Genetically Modified PSC-Derived NK Cells for Stable Gene Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current gene editing strategies for pluripotent stem cell-derived natural killer (iNK) cells face challenges such as gene silencing and reduced gene expression, which affect their cytotoxicity and differentiation potential, hindering their effectiveness in cell therapy applications.
Innovation Solution
Integrating an expression cassette at the CISH and/or Rosa26 locus in the genome of pluripotent stem cells using site-specific endonucleases for homologous recombination, ensuring stable and uniform expression of exogenous polynucleotides in genetically-modified iNK cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gene editing is performed at random loci in PSCs, then gene integration can be achieved, but gene silencing and reduced expression occur during differentiation
Solution Approach 1:
The patent identifies and selects safe harbor loci (CISH, Rosa26) in advance where gene integration will not cause silencing. By pre-determining these safe locations through systematic investigation of various target sites, the patent ensures stable expression before differentiation occurs, avoiding the need for random integration that leads to silencing.
Solution Approach 2:
The patent changes the integration location parameter from random to specific predetermined loci (CISH, Rosa26). This parameter change transforms the gene editing outcome from unstable and silencing-prone to stable and reliable, while the use of CRISPR/Cas9 maintains reasonable ease of manufacture.
2Reliability
If gene editing is performed at highly methylated loci in PSCs, then integration can occur, but expression of target gene cannot be observed or detected
Solution Approach 1:
The patent performs preliminary investigation of the epigenetic state of various loci to identify those that are not highly methylated. By selecting loci like CISH and Rosa26 that have been pre-validated to lack high methylation, the patent ensures gene expression detectability without needing to perform complex epigenetic modifications at the time of integration.
Solution Approach 2:
The patent applies the principle of local quality by selecting specific loci with favorable epigenetic characteristics (low methylation) for gene integration. Rather than attempting to modify the epigenetic state of all loci, the patent focuses on integrating genes at locations that naturally possess the desired quality of low methylation, ensuring expression detectability.
3Reliability
If gene editing is performed at certain loci to enhance expression, then cytotoxicity can be improved, but pluripotency and differentiation potential of PSCs are adversely affected
Solution Approach 1:
The patent performs preliminary testing to identify loci that allow high gene expression without compromising PSC characteristics. By pre-identifying safe harbor loci like CISH and Rosa26 that are permissive for gene expression but neutral for pluripotency and differentiation, the patent achieves both improved cytotoxicity and preserved differentiation potential.
Solution Approach 2:
The patent changes the integration location parameter to specific safe harbor loci that are decoupled from pluripotency and differentiation control regions. This parameter change allows high-level gene expression for improved cytotoxicity while maintaining the adaptability and differentiation potential of PSCs, as the selected loci do not interfere with key developmental pathways.
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 enhances the expression level and uniformity of exogenous polynucleotides in iNK cells, improving their cytotoxicity and differentiation efficiency, addressing the limitations of previous methods.
Implementation Method 1
integrating the expression cassette comprising the one or more exogenous polynucleotides of interest and the one or more promoters into the genome of the PSCs at the selected locus via homologous recombination
Data Source
AI summary
Provided herein are genetically-modified cells such as pluripotent stem cells and derived NK cells and methods for producing the same. The gene editing strategy of the present disclosure can achieve higher expression of the transgene.