Transgenic ungulates expressing CTLA4-IG and uses thereof
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Publication Number
US-10300112-B2
Publication Date
2019-05-28
Expiration Date
Abstract
The present invention provides ungulates, including pigs, expressing CTLA4-Ig, as well as tissue, organs, cells and cell lines derived from such animals. Such animals, tissues, organs and cells can be used in research and medical therapy, including xenotransplanation. In addition, methods are provided to prepare organs, tissues and cells expressing the CTLA4-Ig for use in xenotransplantation, and nucleic acid constructs and vectors useful therein.
Core Innovation
The disclosed invention concerns genetically modified porcine cells for xenotransplantation, where cell mediated rejection is reduced without suppression of the recipient's normal immune function. The genetically modified cells lack expression of the alpha-1,3-galactosyltransferase (alpha-1,3-GT) gene and express a transgene encoding a porcine CTLA4 peptide fused to a human immunoglobulin peptide (pCTLA4-Ig).
In one described aspect, the genetically modified cells are directly obtained from a genetically modified porcine animal engineered by insertion of the pCTLA4-Ig transgene into the genome of the animal, together with loss of alpha-1,3-GT expression. The patent text further describes CTLA4 fused to an immunoglobulin, including variants such as extracellular or truncated forms, and additional CTLA4 peptide modifications.
The disclosed construct and regulatory concepts include nucleic acid constructs and vectors encoding CTLA4 or CTLA4-Ig, with regulatory control using tissue-specific or regulatable promoters. The document also discusses optional additional genetic modifications and control of transgene expression, in the context of reducing cell mediated rejection and addressing hyperacute and delayed xenograft rejection.
Claims Coverage
The document includes one independent claim directed to a method for reducing cell mediated rejection in a xenotransplantation setting, comprising three core inventive requirements: immune-preserving reduction of rejection, alpha-1,3-GT-negative pCTLA4-Ig expression in genetically modified porcine cells, and sourcing of those cells directly from a corresponding genome-engineered porcine animal. Dependent claims refine the CTLA4-Ig component, select immunoglobulin subclass options, optionally add soluble CTLA4, and limit the source context to cells obtained from porcine tissue or organ.
Immune-preserving reduction of cell mediated rejection during xenotransplantation
A method of reducing cell mediated rejection of xenotransplanted genetically modified porcine cells in a recipient, wherein the cell mediated rejection is reduced without suppression of the recipient's normal immune function.
Alpha-1,3-GT loss combined with porcine CTLA4-human immunoglobulin fusion expression
The genetically modified cells lack expression of the alpha-1,3-galactosyltransferase (alpha-1,3-GT) gene and express a transgene encoding a porcine CTLA4 peptide fused to a human immunoglobulin peptide (pCTLA4-Ig).
Direct sourcing of xenotransplanted genetically modified cells from a genome-engineered porcine animal
The genetically modified cells are directly obtained from a genetically modified porcine animal that lacks expression of the alpha-1,3-GT gene and expresses the pCTLA4-Ig peptide by insertion of the pCTLA4-Ig transgene into the genome of the animal.
CTLA4 peptide amino-acid substitutions
The CTLA4 peptide is mutated by substituting alanine at position +29 with tryptophan and substituting leucine at position +104 with glutamic acid.
CTLA4 intracellular retention signal
The CTLA4 peptide is modified by adding an intracellular retention signal.
Immunoglobulin subclass selection
The pCTLA4-Ig immunoglobulin peptide is an IgG selected from IgG1 or IgG4.
Optional administration of soluble CTLA4 to the recipient
The method includes administering soluble CTLA4 to the recipient in addition to the genetically modified cells.
Porcine tissue or organ sourcing of the genetically modified cells
The method further specifies that the cells used are obtained from a porcine tissue or organ.
Overall, the claim set is directed to xenotransplantation of alpha-1,3-GT-negative porcine cells engineered to express pCTLA4-Ig, with a requirement that the xenotransplanted cells are directly obtained from a corresponding genome-engineered porcine animal. Dependent claims further constrain CTLA4-Ig features, immunoglobulin subclass choice, optional soluble CTLA4 administration, and the porcine tissue or organ source context.
Stated Advantages
Reduces cell mediated rejection of xenotransplanted genetically modified porcine cells without suppression of the recipient's normal immune function.
Documented Applications
Xenotransplantation of genetically modified porcine cells into a recipient to reduce cell mediated rejection while aiming to preserve the recipient's normal immune function.
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