Expression Cassette Architecture for Stable Therapeutic C1-INH Output
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Solution Overview
Problem
Current gene therapy methods face challenges in achieving high and stable expression of target genes, such as C1-INH protein, required for treating conditions like hereditary angioedema, with existing expression cassettes failing to reach therapeutic levels and causing immune responses and side effects.
Innovation Solution
Development of an expression cassette with a specific structure (Formula I) comprising elements like HCR, DSE, TPL, eMlp, intron, and poly(A) sequences, optimized for high expression and integration into host chromosomes, using AAV vectors for delivery, enabling long-term expression with low side effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional expression cassettes are used, then gene delivery can be achieved, but the expression level of target genes is insufficient to reach therapeutic levels
Solution Approach 1:
The patent combines multiple enhancer elements (HCR, DSE, TPL, eMlp) and intron elements into a single expression cassette structure. This merging of multiple functional elements works synergistically to achieve high-level expression of target genes, resolving the contradiction between achieving sufficient expression quantity and maintaining therapeutic reliability.
Solution Approach 2:
The expression cassette is constructed as a composite structure containing multiple different functional elements (promoters, enhancers, introns, poly(A) signals) that work together. This composite design enables the cassette to simultaneously achieve high expression levels and stable, reliable therapeutic effects by integrating the functions of various genomic elements.
2Quantity of substance
If high expression of target genes is achieved, then therapeutic levels can be reached, but immune responses and side effects occur
Solution Approach 1:
The patent optimizes the expression cassette structure by selecting and combining specific enhancer elements (HCR, DSE, TPL, eMlp) and intron elements that enable high expression at physiological parameters. This parameter optimization allows achieving therapeutic protein levels without triggering immune responses, as the expression remains within physiological ranges despite high production.
3Duration of action of stationary object
If gene therapy provides long-term expression, then lifelong benefits can be achieved, but integration into host chromosome may cause insertional mutagenesis
Solution Approach 1:
The patent uses AAV vectors as intermediary carriers to deliver the expression cassette. AAV vectors enable long-term gene expression through episomal persistence or safe integration mechanisms, acting as an intermediary that reduces the direct risk of harmful chromosomal integration while maintaining durable expression benefits.
4Quantity of substance
If expression cassette structure is optimized for high expression, then therapeutic levels are achieved, but preparation complexity increases
Solution Approach 1:
The complex expression cassette is segmented into distinct functional modules: promoter region, enhancer elements (HCR, DSE, TPL, eMlp), intron elements, target gene, and poly(A) signal. This segmentation allows for systematic construction and optimization of each element independently while maintaining overall high expression capability, making the complex structure more manageable and preparable.
Data Source
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.