Double knockout (gt/cmah-ko) pigs, organs and tissues

Abstract

The invention provides double knockout transgenic pigs (GT / CMAH-KO pigs) lacking expression of any functional αGAL and CMAH. Double knockout GT / CMAH-KO transgenic organs, tissues and cells are also provided. Methods of making and using the GT / CMAH-KO pigs and tissue are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to, and the benefit of, U.S. Provisional Patent Application No: 61 / 717,845, filed Oct. 24, 2012, which is incorporated herein by reference in its entirety for all purposes.INCORPORATION OF SEQUENCE LISTING[0002]The sequence listing in text format submitted herewith is herein incorporated by reference in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0003]Not applicable.FIELD OF THE INVENTION[0004]The present invention is generally in the field of xenotransplantation and genetic modification to produce transgenic animals, organs tissue, or cells suitable for transplantation into a human.BACKGROUND[0005]It is well known that transplants from one animal into another animal of the same species, such as human to human, are a routine treatment option for many serious conditions, including kidney, heart, lung, liver and other organ disease. However, it is also well k...

AI Technical Summary

Benefits of technology

The present patent provides a knockout pig with disrupted genes for α(1,3)-galactosyltransferase (αGal) and CMAH. This reduces the amount of these molecules in the pig compared to a wild-type pig, which helps decrease hyperacute rejection when transplanting pig tissue into a human. The knockout pig can also be used to produce glycoproteins with lower amounts of Neu5Gc or αGal epitopes compared to a wild-type pig. The disruptions in the genes are caused by a three base pair deletion adjacent to a G to A substitution for αGal and a four base pair insertion for CMAH. Overall, this invention provides a way to improve the safety and efficiency of pig-derived organs for transplantation.

Problems solved by technology

However, it is also well known that there are not enough suitable organs available for transplant to meet current or expected clinical demands for organ transplants.

However, xenotransplantation using standard, unmodified pig tissue into a human (or other primate) is accompanied by severe rejection of the transplanted tissue.

The hyperacute response to the pig antibodies present on the transplanted tissue is so strong that the transplant is typically damaged by the human immune system within minutes or hours of transplant into the human recipient.

The antibodies are present in the patient's blood prior to implantation of the tissue, resulting in the intense and immediate rejection of the implanted tissue.

Unfortunately, developing homozygous knockout pigs is a slow process, requiring as long as three years using homologous recombination in fetal fibroblasts followed by somatic cell nuclear transfer (SCNT), and then breeding of heterozygous knockout animals.

The development of new knockout pigs for xenotransplantation has been hampered by the lack of pluripotent stem cells, relying instead on the fetal fibroblast as the cell upon which genetic engineering was carried out.

Unfortunately, while the GTKO pig may have eliminated anti-αGal antibodies as a barrier to xenotransplantation, studies using GTKO cardiac and renal xenografts in baboons show that the GTKO organs still trigger an immunogenic response, resulting in rejection or damage to the transplanted organ.

Some negative control histograms are difficult to see because of significant overlap with the experimental group.

Negative controls are shown (*) but are difficult to see in some panels because of significant overlap with the experimental group.

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