Porcine reproductive and respiratory syndrome virus resistant animals

Patent metadata

Field	Value
Publication number	US-10080353-B2
Application number	US-201715783417-A
Country	US
Kind code	B2
Filing date	Oct 13, 2017
Priority date	May 16, 2011
Publication date	Sep 25, 2018
Grant date	Sep 25, 2018

Abstract

The present invention generally relates to genetically modified swine wherein at least one allele of a SIGLEC1 gene has been inactivated and/or at least one allele of a CD163 gene has been inactivated. Genetically modified swine having both alleles of the SIGLEC1 gene and/or both alleles CD163 gene inactivated are resistant to porcine reproductive and respiratory syndrome virus (PRRSV). Methods for producing such transgenic swine are also provided.

First claim

What is claimed is:

1. A genetically modified swine comprising an inactivating mutation in an allele of a CD163 gene.

2. The genetically modified swine of claim 1 , wherein the swine comprises inactivating mutations in both alleles of the CD163 gene.

3. The genetically modified swine of claim 1 , wherein the mutation alters the expression or activity of CD163.

4. The genetically modified swine of claim 2 , wherein the mutations alter the expression or activity of CD163.

5. The genetically modified swine of claim 1 , wherein the mutation comprises an insertion, a deletion, a frame shift mutation, a nonsense mutation, a missense mutation, or a point mutation.

6. The genetically modified swine of claim 2 , wherein the mutations comprise insertions, deletions, frame shift mutations, nonsense mutations, missense mutations, point mutations, or a combination thereof.

7. The genetically modified swine of claim 1 , wherein the mutation comprises a partial deletion or a complete deletion of the allele.

8. The genetically modified swine of claim 2 , wherein the mutations comprise partial deletions of the alleles, complete deletions of the alleles, or a combination thereof.

9. The genetically modified swine of claim 1 , wherein the mutation comprises a complete knockout of the CD163 allele.

10. The genetically modified swine of claim 2 , wherein the mutations comprise complete knockouts of the CD163 alleles.

11. The genetically modified swine of claim 1 , wherein the inactivating mutation results in a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

12. The genetically modified swine of claim 2 , wherein at least one of the inactivating mutations results in a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

13. A method for producing a genetically modified swine of claim 1 , the method comprising:
editing the genetic content of a porcine cell to create a modification which alters the expression or activity of CD163; and
generating an animal from the cell.

14. The method of claim 13 , wherein the editing comprises the use of a zinc-finger nuclease or a TAL effector nuclease.

15. The method of claim 13 , wherein the editing results in partial or complete deletion of a CD163 allele.

16. The method of claim 13 , wherein the editing results in an animal that produces a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

17. The method of claim 13 , wherein the method further comprises:
mating a female animal produced by the method of claim 13 with a male animal produced by the method of claim 13 to produce F1 progeny; and
screening the F1 progeny to identify animals having a modification in both alleles of a CD163 gene.

18. The method of claim 17 , wherein the screening comprises using a polymerase chain reaction (PCR) or Southern blot.

19. A method for producing a genetically modified swine of claim 1 , the method comprising:
introducing a site-specific nuclease into a porcine cell, the nuclease being adapted to bind to a target sequence in an allele of the CD163 gene; and
incubating the cell under conditions that permit the nuclease to act upon the DNA at or near the target sequence and thereby induce recombination, homology-directed repair, or non-homologous end joining at or near the target site.

20. The method of claim 19 , wherein the site-specific nuclease comprises a zinc-finger nuclease or a TAL effector nuclease.

21. The method of claim 19 , wherein introducing the nuclease into the cell results in partial or complete deletion of a CD163 allele.

22. The method of claim 19 , wherein introducing the nuclease into the cell results in a cell that produces a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

23. The method of claim 19 , wherein the method further comprises generating an animal from the cell.

24. The method of claim 23 , wherein the method further comprises mating a female animal produced by the method of claim 23 with a male animal produced by the method of claim 23 to produce F1 progeny; and
screening the F1 progeny to identify animals having a modification in both alleles of a CD163 gene.

25. The method of claim 24 , wherein the screening comprises using a polymerase chain reaction (PCR) or Southern blot.

Assignees

• Univ Missouri

Inventors

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

Classifications

• A01K2267/02

  Animal zootechnically ameliorated · CPC title

• A01K2227/108

  Swine · CPC title

• A01K67/0276 Primary

  Knock-out vertebrates · CPC title

• C12N5/10

  Cells modified by introduction of foreign genetic material · CPC title

• C07K14/70596

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