof of concept demonstrates two complementary strategies: (1) ASOs can suppress stress granule proteins and reduce the molecular environment that drives TDP-43 aggregation, and (2) RNA aptamers hold potential as a fail-safe mechanism by directly targeting TDP-43. While ASO results were encouraging, aptamer binding requires further optimization.

Next steps include:

  • Optimizing ASO concentrations and testing in stressed cells.
  • Expanding aptamer libraries and refining staining protocols.
  • Assessing combined ASO + aptamer treatments under oxidative stress.

Ultimately, this layered approach provides a promising framework for developing RNA-based therapeutics that intervene in TDP-43 pathology at its earliest stages, with broad implications for ALS and other neurodegenerative diseases.