Compositions and methods for epigenetic regulation of HBV gene expression
Inventors
Jaffe, Aron Brandon • Abubucker, Noorussahar • Anglero-Rodriguez, Yesseinia • Myer, Vic • Lombardo, Angelo Leone • Cappelluti, Martino Alfredo
Assignees
Fondazione Telethon • Ospedale San Raffaele SRL • Nchroma Bio Inc • Chroma Medicine Inc
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Publication Number
US-12390538-B2
Publication Date
2025-08-19
Expiration Date
Abstract
This invention relates to compositions, methods, strategies, and treatment modalities related to the epigenetic modification of hepatitis B virus (HBV) genes.
Core Innovation
The invention relates to HBV epigenetic editing by administering an epigenetic editing system to a subject comprising detectable levels of HBV DNA, HBsAg, and/or HBeAg in plasma. The epigenetic editing system represses HBV gene expression, including reducing HBV episomes and/or HBV replication and expression of HBV gene products such as HBsAg and HBeAg.
The system includes a fusion protein comprising a dCas9 protein domain, a DNMT3A domain, and a human KRAB domain, or one or more nucleic acid molecules encoding the fusion protein. The system also includes a guide RNA (gRNA) comprising a nucleic acid base sequence selected from specified SEQ ID NOs, where the nucleic acid base sequence comprises a region complementary to a strand of a target region having a sequence selected from specified SEQ ID NOs.
The described editing architecture includes programmable DNA-binding using dCas9, with guide RNA targeting defined by selectable gRNA sequences and corresponding target-region sequences. Effector activity is provided by transcriptional repressors and DNA methyltransferase domains, including DNMT3A and optionally DNMT3L, to induce durable epigenetic silencing of HBV-associated nucleic acid regions such as CpG islands and promoter regions, including promoter and enhancer regions.
Claims Coverage
The independent claim requires administering an epigenetic editing system to a subject with detectable plasma HBV DNA, HBsAg, and/or HBeAg, using a specific fusion protein architecture and a gRNA selected by SEQ ID NO-based complementarity to HBV target regions. The claim contains 2 main inventive elements: the dCas9–DNMT3A–human KRAB fusion protein (or encoding nucleic acid) and the gRNA with SEQ ID NO-defined base sequence complementary to SEQ ID NO-defined target regions.
Administering an HBV epigenetic editing system to repress HBV gene expression
A method comprising administering an epigenetic editing system to a subject wherein the subject comprises detectable levels of HBV DNA, HBsAg, and/or HBeAg in plasma of the subject.
dCas9–DNMT3A–human KRAB fusion protein or encoding nucleic acid
The epigenetic editing system comprises a fusion protein comprising a dCas9 protein domain, a DNMT3A domain, and a human KRAB domain or one or more nucleic acid molecules encoding the fusion protein.
SEQ ID NO-based gRNA complementary to a SEQ ID NO-defined HBV target region
The epigenetic editing system comprises a guide RNA (gRNA) comprising a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 1093-1110, 1130-1164, 1166-1173, 1175-1199, 1201-1235, and 1249, wherein the nucleic acid base sequence comprises a region complementary to a strand of a target region wherein the target region comprises a sequence selected from the group consisting of SEQ ID NOs: 333-350, 370-404, 406-413, 415-439, and 441-475.
Across the independent claim, coverage is directed to HBV epigenetic editing in plasma HBV contexts using a dCas9 protein domain fused to DNMT3A and a human KRAB domain, together with a gRNA whose nucleic acid base sequence is selected from specified SEQ ID NOs to be complementary to specified SEQ ID NO-defined HBV target regions.
Stated Advantages
Durable reductions in HBV biomarkers are described.
Many gRNAs reduce HBeAg and HBsAg versus non-targeting controls.
Specificity and methylation assessment are described.
Reduction of HBV biomarkers in plasma, including at least a 1-log (≥90%) reduction of HBV DNA, HBsAg, and/or HBeAg maintained for ≥14 days.
Embodiments include higher log reductions (e.g., ≥99%) and maintenance durations.
Represses HBV gene expression, including reducing HBV episomes/replication and expression of HBV gene products such as HBsAg and HBeAg.
Documented Applications
In vitro HepG2-NTCP silencing using CRISPR-Off constructs with reductions in HBeAg and HBsAg versus non-targeting controls.
Further in vitro and ex vivo testing across cell models including PLC/PRF/5 and primary human hepatocytes.
In vivo HBV mouse model efficacy and durability, including redosing/multiplexing and dose-response.
Assessment of specificity and methylation, including comparison to WT-Cas9 controls.
Combination treatment with entecavir (ETV).
HBV genome methylation profiling and related transcriptional readouts.
Treating chronic HBV in subjects with detectable plasma HBV DNA, HBsAg, and/or HBeAg using an administered epigenetic editing system.
A method of administering an HBV epigenetic editing system to subjects, including subjects with chronic HBV infection, to reduce plasma HBV biomarkers and associated outcomes such as viral episomes, cccDNA, total HBV DNA, replication, and antigen/protein expression [procedural detail omitted for safety].
Treating HBV by administering an epigenetic editing system to subjects with detectable plasma HBV DNA, HBsAg, and/or HBeAg to repress HBV gene expression.
Treating HDV by decreasing HBsAg dependent on HBV S-antigen.
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