Cell-Type-Specific Gene Editing via Splice-Capture Donor Cassette
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Solution Overview
Problem
Current technologies lack the ability to achieve simple and selective functional control over the majority of distinct cell types in the body, particularly in non-transgenic organisms, due to limitations in gene expression targeting and delivery methods.
Innovation Solution
The method involves introducing a programmable nuclease and a donor nucleic acid with a recognition site, splice acceptor site, self-cleaving peptide sequence, and effector gene into cells, utilizing non-homologous end joining (NHEJ)-dependent DNA repair to incorporate the effector gene specifically into cells with a target gene, ensuring cell-type-specific expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If promoter driver constructs are used for cell-type-specific gene expression, then cell type specificity is achieved, but the method is labor intensive and time consuming
Solution Approach 1:
The effector gene is divided into modular components: a splice acceptor site, a self-cleaving peptide sequence, and the effector coding sequence. This segmentation allows the gene to be delivered as a compact cassette that can be efficiently integrated into the genome without requiring large promoter constructs, thereby reducing delivery time while maintaining cell-type-specific expression through the splice-capture mechanism.
Solution Approach 2:
The effector gene is pre-configured with a splice acceptor site and self-cleaving peptide sequence before delivery. This preliminary preparation enables the gene to be immediately functional upon integration into the intron of a cell-type-specific gene, eliminating the need for time-consuming post-integration modifications and accelerating the overall process.
2Ease of operation
If viral vectors with capsid composition bias are used for delivery, then uptake is biased toward desired cells, but the method is not suitable for reliable differential targeting of most cell types
Solution Approach 1:
The invention uses the cell's own splicing machinery as an intermediary to achieve cell-type-specific expression. The effector gene is delivered with a splice acceptor site that captures the splicing machinery of actively transcribing cells. This intermediary mechanism bypasses the need for cell-type-specific delivery bias, allowing reliable targeting of most cell types through their universal splicing apparatus rather than relying on capsid composition bias.
3Reliability
If effector genes are delivered under control of cell-type-specific promoters, then functional control is achieved, but size limitations of viral delivery routes prevent reliable targeting of most cells
Solution Approach 1:
The invention extracts the cell-type-specificity function from the promoter region and relocates it to the splicing machinery. By removing the requirement for large promoter sequences and placing the effector gene under the control of constitutive promoters or even without promoters (using the splice acceptor site for transcription initiation), the construct size is dramatically reduced to fit within viral delivery vectors while maintaining reliable cell-type-specific functional control.
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 achieves high targeting efficiency and specificity, allowing for precise manipulation of molecularly distinct cell types and states, even in non-transgenic organisms, and is applicable for therapeutic interventions and tissue engineering.
Implementation Method 1
whereby the donor nucleic acid is capable of being incorporated into the intron through non-homologous end joining (NHEJ)-dependent DNA repair
Data Source
AI summary
Disclosed herein include methods and compositions for incorporating an effector gene into the genome of a cell. The method can comprise introducing into a cell a donor nucleic acid comprising a recognition site, a splice acceptor site, a self-cleaving peptide sequence, an effector gene, and an optional transcript stabilization element. The donor nucleic acid can be incorporated into the intron of a target gene differentially expressed in a unique cell type and/or in a cell during a unique cell state via non-homologous end joining (NHEJ)-dependent DNA repair. There are also provided, in some embodiments, methods and compositions for treating a disease or disorder in a subject.