Human Artificial Chromosome Vector for Stable Gene Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional vectors used for gene introduction into mammalian cells, such as plasmids and virus vectors, face issues like transient gene expression, chromosomal disruption, unregulated copy numbers, and interference from host chromosome control sequences, limiting their stability and efficiency in gene therapy applications.
Innovation Solution
A human artificial chromosome (HAC) vector is developed, comprising a human chromosome-derived centromere, telomere, and subtelomere sequences, which allows for stable integration and regulated expression of foreign genes by deleting distal regions of human chromosomes 14 and 21, enhancing gene expression and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional vectors (plasmid, cosmid, BAC, YAC, or virus vectors) are used to introduce genes into mammalian cells, then gene introduction is achieved, but transient expression occurs and gene stability is poor
Solution Approach 1:
The invention segments the chromosome into functional modules: centromere for replication control, telomere for stability, and gene insertion region for expression. This modular HAC vector structure enables stable long-term gene expression by separating essential chromosomal functions from the therapeutic gene, avoiding the transient expression problem of conventional vectors.
Solution Approach 2:
The HAC vector acts as an intermediary chromosome structure that bridges conventional vectors and host chromosomes. It provides chromosomal-level stability and long-term expression without requiring integration into host chromosomes, thus avoiding disruption of host genome while achieving stable gene expression.
2Stability of the object's composition
If genes are integrated into host cell chromosomes, then stable expression is achieved, but host chromosome genes are disrupted and copy number cannot be regulated
Solution Approach 1:
The invention extracts essential chromosomal elements (centromere, telomere, origin of replication) from host chromosomes to construct an autonomous HAC vector. This extracted chromosomal framework enables stable gene expression without requiring integration into host chromosomes, thereby eliminating the harmful effect of host chromosome disruption while maintaining gene stability.
3Quantity of substance
If conventional vectors are used, then gene introduction is achieved, but copy number of introduced genes is not regulated
Solution Approach 1:
The HAC vector incorporates a centromere with origin of replication that provides feedback control for copy number maintenance. The centromeric origin recognizes and binds replication initiators, automatically regulating the copy number of the introduced gene to remain consistent with the host cell's chromosomal copy number, thus achieving precise copy number control without complex external regulation systems.
4Stability of the object's composition
If genes are integrated into host chromosomes, then stable expression is achieved, but gene expression is affected by host chromosome control sequences
Solution Approach 1:
The HAC vector applies local quality control by providing a dedicated gene expression region with its own control sequences, isolated from host chromosome control sequences. The telomere-proximal gene insertion region allows genes to be expressed under their own promoters without being influenced by distant host chromosome regulatory elements, ensuring predictable and controllable gene expression while maintaining chromosomal stability.
Data Source
Figure 1
Figure 2
Figure 3
AI summary
This invention relates to a human artificial chromosome (HAC) vector carrying a human chromosome-derived centromere, a subtelomere sequence, and a telomere sequence, to a human cell medicine or human cells comprising the HAC vector, to methods for preparing the HAC vector and human cells, and to methods for producing a therapeutic protein using the HAC vector.