LDHA and PDK Knockout in Mammalian Cells

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Solution Overview

Problem

Current methods fail to completely eliminate lactate dehydrogenase activity in mammalian cells, leading to lactate production that limits cell growth and affects recombinant protein production, requiring base addition that increases osmolarity and impacts bioprocesses.

Innovation Solution

Mammalian cells are engineered to be completely devoid of lactate dehydrogenase activity by homozygously knocking out or rendering non-functional genes for LDHA, LDHB, and pyruvate dehydrogenase kinase isoforms PDK1, PDK2, PDK3, and PDK4, allowing for the production of recombinant proteins without detectable lactate.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Stability of the object's composition

If base is added to control pH during bioprocess, then pH regulation is improved, but osmolarity increases which inhibits cell growth and shortens production time

Engineering Contradiction:

ImprovepH controlVSAvoidosmolarity increase

Core Design Contradiction:

Stability of the object's compositionVSObject-affected harmful factors

Solution Approach 1:

The patent removes the harmful metabolic pathway by knocking out LDHA and PDK genes, extracting the lactate production capability from the cell. This eliminates the source of pH deviation, making base addition unnecessary and avoiding osmolarity increase while maintaining pH stability

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent performs preliminary genetic modification by knocking out LDHA and PDK genes before bioprocess begins. This preliminary action prevents lactate accumulation from occurring, thereby preventing the need for pH correction and avoiding subsequent osmolarity problems

Inventive Principle:

Principle #10Preliminary action

2Object-generated harmful factors

If LDHA is knocked out to eliminate lactate production, then lactate accumulation is reduced, but complete knockout is lethal in CHO cells when PDK is not down-regulated

Engineering Contradiction:

Improvelactate productionVSAvoidcell viability

Core Design Contradiction:

Object-generated harmful factorsVSReliability

Solution Approach 1:

The patent segments the metabolic regulation by targeting multiple separate genes (LDHA and PDK1/PDK2/PDK3) rather than a single gene. This segmentation allows precise control of metabolic pathways while maintaining cell viability through coordinated gene knockout

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent combines knockout of LDHA with down-regulation of multiple PDK isoforms to achieve complete lactate elimination while maintaining cell viability. This combined approach addresses both the harmful lactate production and the viability issue simultaneously

Inventive Principle:

Principle #5Merging (Combining)

Data Source

PatentUS11242510B2Mammalian cells devoid of lactate dehydrogenase activity

Publication Date: 2022.02.08 RGT UNIV OF CALIFORNIA

AI summary

Provided are mammalian cells devoid of lactate dehydrogenase activity.