CRISPR/Cas9 Mediated Class Switch Recombination in Antibody Production
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Solution Overview
Problem
Current methods for class switch recombination (CSR) of immunoglobulin heavy chain genes require activation of B cells, intracellular signaling, and T cell cytokines, limiting the ability to produce specific antibody subclasses on demand.
Innovation Solution
The CRISPR/Cas9 system is used to edit the immunoglobulin heavy chain genes in mammalian cells, allowing for targeted CSR independent of B cell activation and T cell cytokines, enabling the production of desired antibody subclasses by delivering Cas9 enzyme and guide RNAs via vectors like retrovirus or lentivirus.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR/Cas9 system is used to edit immunoglobulin heavy chain genes, then class switch recombination efficiency is improved and can be performed without B cell activation, but the device complexity increases due to requiring vector delivery systems
Solution Approach 1:
The patent uses viral vectors (retrovirus or lentivirus) as intermediary carriers to deliver the CRISPR/Cas9 system components (Cas9 enzyme and guide RNAs) into mammalian cells. This mediator approach enables efficient gene editing without requiring complex in vivo manipulation or B cell activation, resolving the contradiction by providing a controlled delivery mechanism that achieves high CSR efficiency while managing the complexity through standardized vector systems.
2Adaptability or versatility
If traditional CSR methods using B cell activation and T cell cytokines are used, then the process follows natural physiological pathways, but the ability to produce specific antibody subclasses on demand is limited
Solution Approach 1:
The patent extracts the essential function of class switch recombination from its complex physiological context by using CRISPR/Cas9 to directly target and edit the immunoglobulin heavy chain gene locus. This removes the need for B cell activation, T cell cytokine signaling, and other complex physiological processes, allowing specific antibody subclasses to be produced on demand through simple gene editing while maintaining the core CSR function.
Solution Approach 2:
The patent changes the fundamental parameters of the CSR process by replacing biological signaling parameters (cytokines, cell activation states) with precise molecular targeting parameters (guide RNA sequences that direct Cas9 to specific switch regions). This parameter transformation enables controlled production of specific antibody subclasses by simply changing the guide RNA target sequence, greatly enhancing adaptability while simplifying operation.
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 efficient and specific induction of CSR in both mouse and human cells, allowing for rapid production of desired antibody subclasses, including Fab or Fab′ fragments, without the need for B cell activation or cytokine signaling.
Implementation Method 1
The CRISPR/Cas9 system is used to edit the immunoglobulin heavy chain genes in mammalian cells, allowing for targeted CSR independent of B cell activation and T cell cytokines
Implementation Method 2
DSBs are introduced at the Ig genes by the activity of B lymphocyte cell specific enzymes such as recombinase activating gene 1/2 (RAG1/2) and activation-induced cytidine deaminase, also known as AICDA and AID
Implementation Method 3
Paired DSBs in the switch regions are then joined by the classical and alternative non-homologous end joining (NHEJ) pathways to generate a switch of the IgH
Data Source
AI summary
The present disclosure provides genetically modified antibody-producing cells comprising edited chromosomal sequences associated with immunoglobulin heavy chain constant region, the IgH locus. In particular, these cells are generated using a CRISPR/Cas 9-mediated editing process. The disclosure also provides specific guide RNA (gRNA) guide sequences that target the chromosomal sequence of immunoglobulin heavy chain constant region in the Switch regions.