SHMT2 Knockout Cell Lines for Dual Vector Selection

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Solution Overview

Problem

Existing mammalian cell lines for biomanufacturing lack efficient multiple selection methods for introducing multiple vectors, limiting the production of molecules like bispecific antibodies and proteins requiring effector proteins.

Innovation Solution

Engineer mammalian cell lines with reduced or eliminated expression of the endogenous Serine hydroxymethyl transferase 2 (SHMT2) gene and optionally Glutamine Synthetase (GS) gene, using targeted endonuclease-mediated genome editing to introduce a glycine auxotrophy, enabling dual selection systems with glycine-formate supplementation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If traditional single selection method is used, then cell line is simple to maintain, but multiple vectors cannot be introduced for producing bispecific antibodies and multispecific proteins

Engineering Contradiction:

Improveselection method versatilityVSAvoidcell line engineering complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent changes the metabolic parameters of the cell line by knocking out the SHMT2 gene, which blocks the glycine-formate biosynthesis pathway. This creates a new selectable phenotype (glycine auxotrophy) that enables a second selection method independent of the traditional GS system, allowing dual vector introduction without increasing overall system complexity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent segments the selection system into two independent pathways: (1) GS-based selection for one vector, and (2) glycine-formate pathway selection for another vector. By dividing the selection functionality into separate metabolic pathways, the system can independently select for multiple different vectors simultaneously

Inventive Principle:

Principle #1Segmentation

2Adaptability or versatility

If SHMT2 gene expression is eliminated, then dual selection system is enabled, but cells require exogenous glycine and formate supplementation

Engineering Contradiction:

Improveselection system capabilityVSAvoidmedia supplementation requirement

Core Design Contradiction:

Adaptability or versatilityVSEase of operation

Solution Approach 1:

The patent applies self-service by providing the cell line with constitutive expression of the formate transporter gene (SLC19A3), enabling the cells to autonomously import formate from the media. This built-in transport capability eliminates the need for external formate supplementation, as the cells self-regulate their formate uptake to maintain the selection pressure

Inventive Principle:

Principle #25Self-service

3Manufacturing precision

If CRISPR RNP complexes are used for genome modification, then precise SHMT2 knockout is achieved, but additional reagents and procedures are required

Engineering Contradiction:

Improvegenome editing precisionVSAvoidcell line engineering process

Core Design Contradiction:

Manufacturing precisionVSEase of manufacture

Solution Approach 1:

The patent applies preliminary action by delivering pre-assembled CRISPR ribonucleoprotein (RNP) complexes directly into the cells. The CRISPR guide RNA and Cas9 protein are pre-complexed before delivery, allowing the editing machinery to be immediately functional upon entry. This pre-preparation eliminates the need for cells to transcribe and translate CRISPR components, streamlining the process while maintaining high precision

Inventive Principle:

Principle #10Preliminary action

Data Source

PatentUS20260098081A1Metabolic selection via the glycine-formate biosynthesis pathway

Publication Date: 2026.04.09 EMD MILLIPORE CORP

AI summary

The present disclosure provides an isolated mammalian cell comprising a reduced or eliminated expression of Serine Hydroxymethyltransferase 2 (SHMT2). Further provided are methods for preparing such cells and methods for using such cells for the production of recombinant proteins.