Base Editing for Beta-Hemoglobinopathies
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Solution Overview
Problem
Current genome editing techniques for treating β-hemoglobinopathies, such as β-thalassemia and sickle cell disease, face challenges including DNA double-strand breaks-induced toxicity and off-target effects, which can lead to apoptosis and genetic instability, limiting their efficacy and safety.
Innovation Solution
The use of base editing approaches with cytosine and adenine base-editing enzymes (CBEs and ABEs) that make pinpoint changes in DNA without inducing double-strand breaks, allowing for precise DNA repair and reducing off-target activity, enabling the recreation of HPFH mutations to reactivate fetal hemoglobin expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR/Cas9 system is used to induce DNA double-strand breaks for gene correction, then gene editing capability is improved, but DNA damage response and apoptosis are induced reducing cell viability
Solution Approach 1:
The patent extracts and removes the harmful nuclease activity from the CRISPR system by using catalytically inactive Cas9 (dCas9) fused to base editing enzymes (cytosine deaminase or adenine deaminase). This separation allows the system to perform base editing without inducing DNA double-strand breaks, thereby eliminating the DNA damage response and apoptosis while maintaining gene editing capability.
Solution Approach 2:
The patent replaces the mechanical DNA cleavage mechanism of Cas9 nuclease with a chemical base modification mechanism. Instead of cutting DNA strands, the system uses deaminase enzymes to chemically convert cytosine to uracil or adenine to inosine, which are then processed by cellular repair mechanisms to achieve permanent base changes without DNA breaks.
2Productivity
If multiple on-target DSBs are generated to edit multiple sites, then gene editing efficiency is improved, but risk of deletions, inversions and translocations increases
Solution Approach 1:
The patent removes the harmful DNA cleavage function from the editing system by employing catalytically inactive Cas9 (dCas9). This allows multiple target sites to be edited simultaneously through base modification without generating DNA double-strand breaks, thereby eliminating the risk of large-scale genomic rearrangements such as deletions, inversions, and translocations while maintaining multi-site editing capability.
3Reliability
If CRISPR/Cas9 induces DNA double-strand breaks for genome editing, then therapeutic effect is improved, but off-target effects and genetic instability occur reducing safety
Solution Approach 1:
The patent replaces the DNA cleavage mechanism with a base modification mechanism using deaminase enzymes. This chemical approach allows for more precise and localized changes at the target site without creating DNA breaks that could lead to off-target effects. The base editing enzymes have higher specificity and do not generate the same level of genomic instability as nuclease-based systems.
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 method avoids the toxicity associated with double-strand breaks, achieves homogeneous and predictable base changes, and effectively increases fetal hemoglobin levels, potentially offering a safer and more effective treatment for β-hemoglobinopathies by disrupting repressor binding sites or creating activator binding sites in the γ-globin promoters.
Implementation Method 1
The basic components of base-editing enzymes are a catalytically disabled Cas9 nuclease and a deaminase; these eventually produce a C-G to T-A or A-T to G-C conversion
Data Source
AI summary
The clinical history of β-hemoglobinopathies shows that the severity is mitigated by the synthesis of the fetal γ-globin in adulthood, typically associated with genetic variants the HBB cluster known as hereditary persistence of fetal hemoglobin (HPFH) mutations. The inventors identified that most of the known HPFH mutations in the γ-globin promoters (C>T, G>A, T>C or A>G) can be recapitulated using CBE- and ABE-mediatedbase-editing approaches. In particular, the inventors designed gRNAs that, when combined with CBEs or ABEs, generate HPFH mutations, and either disrupt binding sites for transcriptional repressors (-200 and -115 sites) or generate de novo DNA motifs recognized by transcriptional activators (e.g., -198 T>C, the -175 T>C and -113 A>G). It is noteworthy that a subset of the gRNAs targeting the -200 and the 115 regions are predicted to generate simultaneously HPFH mutations and also to make base changes other than HPFH mutations in or around the LRF and BCL11A binding sites, which might further reduce LRF and BCL11A occupancy. Accordingly, the present invention relates to base editing approaches for the treatment of β-hemoglobinopathies.