NK Cell Gene Inactivation via Endonuclease for Enhanced Cytotoxicity
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Solution Overview
Problem
Current NK cell-based therapies for cancer have shown modest clinical success due to limitations in enhancing the cytotoxicity and durability of NK cells, necessitating improved methods to boost their functionality and cytolytic activity.
Innovation Solution
The method involves genetically engineering NK cells using specific rare-cutting endonucleases like TAL-nucleases, CRISPR, or Argonaute to inactivate genes such as TGF-β receptor, Cbl-B, and PD-1, and overexpressing genes like IL-2 receptor or IL-15 to enhance cytotoxicity and engraftment, while introducing Chimeric Antigen Receptors (CARs) to target malignant cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional NK cell-based therapies are used, then the treatment can be administered, but the cytotoxicity and durability of NK cells remain limited
Solution Approach 1:
The patent applies parameter changes by genetically modifying NK cells through gene inactivation (using endonucleases to disable inhibitory genes like TGF-βR, Cbl-B, PD-1) and gene overexpression (introducing activating genes like IL-2R, IL-15, CARs). These genetic parameter modifications fundamentally alter the functional parameters of NK cells, enhancing their cytotoxicity, persistence, and therapeutic efficacy while resolving the contradiction between limited durability and needed productivity.
2Reliability
If gene inactivation using endonucleases is performed, then NK cell functionality is enhanced, but the genetic modification complexity increases
Solution Approach 1:
The patent applies the extraction principle by specifically removing or inactivating problematic genetic elements (inhibitory genes such as TGF-βR, Cbl-B, PD-1, KIRs) from the NK cell genome using endonucleases. By extracting or disabling these specific inhibitory components while preserving the rest of the cell's functionality, the method enhances NK cell performance without requiring complete genetic redesign, thus managing complexity while improving reliability.
3Productivity
If multiple genes are modified to enhance cytotoxicity, then therapeutic activity improves, but the risk of off-target effects increases
Solution Approach 1:
The patent employs endonucleases (such as TALENs, CRISPR-Cas9, or meganucleases) as intermediary tools to achieve precise gene inactivation. These intermediary enzymes provide high specificity for their target DNA sequences, enabling selective modification of intended genes (TGF-βR, Cbl-B, PD-1, etc.) while minimizing off-target effects. The use of these specialized intermediary molecules allows multiple gene modifications to be performed safely, enhancing therapeutic activity without proportionally increasing harmful effects.
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 leads to a significant enhancement of NK cell cytotoxicity, engraftment, and drug resistance, resulting in a more durable and efficient antitumor response with improved therapeutic activity and prolonged persistence of engineered NK cells.
Implementation Method 1
Inactivating the expression of at least one gene in said NK cells by the use of a specific endonuclease such as TAL-nucleases, CRISPR or Argonaute
Data Source
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Figure 2A~2B
AI summary
The present invention relates to methods for improving therapeutic activity of NK cell, such as their cytotoxic/cytolytic activity, to be used in immunotherapy, by gene editing. In particular, these methods comprise a step of reduction or inactivation of gene expression using specific endonuclease such as TAL-nuclease, CRISPR or Argonaute. An additional genetic modification can be performed by (over)expressing at least one gene involved in N K function. The present invention encompasses also engineered NK cell, pharmaceutical composition containing the same.