Engineering B Lymphocytes via Endogenous AID Activation
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Solution Overview
Problem
Current methods for engineering B cells to produce therapeutic antibodies face challenges such as off-target mutations, immunogenicity, and safety concerns due to the use of exogenous nucleases like CRISPR/Cas9, which can lead to unintended effects and immune responses.
Innovation Solution
A method involving the activation of endogenous activation-induced cytidine deaminase in B lymphocytes to introduce a DNA molecule encoding a peptide of interest, allowing for genome editing without the need for exogenous nucleases, thereby reducing the risk of off-target mutations and immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If exogenous nucleases like CRISPR/Cas9 are used to edit B cell genomes, then genome editing capability is improved, but off-target mutations and immunogenicity increase
Solution Approach 1:
The patent utilizes the B cell's own endogenous AID enzyme to perform genome editing functions. By providing a DNA template with homology arms that targets the switch region, the endogenous AID-mediated mechanisms (normally used for class switch recombination) are harnessed to achieve safe and efficient genome editing without introducing exogenous nucleases, thereby eliminating off-target effects and immunogenicity concerns
Solution Approach 2:
The patent introduces a DNA template molecule as an intermediary that contains the desired genetic modification and homology arms. This template serves as the mediator between the endogenous AID enzyme and the target genome, directing the editing activity to the specific switch region while avoiding random off-target mutations
2Manufacturing precision
If exogenous nucleases are introduced into B cells, then editing precision is improved, but safety and immunogenicity worsen
Solution Approach 1:
The invention replaces exogenous nuclease systems with the B cell's endogenous AID enzyme, which naturally targets switch regions for class switch recombination. This self-service approach maintains editing precision at the intended locus while eliminating safety concerns associated with foreign nucleases, including off-target effects and immune responses
3Reliability
If endogenous AID is activated for genome editing, then safety and reduced immunogenicity are improved, but editing efficiency may be limited compared to exogenous nucleases
Solution Approach 1:
The patent optimizes parameters including the design of homology arms on the DNA template, the choice of target switch regions, and culture conditions to enhance the efficiency of endogenous AID-mediated editing. By carefully tuning these parameters, the method achieves reliable safety profiles while maintaining practical editing efficiency for therapeutic antibody production
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 enables safer and more precise editing of B cell genomes, potentially lowering the risk of off-target effects and immune responses, while allowing for the production of customized antibodies.
Implementation Method 1
activating endogenous activation-induced cytidine deaminase of the B lymphocyte; introducing a DNA molecule comprising a nucleotide sequence encoding a (poly)peptide of interest into the B lymphocyte
Data Source
AI summary
The present invention provides a method for engineering B lymphocytes by utilizing activation-induced cytidine deaminase of the B lymphocyte. Thereby, use of engineered nucleases, such as Cas nuclease, can be avoided. Engineered B cells are useful to produce customized antibodies and for B cell therapy. Accordingly, the present invention also provides engineered B cells and customized antibodies produced by engineered B cells.