Metabolically Competent Cells via CRISPR-Cas9 SAM
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Solution Overview
Problem
Current in vitro toxicity assays using immortalized cell lines are limited by their metabolic inaccuracies compared to in vivo cells, particularly in expressing genes involved in chemical metabolism, such as cytochrome P450 enzymes, which affects the accuracy of toxicity testing for industrial, pharmaceutical, and environmental chemicals.
Innovation Solution
Genetically engineered metabolically competent cells are developed using a CRISPR-Cas9 system, specifically the synergistic activation mediator (SAM) approach, to enhance the expression of cytochrome P450 and other metabolic genes, creating cells that mimic in vivo metabolic profiles for improved high-throughput screening assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If immortalized cell lines are used for in vitro toxicity assays, then the assays are easier to perform and more reproducible, but the metabolic accuracy and reliability of toxicity data are reduced
Solution Approach 1:
The patent changes the metabolic state parameters of immortalized cell lines by introducing exogenous metabolic genes (CYP450 enzymes, phase II enzymes) through genetic engineering. This transforms the cells from metabolically inactive to metabolically active, enabling them to perform chemical metabolism functions similar to primary cells while retaining the operational advantages of immortalized lines.
Solution Approach 2:
The patent creates composite cell systems by combining immortalized cell line frameworks with exogenously introduced metabolic gene components. The resulting cells contain both the stable, reproducible characteristics of immortalized lines and the metabolic capabilities of primary cells, forming a hybrid system that achieves both ease of operation and reliability.
2Ease of manufacture
If standard immortalized cell lines are used, then the cell culture procedures are simpler and more standardized, but the expression of metabolic genes such as cytochrome P450 is insufficient
Solution Approach 1:
The patent performs preliminary genetic engineering of immortalized cell lines before toxicity testing to establish metabolic competence. By pre-introducing and expressing metabolic genes in the cell line, the cells are prepared in advance to perform metabolic transformations, eliminating the need for complex metabolic activation steps during the actual toxicity assay procedures.
3Device complexity
If in vitro assays use cells with low metabolic activity, then the assay procedures are more straightforward, but the results do not accurately reflect in vivo metabolic transformations
Solution Approach 1:
The patent enables the immortalized cell lines to self-perform metabolic transformations by endogenously expressing CYP450 enzymes and phase II metabolic enzymes. The cells autonomously carry out Phase I and Phase II metabolic reactions on test chemicals without requiring external metabolic activation systems, thereby achieving accurate in vivo-like metabolic assessment while maintaining simple assay procedures.
Data Source
AI summary
Provided herein are genetically engineered cells containing one or more modulated metabolic genes, where the expression of the modulated metabolic gene(s) can be greater than that of an unmodified control. Also provided herein are methods of making the genetically engineered cells using synergistic activation mediator CRISPR-Cas9. Further provided herein are high throughput assays that can employ the genetically engineered cells provided herein.