Splice-Responsive Gene Constructs for TDP-43 Depleted Cells
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Solution Overview
Problem
Current therapies for neurodegenerative diseases targeting RNA-binding proteins like TDP-43 face challenges due to widespread expression in both diseased and healthy cells, leading to adverse effects and reduced efficacy, necessitating the development of tools to selectively target and correct dysregulated molecular mechanisms.
Innovation Solution
A construct comprising a start codon, regulatory domain with splice acceptor and donor sites, and a binding domain for hnRNP splicing factors, configured to repress or allow splicing based on nuclear depletion of the factor, ensuring functional protein expression only in diseased cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If transgenic expression of proteins is used to treat neurodegenerative diseases, then therapeutic effect is achieved in diseased cells, but expression occurs in both diseased and non-diseased cells causing adverse effects
Solution Approach 1:
The construct employs cell-type-specific promoters (e.g., synapsin promoter for neuronal cells) to ensure that transgenic protein expression occurs only in specific cell types relevant to the disease, such as neurons in ALS, rather than uniformly across all cell types. This localizes the therapeutic effect to diseased cells while sparing healthy cells from exposure to the transgenic protein.
Solution Approach 2:
The construct incorporates regulatory elements that enable dynamic control of gene expression. The promoter activity can be modulated based on cellular conditions, allowing the system to activate therapeutic protein expression specifically when needed in diseased cells while remaining inactive or suppressed in healthy cells, thereby adapting expression to disease state.
2Object-affected harmful factors
If expression of transgenic protein is decreased to lower adverse effects, then safety is improved, but efficacy within diseased cells is decreased
Solution Approach 1:
By using cell-type-specific promoters, the construct achieves high expression levels specifically in the target cell type (e.g., neurons) where the disease occurs, while maintaining low or zero expression in non-target cells. This spatial differentiation allows maximum efficacy in diseased cells without exposing healthy cells to potential adverse effects, eliminating the need to reduce overall expression levels.
3Productivity
If widespread expression of therapeutic proteins is achieved, then coverage of diseased cells is improved, but risk of adverse events increases
Solution Approach 1:
The construct employs tissue-specific or cell-type-specific promoters that drive expression only in particular organs or cell types relevant to the neurodegenerative disease (e.g., brain-specific promoters for neuronal diseases). This ensures high expression coverage within the affected tissue while completely avoiding expression in other tissues, thereby maximizing therapeutic coverage of diseased cells without increasing adverse events in healthy tissues.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables selective expression of therapeutic proteins in cells with hnRNP splicing factor depletion, improving safety and efficacy while minimizing expression in healthy cells, and allowing pre-emptive treatment by activating only in diseased cells.
Implementation Method 1
a binding domain for a splicing factor of the heterogenous nuclear ribonucleoprotein (hnRNP) family, located within 150 nucleotides of the first splice donor site and/or first splice acceptor site
Implementation Method 2
if placed in a cell with nuclear depletion of the splicing factor, splicing of the first splice acceptor site and first donor site is not repressed, such that a functional protein is produced from the transgene sequence
Data Source
AI summary
A construct comprising a start codon, a regulatory domain comprising a first splice acceptor site and a first splice donor site, a binding domain for a splicing factor of the hnRNP family, located within 150 nucleotides of the first splice donor site and/or first splice acceptor site and/or located between the first splice acceptor site and first splice donor site; and a transgene sequence, wherein the construct is configured such that (i) if placed in a cell with nuclear depletion of the splicing factor, splicing of the first splice acceptor site and first donor site is not repressed, such that a functional protein is produced from the transgene sequence, and (ii) if placed in a cell without nuclear depletion of the splicing factor, splicing of the first splice acceptor site and/or first donor site is repressed such that no functional protein is produced from the transgene sequence. A vector comprising the construct, as well as a system comprising the constructs or vector and a cell are also described. The splicing factor of the hnRNP family may be TDP-43. The construct and vector may be used in therapy, for example, in diseases associated with depletion of a hnRNP splicing factor.