Conditional TDP-43 Mutant Animal Models for ALS Pathogenesis
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Solution Overview
Problem
There is a need for animal models of TDP-43 proteinopathy to understand the role of TDP-43 in ALS pathogenesis, as existing models do not adequately replicate the redistribution and accumulation of TDP-43 protein associated with ALS.
Innovation Solution
Development of non-human animals, such as rodents, that express only a mutant form of TDP-43 lacking a functional nuclear localization signal or prion-like domain, mimicking ALS-like symptoms by introducing specific mutations in the TARDBP gene, allowing for the creation of animal models with TDP-43 proteinopathies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing animal models are used to study TDP-43 in ALS, then general ALS research can be conducted, but they fail to adequately replicate the specific redistribution and accumulation of TDP-43 protein associated with ALS pathogenesis
Solution Approach 1:
The patent introduces specific mutations in the TARDBP gene (such as A315T, P360S, G368S, and C-terminal fragment mutations) to alter the biochemical properties of TDP-43 protein. These parameter changes in the protein sequence cause it to mislocalize from the nucleus to the cytoplasm and form aggregates, accurately replicating the pathological features of ALS while maintaining the ability to study disease mechanisms.
2Reliability
If transgenic animals overexpressing mutant TDP-43 are created, then ALS-like symptoms can be observed, but the models may not accurately represent the loss-of-function and gain-of-toxic-function mechanisms in human ALS
Solution Approach 1:
The patent segments the TARDBP gene into different allelic forms: one allele carries the mutant TDP-43 sequence while the other allele is knocked out or carries a conditional knockout. This segmentation allows selective expression of mutant protein without wild-type competition, accurately modeling the pathological conditions while using standardized genetic tools like Cre-loxP systems.
Solution Approach 2:
The patent uses conditional knockout systems with Cre recombinase as an intermediary mechanism. The mutant TDP-43 expression is controlled through loxP-flanked sequences that can be conditionally deleted or activated. This intermediary system provides precise spatial and temporal control over mutant protein expression, improving model accuracy while using well-established genetic tools.
3Ease of operation
If conditional knockout systems are used to control TDP-43 expression, then temporal and spatial control is achieved, but the model development process becomes more complex
Solution Approach 1:
The patent incorporates loxP recognition sequences flanking the stop cassette or mutant TDP-43 coding sequence during initial genetic engineering. This preliminary arrangement of genetic elements allows for future conditional activation or deletion using Cre recombinase. The preparatory genetic architecture enables precise temporal and spatial control without requiring complex real-time manipulation during experiments.
Data Source
AI summary
Described herein is the discovery that TDP-43 proteinopathies may be induced in adult or neonatal animals bearing on one chromosome a mutant TARDBP gene encoding a mutant TDP-43 protein that lacks a functional nuclear localization signal (NLS) or a mutant TARDBP gene encoding a mutant TDP-43 protein that lacks a prion-like domain (PLD) and on the other homologous chromosome a TARDBP gene comprising a conditional knockout mutation. Knockout of the TARDBP gene comprising the conditional knockout mutation, e.g., using Cre recombinase, during the neonatal stage, e.g., at P0-P10, or during adulthood, e.g., at about 5 months of age, results in the mice exhibiting neuromuscular phenotypes such as early lethality, paralysis, weight loss, etc. These animals exhibit hallmark symptoms of ALS and that may be used in testing candidate agents useful in treating TDP-43 proteinopathies.