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

Tune Therapeutics Inc

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 document describes epigenetic-modifying, DNA-targeting systems for repressing Hepatitis B virus (HBV) transcriptional activity without DNA breaks. The systems use a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein fused to transcriptional repressor effector domains that include a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain having DNA methyltransferase activity.

A plurality of polynucleotides comprises a gRNA targeting a target site in a Hepatitis B viral DNA sequence, where the target site is in a regulatory element positioned between residue 1033 and a transcription start site of the HBx gene at a residue base pair 1376 of a Hepatitis B viral sequence with a reference to nucleotide positions of SEQ ID NO: 650. The document also describes engineered zinc finger protein (eZFP) architectures for transcriptional repression of HBV, including eZFP and eZFP-KRAB fusion protein sequence references.

Transcriptional repression is applied to HBV genes present in both covalently closed circular DNA (cccDNA) form and HBV viral DNA integrated in human genomic DNA, and total HBV viral RNA transcript levels are repressed. The document further provides example embodiments combining engineered targeting components with KRAB and DNA methyltransferase domains, including dCas9-KRAB and DNMT3A/L fusions for repression.

Claims Coverage

The independent claims cover treatment and reduction/repression methods for HBV by administering a plurality of polynucleotides that include an HBx regulatory-element targeting gRNA and a dSpCas9 fusion protein with KRAB and DNMT3 DNA methyltransferase activity. The claims also extend to engineered zinc finger protein embodiments and to outcomes including repression of HBV transcription, repression of total HBV viral RNA transcript levels, and reduction of HBsAg.

Across the independent claims, the coverage is directed to administering polynucleotides encoding an HBx regulatory-element targeting gRNA and a dSpCas9-based fusion repressor comprising a KRAB domain and a DNMT3 domain with DNA methyltransferase activity. The claims cover repression of HBV gene transcription from both cccDNA and integrated HBV DNA, repression of total HBV viral RNA transcript levels, reduction of HBsAg, and engineered zinc finger protein embodiments.

Stated Advantages

Documented Applications