US 12,325,858 B1
Compositions, systems, and methods for regulation of Hepatitis B virus through targeted gene repression
Brian Cosgrove, Durham, NC (US); Kendra Congdon, Durham, NC (US); Jason Dean, Seattle, WA (US); Veronica Gough, Durham, NC (US); Joshua B. Black, Durham, NC (US); and Britta Jones, Durham, NC (US)
Assigned to Tune Therapeutics, Inc., Seattle, WA (US)
Filed by Tune Therapeutics, Inc., Seattle, WA (US)
Filed on Feb. 14, 2025, as Appl. No. 19/054,630.
Application 19/054,630 is a continuation of application No. 19/007,236, filed on Dec. 31, 2024.
Application 19/007,236 is a continuation of application No. 18/452,508, filed on Aug. 18, 2023, granted, now 12,221,608, issued on Feb. 11, 2025.
Claims priority of provisional application 63/531,309, filed on Aug. 7, 2023.
Claims priority of provisional application 63/472,236, filed on Jun. 9, 2023.
Claims priority of provisional application 63/399,634, filed on Aug. 19, 2022.
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/113 (2010.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/88 (2006.01)
CPC C12N 15/1131 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/88 (2013.01); C12N 2310/11 (2013.01); C12N 2310/20 (2017.05); C12N 2310/315 (2013.01); C12N 2310/321 (2013.01); C12N 2310/351 (2013.01)] 30 Claims
 
1. A method of treating a Hepatitis B virus (HBV) infection in a subject comprising administering to the subject a therapeutically effective amount of a plurality of polynucleotides comprising:
(a) a gRNA for targeting to a target site in a Hepatitis B viral DNA sequence, wherein 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; and
(b) a polynucleotide encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity,
wherein transcription of one or more HBV genes present in both a covalently closed circular DNA (cccDNA) form and in HBV viral DNA integrated in human genomic DNA are repressed in the subject, and transcription of total HBV viral RNA transcript levels are repressed in the subject.