Compositions, systems, and methods for regulation of hepatitis B virus through targeted gene repression
Inventors
COSGROVE, Brian • CONGDON, Kendra • Dean, Jason • Gough, Veronica • Black, Joshua B. • Jones, Britta
Assignees
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Publication Number
US-12252692-B2
Publication Date
2025-03-18
Expiration Date
Abstract
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.
Core Innovation
The invention is an epigenetic-modifying DNA-targeting system for repressing transcription of one or more Hepatitis B viral (HBV) genes. The DNA-targeting module comprises a gRNA that targets a target site in a Hepatitis B viral DNA sequence and a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein with transcriptional repressor effector domains comprising a KRAB domain and a DNMT3A/L domain.
The disclosed system targets HBV DNA sequences and regulatory elements, including cccDNA/rcDNA, promoters and enhancers, and CpG islands. The target site specification is grounded in sequence identifiers and target-site sequence constraints across HBV genotypes and genomes, including homology or identity constraints and mismatch constraints, with optional zero-mismatch cases.
The system supports multiplexed epigenetic-modifying DNA-targeting systems using multiple gRNAs to repress multiple HBV genes and regulatory elements, including HBx promoter/enhancer and CpG islands. The disclosure also describes sequence-defined fusion protein configurations and nuclear localization signals positioned in defined locations.
Claims Coverage
The independent claims across the provided items cover an epigenetic-modifying DNA-targeting system for repressing transcription of one or more HBV genes using a gRNA that targets a specified HBV DNA sequence and a dSpCas9 fusion protein that includes KRAB and DNMT3A/L transcriptional repressor effector domains, with additional sequence and architecture refinements in dependent claims.
HBV DNA targeting with specified gRNA sequences
A gRNA for targeting to a target site in a Hepatitis B viral DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 22, or the gRNA comprises the sequence set forth in SEQ ID NO: 217, or the gRNA comprises the sequence set forth in SEQ ID NO: 412.
dSpCas9 KRAB-DNMT3A/L transcriptional repressor fusion protein
A fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and a transcriptional repressor effector domain comprising a KRAB domain and a DNMT3A/L domain, with KRAB and DNMT3A/L sequences defined by the cited SEQ ID NOs.
Fusion protein architecture and nuclear localization signals
Dependent claim refinements specify dSpCas9 sequence identity and amino acid positions, the N- to C-terminal order of DNMT3A/L, dSpCas9, and KRAB domains, and one or more nuclear localization signals (NLS) with defined placement and listed sequence options.
Target coverage across HBV genomes
Dependent refinements require the target site to be at least 70% homologous to at least 1000 Hepatitis B viral genomes and/or the system to target at least 70% of Hepatitis B viral genomes.
Across the independent claims, coverage centers on repressing transcription of one or more HBV genes using a gRNA targeting specified HBV DNA sequences together with a dSpCas9-based fusion protein containing KRAB and DNMT3A/L effector domains. Dependent claims further constrain sequence identity, domain ordering, NLS placement, and HBV genome targeting coverage.
Stated Advantages
Durable repression of HBV replication and expression across cccDNA, rcDNA, and integrated HBV DNA.
Represses HBV transcription without DNA breakage.
Promotes CpG methylation at targeted HBV DNA sites.
Reduces HBV RNA and HBV protein readouts including total HBV RNA, HBsAg, HBeAg, HBx, and HbcrAg.
Minimal off-target differential gene expression assessed by RNA-seq.
Documented Applications
Treatment of HBV infection using the epigenetic-modifying DNA-targeting system and associated pharmaceutical compositions to reduce HBV RNA and protein readouts.
Use in humanized mouse models, including humanized liver mice and FRG, with documented in vivo repression.
Targeted repression of HBV genes and regulatory elements including HBx promoter/enhancer and CpG islands.
Application in liver cells including hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, including in vivo in a human subject.
Comparative repression testing of eZFP-KRAB versus dSpCas9-KRAB using specific gRNAs in the described examples.
True infection screening in HepG2.NTCP cells for HBV repression constructs.
Durable repression assessment with DNMT3A/L-eZFP-KRAB, with minimal off-target differential expression assessed by RNA-seq in the described examples.
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