Hypoimmunogenic Cell Engineering With Integrated B2M:HLA Kill Switch
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Solution Overview
Problem
Existing genetic engineering methods struggle to create hypoimmunogenic cells that prevent HLA class II function and ensure proper integration of a kill switch gene, leading to potential immune rejection and loss of cell functionality.
Innovation Solution
Integrate an insertion sequence into the B2M gene to create a B2M:HLA fusion gene in the same open reading frame with a kill switch gene, using selectable markers for verification, and a multicistronic element to ensure separate protein functionality, thereby creating a safeguarded hypoimmunogenic cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing genetic engineering methods are used to create hypoimmunogenic cells, then HLA class II function can be prevented, but proper integration of kill switch gene and prevention of immune rejection cannot be ensured
Solution Approach 1:
The patent combines the kill switch gene and B2M:HLA fusion gene into a single integrated genetic construct. This merging ensures that both the hypoimmunogenic function (through B2M:HLA fusion) and the safety mechanism (through kill switch) are co-delivered and co-integrated into the cell genome, eliminating the risk of separate integration failures and ensuring coordinated expression of both functional elements.
Solution Approach 2:
The patent introduces a selectable marker gene as an intermediary element in the integration process. This marker serves as a mediator to verify successful integration of the B2M:HLA fusion gene and kill switch gene into the cell genome. The selectable marker allows for identification and selection of cells that have properly integrated the therapeutic construct, ensuring manufacturing precision before proceeding to further differentiation or implantation.
2Adaptability or versatility
If B2M gene is edited to create hypoimmunogenic cells, then HLA class I functionality can be maintained, but proper integration and separate protein functionality cannot be ensured
Solution Approach 1:
The patent segments the genetic construct into distinct functional modules: the B2M:HLA fusion gene portion, the selectable marker gene, and the kill switch gene. These segmented elements are organized in a specific arrangement where the B2M:HLA fusion gene is positioned to maintain HLA class I functionality, the selectable marker serves as an independent verification module, and the kill switch remains as a separate safety module. This segmentation allows each element to function independently while being integrated into a cohesive therapeutic construct.
3Manufacturing precision
If selectable markers are used for verification, then integration accuracy can be confirmed, but additional genetic elements and process complexity are introduced
Solution Approach 1:
The patent employs the selectable marker gene with the intent of discarding it after its verification function is fulfilled. The marker serves its purpose during the integration verification and cell selection phase, allowing researchers to identify successfully integrated cells. Once verification is complete, the marker can be removed through additional genetic editing, thereby eliminating the temporary complexity it introduced while retaining the benefits of accurate integration verification.
Data Source
AI summary
In variants, the method of generating a hypoimmunogenic cell can include: integrating an insertion sequence into a cell genome (e.g., into a Beta-2-Microglobulin (B2M) gene) and performing a cell selection. The method can optionally include: integrating a selection sequence into the cell genome, removing a selectable marker, editing an additional gene, and/or any other suitable steps. The hypoimmunogenic cell can include a genetically engineered sequence including: a Beta-2-Microglobulin:human leukocyte antigen (B2M:HLA) fusion gene, a multicistronic element, and a kill switch gene.