Methods for protecting porcine fetuses from infection with porcine reproductive and respiratory syndrome virus (PRRSV)

Patent metadata

Field	Value
Publication number	US-11160260-B2
Application number	US-201916386901-A
Country	US
Kind code	B2
Filing date	Apr 17, 2019
Priority date	Apr 17, 2018
Publication date	Nov 2, 2021
Grant date	Nov 2, 2021

Abstract

Methods for protecting porcine fetuses from infection with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The methods comprise breeding a female porcine animal with a male porcine animal. The female porcine animal comprises modified chromosomal sequences in both alleles of its CD163 gene, wherein the modified chromosomal sequences reduce the susceptibility of the female porcine animal to infection by PRRSV, as compared to the susceptibility to infection by PRRSV of a female porcine animal that does not comprise any modified chromosomal sequences in the alleles of its CD163 gene. The male porcine animal comprises at least one wild-type CD163 allele.

First claim

What is claimed is:

1. A method for protecting a porcine fetus from infection with porcine reproductive and respiratory syndrome virus (PRRSV) in utero, the method comprising breeding a female porcine animal with a male porcine animal, wherein:
the female porcine animal comprises modified chromosomal sequences in both alleles of its CD163 gene, wherein the modified chromosomal sequences reduce the susceptibility of the female porcine animal to infection by PRRSV, as compared to the susceptibility to infection by PRRSV of a female porcine animal that does not comprise any modified chromosomal sequences in the alleles of its CD163 gene; and
the male porcine animal comprises two wild-type CD163 alleles, and
wherein the porcine fetus is protected from PRRSV infection in utero.

2. The method of claim 1 , wherein the female porcine animal comprises a genetically edited female porcine animal, wherein the female porcine animal has been genetically edited using a homing endonuclease.

3. The method of claim 2 , wherein the homing endonuclease comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), a recombinase fusion protein, a meganuclease, or a combination of any thereof.

4. The method of claim 1 , wherein the female porcine animal comprises the same modified chromosomal sequence in both alleles of the CD163 gene.

5. The method of claim 1 , wherein the female porcine animal comprises a first modified chromosomal sequence in a first allele of the CD163 gene and a second modified chromosomal sequence in a second allele of the CD163 gene, the first and second modified chromosomal sequences being different from each other.

6. The method of claim 1 , wherein each allele of the CD163 gene of the female porcine animal comprises an insertion, a deletion, or a combination thereof.

7. The method of claim 1 , wherein the modified chromosomal sequences cause CD163 protein production or activity to be reduced, as compared to CD163 protein production or activity in a female porcine animal that lacks the modified chromosomal sequences.

8. The method of claim 1 , wherein the modified chromosomal sequences result in production of substantially no functional CD163 protein by the female porcine animal.

9. The method of claim 1 , wherein each allele of the CD163 gene of the female porcine animal comprises a modification in exon 7, a modification in exon 8, a modification in an intron that is contiguous with exon 7 or exon 8, or a combination of any thereof.

10. The method of claim 1 , wherein each allele of the CD163 gene of the female animal comprises a modification in exon 7 of the CD163 gene, and the modification in exon 7 comprises a deletion or an insertion.

11. The method of claim 6 , wherein the deletion comprises an in-frame deletion.

12. The method of claim 1 , wherein the modified chromosomal sequences in one or both of the alleles of the CD163 gene of the female porcine animal result in a miscoding.

13. The method of claim 12 , wherein the miscoding results in a premature stop codon downstream of the miscoding in the allele of the CD163 gene.

14. The method of claim 1 , wherein at least one of the alleles of the CD163 gene in the female porcine animal comprises a modification selected from the group consisting of:
an 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 as compared to reference sequence SEQ ID NO: 47;
a 2 base pair insertion between nucleotides 3,149 and 3,150 as compared to reference sequence SEQ ID NO: 47, with a 377 base pair deletion from nucleotide 2,573 to nucleotide 2,949 as compared to reference sequence SEQ ID NO: 47 on the same allele;
a 124 base pair deletion from nucleotide 3,024 to nucleotide 3,147 as compared to reference sequence SEQ ID NO: 47;
a 123 base pair deletion from nucleotide 3,024 to nucleotide 3,146 as compared to reference sequence SEQ ID NO: 47;
a 1 base pair insertion between nucleotides 3,147 and 3,148 as compared to reference sequence SEQ ID NO: 47;
a 130 base pair deletion from nucleotide 3,030 to nucleotide 3,159 as compared to reference sequence SEQ ID NO: 47;
a 132 base pair deletion from nucleotide 3,030 to nucleotide 3,161 as compared to reference sequence SEQ ID NO: 47;
a 1506 base pair deletion from nucleotide 1,525 to nucleotide 3,030 as compared to reference sequence SEQ ID NO: 47;
a 7 base pair insertion between nucleotide 3,148 and nucleotide 3,149 as compared to reference sequence SEQ ID NO: 47;
a 1280 base pair deletion from nucleotide 2,818 to nucleotide 4,097 as compared to reference sequence SEQ ID NO: 47;
a 1373 base pair deletion from nucleotide 2,724 to nucleotide 4,096 as compared to reference sequence SEQ ID NO: 47;
a 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897 as compared to reference sequence SEQ ID NO: 47;
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 wherein there is a further 129 base pair deletion in exon 7 from nucleotide 3,044 to nucleotide 3,172 as compared to reference sequence SEQ ID NO: 47;
a 28 base pair deletion from nucleotide 3,145 to nucleotide 3,172 as compared to reference sequence SEQ ID NO: 47;
a 1387 base pair deletion from nucleotide 3,145 to nucleotide 4,531 as compared to reference sequence SEQ ID NO: 47;
a 1382 base pair deletion from nucleotide 3,113 to nucleotide 4,494 as compared to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced with an 11 base pair insertion beginning at nucleotide 3,113;
a 1720 base pair deletion from nucleotide 2,440 to nucleotide 4,160 as compared to reference sequence SEQ ID NO: 47;
a 452 base pair deletion from nucleotide 3,015 to nucleotide 3,466 as compared to reference sequence SEQ ID NO: 47;
and combinations of any thereof.

15. The method of claim 6 , wherein the alleles of the CD163 gene of the female porcine animal 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 insertion or deletion.

16. The method of claim 1 , wherein the female porcine animal comprises a chromosomal sequence comprising SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 119 in one or both alleles of the CD163 gene.

17. The method of claim 1 , wherein the breeding produces one or more fetuses that comprise a modified chromosomal sequence in a single allele of the CD163 gene.

18. The method of claim 17 , wherein the fetuses have reduced susceptibility to infection by PRRSV while in utero, as compared to fetuses in utero in a wild-type female porcine animal.

19. The method of claim 1 , wherein the breeding comprises: mating of the female porcine animal with the male porcine animal; artificial insemination of the female animal with sperm obtained from the male animal; or transferring a fertilized oocyte into the reproductive tract of the female porcine animal.

Assignees

• Univ Missouri

Inventors

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

Classifications

• C12N15/11

  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

• A01K2217/072

  maintaining or altering function, i.e. knock in · CPC title

• A01K67/0276 Primary

  Knock-out vertebrates · CPC title

• A01K2227/108

  Swine · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title