ABCA4 Split Expression Cassettes Using GP41-1 Intein Splicing
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Solution Overview
Problem
Existing intein-based strategies for expressing ABCA4 protein suffer from inefficiencies in cleavage and produce unwanted protein intermediates, leading to potential safety risks and immune responses, particularly when using split intein dual AAV vectors for treating retinal degenerative diseases.
Innovation Solution
The use of specific cleavage sites for ABCA4 protein truncation, combined with GP41-1 intein, to create expression cassettes that form a complete ABCA4 protein through fusion with N-terminal and C-terminal truncated forms, allowing efficient expression and reduced intein production, thereby minimizing immune response.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If split intein dual AAV vectors are used to express ABCA4 protein, then the expression of ABCA4 can be achieved, but unwanted protein intermediates accumulate leading to immune responses and safety risks
Solution Approach 1:
The patent extracts and removes the problematic intein sequences from the expression system. By using aintein-free expression cassettes that directly express the N-terminal and C-terminal fragments of ABCA4 without intein components, the source of unwanted protein intermediates and immune responses is eliminated while maintaining the ability to express functional ABCA4 protein.
2Productivity
Ifinteins are used to facilitate protein splicing, then larger protein fragments can be expressed, but the excised inteins and protein intermediates accumulate causing adverse side effects
Solution Approach 1:
The patent converts the potential harm of intein accumulation into a benefit by completely eliminating inteins from the system. Instead of using inteins that require excision and potentially accumulate, the design uses direct expression cassettes that produce only the desired ABCA4 fragments, transforming the problem of intein management into a clean expression system with no harmful byproducts.
3Device complexity
If full-length ABCA4 is cleaved into two fragments using p1 150Cys as cleavage site, then the split intein dual AAV strategy can be implemented, but N-terminal and C-terminal by-products that are uneffectively spliced create safety risks
Solution Approach 1:
The patent employs a disposable expression approach where N-terminal and C-terminal fragments of ABCA4 are expressed separately by AAV vectors and naturally accumulate in the target cells. These fragments are designed to be stable and functional without requiring efficient splicing, effectively making the splicing step unnecessary while maintaining therapeutic benefit.
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 expression efficiency of the complete ABCA4 protein, effectively restoring retinal function in mouse models and reducing toxic bisretinoid derivatives, thus addressing the limitations of previous methods.
Implementation Method 1
When transformed into mature proteins, they are excised through non-enzymatic transpeptidation reactions, and the corresponding exteins are retained in the mature protein.
Data Source
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AI summary
An expression cassette combination and use thereof. The expression cassette combination comprises the first expression cassette and the second expression cassette, wherein the first expression cassette can express a fusion protein of the N-terminal truncated form of ABCA4 protein and the N-terminal of intein, and the second expression cassette can express a fusion protein of the C-terminal truncated form of ABCA4 protein and the C-terminal of intein. When expressed, the N-terminal truncated form of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncated form of the ABCA4 protein expressed by the second expression cassette can form a complete full-length ABCA4 protein through the cleavage function of the N-terminal and C-terminal of the intein.