Metazoan Replicator Sequences Stabilize Transgene Expression
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Solution Overview
Problem
Gene silencing remains a significant impediment in gene therapy, as introduced transgene constructs often experience a decline in expression to undetectable levels without DNA sequence changes, due to eukaryotic genome protection mechanisms.
Innovation Solution
Incorporating metazoan replicator nucleic acid sequences into transgene constructs that integrate into the host genome, altering DNA replication timing from late S phase to early S phase, thereby inhibiting silencing and maintaining chromatin decondensation and histone acetylation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If transgene constructs are introduced into host cells for gene therapy, then therapeutic gene expression is achieved, but gene silencing occurs over time causing expression to decline to undetectable levels
Solution Approach 1:
The patent applies preliminary action by incorporating replicator sequences into the transgene construct before introduction into host cells. These replicator sequences pre-establish early S phase replication timing and open chromatin structure, creating a protective environment that prevents subsequent gene silencing and maintains long-term expression stability
Solution Approach 2:
The patent changes the replication timing parameter from late S phase to early S phase by incorporating replicator sequences. This parameter change correlates with maintained gene expression, as early replicating regions are associated with open chromatin structure and active transcription, thereby preventing gene silencing over time
2Stability of the object's composition
If eukaryotic genome protection mechanisms are activated to protect against foreign DNA, then genome stability is maintained, but transgene expression is silenced
Solution Approach 1:
The patent uses replicator sequences as intermediary elements that mediate between the host genome protection mechanisms and the introduced transgene. These replicator sequences facilitate the acceptance of foreign DNA by establishing early replication timing and open chromatin structure, allowing transgene expression without triggering silencing while maintaining genome stability
Solution Approach 2:
The patent converts the potentially harmful effect of genome protection mechanisms (which normally silence foreign DNA) into a benefit by using replicator sequences to establish a permissive chromatin environment. The same mechanisms that protect the genome are redirected to protect and maintain transgene expression through early replication timing and open chromatin structure
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 effectively delays or prevents gene silencing, stabilizes transgene expression, and maintains early replicating chromatin structure, enhancing the longevity and level of gene expression in mammalian cells.
Implementation Method 1
The replicator nucleic acid sequence alters the timing of DNA replication of the transgene construct, e.g., from late S phase to early S phase, thereby inhibiting silencing of the coding nucleic acid sequence
Data Source
AI summary
Regulatory elements, specifically replicators and transgene constructs containing replicator nucleic acid sequences, are disclosed herein. Methods of using replicators and transgene constructs including replicators to inhibit, delay, or prevent gene silencing are also disclosed herein.