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-12398394-B2
Publication Date
2025-08-26
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 disclosure describes a plurality of polynucleotides comprising a guide RNA and a polynucleotide encoding a fusion protein. The guide RNA targets an HBV regulatory element positioned between residue 1033 and the HBx gene transcription start site at residue base pair 1376. The encoded fusion protein comprises deactivated Streptococcus pyogenes Cas9 combined with transcriptional repressor effector domains comprising a KRAB domain and a DNA methyltransferase 3 domain with DNA methyltransferase activity.
When delivered to an HBV-infected cell, the system directs the fusion protein to repress HBV genes present in both covalently closed circular DNA and integrated viral DNA. This system represses total HBV RNA transcript levels without DNA cleavage. Reported screening results demonstrate targeted HBV DNA methylation, durable repression in cell models and primary human hepatocytes, and reduced viral biomarkers in humanized-liver mice.
Claims Coverage
The claims identify one independent claim, claim 1, comprising three inventive features directed to targeting and repressing HBV transcription.
gRNA targeting an HBV regulatory element
A gRNA targets a site in an HBV DNA regulatory element positioned between residue 1033 and the HBx gene transcription start site at residue base pair 1376, with reference to SEQ ID NO: 650.
dSpCas9 fusion with transcriptional repressor domains
A polynucleotide encodes a fusion protein comprising deactivated Streptococcus pyogenes Cas9 and transcriptional repressor effector domains comprising a KRAB domain and a DNA methyltransferase 3 domain with DNA methyltransferase activity.
Repression of HBV genes and total viral RNA
When delivered to an HBV-infected cell, the gRNA targets the encoded fusion protein to repress one or more HBV genes present in both covalently closed circular DNA and HBV viral DNA integrated in human genomic DNA, repressing total HBV RNA transcript levels.
Claim 1 covers the combination of an HBV regulatory element-targeting gRNA with a deactivated Cas9 fusion containing KRAB and DNMT3 repressor domains to repress HBV gene transcription and total viral RNA transcript levels across both cccDNA and integrated viral DNA.
Stated Advantages
The encoded fusion protein represses total HBV RNA transcript levels, viral RNA, antigen expression, and viral replication without DNA cleavage.
Selected guides produce durable repression lasting up to 248 days in cell models and strong repression in primary human hepatocytes.
Targeted administration achieves reduced viral biomarkers in humanized-liver mouse models.
Reported DNMT3A/L-eZFP-KRAB variants produce durable repression through day 15.
Reported variants show minimal off-target differential gene expression.
Reported eZFP-KRAB repressors achieve repression comparable to dSpCas9-KRAB/gRNA controls.
Documented Applications
Delivery to HBV-infected cells to target an HBV regulatory element and repress HBV genes in covalently closed circular DNA and integrated HBV viral DNA, including therapeutic administration to infected subjects.
Testing HBV-targeted epigenetic repression in primary human hepatocytes and humanized-liver mouse models, including formulation in lipid nanoparticles.
Evaluation of repression and methylation across HBV targets and guide combinations in cell models, such as Hep3B cells, and animal infection models.
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