Engineered Meganucleases for HBV-Specific Genome Cleavage
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Solution Overview
Problem
Current anti-HBV drugs, such as interferon alpha and nucleoside analogues, have limitations including severe adverse reactions and require long-term administration, while HBV exhibits genetic variability leading to multiple genotypes and challenges in developing effective therapeutic meganucleases with enhanced on-target specificity and reduced off-target cutting.
Innovation Solution
Development of engineered meganucleases with enhanced specificity for the HBV 11-12 recognition sequence, comprising first and second subunits with hypervariable regions, and a linker, to increase on-target cutting and reduce off-target effects in the host cell genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional anti-HBV drugs (interferon alpha, nucleoside analogues) are used, then viral replication is suppressed, but severe adverse reactions occur and long-term administration is required
Solution Approach 1:
The patent replaces conventional pharmacological approaches (interferon alpha, nucleoside analogues) with an engineered enzymatic system (meganuclease). This substitution transitions from chemical inhibition of viral replication to precise enzymatic cleavage of viral DNA, aiming to eliminate the need for long-term administration and reduce adverse reactions associated with conventional drugs.
Solution Approach 2:
The invention modifies the recognition specificity parameters of meganucleases through engineered mutations in the hypervariable regions. By changing amino acid sequences in HVR1 and HVR2, the meganuclease is tailored to recognize specific HBV genotypes, enabling selective viral DNA cleavage while preserving host cell genome integrity, thus reducing off-target effects.
2Measurement precision
If meganuclease specificity is enhanced for HBV recognition sequence, then on-target cutting increases, but off-target effects in host cell genome may still occur
Solution Approach 1:
The patent applies local quality by engineering specific amino acid substitutions in the hypervariable regions (HVR1 and HVR2) of the meganuclease. These localized changes in sequence composition and properties enable the enzyme to distinguish between HBV recognition sequences and host cell genomic sequences, thereby enhancing on-target specificity while minimizing off-target effects.
Solution Approach 2:
The invention creates multiple meganuclease variants with different specificity profiles by copying and modifying the parent meganuclease sequence. These engineered variants are designed to recognize different HBV genotypes while maintaining reduced off-target activity, providing a toolkit for selective viral genome cleavage.
3Adaptability or versatility
If HBV genetic variability is considered across genotypes A-G, then broad virus coverage is achieved, but developing effective therapeutic meganucleases becomes more challenging
Solution Approach 1:
The patent develops a universal meganuclease platform based on I-CreI that can be engineered to target multiple HBV genotypes (A-G) by modifying the hypervariable regions. This universal platform approach allows a single core enzyme structure to serve multiple functions across different viral genotypes, reducing the need to design entirely new enzymes for each genotype.
Solution Approach 2:
The invention segments the meganuclease into functional domains with distinct roles: the core catalytic domain (LAGLIDADG motif) remains unchanged across variants, while the hypervariable regions (HVR1, HVR2) are engineered to provide genotype-specific recognition. This segmentation allows independent optimization of catalytic activity and specificity.
Data Source
AI summary
The present disclosure encompasses engineered nucleases which recognize and cleave a recognition sequence within a Hepatitis B virus (HBV) genome. The engineered meganucleases described herein can exhibit improved characteristics, such as enhanced specificity and/or efficiency of indel formation, when compared to previously described HBV meganucleases. Further, the disclosure encompasses pharmaceutical compositions comprising engineered meganuclease proteins, nucleic acids encoding engineered meganucleases, and the use of such compositions for treating HBV infections or diseases associated with HBV infections.