HSD17B13 variants and uses thereof

Patent metadata

Field	Value
Publication number	US-10787647-B2
Application number	US-201815875514-A
Country	US
Kind code	B2
Filing date	Jan 19, 2018
Priority date	Jan 23, 2017
Publication date	Sep 29, 2020
Grant date	Sep 29, 2020

Abstract

Provided are compositions related to HSD17B13 variants, including isolated nucleic acids and proteins related to variants of HSD17B13, and cells comprising those nucleic acids and proteins. Also provided are methods related to HSD17B13 variants. Such methods include methods for modifying a cell through use of any combination of nuclease agents, exogenous donor sequences, transcriptional activators, transcriptional repressors, and expression vectors for expressing a recombinant HSD17B13 gene or a nucleic acid encoding an HSD17B13 protein. Also provided are therapeutic and prophylactic methods for treating a subject having or at risk of developing chronic liver disease.

First claim

We claim:

1. A method of modifying or altering expression of an HSD17B13 gene in a cell, comprising introducing into the cell:
(a) a Cas9 protein or a nucleic acid encoding the Cas9 protein; and
(b) a first guide RNA or a DNA encoding the first guide RNA, wherein the first guide RNA comprises a first CRISPR RNA (crRNA) portion and a first trans-activating CRISPR RNA (tracrRNA) portion, wherein the first guide RNA forms a complex with the Cas9 protein and targets a first guide RNA target sequence within the HSD17B13 gene, wherein the first guide RNA target sequence includes or is proximate to the start codon for the HSD17B13 gene, and
wherein the Cas9 protein cleaves the first guide RNA target sequence to generate a targeted genetic modification in the HSD17B13 gene or the Cas9 protein binds the first guide RNA target sequence and alters expression of the HSD17B13 gene.

2. The method of claim 1 , wherein:
(a) the first guide RNA target sequence comprises any one of SEQ ID NOS: 20-81 and 259-263; and/or
(b) the first guide RNA comprises a DNA-targeting segment that comprises any one of SEQ ID NOS: 1423-1484 and 1643-1647; and/or
(c) the first guide RNA comprises any one of SEQ ID NOS: 500-561, 730-791, 960-1021, 1190-1251, 720-724, 950-954, 1180-1184, and 1410-1414.

3. The method of claim 2 , wherein:
(a) the first guide RNA target sequence comprises any one of SEQ ID NOS: 20-41, any one of SEQ ID NOS: 21-23, 33, and 35, or any one of SEQ ID NOS: 33 and 35; and/or
(b) the first guide RNA comprises a DNA-targeting segment that comprises any one of SEQ ID NOS: 1447-1468, any one of SEQ ID NOS: 1448-1450, 1460, and 1462; or any one of SEQ ID NOS: 1460 and 1462; and/or
(c) the first guide RNA comprises any one of SEQ ID NOS: 524-545, 754-775, 984-1005, and 1214-1235, or any one of SEQ ID NOS: 295-297, 525-527, 755-757, 985-987, 1215-1217, 307, 309, 537, 539, 767, 769, 997, 999, 1227, and 1229, or any one of SEQ ID NOS: 307, 309, 537, 539, 767, 769, 997, 999, 1227, and 1229.

4. The method of claim 1 , wherein the Cas9 protein is a nuclease-active Cas9 protein.

5. The method of claim 1 , wherein the Cas9 protein is a nuclease-inactive Cas9 protein fused to a transcriptional repressor domain.

6. The method of claim 1 , wherein the Cas9 protein is a nuclease-inactive Cas9 protein fused to a transcriptional activator domain, and wherein the HSD17B13 gene comprises a thymine inserted between nucleotides corresponding to positions 12665 and 12666 of SEQ ID NO: 1 when the HSD17B13 gene is optimally aligned with SEQ ID NO: 1.

7. The method of claim 1 , further comprising introducing into the cell a second guide RNA or a DNA encoding the second guide RNA, wherein the second guide RNA comprises a second CRISPR RNA (crRNA) portion and a second trans-activating CRISPR RNA (tracrRNA) portion, wherein the second guide RNA forms a complex with the Cas9 protein and targets a second guide RNA target sequence within the HSD17B13 gene, wherein the second guide RNA target sequence includes or is proximate to the stop codon for the HSD17B13 gene, wherein the cell is modified to comprise a deletion between the first guide RNA target sequence and the second guide RNA target sequence.

8. The method of claim 7 , wherein:
(a) the second guide RNA target sequence comprises any one of SEQ ID NOS: 82-225; and/or
(b) the second guide RNA comprises a DNA-targeting segment that comprises any one of SEQ ID NOS: 1485-1628; and/or
(c) the second guide RNA comprises any one of SEQ ID NOS: 562-705, 792-935, 1022-1165, and 1252-1395.

9. The method of claim 1 , further comprising introducing an expression vector into the cell, wherein the expression vector comprises a recombinant HSD17B13 gene comprising a thymine inserted between nucleotides corresponding to positions 12665 and 12666 of SEQ ID NO: 1 when the recombinant HSD17B13 gene is optimally aligned with SEQ ID NO: 1.

10. The method of claim 9 , wherein the recombinant HSD17B13 gene is a human gene.

11. The method of claim 9 , wherein the recombinant HSD17B13 gene is an HSD17B13 minigene in which one or more nonessential segments of the gene have been deleted with respect to a corresponding wild type HSD17B13 gene.

12. The method of claim 11 , wherein the deleted segments comprise one or more intronic sequences.

13. The method of claim 11 , wherein the HSD17B13 minigene comprises an intron corresponding to intron 6 of SEQ ID NO: 2 when optimally aligned with SEQ ID NO: 2.

14. The method of claim 1 , further comprising introducing an expression vector into the cell, wherein the expression vector comprises a nucleic acid encoding an HSD17B13 protein that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15 (HSD17B13 Isoform D).

15. The method of claim 14 , wherein the nucleic acid encoding the HSD17B13 protein is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7 (HSD17B13 Transcript D) when optimally aligned with SEQ ID NO: 7.

16. The method of claim 1 , further comprising introducing an HSD17B13 protein or fragment thereof into the cell, wherein the HSD17B13 protein or fragment thereof is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15 (HSD17B13 Isoform D).

17. The method of claim 1 , wherein the cell is a mouse cell, a rat cell, or a human cell.

18. The method of claim 1 , wherein the cell is a human liver cell, a mouse liver cell, a rat liver cell, a mouse pluripotent cell, or a rat pluripotent cell.

19. The method of claim 1 , wherein the cell is ex vivo or in vivo.

20. The method of claim 18 , wherein the cell is the human liver cell.

21. The method of claim 20 , wherein the cell is in vivo.

22. The method of claim 1 , wherein the method results in loss of function of the HSD17B13 gene.

23. The method of claim 1 , wherein the method results in disruption of the start codon of the HSD17B13 gene.

24. The method of claim 1 , wherein the first guide RNA target sequence is within a region corresponding to exon 1 of SEQ ID NO: 1 or 2 when the HSD17B13 gene is optimally aligned with SEQ ID NO: 1 or 2.

25. The method of claim 1 , wherein the method further comprises introducing into the cell a second guide RNA or a DNA encoding the second guide RNA, wherein the second guide RNA forms a complex with the Cas9 protein and targets a second guide RNA target sequence within the HSD17B13 gene, wherein the second guide RNA target sequence includes or is proximate to the start codon for the HSD17B13 gene.

26. The method of claim 7 , wherein the coding sequence of the HSD17B13 gene is deleted.

27. The method of claim 7 , wherein the second guide RNA target sequence is within a region corresponding to exon 7 of SEQ ID NO: 1 or 2 when the HSD17B13 gene is optimally aligned with SEQ ID NO: 1 or 2.

28. The method of claim 1 , wherein the method comprises introducing into the cell the nucleic acid encoding the Cas9 protein.

29. The method of claim 28 , wherein the method comprises introducing into the cell the nucleic acid encoding the Cas9 protein, wherein the nucleic acid encoding the Cas9 protein comprises DNA.

30. The method of claim 28 , wherein the method comprises introducing into the cell the nucleic acid e

Assignees

• Regeneron Pharma

Inventors

• Abul-Husn Noura S
• Gottesman Omri
• Li Alexander
• Cheng Xiping
• Xin Yurong
• Pefanis Evangelos
• Hartford Suzanne
• Gromada Jesper
• Dewey Frederick E
• Baras Aris
• Shuldiner Alan

Classifications

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N2320/30

  Special therapeutic applications · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• G01N2333/4704

  Inhibitors; Supressors · CPC title

• G01N33/5067

  Liver cells · CPC title