S/MAR-Flanked Polynucleotide Construct for Stable Non-Integrative Gene Expression
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Solution Overview
Problem
Current genetic modification techniques for treating inherited diseases often result in transient modifications and risk inducing deleterious mutations, such as cancer, due to the integration of transgenes into the host cell genome, while stable non-integrative vectors based on S/MAR elements are not yet available for gene therapy.
Innovation Solution
A polynucleotide construct comprising a promoter, an expressible construct, and an S/MAR element, where the S/MAR element is located downstream of the promoter and flanked by a splice donor and a splice acceptor, providing stable expression and minimizing integration risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If transgenes are integrated into the host cell genome to achieve stable modification, then stable transgene expression is achieved, but the risk of inducing deleterious mutations increases
Solution Approach 1:
The construct is divided into functional modules: promoter region, expressible construct, and S/MAR element. This segmentation allows the transgene to be maintained as a stable episomal unit rather than integrating into the genome, thus achieving stable expression without genotoxicity
Solution Approach 2:
The S/MAR element acts as an intermediary that mediates stable maintenance of the episomal construct by facilitating attachment to the nuclear matrix. This intermediary mechanism enables long-term stable expression without requiring genomic integration
2Object-affected harmful factors
If transient transfection protocols are used to avoid genome integration, then the risk of deleterious mutations is reduced, but stable transgene expression is not achieved
Solution Approach 1:
The construct is designed with pre-configured S/MAR elements that enable stable episomal maintenance from the outset. The splicing signals are预先 arranged to ensure proper processing and stable expression without requiring subsequent integration events
Solution Approach 2:
The invention changes the fundamental parameter of transgene maintenance from transient to stable by incorporating S/MAR elements. This parameter change enables long-term expression while maintaining the non-integrative nature of the delivery
3Stability of the object's composition
If S/MAR-based vectors are used for stable maintenance, then stable transgene expression is achieved, but satisfactory long-term expression has not been reliably obtained
Solution Approach 1:
The construct combines multiple functional elements (promoter, expressible construct, S/MAR element with flanking splice sites) into a composite structure. This composite design integrates the stabilizing effect of S/MARs with the expression machinery, achieving reliable long-term expression
4Reliability
If the S/MAR element is flanked by splice donor and splice acceptor sites, then proper splicing and expression are achieved, but the construct complexity increases
Solution Approach 1:
The splice donor and acceptor sites are merged with the S/MAR element boundaries, creating a unified functional unit. This merging eliminates the need for separate splicing signals and integrates the stability function with the expression function in a single compact design
Data Source
AI summary
The present invention relates to a polynucleotide comprising at least one promoter, at least one expressible construct, and an S/MAR element, wherein said polynucleotide is an integration construct or a non-integrative vector construct, wherein said S/MAR element is located downstream of said promoter and of said expressible construct, and wherein said S/MAR element is flanked by a splice donor and a splice acceptor. The present invention also relates to a composition and a host cell comprising said polynucleotide, as well as to uses and methods related thereto.