Granted Patent
B2
US 12,325,857 · App. 19/007,278 · Granted Jun 10, 2025
Compositions, systems, and methods for regulation of Hepatitis b virus through targeted gene repression
Inventors:
Brian Cosgrove (Durham, NC); Kendra Congdon (Durham, NC); Jason Dean (Seattle, WA); Veronica Gough (Durham, NC); Joshua B. Black (Durham, NC); Britta Jones (Durham, NC)
Assignee:
Tune Therapeutics, Inc.
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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.
Claims (36)
1. 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 comprises the sequence set forth in SEQ ID NO: 22; and
(b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and a transcriptional repressor effector domain comprising a KRAB domain and a DNMT3A/L domain, wherein the KRAB domain comprises the sequence set forth in SEQ ID NO: 590 and the DNMT3A/L domain comprises the sequence set forth in SEQ ID NO: 651.
2. A vector comprising the plurality of polynucleotides of claim 1 .
3. The vector of claim 2 that is a lipid nanoparticle.
4. The plurality of polynucleotides of claim 1 , wherein the KRAB domain comprises the sequence set forth in SEQ ID NO: 669.
5. The plurality of polynucleotides of claim 1 , wherein the gRNA comprises the sequence set forth in SEQ ID NO: 217.
6. The plurality of polynucleotides of claim 1 , wherein the dSpCas9 comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 598.
7. The plurality of polynucleotides of claim 1 , wherein the dSpCas9 comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 599 and comprises an alanine (A) at amino acid position 9 and an alanine at position 839 corresponding to amino acid residues in SEQ ID NO:599.
8. The plurality of polynucleotides of claim 1 , wherein the fusion protein comprises, from N- to C-terminal order: the DNMT3A/L fusion domain set forth in SEQ ID NO: 651, the dSpCas9 set forth in SEQ ID NO: 599, and the KRAB domain set forth in SEQ ID NO: 590.
9. The plurality of polynucleotides of claim 8 , wherein the gRNA comprises the sequence set forth in SEQ ID NO: 412.
10. The plurality of polynucleotides of claim 8 , wherein the fusion protein further comprises one or more nuclear localization signals (NLS), wherein the one or more NLS is at the N- or C-terminus of the fusion protein, between the dSpCas9 and the DNMT3A/L domain, or between the dSpCas9Cas and the KRAB domain.
11. The plurality of polynucleotides of claim 10 , wherein the one or more NLS is selected from the group consisting of the sequences PKKKRKV (SEQ ID NO: 593), KRPAATKKAGQAKKKK (SEQ ID NO: 592), and PKQKKRK (SEQ ID NO: 624).
12. The plurality of polynucleotides of claim 11 , wherein the fusion protein comprises, from N- to C-terminal order: the DNMT3A/L domain set forth in SEQ ID NO: 651, the sequence set forth in SEQ ID NO:672, the sequence set forth in SEQ ID NO: 670, the dSpCas9 set forth in SEQ ID NO: 599, the sequence set forth in SEQ ID NO: 677, the KRAB domain set forth in SEQ ID NO: 669, and the sequence set forth in SEQ ID NO: 593.
13. A vector comprising the plurality of polynucleotides of claim 12 .
14. The vector of claim 13 that is a lipid nanoparticle.
15. The plurality of polynucleotides of claim 12 , wherein the gRNA comprises the sequence set forth in SEQ ID NO:412.
16. A vector comprising the plurality of polynucleotides of claim 15 .
17. The vector of claim 16 that is a lipid nanoparticle.
18. A plurality of polynucleotides comprising:
(a) a gRNA for targeting to a target site in a Hepatitis B viral DNA sequence, wherein the gRNA comprises the sequence set forth in SEQ ID NO: 217; and
(b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and a transcriptional repressor effector domain comprising a KRAB domain and a DNMT3A/L domain, wherein the KRAB domain comprises the sequence set forth in SEQ ID NO: 669 and the DNMT3A/L domain comprises the sequence set forth in SEQ ID NO: 651.
19. A vector comprising the plurality of polynucleotides of claim 18 .
20. The vector of claim 19 that is a lipid nanoparticle.
21. The plurality of polynucleotides of claim 18 , wherein the dSpCas9 comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 599 and comprises an alanine (A) at amino acid position 9 and an alanine at position 839 corresponding to amino acid residues in SEQ ID NO:599.
22. The plurality of polynucleotides of claim 18 , wherein the fusion protein comprises, from N- to C-terminal order: the DNMT3A/L domain set forth in SEQ ID NO: 651, the dSpCas9 set forth in SEQ ID NO: 599, and the KRAB domain set forth in SEQ ID NO: 669.
23. A vector comprising the plurality of polynucleotides of claim 22 .
24. The vector of claim 23 that is a lipid nanoparticle.
25. The plurality of polynucleotides of claim 22 , wherein the gRNA comprises the sequence set forth in SEQ ID NO:412.
26. The plurality of polynucleotides of claim 22 , wherein the fusion protein further comprises one or more nuclear localization signals (NLS), wherein the one or more NLS is at the N- or C-terminus of the fusion protein, between the dSpCas9Cas and the DNMT3A/L domain, or between the dSpCas9 and the KRAB domain.
27. The plurality of polynucleotides of claim 26 , wherein the one or more NLS is selected from the group consisting of the sequences PKKKRKV (SEQ ID NO: 593), KRPAATKKAGQAKKKK (SEQ ID NO: 592), and PKQKKRK (SEQ ID NO: 624).
28. A plurality of polynucleotides comprising,
(a) an mRNA encoding a fusion protein comprising (i) a Streptococcus pyogenes dCas9 (dSpCas9) protein set forth in SEQ ID NO:599 and (ii) a transcriptional repressor effector domain comprising a KRAB domain and a DNMT3A/L domain, wherein the KRAB domain comprises the amino acid sequence set forth in SEQ ID NO: 669 and the DNMT3A/L domain comprises the amino acid sequence set forth in SEQ ID NO:651; and
(b) a gRNA for targeting to a target site in a Hepatitis B viral DNA sequence, wherein the gRNA comprises the sequence set forth in SEQ ID NO: 217.
29. The plurality of polynucleotides of claim 28 , wherein the gRNA comprises the sequence set forth in SEQ ID NO: 412.
30. The plurality of polynucleotides of claim 28 , wherein the fusion protein comprises, from N- to C-terminal order: the DNMT3A/L domain set forth in SEQ ID NO: 651, the sequence set forth in SEQ ID NO:672, the sequence set forth in SEQ ID NO: 670, the dSpCas9 set forth in SEQ ID NO: 599, the sequence set forth in SEQ ID NO: 677, the KRAB domain set forth in SEQ ID NO: 669, and the sequence set forth in SEQ ID NO: 593.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Feb 4, 2025
From: COSGROVE, BRIAN; CONGDON, KENDRA; DEAN, JASON; GOUGH, VERONICA; BLACK, JOSHUA B.; JONES, BRITTA
To: TUNE THERAPEUTICS, INC.
Reel/Frame 070108/0538 →
Continuity (5)
Provisional Application
63531309
· Aug 7, 2023
Provisional Application
63472236
· Jun 9, 2023
Provisional Application
63399634
· Aug 19, 2022
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