Self-limiting rAAV Vectors for Controlled Genome Editing
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Solution Overview
Problem
Current rAAV vectors have long persistence times in cells, leading to immune responses and off-target DNA cutting due to prolonged expression of site-specific endonucleases, which is undesirable for genome editing applications.
Innovation Solution
Development of self-limiting rAAV vectors that incorporate a recognition sequence for a site-specific endonuclease within the vector, allowing the endonuclease to cut the vector and limit its persistence by exonuclease degradation, ensuring controlled expression and reduced immune response.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If rAAV vectors are used to deliver site-specific endonucleases for genome editing, then precise genome editing is achieved, but the vectors persist too long in cells causing immune responses and off-target DNA cutting
Solution Approach 1:
The patent incorporates a recognition sequence for the site-specific endonuclease directly into the rAAV vector genome before delivery. This preliminary action ensures that once the endonuclease is expressed, it will automatically cleave the vector DNA at the recognition site, limiting persistence time without requiring external intervention or complex regulatory elements
Solution Approach 2:
The patent converts the potential harmful effect of endonuclease activity (which could cut any DNA it encounters) into a beneficial self-limiting mechanism. By including the recognition sequence in the vector, the endonuclease's cutting activity is redirected to eliminate the vector itself after delivering the editing function, thereby preventing prolonged expression and off-target effects
2Productivity
If the endonuclease expression is prolonged to ensure adequate genome editing, then editing efficiency improves, but immune responses and off-target cutting increase
Solution Approach 1:
The patent implements a self-service mechanism where the endonuclease expressed from the rAAV vector automatically cleaves the vector's own recognition sequence. This self-limiting action ensures the endonuclease is expressed long enough to perform genome editing but is then automatically terminated, eliminating the need for external control mechanisms or complex regulatory elements
Solution Approach 2:
The patent employs a mechanism where the vector DNA is deliberately discarded after serving its purpose of delivering the endonuclease. The recognition sequence embedded in the vector allows the endonuclease to cleave and eliminate the vector DNA once editing is complete, thereby recovering the cell from prolonged foreign protein expression and reducing immune activation
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 self-limiting rAAV vectors effectively limit vector persistence, preventing concatamer formation and immune activation, while ensuring precise and controlled genome editing by attenuating endonuclease expression, thereby enhancing therapeutic safety and efficacy.
Implementation Method 1
expression of the endonuclease in a cell cleaves the viral vector and limits its persistence time
Data Source
AI summary
Disclosed herein are viral vectors for use in recombinant molecular biology techniques. In particular, the present disclosure relates to self-limiting viral vectors comprising genes encoding site-specific endonucleases as well as recognition sequences for site-specific endonucleases such that expression of the endonuclease in a cell cleaves the viral vector and limits its persistence time. In some embodiments, the viral vectors disclosed herein also carry directives to delete, insert, or change a target sequence.