Universal Donor Cells With MHC Editing and NK Cell Evasion
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Solution Overview
Problem
Existing methods for generating universal donor cells face challenges in effectively evading immune rejection and ensuring cell survival post-engraftment, with potential risks from off-target cleavage and residual MHC-I expression, and susceptibility to natural killer cells.
Innovation Solution
Genetic modification of cells by introducing deletions and insertions in MHC-I and MHC-II genes, combined with the introduction of tolerogenic factors like PD-L1 and HLA-E, using CRISPR systems to enhance immune evasion and cell survival.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If MHC-I and MHC-II genes are disrupted to reduce immunogenicity, then immune evasion is improved, but residual MHC-I expression may still occur causing rejection
Solution Approach 1:
The patent segments the MHC-I gene disruption into multiple targeted deletions across different exons (exon 1, exon 2, exon 3) to ensure complete elimination of MHC-I expression. This multi-site segmentation approach prevents residual expression that would occur with single-point mutations, thereby resolving the contradiction between achieving complete immune evasion and maintaining manufacturing precision.
Solution Approach 2:
The patent changes the genetic parameters by introducing specific deletions (e.g., 12 bp deletion in exon 1, 48 bp deletion in exon 2) that fundamentally alter the MHC-I gene structure. These parameter changes ensure complete gene disruption and eliminate residual expression, thereby achieving reliable immune evasion while maintaining precise control over the genetic modification process.
2Reliability
If B2M is knocked out to reduce MHC-I surface expression, then immunogenicity is reduced, but off-target cleavage events may occur causing safety concerns
Solution Approach 1:
The patent uses a carefully designed gRNA sequence as an intermediary that specifically targets the B2M gene without causing off-target effects. The gRNA acts as a mediator between the CRISPR-Cas9 system and the B2M gene, ensuring precise targeting while avoiding harmful off-target cleavage events. This resolves the contradiction by enabling reliable reduction of immunogenicity through safe, specific gene editing.
3Reliability
If MHC class I-negative cells are created to evade immune rejection, then allogeneic recognition is reduced, but susceptibility to natural killer cells increases
Solution Approach 1:
The patent converts the harmful effect of MHC-I deficiency (susceptibility to NK cells) into a benefit by simultaneously introducing HLA-E expression. The HLA-E molecule binds to NK cell inhibitory receptors, thereby protecting the MHC-I-negative cells from NK cell lysis. This resolves the contradiction by transforming the vulnerability created by MHC-I knockout into a protected state through compensatory HLA-E expression.
4Adaptability or versatility
If multiple genetic modifications are introduced to achieve universal donor status, then immune compatibility is improved, but device complexity increases
Solution Approach 1:
The patent merges multiple genetic modification objectives into a unified CRISPR-Cas9 editing strategy. By combining MHC-I gene deletions, B2M knockout, and HLA-E introduction into a single coordinated genetic engineering process, the patent achieves universal donor status while managing complexity through integrated design. This resolves the contradiction by achieving high immune compatibility through a consolidated rather than fragmented approach.
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 one or more MHC-I or MHC-II human leukocyte antigens or component or transcriptional regulator of the MHC-I or MHC-II complex, at least one genetic modification that increases the expression of at least one polynucleotide that encodes a tolerogenic factor, and optionally at least one genetic modification that increases or decreases the expression of at least one gene that encodes a survival factor.