Invented by David Ayares, Paul Rohricht, Revivicor Inc

The market for tissue products derived from animals lacking any expression of functional alpha 1,3 galactosyltransferase is a rapidly growing industry that holds immense potential for various medical applications. Alpha 1,3 galactosyltransferase (?1,3GT) is an enzyme responsible for the production of a sugar molecule called galactose-alpha-1,3-galactose (?-Gal), which is found in the tissues of most mammals, including humans. However, some individuals have natural antibodies against ?-Gal, leading to potential complications in medical procedures involving animal-derived tissues. The development of animals lacking ?1,3GT expression has opened up new possibilities in the field of regenerative medicine and xenotransplantation. Xenotransplantation refers to the transplantation of organs or tissues from one species to another, and it has the potential to address the shortage of human organs available for transplantation. However, the presence of ?-Gal in animal tissues has been a major hurdle in xenotransplantation, as it triggers an immune response in humans, leading to organ rejection. By using gene-editing techniques such as CRISPR-Cas9, scientists have successfully created animals lacking ?1,3GT expression. These genetically modified animals, such as pigs, are known as ?-Gal knockout animals. The absence of ?-Gal in their tissues makes them more compatible with human recipients, reducing the risk of organ rejection and increasing the chances of successful xenotransplantation. The market for tissue products derived from ?-Gal knockout animals is primarily driven by the increasing demand for organs and tissues for transplantation. According to the World Health Organization, the demand for organ transplantation far exceeds the available supply, resulting in long waiting lists and high mortality rates for patients in need. The use of ?-Gal knockout animals could potentially alleviate this problem by providing a sustainable source of organs and tissues for transplantation. In addition to xenotransplantation, tissue products derived from ?-Gal knockout animals have other medical applications. These tissues can be used in the development of bioengineered organs, tissue engineering, and regenerative medicine. The ability to modify animal tissues to be more compatible with humans opens up new avenues for research and development in these fields. Furthermore, the market for tissue products derived from ?-Gal knockout animals also has potential in the pharmaceutical industry. Animal tissues can be used for drug testing and research, as well as the production of biologics and therapeutic proteins. The absence of ?-Gal in these tissues reduces the risk of adverse immune reactions, making them more suitable for pharmaceutical applications. However, it is important to consider the ethical implications and regulatory challenges associated with the use of genetically modified animals. The development and commercialization of tissue products derived from ?-Gal knockout animals require careful consideration of animal welfare, safety, and public acceptance. In conclusion, the market for tissue products derived from animals lacking any expression of functional alpha 1,3 galactosyltransferase is a promising and rapidly growing industry. The ability to modify animal tissues to be more compatible with humans has the potential to revolutionize xenotransplantation, regenerative medicine, and pharmaceutical research. However, it is crucial to address ethical concerns and regulatory frameworks to ensure the responsible and sustainable development of this market.

The Revivicor Inc invention works as follows

The present invention provides tissues derived from animals, which lack any expression of functional alpha 1,3 galactosyltransferase (alpha-1,3-GT). These tissues can be used for xenotransplantation in areas such as orthopedic reconstruction, skin repair, internal tissue repair and medical devices.

Background for Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase

Ruminant species, including porcine, bovine, and ovine, are likely to be sources of xenograft tissues and organs.” Porcine xenografts are the most popular because of the abundance of pigs, the well-established breeding program, and the size and physiology that are compatible with human beings. Bovine and ovine ruminants have also been suggested for xenografts of soft and hard tissue. There are still several obstacles to overcome before these organs and tissues can be successfully transferred into humans. Immune rejection is the most important. The first hurdle immunologically is “hyperacute rejection” (HAR). The presence of large amounts of natural antibodies that have been preformed and bind to foreign tissue is what defines HAR. This binding of natural antibodies to epitopes found on the endothelium of donor tissue is thought to be what initiates HAR. Within minutes of perfusion, the binding of these natural antibodies to target epitopes on the donor tissue endothelium is followed by complement activation and platelet and fibrin deposits, and finally by interstitial hemorrhage and edema in the donor organ. All of this leads to rejection of tissue by the recipient. (1996) Frontiers in Bioscience, 1, e34-41).

The most commonly transplanted human tissue is bone. (J. M. Lane and colleagues). Current Approaches To Experimental Bone Grafting (18 Orthopedic Clinics Of North America (2) 213, (1987)). Every year, more than 100,000 bone implants or grafts are performed in the United States to repair or replace osseous deformities caused by trauma, infection or malignancy. The human bone is a connective tissue made up of cells that are embedded in a matrix of collagen fibers and mineralized ground substance (Stedman?s Medical Dictionary, Williams & Wilkins Baltimore, Md. (1995)).

Bone implants and grafts are frequently made from autologous bones. Unfortunately, autologous tissue is not always available for large defects in children. Autologous bone implants can also cause postoperative morbidity, such as pain, bleeding, cosmetic disabilities, infections, or nerve damage. In addition, the difficulty in obtaining the desired functional shape using the autologous tissue transplanted can lead to less than optimal filling.

Soft tissues such as skin, tendons, cartilage and heart tissue, valves and submucosal tissue are commonly transplanted in humans. By using xenografts, it is possible to retain much of the original structure and properties in the transplanted tissue. “If xenograft tissues can be safely processed for human transplantation, they represent an unlimited source of material.

Once implanted, a xenograft can cause immunogenic reactions like chronic and hypercracute rejection. Bone xenografts are more likely to fracture, undergo resorption, and fail to unite due this rejection. The natural anti-galactose 1,3-galactose antibodies are the major obstacle to the use of animal tissue as implants for humans, including porcine, ovine or bovine. These antibodies make up approximately 1% of the antibodies in humans and in monkeys.

Except for Old World Monkeys, Apes and Humans, most mammals have glycoproteins that carry the galactose 1,3-galactose epitope (Galili, et al. J. Biol. Chem. 263: 17755-17762, 1988). Other mammals such as pigs, however, have large amounts of glycoproteins containing galactose 1,3-galactose. Humans, old world monkeys, and apes don’t have galactose 1,3-galactose. Instead, they have an anti-galactose 1,3-galactose antibodies that are produced in large quantities (Cooper, et. al., Lancet 342, 682-683, 1993). It binds to glycoproteins or glycolipids that contain galactose alpha-1.3,3 galactose.

This differential distribution of?alpha-1.3, GT epitopes? This differential distribution of the?alpha-1,3 GT epitope? mutated) alpha-1,3-galactosyltransferase in ancestral Old World primates and humans. Humans are therefore ‘natural knockouts’ The alpha-1.3,3-GT is a natural knockout. This rejection is a direct result of the xenograft, as in the case of the pig organs that were first transplanted to humans via HAR.

Chem. Chem. Chem. Exp. Immunol. 86: 31-35, 1991, Dalmasso et al. Transplantation 52, 530-533 (1991). Costa et al. FASEB J 13,1762 (1999) reported that the competitive inhibition of alpha-1.3,3-GT results in a partial reduction in epitope number in H-transferase-transgenic pigs. Similarly, Miyagawa et al. (J Biol. Chem 276, 39310 (2001)) reported that attempts to block expression of gal epitopes in N-acetylglucosaminyltransferase III transgenic pigs also resulted in only partial reduction of gal epitopes numbers and failed to significantly extend graft survival in primate recipients.

Badylak et. al. Al. developed a method to isolate submucosa tissues from pigs’ small intestines. These can be used in tissue grafts, such as connective tissue to repair knee ligaments and shoulder rotator-cuff repairs. The material from the small intestine (SIS) is stripped of its viable cells using enzymatic and chemical steps, leaving a acellular extracellular matrice that promotes tissue regeneration and in-growth. Nos. Nos. This method is used for human tissue transplants. “However, despite chemical treatment, galactose 1,3-galactose residues are still embedded in the graft, and can cause inflammation and immune activation in humans (Allman and Mcpherson, 2001, Transplantation, 71, 1631-1640, Mcpherson and Mcpherson, 2000, Tissue Engineering, 6(3), 233-239).

Stone et al. developed a process to treat porcine soft tissue and bone tissue to remove cellular material followed by treatment with alpha-galactosylsidase to remove the galactose alpha 1,3-galactose from the tissue prior to transplantation (Stone et al. Transplantation 1997: 63: 646-651; Stone et al. Transplantation 1998, 65:1577-83). Numerous patent applications have been filed on this process, discussing the use of tissue in a variety applications such as anterior cruciate repair, meniscal repairs, articular cartilage and submucosal tissue, bone and bone matrix tissue, heart valve replacement, and soft tissue tissue. Nos. Nos. 2002/0087211; 2001/0051828; 2003/0039678, and 2003/0023304. “WO 00/47131 and WO/47132; WO 1999/44533; WO 02/076337; WO99/51170; WO99/47080. WO 03/097809 WO 02/089711 WO 01/91671 and WO03/105737.

There is therefore a need to develop tissue grafts which do not have deleterious effects on humans.

Costa et al. (FASEB (2003) 17: 109-111) reported that the delayed rejection of porcine cartilage transplanted into wild-type and ?-1,3-galactosyltransferase knockout mice is reduced by transgenic expression of ?1,2-fucosyltransferase (HT transgenic) in the cartilage.

Single allele knockouts have been reported in both porcine cells as well as live animals. Denning et al. Nature Biotechnology (19: 559-562 2001) reported a targeted gene deletion in sheep of an allele of alpha-1.3,3-GT. Harrison et al. Transgenics Research 11, 143-150 (2002) reported on the production of heterozygous, alpha-1.3,3-GT knockout somatic porcine fetal fibroblasts. In 2002, Lai et al. In 2002, Lai et. Nature Biotechnology 20:251-255,2002) reported on the production of pigs in which an allele of alpha-1.3-GT was successfully rendered inactive. Ramsoondar et al. (Biol of Reproduc 69, 437-445 (2003)) reported the generation of heterozygous alpha-1,3-GT knockout pigs that also express human alpha-1,2-fucosyltransferase (HT), which expressed both the HT and alpha-1,3-GT epitopes.

PCT publication No. “PCT publication No. No. Bresatec received PCT publication No. Bresatec received PCT publication No. WO 95/28412 U.S. Pat. Nos. US publication no. BioTransplant Inc. and The General Hospital Corporation, 2003/0014770, provide a discussion on the production of alpha-1.3,3-GT-negative porcine cells using the cDNA for the alpha-1.3,3-GT gene, without knowing the genomic organization or the sequence. There was no proof that these cells had been produced before the filing date for the applications, and all the examples were prophetic.

The first public disclosure that a heterozygous, alpha-1,3GT-negative porcine cell was successfully produced occurred at the Lake Tahoe Transgenic Animal Confernce in July 1999 (David Ayares, PPL Therapeutics, Inc., “Gene Targeting In Livestock”, Transgenic Animal Research Cinference, Jul 1999, Abstract, page. Ayares IBS News Report November 1999, p. 5-6). Up until recently, there was no public disclosure or publication of the production of homozygous alpha 1,3GT-negative porcine cells. Since porcine embryonic cells are not available, it was impossible to create a homozygous alpha 1,3GT knockout pig using an alpha-1.3,3-GT homozygous embryonic cell.

On Feb. 27, 2003, Sharma et al. Transplantation 75:430-436, (2003) published an article demonstrating the successful production of fetal porcine fibroblasts homozygous with the knockout gene alpha-1.3,3-GT.

PCT publication no. PPL Therapeutics’ WO 00/51424 describes genetic modification of cells for nuclear transfers. This patent application discloses genetic disruption of alpha-1.3,3-GT in porcine somatic cell nuclei, and subsequent use of these cells’ nuclei lacking at least one alpha-1.3,3-GT copy for nuclear transfer.

U.S. Pat. No. No. 6,331,658 Cooper & Koren’s claims, but it does not confirm the production of genetically modified mammals that express a protein sialyltransferase. The patent states that genetically engineered mammal cells would show a reduction in galactosylated epitopes.

PCT publication no. The Curators of The University of Missouri have confirmed the production of heterozygous miniature swine with alpha 1,3GT gene knockout for use in xenotransplantation. This application is directed at a knockout miniature swine with a disrupted gene alpha-1.3,3-GT, where the expression of functional alpha-1.3,3-GT is reduced in comparison to wildtype. This application doesn’t provide any information on how much the alpha-1.3,3-GT should be reduced to make the swine suitable for xenotransplantation. This application also does not prove that the heterozygous animals that were raised showed a reduced expression of alpha1,3GT. Further, while the application refers to homozygous alpha 1,3GT knockout swine, there is no evidence in the application that any were actually produced or producible, much less whether the resultant offspring would be viable or phenotypically useful for xenotransplantation.

The best way to produce porcine animals suitable for xenotransplantation is by depleting the glycoproteins containing galactose, alpha 1,3-galactose. Theoretically, double knockouts (the disruption of both copies) of the alpha 1,3GT genes could be produced in two ways: either by breeding two single allele knockouts to produce progeny in which case one would predict that one out of four would be double knockouts based on Mendelian Genetics, or 2) by genetic modification of a cell that already has a single knockout. This has actually been a difficult task, as demonstrated by the fact the first patent on knock-out cells for porcine was filed in 1993. However, the first homozygous 1,3GT knockout pig was only produced in July 2002.

Transgenic mice, and not pigs, have been historically the preferred model for studying the effects of genetic modification on mammalian physiological function. This is due to a variety of factors, including the fact that porcine embryonic cells were not available. The mice are the ideal animal for basic research because they are easy to handle and reproduce quickly. They can also be genetically modified at the molecular levels. Scientists study molecular pathologies in a wide range of genetically-based diseases including colon cancer and mental retardation using mouse models. To date, thousands of genetically altered mice have been produced. A “Mouse Knockout and Mutation Database” has been created by BioMedNet to provide a comprehensive database of phenotypic and genotypic information on mouse knockouts and classical mutations (http://research.bmn.com/mkmd; Brandon et al Current Biology 5 [7]:758-765(1995); Brandon et al Current Biology 5[8]:873-881(1995)), this database provides information on over 3,000 unique genes, which have been targeted in the mouse genome to date.

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