Animal tissue with carbohydrate antigens compatible for human transplantation and a carbohydrate determinant selection system for homologous recombination

• US 20030068818A1
• Filed: 03/21/2002
• Published: 04/10/2003
• Est. Priority Date: 03/21/2001
• Status: Active Grant

• Current Assignee: Geron Corporation
• Original Assignee: Geron Corporation
• Inventors: Clark, A. John, Denning, Chris, Schiff, J. Michael
• Granted Patent: US 7,126,039 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 2267/025 Animal producing cells or organs for transplantation
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A61K 2035/122 for inducing tolerance or supression of immune responses
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 2517/02 Cells from transgenic animals
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer
• CPC Class Codes: C12N 2517/10 Conditioning of cells for in vitro fecondation or nuclear transfer
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase

Abstract

This disclosure provides a system for generating animal tissue with carbohydrate antigens that are compatible for transplantation into human patients. The tissue is inactivated homozygously for expression of α(1,3)galactosyltransferase, and comprises a transgene for α(1,2)fucosyltransferase. As a result, cell-surface N-acetyl lactosamine is not converted to the Galα(1,3)Gal xenoantigen. Instead, it is converted to Fucα(1,2)Gal, which is H substance, a self-antigen in humans. The tissue may also contain A or B-transferase, which will cause H substance to be converted into other ABO blood group antigens for compatibility with patients of the same blood type. This invention improves transplant compatibility of the xenograft tissue by lessening the risk of reactions resulting from xenoantigen and unconverted N-acetyl lactosamine acceptor determinants.

Claims

1. A mammalian cell that is homozygous for inactivation of the α

(1,3)galactosyltransferase (α

1,3GT) gene, and expresses an α

(1,2)fucosyltransferase (α

1,2FT) transgene.

2. The cell of claim 1, wherein at least part of the encoding sequence in the α

1,3GT gene is replaced with an α

1,2FT encoding sequence.

3. The cell of claim 1, wherein the α

1,2FT encoding sequence is placed under control of the endogenous α

1,3GT promoter.

4. The cell of claim 1, containing a transgene for blood group A- or B-transferase.

5. The cell of claim 1, which is an ovine cell.

6. The cell of claim 1, which is a porcine cell.

7. Tissue from a non-rodent non-cattharine mammal devoid of antibody-detectable Galα

(1,3)Gal determinants and expressing at least one ABO blood group substance.

8. The tissue of claim 7, wherein the ABO blood group substance is H substance.

9. The tissue of claim 7, wherein the ABO blood group substance is A substance or B substance.

10. The tissue of claim 7, which is selected from the group consisting of lung tissue, kidney tissue, liver tissue, cardiac tissue, pancreatic tissue, and ocular tissue.

11. Tissue from a non-rodent non-cattharine mammal that expresses α

(1,2)fucosyltransferase (α

1,2FT) but does not detectably express α

(1,3)galactosyltransferase (α

1,3GT).

12. The tissue of claim 11, which also expresses blood group A- or B-transferase, or both.

13. A non-human mammal that is homozygous for inactivation of the α

1,3GT gene, and expresses an α

1,2FT transgene.

14. A method for producing the tissue of claim 13, comprising transferring the nucleus of a donor cell wherein at least part of the encoding sequence of an α

1,3GT gene is replaced with an α

1,2FT encoding sequence, to a suitable recipient cell;

engrafting the cell into the uterus of a surrogate host;

obtaining an embryo or neonate from the host that has grown from the engrafted cell; and

harvesting the tissue from the embryo or neonate, or its progeny.

15. The method of claim 14, wherein the donor cell is produced by a process comprising telomerizing a cell, and targeting the cell or its progeny to inactivate or replace at least part of the encoding sequence of an α

1,3GT gene.

16. The method of claim 14, wherein the genetic targeting involves at least one of the following:

inactivation of an α

1,3GT gene by homologous recombination with a vector that eliminates, replaces or alters an α

1,3GT encoding sequence or a transcription or translation control element within the gene;

inactivation of an α

1,3GT gene by targeting with an oligonucleotide that alters the α

1,3GT encoding region or a transcription or translation control element within the gene;

substitution of an α

1,3GT encoding sequence with an α

1,2FT encoding sequence.

17. A method for selecting a mammalian cell that has undergone genetic alteration by homologous recombination from amongst a population of cells that do not have the alteration, comprising separating cells according to a surface carbohydrate determinant that has changed as a result of the homologous recombination.

18. The method of claim 17, wherein the homologous recombination inactivates an endogenous gene in the cell.

19. The method of claim 17, wherein the homologous recombination introduces a transgene into the genome of the cell.

20. The method of claim 17, wherein the homologous recombination introduces a site-specific recombinase recognition sequence into the cell.

21. The method of claim 18, wherein the homologous recombination introduces a transgene into the genome of the cell.

22. The method of claim 21, wherein the endogenous gene encodes a glycosyltransferase, but the transgene does not.

23. The method of claim 21, wherein the transgene encodes a glycosyltransferase, but the endogenous gene does not.

24. The method of claim 21, wherein both the endogenous gene and the transgene encode different glycosyltransferases.

25. The method of claim 18, wherein the endogenous gene encodes α

(1,3)galactosyltransferase (α

1,3GT), A-transferase, or B-transferase.

26. The method of claim 19, wherein the transgene encodes α

(1,2)fucosyltransferase (α

1,2FT), A-transferase, or B-transferase.

27. The method of claim 21, wherein the endogenous gene encodes α

1,3GT, and the transgene encodes α

1,2FT.

28. The method of claim 17, further comprising removing the transgene from the cell having the genetic alteration subsequent to separating it from cells without the genetic alteration.

29. The method of claim 17, wherein the cell population is a population of human pluripotent stem cells.

30. The method of claim 17, wherein the cell population is a population of non-human cells suitable as donors for nuclear transfer.