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-10717991-B2
Application number	US-201615775751-A
Country	US
Kind code	B2
Filing date	Nov 11, 2016
Priority date	Nov 13, 2015
Publication date	Jul 21, 2020
Grant date	Jul 21, 2020

Abstract

The present invention relates to a transgenic pig in which an immune rejection response is suppressed during xenotransplantation, wherein a gene coding for heme oxygenase-1 (HO-1) and a gene coding for tumor necrosis factor receptor 1-Fc (TNFR1-Fc) are simultaneously expressed and a gene coding for α-1,3-galactosyltransferase (GGTA1) is knocked out; and a method for producing the same.

First claim

The invention claimed is:

1. A method of producing a pig nuclear transfer embryo comprising: (a) obtaining a somatic cell from a pig; (b) introducing a transgene encoding a heme oxygenase-1 (HO-1) and a transgene encoding a tumor necrosis factor 1-Fc (TNFR1-Fc) into the somatic cell, wherein the transgenes incorporate into the genome of the somatic cell, and introducing a homozygous disruption into endogenous genes encoding α-1,3-galactosyltransferase (GGTA1) in the somatic cell, thereby producing a genetically modified pig somatic cell comprising in its genome a transgene encoding HO-1, a transgene encoding a TNFR1-Fc, and a homozygous disruption of the endogenous GGTA1 genes; and (c) introducing the genetically modified pig somatic cell into an enucleated pig oocyte to produce a pig nuclear transfer embryo.

2. The method according to claim 1 , wherein the transgene encoding the HO-1 comprises the nucleic acid sequence of SEQ ID NO: 1.

3. The method according to claim 1 , wherein the transgene encoding the TNFR1-Fc comprises the nucleic acid sequence of SEQ ID NO: 8.

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

5. The method according to claim 1 , wherein introducing the homozygous disruption into the endogenous genes comprises introducing a TALEN that specifically binds and cleaves a nucleic acid sequence in the endogenous genes.

6. The method according to claim 5 , wherein the TALEN specifically binds to and causes a double stranded break in exon 9 of the endogenous GGTA1 gene.

7. The method according to claim 1 , further comprising:
d) transferring the pig nuclear transfer embryo into a recipient female pig; and
e) allowing the recipient female pig to gestate until offspring are produced.

8. A transgenic pig comprising in its genome (i) an exogenous nucleic acid encoding a human heme oxygenase-1 (HO-1) gene; (ii) an exogenous nucleic acid encoding a human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc); and (iii) a homozygous disruption of the endogenous gene encoding alpha-1,3, galactosyltransferase (GGTA1), wherein the transgenic pig simultaneously expresses the HO-1 and TNFR1-Fc and lacks expression of the GGTA1, and wherein (i) cells of the transgenic pig have greater resistance to apoptotic and oxidative stress stimulation compared to wild type cells; (ii) cells are more resistant to the human serum and complement compared to those derived from a transgenic pigs comprising in its genome exogenous nucleic acids encoding HO-1 and TNFR1-Fc but the endogenous GGTA1 is intact; and (iii) the antibody/complement-mediated lytic reaction is significantly reduced compared to the transgenic pigs comprising in its genome exogenous nucleic acids encoding HO-1 and TNFR1-Fc but the endogenous GGTA1 is intact.

9. A somatic cell line derived from 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 7 ; and
isolating a transplantable organ from the transgenic pig.

11. The method according to claim 10 , wherein the organ is at least one selected form the group consisting of 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 Curie
• Kim Geon A
• Yeom Su Cheong
• Kim Su Jin
• Cho Bumrae
• Lee Eun Mi
• Lee Sang Hoon
• Hwang In Chang
• Hong Hye Jin

Classifications

• C12Y204/01087

  N-Acetyllactosaminide 3-alpha-galactosyltransferase (2.4.1.87), i.e. alpha-1,3-galactosyltransferase · CPC title

• C12N15/8778

  Swine embryos · CPC title

• C12Y114/14001

  Unspecific monooxygenase (1.14.14.1) · CPC title

• A01K67/027

  New or modified breeds of vertebrates · CPC title

• A01K2217/052

  inducing gain of function · CPC title