PLN Guide RNA and AAV9 Vector Design for Mutation-Specific Editing
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Solution Overview
Problem
Current gene therapy approaches are ineffective for treating heart diseases caused by PLN gene mutations, particularly the PLN-R14Del mutation, due to challenges in identifying guide RNAs and vectors with high editing efficiency, delivering large proteins like Cas9 enzymes, and targeting specific mutations without affecting wild-type alleles.
Innovation Solution
Development of guide RNAs, expression cassettes, and vectors, including AAV9 virions, that target the PLN gene with high editing efficiency, specifically targeting the PLN-R14Del mutation, using saCas9 and gRNAs linked to human TNNT2 and U6 promoters, and ensuring minimal editing of wild-type PLN genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR/Cas9 methodology is used to target specific pathogenic mutations, then editing specificity is improved, but the complexity of identifying and delivering appropriate guide RNAs and vectors increases
Solution Approach 1:
The patent pre-identifies and provides specific guide RNA sequences targeted at the PLN-R14Del mutation site, along with optimized vector designs containing these gRNAs. This preliminary preparation of mutation-specific editing components resolves the complexity of identifying appropriate gRNAs during treatment, enabling precise targeting of pathogenic mutations without affecting wild-type alleles.
2Productivity
If viral vectors are used to deliver Cas9 enzymes, then delivery efficiency is improved, but the packaging limit for large proteins restricts the amount of genetic material that can be delivered
Solution Approach 1:
The patent segments the delivery system into separate components: the viral vector delivers only the compact guide RNA sequences, while the Cas9 enzyme is provided as a separate protein or mRNA component. This segmentation allows efficient viral delivery of minimal genetic material while avoiding the packaging limit restriction, as the bulkier Cas9 protein is delivered through alternative means that do not constrain the vector capacity.
3Productivity
If high editing efficiency is achieved, then treatment effectiveness is improved, but the risk of off-target effects and loss of wild-type allele increases
Solution Approach 1:
The patent designs guide RNAs with specific local characteristics tailored to the PLN-R14Del mutation site, including optimized seed regions and careful selection of PAM sequences. This local optimization ensures that the guide RNAs bind specifically to the mutant allele sequence while minimizing off-target binding to wild-type sequences or other genomic locations, thereby achieving high editing efficiency with reduced harmful off-target effects.
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
Achieves editing efficiencies of at least 50% in PLN-R14Del mutations while avoiding editing of wild-type PLN genes, effectively treating conditions like cardiomyopathy and heart failure.
Implementation Method 1
guide RNAs, vectors, and virions for targeting mutations in the PLN gene... CRISPR/Cas9 methodology to target a specific, pathogenic mutation
Implementation Method 2
delivery of polynucleotides encoding large proteins, such as Cas9 enzymes... vectors, including AAV9 virions
Data Source
AI summary
The present disclosure provides gRNAs targeting the phospholamban (PLN) gene, expression cassettes, vectors, virions and compositions comprising the same, as well as methods useful for the treatment or prevention of heart disease. In some embodiments, the present disclosure provides expression cassettes and vectors comprising a polynucleotide encoding a Cas endonuclease protein operably linked to a protein expression-driving promoter (e.g., a human troponin T promoter) and/or a polynucleotide encoding a gRNA targeting a sequence of the PLN gene comprising a mutation or deletion operably linked to an RNA expression-driving promoter. The present disclosure also provides guide RNAs, expression cassettes, vectors, virions and compositions for specifically targeting PLN gene comprising a deletion of Arg14.