Composite Expression Cassette for High-Level Target Gene Expression
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Solution Overview
Problem
Current gene therapy methods face challenges in achieving high titer, efficient expression of target genes in target cells, specific integration into host chromosomes, and reducing immune responses, particularly for conditions like hereditary angioedema, which requires a minimum C1-INH protein concentration of 112 µg/ml for therapeutic effect.
Innovation Solution
An expression cassette with a specific structure (Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8) is developed, comprising elements like HCR, DSE, TPL, eMlp, intron, and poly(A), optimized for high expression and low side-effects, integrated into an AAV-based gene delivery system for long-term expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional gene therapy methods are used, then gene delivery can be achieved, but the expression level of target genes is insufficient to reach therapeutic concentration
Solution Approach 1:
The patent combines multiple enhancer elements (HCR, DSE, TPL, eMlp) and intron elements into a single expression cassette to achieve synergistic enhancement of gene expression. This merging of multiple functional elements allows the system to overcome the limitation of individual elements and achieve the high expression levels needed for therapeutic effectiveness.
Solution Approach 2:
The expression cassette is constructed as a composite structure containing multiple different functional elements (enhancers, introns, promoter regions) that work together to maximize gene expression. This composite approach allows each element to contribute its specific function, resulting in cumulative enhancement that achieves therapeutic protein concentrations.
2Quantity of substance
If high dose gene therapy is administered to achieve therapeutic protein levels, then expression level increases, but immune responses are triggered
Solution Approach 1:
The patent optimizes the expression cassette parameters including enhancer combinations, intron sequences, and promoter regions to achieve high expression efficiency at lower dosages. By changing these molecular parameters, the system can reach therapeutic protein levels without requiring high doses that would trigger immune responses.
3Quantity of substance
If gene therapy is administered to achieve curative effect, then therapeutic benefit is achieved, but long-term stable expression and specific integration are not achieved
Solution Approach 1:
The patent uses AAV vectors as intermediaries to deliver the optimized expression cassette to target cells. AAV provides site-specific integration capability and long-term stable expression, acting as a mediator between the therapeutic gene and the host genome, thereby achieving both therapeutic effectiveness and long-term stability.
Data Source
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AI summary
The present invention provides an expression cassette for a target gene and use thereof. Specifically, the present invention provides an expression cassette having elements such as HCR, DSE, TPL and eMlp, an encoding nucleic acid thereof, an expression vector thereof, a host cell thereof, a pharmaceutical composition thereof, a gene delivery system thereof, and use thereof. The present invention further provides a C1-INH protein-encoding nucleic acid molecule and use thereof.