Genomic Safe Harbor Selection for Stable Transgene Expression
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Solution Overview
Problem
Current methods for inserting transgenes into the human genome, such as retroviral and lentiviral vectors, often result in mutations due to random integration near actively transcribed genes, posing risks like cancer development, while existing safe harbor sites like AAVS1 may disrupt adjacent gene transcription and suppress transgene expression.
Innovation Solution
A novel genomic safe harbor (GSH) is identified at specific regions on chromosomes 9, 3, and 4, utilizing CRISPR-Cas9 and homology-directed repair to precisely insert exogenous polynucleotides, ensuring stable transgene expression without disrupting adjacent genes, using nucleases like Cas9 and guide RNAs, and incorporating left and right homology arms for precise integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If retroviral or lentiviral vectors are used for gene insertion, then gene delivery efficiency is improved, but insertion accuracy deteriorates and safety risks increase due to random integration near actively transcribed genes
Solution Approach 1:
The patent introduces a site-specific recombinase system as an intermediary mechanism between the viral vector and the genome. The recombinase enzyme acts as a mediator that recognizes specific attachment sites (attP and attL) and catalyzes precise recombination events, ensuring that transgene insertion occurs only at predetermined safe harbor locations rather than randomly near actively transcribed genes.
2Reliability
If AAVS1 is used as a genomic safe harbor, then transgene integration is achieved, but adjacent gene transcription is disrupted and transgene expression is suppressed
Solution Approach 1:
The patent applies the local quality principle by identifying and utilizing multiple distinct genomic safe harbor locations (AAVS1, CCR5, TRPS1, LMO2, and novel sites) throughout the genome. Each location has been specifically characterized to have unique properties that support transgene expression without disrupting adjacent genes. The system allows selection of the most appropriate local site based on the specific application requirements.
3Manufacturing precision
If CRISPR-Cas9 is used for precise gene insertion, then insertion accuracy is improved, but the complexity of the system increases
Solution Approach 1:
The patent merges multiple gene editing approaches into a unified system. It combines the site-specific recombination mechanism (using Cre or Flp recombinases) with homology-directed repair (HDR) pathways, and can be integrated with CRISPR-Cas9 or other nuclease systems. This merged approach leverages the strengths of each mechanism: the precision of site-specific recombination, the efficiency of HDR, and the programmability of CRISPR, while reducing overall system complexity through coordinated action.
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
The novel GSH maintains stable transgene expression without disturbing adjacent gene transcription, enabling safe and efficient intracellular expression of various transgenes.
Implementation Method 1
a site-specific gene insertion system based on CRISPR-Cas9 and homology-directed repair (HDR) is used. CRISPR stands for clustered regularly interspaced short palindromic repeats and is a genetic sequence that acts as an adaptive immune system in bacteria. When bacteria are infected with a virus, the guide RNA (gRNA) expressed from CRISPR binds to the nuclease Cas9, and recognizes and cleaves the part of the virus genome that has a sequence complementary to the gRNA
Implementation Method 2
HDR is a process of repairing damaged DNA using DNA fragments that are homologous to the corresponding position sequence when a double strand break (DSB) occurs. By inducing DSB in the genome through gRNA and Cas9, and simultaneously introducing a donor vector that has a sequence homologous to transgenes, gene insertion can be precisely induced
Data Source
AI summary
The present invention relates to a novel genomic safe harbor (GSH), a method for expressing a transgene using same, and cells introduced with a transgene by the same method. The method for expressing a transgene using the novel genomic safe harbor according to the present invention allows the safe introduction and long-term stable expression of a transgene without disrupting the transcription of adjacent genes, thereby enabling the safe expression of various transgenes within cells without safety concerns.