Base Editing Primary Human NK Cells for Cancer Immunotherapy
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Solution Overview
Problem
Natural killer (NK) cells are difficult to genetically modify effectively due to their hard-to-edit nature, which hinders their tumor-killing activity in cancer immunotherapies, as cancer manipulates inhibitory and activating signals, impairing their function and migration into tumor sites.
Innovation Solution
The use of base editors and guide RNAs, combined with an RNase inhibitor and optimized electroporation settings, to introduce genetic modifications into NK cells, achieving high editing efficiencies and improved cytotoxicity against cancer cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional gene editing methods are used on NK cells, then genetic modification is achieved, but editing efficiency is low and cell viability is poor
Solution Approach 1:
The patent optimizes multiple parameters including electroporation voltage (1100-1500V), pulse duration (5-20ms), RNA-to-cell ratio, and base editor concentration to achieve high editing efficiency while maintaining cell viability. This systematic parameter optimization resolves the contradiction between editing efficiency and cell survival.
Solution Approach 2:
The patent performs preliminary actions by pre-mixing editing reagents with RNase inhibitors and optimizing delivery conditions before electroporation. This preliminary preparation protects the RNA reagents and ensures optimal editing conditions, thereby improving both editing efficiency and cell viability.
2Reliability
If NK cells are genetically modified to enhance tumor-killing activity, then cytotoxicity is improved, but the difficulty of genetic modification increases
Solution Approach 1:
The patent uses electroporation as an intermediary method to deliver base editor mRNA and guide RNA into NK cells. This intermediary approach overcomes the hard-to-edit nature of NK cells while achieving reliable genetic modification for enhanced cytotoxicity.
Solution Approach 2:
The patent replaces traditional mechanical gene delivery methods (such as viral transduction or microinjection) with electroporation, which uses controlled electrical fields to create temporary pores in cell membranes. This substitution simplifies the genetic modification process while maintaining high efficiency.
3Manufacturing precision
If base editors and guide RNAs are introduced into NK cells, then genetic modification efficiency is improved, but process complexity increases
Solution Approach 1:
The patent merges the base editor mRNA and guide RNA into a single electroporation mixture, simplifying the delivery process. This combination approach maintains high genetic modification efficiency while reducing the number of separate steps required.
Solution Approach 2:
The electroporation method serves multiple functions: it delivers both base editor mRNA and guide RNA simultaneously, optimizes RNA stability through controlled parameters, and ensures efficient cellular uptake. This multi-functionality reduces overall process complexity.
Data Source
AI summary
The present invention provides efficient methods for producing genetically modified natural killer (NK) cells using a base editor and guide RNA(s). Genetically modified NK cells produced by these methods and the use of these cells in the treatment of cancer are also provided.