Engineered B-Cell Protein Factories Using β2M Locus Integration

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Solution Overview

Problem

Existing methods for genetically modifying B cells for therapeutic protein expression are not well-established, limiting their use in treating diseases such as cancer, heart disease, inflammatory diseases, and neurological disorders.

Innovation Solution

Engineering B cells to express therapeutic proteins by inserting genes into the β2M locus, either in exons or introns, while disrupting or minimally disrupting β2M expression, using RNA-guided nucleases and gRNA to achieve targeted integration and reduced endogenous β2M levels.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If genes are inserted into the β2M locus for therapeutic protein expression, then therapeutic protein expression level is improved, but β2M gene function is disrupted

Engineering Contradiction:

Improvetherapeutic protein expression levelVSAvoidβ2M gene function

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent applies local quality by making differential modifications to different regions of the β2M locus. Specifically, it inserts therapeutic genes into intronic regions while preserving exon integrity, or uses targeted approaches that disrupt only specific portions of the gene. This allows high-level therapeutic protein expression from the modified locus while maintaining sufficient β2M function for cellular viability and MHC class I presentation.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent employs partial action by achieving sufficient rather than complete disruption of β2M. The modification reduces β2M expression to levels that are adequate for maintaining cell function and immune recognition, while still allowing robust therapeutic protein expression. This partial modification strategy balances therapeutic efficacy with cellular health requirements.

Inventive Principle:

Principle #16Partial or excessive action

2Manufacturing precision

If RNA-guided nucleases are used for targeted gene insertion, then integration precision is improved, but immune response risk increases

Engineering Contradiction:

Improvegene integration precisionVSAvoidimmune response

Core Design Contradiction:

Manufacturing precisionVSObject-affected harmful factors

Solution Approach 1:

The patent uses RNA-guided nucleases (such as CRISPR-Cas9) that function by copying and recognizing specific DNA sequences through complementary base pairing. The guide RNA copies the target sequence information, enabling precise navigation to the β2M locus without requiring complex protein-protein recognition systems. This reduces the immunogenicity of the delivery system while maintaining high integration precision.

Inventive Principle:

Principle #26Copying

Solution Approach 2:

The patent employs guide RNA as an intermediary molecule that mediates between the nuclease enzyme and the target DNA sequence. This RNA intermediary provides sequence-specific targeting while being less immunogenic than protein-based recognition systems. The RNA-DNA hybrid formation allows precise localization to the β2M locus with reduced risk of triggering immune responses compared to direct protein-DNA interactions.

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS20250283036A1Engineering b cell-based protein factories to treat serious diseases

Publication Date: 2025.09.11 WALKING FISH THERAPEUTICS INC

AI summary

The invention(s) disclosed herein relate to improved methods for expanding cell populations, particularly B cell populations. The invention further relates comprising improved cell media, compositions thereof, and methods of using such expanded B cells. Wherein a population of cells comprises engineered human B cells, wherein the engineered human B cells comprise a therapeutic protein, whose gene has been inserted into the beta-2M locus.