5'UTR Mirtron Vector Splicing Efficiency
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Solution Overview
Problem
Co-delivery of artificial mirtrons with a transgene for gene therapy is challenging due to inefficient splicing and transgene expression, as existing methods struggle to preserve both efficient splicing and adequate transgene expression.
Innovation Solution
A method involving the use of a vector with a mirtron located in the 5'UTR of a transgene, accompanied by exonic splice enhancers, to facilitate efficient splicing and gene knockdown, utilizing adeno-associated virus (AAV) for delivery and incorporating multiple mirtrons for enhanced efficacy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If artificial mirtrons are co-delivered with a transgene, then gene knockdown efficiency is improved, but splicing efficiency and transgene expression are adversely affected
Solution Approach 1:
The patent segments the transgene expression unit into distinct functional regions: a promoter region, a 5'UTR region containing the mirtron, and a coding region. This segmentation allows the mirtron to be isolated in the 5'UTR where it can efficiently splice and knock down target genes without interfering with the coding sequence and transgene expression.
Solution Approach 2:
The 5'UTR serves as an intermediary region that hosts the mirtron sequence. This intermediary location enables the mirtron to perform its gene knockdown function while the downstream coding region maintains normal transgene expression, thus mediating between the conflicting requirements of efficient splicing and adequate expression.
2Manufacturing precision
If mirtrons are placed in the coding region of a reporter gene, then splicing efficiency is improved, but transgene expression is reduced
Solution Approach 1:
The patent extracts the mirtron from the coding region and relocates it to the 5'UTR. This extraction removes the harmful impact on transgene expression while preserving the mirtron's splicing efficiency, as the 5'UTR provides a suitable environment for mirtron processing without disrupting the coding sequence.
Solution Approach 2:
The patent changes the dimensional location of the mirtron from within the coding sequence (one-dimensional insertion) to the upstream 5'UTR region (spatial relocation). This dimensional change allows the mirtron to function independently in a different genomic context that is favorable for both splicing and non-interference with transgene expression.
3Reliability
If multiple mirtrons are co-delivered from a single transcript, then gene knockdown efficacy is enhanced, but splicing complexity increases
Solution Approach 1:
The patent merges multiple mirtron sequences into a single 5'UTR region of one transcript. This combining approach allows multiple mirtrons to be co-expressed from a single transcriptional unit, enhancing gene knockdown efficacy against multiple targets while utilizing the shared 5'UTR splicing machinery to manage splicing complexity.
Data Source
AI summary
A method of gene therapy in a subject in need thereof, the method comprising administering to the subject a vector that comprises a transgene for expression in the subject and a mirtron for knocking down expression of a gene in the subject, wherein the mirtron is in the 5′UTR of the transgene. Also a vector comprising a transgene for expression from the vector and a mirtron for knocking down expression of a gene, wherein the mirtron is in the 5′UTR of the transgene and wherein the 5′UTR further comprises an exonic splice enhancer motif and/or wherein the mirtron is flanked in the 5′UTR by a fragment of the coding region of a reporter gene, and methods for preparing a vector.