Pathogen-resistant animals having modified CD163 genes

Patent metadata

Field	Value
Publication number	US-10091975-B2
Application number	US-201615216865-A
Country	US
Kind code	B2
Filing date	Jul 22, 2016
Priority date	Aug 6, 2015
Publication date	Oct 9, 2018
Grant date	Oct 9, 2018

Abstract

Non-human animals and offspring thereof comprising at least one modified chromosomal sequence in a gene encoding a CD163 protein are provided. Animal cells that contain such modified chromosomal sequences are also provided. The animals and cells have increased resistance to pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). The animals and offspring have chromosomal modifications of a CD163 gene. The invention further relates to methods of breeding to create pathogen-resistant animals and populations of animals made using such methods.

First claim

What is claimed is:

1. A porcine animal or offspring thereof or a porcine cell comprising a modified chromosomal sequence in at least one allele of a gene encoding a CD163 protein, wherein the modified chromosomal sequence in the at least one allele comprises a deletion and the deletion consists of
an 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 as compared to reference sequence SEQ ID NO: 47.

2. The animal, offspring, or cell of claim 1 , wherein the modification reduces the susceptibility of the animal, offspring, or cell to infection by a virus, as compared to the susceptibility of an animal, offspring, or cell that does not comprise a modified chromosomal sequence in a gene encoding a CD163 protein to infection by the virus.

3. The animal, offspring, or cell of claim 2 , wherein the virus comprises a porcine reproductive and respiratory syndrome virus (PRRSV).

4. The animal, offspring, or cell of claim 3 , wherein the modification reduces the susceptibility of the animal, offspring, or cell to a Type 1 PRRSV virus, a Type 2 PRRSV, or to both Type 1 and Type 2 PRRSV viruses.

5. The animal, offspring, or cell of claim 4 , wherein the modification reduces the susceptibility of the animal, offspring or cell to a PRRSV isolate selected from the group consisting of NVSL 97-7895, KS06-72109, P129, VR2332, CO90, AZ25, MLV-ResPRRS, KS62-06274, KS483 (SD23983), CO84, SD13-15, Lelystad, 03-1059, 03-1060, SD01-08, 4353PZ, and combinations of any thereof.

6. The animal, offspring, or cell of claim 1 , wherein the animal or offspring comprises a genetically edited animal or offspring or wherein the cell comprises a genetically edited cell.

7. The animal, offspring, or cell of claim 6 , wherein the animal or cell has been genetically edited using a designed homing endonuclease, wherein the homing endonuclease comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), a recombinase fusion protein, a meganuclease, or a combination thereof.

8. The animal, offspring, or cell of claim 7 , wherein the animal or cell has been genetically edited using a CRISPR/Cas system.

9. The animal, offspring, or cell of claim 8 , wherein the CRISPR/Cas system comprises a CRISPR/Cas9 system.

10. The animal, offspring, or cell of claim 8 , wherein said edited animal, offspring, or cell displays increased resistance to PRRSV as compared to a non-edited animal.

11. The animal, offspring, or cell of claim 1 , wherein the deletion causes CD163 protein production or activity to be reduced, as compared to CD163 protein production or activity in an animal, offspring, or cell that lacks the deletion.

12. The animal, offspring, or cell of claim 1 , wherein the deletion results in production of substantially no functional CD163 protein by the animal, offspring, or cell.

13. The porcine animal, offspring, or cell of claim 1 , wherein the animal, offspring, or cell comprises a chromosomal sequence having at least 80% sequence identity to SEQ ID NO: 47 in the regions of said chromosomal sequence outside of the deletion.

14. The cell of claim 1 .

15. The cell of claim 14 , wherein said cell is a sperm cell.

16. The cell of claim 14 , wherein said cell is an egg cell.

17. The cell of claim 16 , wherein said egg cell is a fertilized egg.

18. The cell of claim 14 , wherein said cell is a somatic cell.

19. The cell of claim 18 , wherein the somatic cell comprises a fibroblast.

20. The cell of claim 19 , wherein the fibroblast comprises a fetal fibroblast.

21. The non-human animal of claim 1 .

22. The offspring of claim 1 .

23. The animal, offspring, or cell of claim 1 , comprising a chromosomal sequence comprising SEQ ID NO: 102.

24. The animal, offspring, or cell of claim 1 , wherein the animal or offspring is a juvenile or an adult or wherein the cell comprises a cell derived from a juvenile animal or a cell derived from an adult animal.

25. The animal, offspring, or cell of claim 1 , wherein the animal or offspring is an embryo or where the cell comprises an embryonic cell.

26. The animal, offspring, or cell of claim 1 , wherein the animal, offspring, or cell is heterozygous for the modified chromosomal sequence.

27. The animal, offspring, or cell of claim 25 , wherein the embryo or the embryonic cell is heterozygous for the modified chromosomal sequence.

28. The animal, offspring, or cell of claim 1 , wherein the animal, offspring, or cell is homozygous for the modified chromosomal sequence.

29. The animal, offspring, or cell of claim 1 , wherein the animal, offspring, or cell comprises:
the 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 as compared to reference sequence SEQ ID NO: 47 in one allele of the gene encoding the CD163 protein; and
an insertion in the other allele of the gene encoding the CD163 protein, the insertion consisting of a 7 base pair insertion between nucleotide 3,148 and nucleotide 3,149 as compared to reference sequence SEQ ID NO: 47.

30. The animal, offspring, or cell of claim 29 , wherein the 7 base pair insertion between nucleotide 3,148 and nucleotide 3,149 as compared to reference sequence SEQ ID NO: 47 consists of the sequence TACTACT (SEQ ID NO: 115).

31. The animal, offspring, or cell of claim 1 , wherein the animal, offspring, or cell comprises:
the 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 as compared to reference sequence SEQ ID NO: 47 in one allele of the gene encoding the CD163 protein; and
a deletion in the other allele of the gene encoding the CD163 protein, the deletion consisting of a 1280 base pair deletion from nucleotide 2,818 to nucleotide 4,097 as compared to reference sequence SEQ ID NO: 47.

32. The animal, offspring, or cell of claim 1 , wherein the animal, offspring, or cell comprises:
the 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 as compared to reference sequence SEQ ID NO: 47 in one allele of the gene encoding the CD163 protein; and
deletions in the other allele of the gene encoding the CD163 protein, the deletions consisting of:
a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 as compared to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced with a 12 base pair insertion beginning at nucleotide 488; and
a 129 base pair deletion in exon 7 from nucleotide 3,044 to nucleotide 3,172 as compared to reference sequence SEQ ID NO: 47.

33. The animal, offspring, or cell of claim 32 , wherein the 12 base pair insertion beginning at nucleotide 488 consists of the sequence TGTGGAGAATTC (SEQ ID NO: 116).

34. The animal, offspring, or cell of claim 1 , wherein the animal, offspring, or cell comprises:
the 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 as compared to reference sequence SEQ ID NO: 47 in one allele of the gene encoding the CD163 protein; and
a deletion in the other allele of the gene encoding the CD163 protein, the deletion consisting of a 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897 as compared to reference sequence SEQ ID NO: 47 in the other allele of the gene encoding the CD163 protein.

35. The animal, offspring, or cell of cl

Assignees

• Univ Missouri

Inventors

• Prather Randall S
• Wells Kevin D
• Whitworth Kristin M

Classifications

• A01K2227/108

  Swine · CPC title

• C12N2770/10011

  Arteriviridae · CPC title

• A01K2267/02

  Animal zootechnically ameliorated · CPC title

• C07K14/70596

  Molecules with a "CD"-designation not provided for elsewhere · CPC title

• C12N15/8778

  Swine embryos · CPC title