Chimeric TDP-43 Proteins for Splicing Recovery Without Aggregation
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Solution Overview
Problem
The precise mechanism underlying TDP-43 pathology in neurodegenerative diseases such as ALS remains unclear, and the role of TDP-43 splicing repression in motor neurons is unknown, complicating therapeutic strategies.
Innovation Solution
A chimeric protein comprising the RNA recognition domain of TDP-43 fused to an unrelated splicing repressor (RAVER1) is delivered using an AAV9 approach to restore splicing repression in motor neurons, addressing the central role of TDP-43 in motor neuron physiology.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If TDP-43 is overexpressed in transgenic models, then motor neuron function is improved, but TDP-43 aggregates in the cytoplasm and depletes from the nucleus
Solution Approach 1:
The TDP-43 protein is segmented into functional domains: the N-terminal domain (residues 1-273) containing RNA recognition motifs is separated from the C-terminal domain (residues 274-414) that is prone to aggregation. This segmentation allows the functional N-terminal domain to be expressed without the aggregation-prone C-terminal region, restoring nuclear TDP-43 function while preventing cytoplasmic aggregation.
Solution Approach 2:
The aggregation-prone C-terminal domain of TDP-43 is extracted and removed from the expressed protein construct. By expressing only the N-terminal domain (residues 1-273) or N-terminal fragment fused to a carrier protein, the patent eliminates the source of cytoplasmic aggregation while preserving the essential RNA binding and splicing repression functions located in the N-terminal region.
2Reliability
If wild-type TDP-43 is expressed to compensate for loss, then splicing repression is restored, but mislocalization and aggregation persist
Solution Approach 1:
The invention applies local quality by modifying only the problematic C-terminal region of TDP-43 while preserving the functional N-terminal domain. The N-terminal domain (residues 1-273) maintains its native RNA recognition and splicing repression capabilities, while the removed C-terminal domain eliminates the source of aggregation. Alternatively, fusion to a carrier protein provides a stable structural context that prevents mislocalization while allowing the TDP-43 N-terminal domain to perform its local splicing repression function.
Solution Approach 2:
A carrier protein or fusion partner acts as an intermediary between the TDP-43 N-terminal domain and the cellular environment. This intermediary provides a stable structural framework that prevents mislocalization and aggregation, while allowing the TDP-43 N-terminal domain to interact with its RNA targets and perform splicing repression. The carrier mediates proper subcellular localization and stability without interfering with TDP-43's biological function.
3Productivity
If adenoviral vectors are used to deliver therapeutic genes, then transduction efficiency is high, but immune response and toxicity increase
Solution Approach 1:
The patent uses adeno-associated virus (AAV) vectors as a copy or alternative to adenoviral vectors. AAV provides similar gene delivery capabilities to neurons but with significantly reduced immunogenicity and toxicity. The AAV vector system replicates the essential function of adenoviral vectors (high transduction efficiency in neural tissue) while eliminating their harmful side effects, making it a safer therapeutic platform for long-term TDP-43 gene delivery.
Data Source
AI summary
An adenovirus or adenoviral vector is described that includes a non-native nucleotide sequence capable of expressing a chimeric protein comprising an N-terminal nucleotide binding domain of transactivation response element DNA-binding protein (TDP-43), a C-terminal domain derived from a splicing repressor, and an autoregulatory element. Methods of using the adenovirus or adenoviral vector to treat degenerative diseases such as inclusion body myocytosis, amyotrophic lateral sclerosis, and frontotemporal dementia are also described.