Transgenic pig which simultaneously expresses ho-1 gene and tnfr1-fc gene, and comprises knocked-out ggta1 gene, and use thereof

Patent metadata

Field	Value
Publication number	US-2019024115-A1
Application number	US-201615775751-A
Country	US
Kind code	A1
Filing date	Nov 11, 2016
Priority date	Nov 13, 2015
Publication date	Jan 24, 2019
Grant date	—

Abstract

The transgenic pig of the present invention, in which the genes coding for human HO-1 and TNFR1-Fc fusion protein are simultaneously expressed and the gene coding for GGTA1 is knocked out, may reduce oxidative stress during organ isolation and in vitro culture by antioxidative reaction, cytoprotective function, etc., and may also reduce a TNF-α-mediated inflammatory response in early transplantation by TNFR1-Fc expression. In addition, the transgenic pig may inhibit the maturation of dendritic cells and regulate the activation and proliferation of T-cells, thereby reducing an acute vascular rejection response to promote early engraftment of a transplanted organ. In addition, the transgenic pig can increase the viability of a transplanted organ by suppressing a hyper-acute immune rejection reaction caused by GGTA1. Accordingly, an organ, in which an immune rejection response is suppressed during xenotransplantation, can be produced using the transgenic pig.

First claim

1 . A method for producing a transgenic pig in which an immune rejection response is suppressed during xenotransplantation, comprising:
a) isolating somatic cells from a pig; b) introducing a gene coding for human heme oxygenase-1 (HO-1) and a gene coding for human tumor necrosis factor receptor 1-Fc (TNFR1-Fc) fusion protein into the somatic cells, and knocking out a gene coding for α-1,3-galactosyltransferase (GGTA1); c) selecting and culturing the somatic cells in which the gene coding for human HO-1 and the gene coding for human TNFR1-Fc fusion protein are introduced and the gene coding for GGTA1 is knocked out; and d) removing the nucleus from an oocyte of the pig and fusing it with the selected somatic cells to prepare an embryo transplanted with a somatic cell nucleus.

2 . The method according to claim 1 , wherein the gene coding for human HO-1 of step b) comprises a nucleotide sequence of SEQ ID NO: 1.

3 . The method according to claim 1 , wherein the gene coding for human TNFR1-Fc fusion protein of step b) comprises a nucleotide sequence of SEQ ID NO: 8.

4 . The method according to claim 1 , wherein in step b), a single vector comprising the gene coding for human HO-1 and a single vector comprising the gene coding for human TNFR1-Fc fusion protein are introduced separately or simultaneously into the somatic cells.

5 . The method according to claim 1 , wherein the gene knock-out is carried out using transcription activator-like effector nuclease (TALEN).

6 . The method according to claim 5 , wherein the TALEN targets exon 9 of the gene coding for GGTA1.

7 . The method according to claim 1 , further comprising implanting the embryo of step d) into a porcine uterus.

8 . A transgenic pig for organ transplantation in which an immune rejection response is suppressed during xenotransplantation, wherein a gene coding for human HO-1 and a gene coding for human TNFR1-Fc fusion protein are introduced and a gene coding for GGTA1 is knocked out.

9 . A somatic donor cell line for producing the transgenic pig of claim 8 .

10 . A method for producing a transplantable organ in which an immune rejection response is suppressed during xenotransplantation, comprising:
producing a transgenic pig by the method of claim 1 ; and isolating a transplantable organ from the transgenic pig.

11 . The method according to claim 10 , wherein the organ is at least one selected from the group consisting of a pancreatic islet, a pancreas, a heart, a kidney, a liver, a lung, and a cornea.

Assignees

• Seoul Nat Univ R&Db Foundation
• Chong Kun Dang Pharmaceutical Corp

Inventors

• Lee Byeong Chun
• Ahn Cu Rie
• Kim Geon A
• Yeom Su Cheong
• Kim Su Jin
• Cho Bum Rae
• Lee Eun Mi
• Lee Sang Hoon
• Hwang In Chang
• Hong Hye Jin

Classifications

• C07K14/70575

  NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525) · CPC title

• A01K2217/075

  inducing loss of function, i.e. knock out · CPC title

• C12N15/8778

  Swine embryos · CPC title

• A01K2217/15

  Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding · CPC title

• A01K67/0271

  Chimeric vertebrates, e.g. comprising exogenous cells · CPC title