dCas9-VPR TTN Activation for Broad DCM Mutation Coverage
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Solution Overview
Problem
Current treatments for dilated cardiomyopathy (DCM) caused by TTN gene variants are inadequate, as they are not generalizable to the diverse range of mutations and do not effectively restore TTN gene function.
Innovation Solution
A CRISPR-Cas9 complex, comprising a nuclease-dead Cas9 protein linked to a eukaryotic transcriptional activator, is delivered to target tissues to increase expression of functional TTN gene products, specifically through regulatory sequences in the promoter or enhancer regions, using guide RNAs to target mutated or wild-type TTN alleles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-Cas9 is used to target and correct specific TTN mutations, then treatment precision for individual mutations is improved, but the approach is not generalizable to the diverse range of TTN mutations found in DCM patients
Solution Approach 1:
Instead of attempting to correct each mutated TTN allele directly (which would require numerous mutation-specific approaches), the invention inverts the strategy by targeting the wild-type TTN allele for activation. This allows a single universal approach to benefit patients with any TTN mutation, as the wild-type allele is present in all DCM patients carrying a mutated allele.
Solution Approach 2:
The invention creates a universal therapeutic approach that applies to all DCM patients with TTN mutations regardless of the specific mutation type. The dCas9-VPR system with wild-type allele-specific gRNA serves as a multi-functional solution that can treat nonsense mutations, frameshift mutations, splice site mutations, and other TTN mutation types with a single platform.
2Reliability
If CRISPR-Cas9 nuclease is used to correct TTN mutations, then gene function restoration is achieved, but off-target effects and DNA damage risks increase
Solution Approach 1:
The invention extracts the transcriptional activation function from the Cas9 nuclease by using a catalytically inactive dCas9 variant. This separates the gene-targeting capability (guide RNA-directed binding) from the DNA-cleaving activity, allowing precise targeting of the TTN promoter without causing DNA double-strand breaks or off-target mutagenesis.
Solution Approach 2:
The dCas9-VPR complex serves as an intermediary that binds to the wild-type TTN promoter and recruits transcriptional activation machinery without directly modifying the DNA sequence. This mediator approach achieves gene activation through protein-protein interactions and chromatin remodeling rather than through DNA cleavage and repair pathways.
3Reliability
If current TTN-targeted therapies are developed for specific mutations, then treatment effectiveness for those mutations is improved, but the complexity of developing multiple mutation-specific treatments increases
Solution Approach 1:
The invention develops a single universal therapeutic platform that treats all TTN mutation types through activation of the wild-type allele. This eliminates the need to develop and maintain multiple mutation-specific therapies, reducing developmental complexity while maintaining treatment effectiveness across diverse patient populations.
Solution Approach 2:
The invention achieves sufficient therapeutic effect by activating only the wild-type TTN allele, without attempting to correct the mutated allele. This partial action approach (activating one allele rather than correcting both) is sufficient to restore adequate TTN function and avoid the complexity of developing tools for each specific mutation type.
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
This approach effectively restores TTN gene function in a broad range of DCM individuals, improving cardiac and skeletal muscle tissue function by increasing TTN protein levels and normalizing contractile deficits.
Implementation Method 1
A CRISPR-Cas9 complex, comprising a nuclease-dead Cas9 protein linked to a eukaryotic transcriptional activator, is delivered to target tissues to increase expression of functional TTN gene products, specifically through regulatory sequences in the promoter or enhancer regions, using guide RNAs to target mutated or wild-type TTN alleles.
Implementation Method 2
A CRISPR-Cas9 complex, comprising a nuclease-dead Cas9 protein linked to a eukaryotic transcriptional activator, is delivered to target tissues to increase expression of functional TTN gene products
Implementation Method 3
This approach effectively restores TTN gene function in a broad range of DCM individuals, improving cardiac and skeletal muscle tissue function by increasing TTN protein levels and normalizing contractile deficits.
Data Source
AI summary
This invention relates to genetic methods for altering gene expression in a human or animal. In particular, the invention relates to increasing expression of the TTN gene in a cell or tissue in an animal or human wherein at least one allele of the gene carries a mutation that results in a truncated Titin protein product. Therapeutic treatments, pharmaceutical compositions and methods of treatment utilizing such pharmaceutical compositions are also provided.