Targeted Nuclear Gene Output via Antisense Oligomers
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Solution Overview
Problem
Genetic diseases caused by haploinsufficiency or mutations result in insufficient production of gene products due to inefficient splicing of introns, leading to reduced expression or activity of target proteins or functional RNAs.
Innovation Solution
The use of antisense oligomers (ASOs) that promote constitutive splicing of retained introns in pre-mRNA, increasing the production of target proteins or functional RNAs by binding to specific regions of the pre-mRNA and inducing complete splicing of introns, thereby enhancing mRNA levels and protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If antisense oligomers are used to promote constitutive splicing of retained introns, then mRNA and protein levels increase, but the complexity of the molecular mechanism increases
Solution Approach 1:
The patent employs antisense oligomers as intermediary molecules that bind to retained introns in pre-mRNA, facilitating splicing by recruiting spliceosomal components. This mediator approach resolves the contradiction by providing a straightforward molecular mechanism (ASO binding→spliceosome recruitment→constitutive splicing) that achieves high productivity without excessive complexity
Solution Approach 2:
The invention changes the splicing parameter from inefficient/partial to constitutive/complete splicing by introducing ASOs that alter the splicing kinetics and thermodynamics at retained intron boundaries. This parameter change enables efficient production of fully-spliced mRNA and protein while maintaining a relatively simple molecular mechanism
2Productivity
If retained introns are spliced out to increase gene expression, then protein production increases, but splicing efficiency must be improved
Solution Approach 1:
The patent implements a feedback mechanism where ASO binding to retained introns recruits spliceosomal components, which then process the pre-mRNA through constitutive splicing. This feedback loop ensures that splicing efficiency is continuously improved until complete removal of retained introns is achieved, reliably increasing protein production
Solution Approach 2:
The antisense oligomers perform preliminary action by binding to retained introns before splicing occurs, pre-positioning spliceosomal components at the correct locations. This preliminary binding event ensures that subsequent splicing proceeds efficiently and reliably, resolving the contradiction between protein production and splicing efficiency
3Productivity
If complete splicing of all introns is achieved, then mature mRNA is produced for translation, but incomplete splicing results in nuclear accumulation and degradation
Solution Approach 1:
The patent extracts the problematic retained introns from the pre-mRNA through ASO-mediated constitutive splicing, removing them completely to prevent nuclear accumulation and degradation. This extraction approach resolves the contradiction by ensuring that only fully-spliced, translation-competent mRNA is produced, eliminating the loss pathway
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
ASOs effectively increase mRNA and protein levels by 1.1 to 10-fold, addressing conditions associated with deficient gene expression and activity, including genetic disorders such as thrombotic thrombocytopenia purpura and cystic fibrosis.
Implementation Method 1
contacting the cells with an ASO complementary to a targeted portion of the RIC pre-mRNA
Data Source
AI summary
Provided herein are methods and compositions for increasing production of a target protein or functional RNA by a cell.