Allele-Specific mHTT Editing Using Poison Exons and NMD
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Solution Overview
Problem
Current methods are inadequate for effectively reducing the level of RNA transcripts from mutant Huntingtin (mHtt) alleles in individuals with Huntington's disease, which contribute to the progression of the disorder.
Innovation Solution
Utilizing CRISPR-Cas systems to introduce a poison exon or mutation that results in a premature stop codon in the mHtt allele, leading to nonsense-mediated mRNA decay (NMD) and subsequent reduction of the RNA transcript levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods are used to reduce mHtt RNA transcript levels, then treatment effectiveness is insufficient, but introducing CRISPR-Cas systems with poison exons increases manufacturing precision and reliability of RNA reduction
Solution Approach 1:
The CRISPR-Cas system is divided into distinct functional components: guide RNA for target recognition, Cas effector polypeptide for cleavage activity, and donor DNA for introducing poison exons. This segmentation allows each component to be optimized independently while working together to achieve reliable mHtt RNA transcript reduction through allele-specific editing and NMD pathway activation.
Solution Approach 2:
The patent introduces an intermediary mechanism (poison exon) that mediates between the CRISPR-Cas editing system and the cellular NMD pathway. The poison exon acts as a intermediary element that, when inserted into the mHtt allele, triggers NMD-mediated degradation of the mutant RNA transcript, thereby translating the genetic edit into effective RNA reduction without directly targeting RNA.
2Measurement precision
If allele-specific editing is performed to reduce mHtt transcripts, then specificity is improved, but the difficulty of detecting and measuring allele-specific effects increases
Solution Approach 1:
The patent employs allele-specific SNP targeting where the guide RNA is designed to recognize unique nucleotide sequences present only in the mutant mHtt allele. This creates a molecular 'signature' that allows specific detection and measurement of allele-specific editing effects through sequencing and other molecular assays, enabling precise monitoring of mutant versus wild-type allele differentiation.
3Productivity
If poison exons are introduced to trigger NMD, then RNA transcript degradation is enhanced, but the loss of time for the editing and degradation process increases
Solution Approach 1:
The CRISPR-Cas system performs preliminary editing by inserting the poison exon into the mHtt allele before the NMD pathway acts on the transcript. This preliminary genetic modification sets up the condition for rapid subsequent degradation, allowing the system to prepare the target in advance so that once the mutant transcript is produced, it is quickly eliminated by the pre-positioned poison exon triggering NMD.
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
The method significantly reduces the level of mHtt RNA transcripts and encoded protein, potentially slowing the progression of Huntington's disease by degrading the spliced mRNA products containing premature stop codons via NMD.
Implementation Method 1
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems comprise a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid. Such CRISPR-Cas systems can bind to and modify a targeted nucleic acid.
Implementation Method 2
modifying the nucleotide sequence of a target nucleic acid in the mHtt allele such that a spliced mRNA product of the modified target nucleic acid comprises a stop codon that was not present in a spliced mRNA transcript of the target nucleic acid prior to the modification and wherein, as a result of the stop codon now present in the spliced mRNA product, the spliced mRNA product of the modified target nucleic acid undergoes nonsense-mediated mRNA decay
Data Source
AI summary
The present disclosure provides methods and compositions for reducing the level of an RNA transcript produced from a mutant Huntingtin (mHtt) allele in a neuron in an individual with Huntington's disease. The present disclosure provides methods for reducing the level of an RNA transcript produced from an mHTT allele in an allele-specific manner. The present disclosure provides systems and compositions for carrying out the methods.