tissues, organs and cells for use in xenotransplantation
Inventors
Phelps, Carol J. • AYARES, DAVID L.
Assignees
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Publication Number
US-7795493-B2
Publication Date
2010-09-14
Expiration Date
Abstract
The present invention is a porcine animal, tissue, organ, cells and cell lines, which lack any expression of functional alpha 1,3 galactosyltransferase (alpha1,3GT). These animals, tissues, organs and cells can be used in xenotransplantation and for other medical purposes.
Core Innovation
The disclosure relates to porcine animals, tissues, organs, and cells that lack any expression of functional alpha-1,3 galactosyltransferase (alpha1,3GT/alpha-1,3-GT) in order to remove the alpha-gal epitope (galactose alpha-1,3-galactose; gal alpha 1-3 gal beta 1-4 GlcNAc) for xenotransplantation use. The problem addressed is hyperacute rejection associated with the alpha-gal epitope, which is driven by binding of anti-alpha-gal antibodies. By producing pigs with functional alpha-1,3GT inactivated, the alpha-gal epitope is eliminated to support xenotransplantation.
The core genetic approach uses complete inactivation of both alpha-1,3GT alleles through genetic targeting and/or a point mutation, including a T-to-G point mutation at exon 9 base 2. The disclosure states that viability is achieved by inactivating both alpha-1,3GT alleles, and includes embodiments that avoid antibiotic-resistance/selectable markers.
Genetic targeting is described via homologous recombination, followed by selection/screening of cells lacking alpha-gal using Clostridium difficile toxin A and lectin/complement assays. Reported outcomes include cloning and production of homozygous alpha-1,3GT double knockout pigs with complete loss of alpha-gal binding, and an in vivo immunogenicity assessment showing functional absence of alpha-gal via lack of anti-alpha-gal IgM response in alpha-gal knockout mice after islet-like cell cluster injection.
Claims Coverage
The document includes one independent claim. It specifies breeding male and female pigs, each heterozygous for inactivation of the alpha-1,3 GT gene, where heterozygosity is due to a point mutation, including a T-to-G point mutation at exon 9 base 2, or a mutation induced or produced by genetic modification.
Breeding heterozygous alpha-1,3 GT inactivation pigs
Breeding a male pig heterozygous for an inactivation of the alpha-1,3 GT gene with a female pig heterozygous for an inactivation of the alpha-1,3 GT gene.
Heterozygosity via exon 9 T-to-G point mutation or genetic modification
Ensuring one or both pigs are heterozygous due to a point mutation in an allele of the alpha-1,3 GT gene, where the point mutation is a T-to-G point mutation at the second base of exon 9 of the alpha-1,3 GT gene or a point mutation induced or produced by a genetic modification in the alpha-1,3 GT gene.
Pig lacks any expression of functional alpha-1,3 galactosyltransferase
Producing a pig that lacks any expression of functional alpha-1,3 galactosyltransferase (alpha-1,3 GT).
Overall, the independent claim coverage centers on breeding male and female pigs heterozygous for alpha-1,3 GT gene inactivation caused by a specified exon 9 T-to-G point mutation or a point mutation produced by genetic modification, to produce a pig lacking expression of functional alpha-1,3 GT.
Stated Advantages
Removes the alpha-gal epitope for xenotransplantation use.
Addresses hyperacute rejection associated with the alpha-gal epitope.
Achieves viability while completely inactivating both alpha-1,3GT alleles.
Maintains embodiments that avoid antibiotic-resistance/selectable markers.
Demonstrates functional absence of alpha-gal in vivo as shown by lack of anti-alpha-gal IgM response.
Documented Applications
Xenotransplantation use of porcine animals, tissues, organs, and cells lacking functional alpha-1,3 galactosyltransferase expression (alpha-gal epitope removal).
In vivo immunogenicity testing involving alpha-gal knockout mice and islet-like cell clusters, assessing anti-alpha-gal IgM response.
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