# US 2019/0313615 A1 · Methods for protecting porcine fetuses from infection with porcine reproductive and respiratory syndrome virus (prrsv)

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- Number: US 2019/0313615 A1
- Document ID: US20190313615A1
- Status: Published application
- Kind: A1
- Application number: 16/386,901
- Filed: 2019-04-17
- Published: 2019-10-17
- Earliest claimed: 2018-04-17
- Assignees: The Curators of the University of Missouri
- Applicants: The Curators of the University of Missouri
- Inventors: Randall S. Prather; Kevin D. Wells; Kristin M. Whitworth
- Web page: https://patentgrep.com/patent/US20190313615A1

## CPC

- A01K 67/0276: Knock-out vertebrates
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- A01K 67/02: Breeding vertebrates
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/11: 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}
- C12N 15/1138: {against receptors or cell surface proteins}
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2217/075: inducing loss of function, i.e. knock out
- A01K 2227/108: Swine
- A01K 2267/02: Animal zootechnically ameliorated
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/30: Special therapeutic applications
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## 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.

## Claims

### Claim 1

A method for protecting a porcine fetus from infection with porcine reproductive and respiratory syndrome virus (PRRSV), 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 at least one wild-type CD163 allele.

### Claim 2 (depends on 1)

The method of claim 1, wherein the male porcine animal comprises two wild-type CD163 alleles.

### Claim at position 3

3-4. (canceled)

### Claim 5 (depends on 1)

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.

### Claim at position 5

6-7. (canceled)

### Claim 8 (depends on 5)

The method of claim 5, 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.

### Claim 9

(canceled)

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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.

### Claim at position 11

13-14. (canceled)

### Claim 15 (depends on 1)

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.

### Claim 16 (depends on 1)

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

### Claim 17

(canceled)

### Claim 18 (depends on 1)

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.

### Claim 19

(canceled)

### Claim 20 (depends on 18)

The method of claim 18, wherein the modification in exon 7 comprises a deletion or an insertion.

### Claim 21 (depends on 12)

The method of claim 12, wherein the deletion comprises an in-frame deletion.

### Claim 22

(canceled)

### Claim 23 (depends on 1)

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.

### Claim 24 (depends on 23)

The method of claim 23, wherein the miscoding results in a premature stop codon downstream of the miscoding in the allele of the CD163 gene.

### Claim 25 (depends on 1)

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.

### Claim at position 23

26-29. (canceled)

### Claim 30 (depends on 12)

The method of claim 12, 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.

### Claim 31 (depends on 1)

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.

### Claim at position 26

32-34. (canceled)

### Claim 35 (depends on 1)

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.

### Claim 36 (depends on 35)

The method of claim 35, 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.

### Claim 37 (depends on 1)

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.

### Claim at position 30

38-41. (canceled)