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
Epsilen Bio Srl • Telethon Foundation • IRCCS Ospedale San Raffaele • nChroma Bio Inc
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Publication Number
US-12649003-B2
Publication Date
2026-06-09
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 disclosure describes HBV-directed epigenetic editing systems and methods of administration for subjects exhibiting detectable HBV DNA, HBsAg, or HBeAg in plasma. The systems comprise a fusion protein containing dCas9, DNMT3A, and human KRAB domains—such as constructs comprising dCas9, DNMT3A, full-length DNMT3L, and a ZIM3 KRAB domain—or nucleic acids encoding the fusion protein, paired with a guide RNA targeting an HBV DNA sequence such as SEQ ID NO: 391. These compositions target episomes including cccDNA as well as integrated DNA across viral promoters, enhancers, and transcript regions to mediate methylation- or chromatin-mediated silencing without DNA cleavage.
The methods achieve repression of viral replication, episomes, circulating viral DNA, and antigen expression across infected cell models and in vivo mouse models. Repeat dosing improves the durability of silencing, while an updated editor and guide modification demonstrates increased potency over prior leads. Profiling indicates durable target silencing, alanine transaminase levels comparable to healthy controls, minimal transcriptomic changes, and no detected differentially methylated off-target genomic regions.
Claims Coverage
The claims provide coverage across three inventive features corresponding to three independent method claims directed to administering an epigenetic editing system to a subject with detectable plasma HBV markers, distinguished by tracrRNA limitations.
Epigenetic editing system with a Cas9 tracrRNA
Claim 1 covers administering to a subject with detectable HBV DNA, HBsAg, and/or HBeAg in plasma an epigenetic editing system comprising a fusion protein containing dCas9, DNMT3A, and a human KRAB domain, or nucleic acid molecules encoding the fusion protein, and a guide RNA targeting a sequence consisting of SEQ ID NO: 391 with a Cas9 tracrRNA.
Epigenetic editing system with a tracrRNA comprising SEQ ID NO: 1089
Claim 18 covers administering to a subject with detectable HBV DNA, HBsAg, and/or HBeAg in plasma an epigenetic editing system comprising a dCas9, DNMT3A, and human KRAB fusion protein, or nucleic acid molecules encoding the fusion protein, and a guide RNA targeting SEQ ID NO: 391 with a tracrRNA comprising SEQ ID NO: 1089.
Epigenetic editing system with a tracrRNA comprising SEQ ID NO: 1087
Claim 19 covers administering to a subject with detectable HBV DNA, HBsAg, and/or HBeAg in plasma an epigenetic editing system comprising a dCas9, DNMT3A, and human KRAB fusion protein, or nucleic acid molecules encoding the fusion protein, and a guide RNA targeting SEQ ID NO: 391 with a tracrRNA comprising SEQ ID NO: 1087.
The three independent claims define methods of administering an HBV-directed dCas9-DNMT3A-human KRAB epigenetic editing system targeting SEQ ID NO: 391 to subjects with detectable HBV markers, differentiated by the inclusion of a general Cas9 tracrRNA (claim 1) or specific tracrRNA sequences comprising SEQ ID NO: 1089 (claim 18) and SEQ ID NO: 1087 (claim 19).
Stated Advantages
The system achieves durable methylation- or chromatin-mediated silencing of HBV genes, regulatory regions, and episomes without DNA cleavage.
Reported assays and animal models demonstrate substantial reductions in HBV episomes, replication, circulating HBV DNA, HBsAg, and HBeAg, with described reductions ranging from 20% to over 99.9%.
Repeat dosing sustains and further improves the durability of HBsAg reduction and viral marker modulation.
An updated editor and guide modification exhibits greater potency relative to the prior lead construct.
Off-target assessments report minimal transcriptomic changes, no detected differentially methylated regions, and alanine transaminase levels comparable to healthy controls.
Documented Applications
Administration of an epigenetic editing system to subjects presenting detectable HBV DNA, HBsAg, and/or HBeAg in plasma.
Treatment of hepatitis B virus (HBV) infection, administered as a monotherapy or in combination with nucleoside/nucleotide analogs or interferon.
Treatment of hepatitis D virus (HDV) infection.
System delivery via viral vectors, lipid nanoparticles, or other nucleic-acid delivery systems into infected hepatocytes, HepG2.2.15 cells, and mouse models.
Evaluation of guide RNA screening result profiles and repression in cell models including HepAD38 and HepG2-NTCP, as well as in vivo AAV-HBV and transgenic HBV mouse models.
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