Epigenetic HBV Gene Repression with Non-Cutting CRISPR-Cas
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Solution Overview
Problem
Current treatments for Hepatitis B infection, such as nucleoside analogs, PEGylated interferon, and siRNA, face challenges in efficacy and stability, necessitating improved methods to suppress Hepatitis B viral replication and expression.
Innovation Solution
An epigenetic-modifying DNA-targeting system using CRISPR-Cas/guide RNA (gRNA) systems to repress Hepatitis B viral gene transcription by binding to specific sites in the HBV DNA sequence, employing fusion proteins with DNA-binding domains and transcriptional repressor effector domains to reduce HBV replication and protein levels without causing genetic disruption.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If nucleoside analogs or siRNA are used to suppress HBV transcription, then viral replication is inhibited, but treatment efficacy and stability are insufficient
Solution Approach 1:
The patent employs a composite epigenetic-modifying system combining DNA-targeting modules (Cas9-gRNA or zinc finger proteins) with effector domains (KRAB repressor, DNMT3A methyltransferase, or HDAC histone deacetylase). This composite approach integrates multiple mechanisms—transcriptional repression, DNA methylation, and chromatin remodeling—to achieve superior and stable suppression of HBV replication compared to single-mechanism therapies like nucleoside analogs or siRNA.
2Reliability
If CRISPR-Cas systems are used to target HBV DNA, then transcriptional repression is achieved, but risk of genetic disruption increases
Solution Approach 1:
The patent extracts the nuclease activity from the Cas9 protein by using catalytically inactive variants (dCas9), thereby separating the DNA-binding function from the DNA-cutting function. This allows the system to target and repress HBV transcription without causing unwanted genetic disruptions. The effector domains are then attached to the inactive Cas9 to provide the repression function independently of DNA cleavage.
3Reliability
If multiple DNA-targeting modules are used to target multiple HBV sites, then repression effectiveness increases, but system complexity increases
Solution Approach 1:
The patent designs a universal epigenetic-modifying platform where a single Cas9-gRNA system can be reprogrammed to target multiple different HBV sites by simply changing the guide RNA sequence. The same effector domain (KRAB, DNMT3A, or HDAC) can be attached to different dCas9 variants to create multiple targeting modules, reducing overall system complexity while maintaining high repression effectiveness across multiple viral sites.
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
The system effectively reduces HBV replication and protein levels by targeting multiple sites in the HBV genome, enhancing treatment efficacy and stability compared to existing therapies.
Implementation Method 1
a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas)-guide RNA (gRNA) combination comprising (a) a Cas protein or a variant thereof and (b) at least one gRNA
Data Source
AI summary
Provided herein are epigenetic-modifying DNA-targeting systems, such as CRISPR-Cas/guide RNA (gRNA) systems, for the transcriptional repression of Hepatitis B viral (HBV) genes to promote a cellular phenotype that leads to the reduction of HBV infection. In some embodiments, the epigenetic-modifying DNA-targeting systems bind to or target a target site of at least one gene or regulatory element thereof in a Hepatitis B viral DNA sequence in cell. In some aspects, the provided systems relate to the transcriptional repression of one or more Hepatitis B viral gene and/or regulatory element thereof. In some aspects, also provided herein are methods and uses related to the provided compositions, for example in repressing Hepatitis B viral replication and expression in connection with Hepatitis B infections.