Pathogen-resistant animals having modified CD163 genes

Patent metadata

Field	Value
Publication number	US-12225889-B2
Application number	US-202318524212-A
Country	US
Kind code	B2
Filing date	Nov 30, 2023
Priority date	Aug 6, 2015
Publication date	Feb 18, 2025
Grant date	Feb 18, 2025

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 method of increasing porcine reproductive and respiratory (PRRS) resistance in a porcine animal, offspring, or cell, relative to a wild-type porcine animal, offspring, or cell, comprising:
modifying at least one chromosomal sequence in a gene encoding a CD163 protein in the porcine animal, offspring, or cell, wherein the modification is selected from the group consisting of:
a modification in exon 7 of the gene encoding the CD163 protein,
a modification in exon 8 of the gene encoding the CD163 protein,
a modification in an intron that is contiguous with exon 7 or exon 8 of the gene encoding the CD163 protein, and
a combination thereof, wherein the modification is selected from the group consisting of
an 11 bp deletion from nucleotide 3137 to nucleotide 3147 relative to the reference sequence SEQ ID NO:47,
a 377 bp deletion from nucleotide 2573 to nucleotide 2949, with a 2 bp addition between nucleotides 3149 to nucleotide 3150 relative to the reference sequence SEQ ID NO:47 on the same allele,
a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417, wherein the deleted sequence is replaced with a 12 base pair insertion beginning at nucleotide 488, with a 129 base pair deletion from nucleotide 3044 to nucleotide 3172 relative to the reference sequence SEQ ID NO: 47 on the same allele,
a 1467 bp deletion from nucleotide 2,431 to nucleotide 3,897 relative to the reference sequence SEQ ID NO:47,
a 1280 bp deletion from nucleotide 2818 to nucleotide 4097 relative to the reference sequence SEQ ID NO:47, and
combinations thereof.

2. The method of claim 1 , wherein the porcine animal, offspring, or cell is heterozygous for the CD163 gene alteration.

3. The method of claim 1 , wherein the porcine animal, offspring, or cell is homozygous for the CD163 gene alteration.

4. The method of claim 1 , wherein the modification is produced by the action of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), Transcription Activator-Like Effector Nuclease (TALEN) or Zinc Finger (ZFN) nuclease.

5. The method of claim 1 , wherein the deletion is selected from the group consisting of an 11 bp deletion from nucleotide 3137 to nucleotide 3147 relative to the reference sequence SEQ ID NO:47 and a 377 bp deletion from nucleotide 2573 to nucleotide 2949, with a 2 bp addition between nucleotides 3149 to nucleotide 3150 relative to the reference sequence SEQ ID NO:47 on the same allele.

6. The method of claim 5 , wherein the deletion deletes a Proline Serine Threonine domain.

7. The method of claim 6 , wherein the Proline Serine Threonine domain is a Proline Serine Threonine domain located after scavenger receptor cysteine-rich domain 6 or a Proline Serine Threonine domain located after scavenger receptor cysteine-rich domain 9.

8. A method of producing a porcine animal with increased porcine reproductive and respiratory (PRRS) resistance relative to a wild-type porcine animal comprising:
enucleating an oocyte;
fusing the oocyte with a donor somatic cell, the genome of the donor somatic cell comprising:
a modification in at least one chromosomal sequence in a gene encoding a CD163 protein, wherein the modification is selected from the group consisting of:
a modification in exon 7 of the gene encoding the CD163 protein,
a modification in exon 8 of the gene encoding the CD163 protein,
a modification in an intron that is contiguous with exon 7 or exon 8 of the gene encoding the CD163 protein, and

a combination thereof, wherein the modification is selected from the group consisting of
an 11 bp deletion from nucleotide 3137 to nucleotide 3147 relative to the reference sequence SEQ ID NO:47,
a 377 bp deletion from nucleotide 2573 to nucleotide 2949, with a 2 bp addition between nucleotides 3149 to nucleotide 3150 relative to the reference sequence SEQ ID NO:47 on the same allele,
a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417, wherein the deleted sequence is replaced with a 12 base pair insertion beginning at nucleotide 488, with a 129 base pair deletion from nucleotide 3044 to nucleotide 3172 relative to the reference sequence SEQ ID NO: 47 on the same allele,
a 1467 bp deletion from nucleotide 2,431 to nucleotide 3,897 relative to the reference sequence SEQ ID NO:47,
a 1280 bp deletion from nucleotide 2818 to nucleotide 4097 relative to the reference sequence SEQ ID NO:47, and
combinations thereof;

activating the oocyte to produce an embryo; and
transferring the embryo into a reproductive tract of a surrogate porcine animal, wherein the surrogate porcine animal has initiated estrus but has not yet completed ovulation; and wherein gestation and term delivery produces a porcine animal with increased porcine reproductive and respiratory (PRRS) resistance relative to a wild-type porcine animal.

9. The method of claim 8 , wherein the porcine animal is homozygous for the modification.

10. The method of claim 8 , wherein the porcine animal is heterozygous for the modification.

11. The method of claim 8 , wherein the donor somatic cell comprises a fibroblast.

12. The method of claim 8 , wherein the deletion is selected from the group consisting of an 11 bp deletion from nucleotide 3137 to nucleotide 3147 relative to the reference sequence SEQ ID NO:47 and a 377 bp deletion from nucleotide 2573 to nucleotide 2949, with a 2 bp addition between nucleotides 3149 to nucleotide 3150 relative to the reference sequence SEQ ID NO:47 on the same allele.

13. The method of claim 12 , wherein the deletion deletes a Proline Serine Threonine domain.

14. The method of claim 13 , wherein the Proline Serine Threonine domain is a Proline Serine Threonine domain located after scavenger receptor cysteine-rich domain 6 or a Proline Serine Threonine domain located after scavenger receptor cysteine-rich domain 9.

15. A method for producing a genetically altered porcine animal comprising a modification in both chromosomal sequences in a gene encoding a CD163 protein, wherein the porcine animal exhibits increased resistance to porcine reproductive and respiratory syndrome virus (PRRSV) relative to a wild-type porcine animal, the method comprising:
mating a female genetically altered porcine animal produced by the method of claim 6 with a male genetically altered porcine animal produced by the method of claim 6 to produce F1 progeny; and
screening the F1 progeny to identify the genetically altered porcine animal comprising a modification in both chromosomal sequences in the gene encoding a CD163 protein, wherein the modification is selected from the group consisting of:
a modification in exon 7 of the gene encoding the CD163 protein,
a modification in exon 8 of the gene encoding the CD163 protein,
a modification in an intron that is contiguous with exon 7 or exon 8 of the gene encoding the CD163 protein, and
a combination thereof, wherein the modification is selected from the group consisting of

an 11 bp deletion from nucleotide 3137 to nucleotide 3147 relative to the reference sequence SEQ ID NO:47,
a 377 bp deletion from nucleotide 2573 to nucleotide 2949, with a 2 bp addition between nucleotides 3149 to nucleotide 3150 relative to the reference sequence SEQ ID NO:47 on the same allele,
a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417, wherein the deleted sequence is replaced with a 12 base pair insertion beginning at nucleotide 488, with a 129 base pair deletion from nucleotide 3044 to nucleotide 3172 relative to the reference sequence SEQ ID NO: 47 on the same allele,
a 1467 bp deletion from nucleotide 2,431 to nucleotide 3,897 relative to the ref

Assignees

• Univ Missouri

Inventors

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

Classifications

• C07K14/70596

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

• C12N9/22

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

• C12N5/10

  Cells modified by introduction of foreign genetic material · CPC title

• C12N5/0609

  Oocytes, oogonia (fertilised oocytes C12N5/0604) · CPC title

• C12N5/061

  Sperm cells, spermatogonia · CPC title