Universal Donor Cells via CRISPR-Mediated MHC-I Reduction
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Solution Overview
Problem
Current methods for generating universal donor cells face challenges in evading immune rejection and ensuring cell survival post-engraftment, as they may still express residual MHC-I molecules and are susceptible to natural killer cell lysis, with concerns about off-target cleavage events using TALENs.
Innovation Solution
The method involves delivering site-directed nucleases, such as CRISPR systems, to target genes encoding survival and tolerogenic factors like TXNIP, HLA-E, and PD-L1, which are flanked by homologous sequences, to generate cells with reduced MHC-I expression and enhanced immune evasion and survival capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If TALEN approach is used to generate B2M-deficient cell lines, then MHC-I expression is reduced, but off-target cleavage events occur causing safety concerns
Solution Approach 1:
The patent replaces the TALEN protein-based genome editing system with the CRISPR-Cas9 system. This substitution maintains the ability to generate B2M-deficient cell lines with reduced MHC-I expression while improving specificity and reducing off-target effects through the RNA-guided mechanism of CRISPR, which offers higher precision in target recognition.
2Reliability
If MHC-I expression is reduced to evade immune rejection, then allogeneic compatibility improves, but cells become susceptible to natural killer cell lysis
Solution Approach 1:
The patent introduces HLA-E as an intermediary molecule that mediates between the reduced MHC-I expression and natural killer cell recognition. HLA-E presents endogenous peptides to NK cell inhibitory receptors, providing a compensatory mechanism that protects B2M-deficient cells from NK cell lysis while maintaining allogeneic compatibility through reduced immunogenicity.
Solution Approach 2:
The patent creates a composite cell line that combines B2M deficiency with HLA-E overexpression. This composite approach integrates two genetic modifications to achieve a balanced immune phenotype: reduced MHC-I expression for allogeneic compatibility combined with HLA-E presence for NK cell protection, thereby resolving the contradiction between these two immune evasion requirements.
3Reliability
If residual B2M mRNA is expressed in TALEN-targeted cells, then MHC-I molecules may still be formed, but complete immune evasion is not achieved
Solution Approach 1:
The patent extracts or removes the residual B2M mRNA through complete knockout of the B2M gene using CRISPR-Cas9. By introducing precise frame-shift mutations or deletions at the B2M locus, the system eliminates transcription of B2M mRNA, thereby preventing any residual protein synthesis and ensuring complete abolition of MHC-I surface expression for optimal immune evasion.
4Reliability
If site-directed nucleases are used to edit multiple genes, then immune evasion and survival capabilities are enhanced, but process complexity increases
Solution Approach 1:
The patent merges multiple genome editing operations into a single CRISPR-Cas9 delivery system. By co-delivering multiple guide RNAs targeting different genes (B2M, HLA-E, PD-L1) along with the Cas9 nuclease, the system achieves simultaneous editing of multiple loci, thereby enhancing immune evasion and survival capabilities while streamlining the overall process compared to sequential editing approaches.
Solution Approach 2:
The patent employs the CRISPR-Cas9 system as a universal platform that can target multiple genes with different functions (MHC-I components, tolerogenic factors) using a common mechanism. This multi-functional approach allows simultaneous modification of B2M for MHC-I reduction, HLA-E for NK cell protection, and PD-L1 for immune checkpoint modulation, achieving comprehensive immune evasion through a single editable system.
Data Source
AI summary
Genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and methods of generating said genetic modified cells are provided herein. The universal donor cells comprise at least one genetic modification within or near at least one gene that encodes a survival factor, wherein the genetic modification comprises an insertion of a polynucleotide encoding a tolerogenic factor. The universal donor cells may further comprise at least one genetic modification within or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or a transcriptional regulator of a MHC-I or MHC-II complex, wherein said genetic modification comprises an insertion of a polynucleotide encoding a second tolerogenic factor.