Deimmunized Type II Cas Polypeptides With Preserved Nuclease Activity
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Solution Overview
Problem
The immunogenicity of Type II Cas polypeptides, such as Cas9 proteins or orthologs, poses a challenge for the clinical translation of CRISPR-based therapeutics, necessitating deimmunization without significantly altering their activity, including nucleic acid binding, complex formation, and target binding.
Innovation Solution
Engineered Type II Cas polypeptides with specific amino acid substitutions at MHC Class I and/or MHC Class II binding sites, such as SaCas9 residues 8-16, 926-934, and 1034-1042, reduce immunogenicity while maintaining nuclease efficiency and RNA binding, optionally combined with additional modifications to enhance activity or reduce off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type Type II Cas polypeptides are used for genome editing, then nuclease efficiency and target binding are maintained, but immunogenicity increases leading to safety concerns
Solution Approach 1:
The patent applies local quality by making specific amino acid substitutions only at MHC Class I and MHC Class II binding sites (epitopes) while leaving the rest of the Cas9 protein structure unchanged. This localized modification approach reduces immunogenicity at specific regions without affecting the overall nuclease efficiency and target binding capabilities of the protein
Solution Approach 2:
The patent changes the amino acid parameters at specific epitopic regions (MHC binding sites) to reduce immunogenicity. By substituting specific amino acids at residues such as 8-16, 926-934, and 1034-1042, the patent modifies the immunogenic properties while preserving the functional parameters of the Cas9 protein
2Reliability
If amino acid substitutions are made at MHC binding sites to reduce immunogenicity, then safety is improved, but nuclease efficiency and RNA binding may be altered
Solution Approach 1:
The patent makes targeted amino acid substitutions only at MHC Class I and MHC Class II binding sites while preserving the catalytic domains and RNA binding regions. This localized approach ensures that immunogenicity is reduced without compromising nuclease efficiency and target binding
Solution Approach 2:
The patent creates deimmunized Cas9 variants that copy the overall structure and function of wild-type Cas9 while making specific local modifications. The engineered polypeptides maintain the essential functional characteristics of the original protein while eliminating immunogenic epitopes
Data Source
AI summary
Engineered Type II Cas polypeptides with reduced immunogenicity, CRISPR-Cas systems thereof, compositions thereof, delivery systems thereof, and methods of use thereof for modifying target polynucleotides, such as, for example, in cells.