迄今,器官短缺仍是同種異體器官移植之主要問題。家豬是最適合做為異種器官移植之器官捐贈者,以緩解器官衰竭末期病人同種異體器官移植等待急器官短缺之急,然而器官捐贈者(donor)與接受者(recipient)之高度免疫不相容性係異種器官移植主要之障礙,此免疫不相容性包括超級性排斥、延遲之異種移植排斥,急性細胞性排斥及慢性排斥等,隨著基因編輯技術之進展迄今之CRISPR/Cas9,針對豬之基因組進行基因組修飾以克服種間免疫不相容問題之效率已提升許多。此技術建立在可促使單或雙股DNA斷裂之基礎上,同時可引起精準基因嵌入及剔除,此種分子層次的改變讓使用基因編輯豬隻之組織或器官做為人類異種器官移植之器官來源燃起希望。異種移植可能發生超急性排斥,甚至導致器官衰竭,超急性異種移植排斥一旦發生,宿主可在幾分鐘或數小時內毀滅了移植物,異種移植物發生超急性排斥主要原因是人類血漿中辨識之上皮細胞表面Galactose 1,3Galactose (Gala1,3Gal)自然發生抗體的出現,Gala(1-3)Gal的合成受1.3-galactosyltransferase (GGTA1)酵素所催化,此酵素基因僅出現在豬,人在演化過程中已失去此酵素之上游基因。尚有其他乙種反應之抗原基因如cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)催化之Neu5Gc (N-glycolylneuraminic acid) 抗原及b1,4-Nacetylgalactosaminyltransferase (B4GALNT2)產生之glycan。因此本三年期計畫之目標係使用CRISPR/Cas9基因編輯技術產製三基因剔除之豬隻,期能緩解擬人化豬器官異種移植之免疫不相容問題。第一年將建立李宋豬之發情同期化及超數排卵之技術平台,做為原核時期豬胚收集及受胚豬之來源;同時使用子計畫所設計之三基因(GGTA1/CMAH/β4GalNT2)剔除之Cas9及gRNA組合進行基因剔除豬囊胚之產製及基因剔除效率分析;第二年將以第一年建立之平台及採用Combi-CRISPR策略進行ROSA26-PAD嵌入李宋豬之基因組中,以產製工具豬隻,同時將產製三基因(GGTA1/CMAH/β4GalNT2)剔除李宋豬;第三年將產生之ROSA26-PAD工具豬及豬胚透過RMCE (recombinase-mediated cassette exchange)方式,應用於替換EGFP或更長免疫調節有關之DNA CARGO。
Up to present, the shortage of allogeneic donor organs for clinical transplantation is a major problem in transplantology. Pigs are the most promising and suitable donors for xenotransplantation caused they are similar to human with respect to their genetics, anatomy, and physiology. However, the immune rejection response due to species disparity between pigs and primates is the major hurdle in successful xenograft transplantation. Xenotransplantation of transplanting pig organs into humans is a promising via development of genetic engineering techniques enables genome modifications in pigs that reduce the cross-species immune barrier. Gene editing (GE) has become a powerful tool in advancing agriculture and/or biomedicine as CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats linked to Cas nuclease 9) techniques have progressively developed. The technology is based on the repair paths of the single or double strand DNA breaks that cause insertions, deletions, or precise integrations of donor DNA. These changes are crucial for many fields of science, one of which is the use of animals (pigs) as a reservoir of tissues and organs for xenotransplantation into humans. The host normally destroys xenografts within minutes or hours through hyperacute xenograft rejection, which inevitably leads to failure. The main reason for hyperacute rejection of xenograft is the presence of naturally occurring antibodies in human plasma that recognizes the Galactose 1,3 Galactose (1,3Gal) antigen on the surface of porcine endothelial cells. Synthesis of 1,3Gal is catalyzed by the enzyme 1,3-galactosyltransferase (GGTA1). Furthermore, other xenoreactive antigens present in pigs but absent in humans have been identified; these antigens include Neu5Gc antigen (N-glycolylneuraminic acid) catalyzed by cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and a glycan produced by b1,4-Nacetylgalactosaminyltransferase (B4GALNT2) (Byrne et al., 2015, 2018). Hence, the aims of this 3-year project is to generate genetically modified pigs for humanized pig organs, tissues and cells by using CRISPR/Cas9 technologies to alleviate the problem of immune incompatibility. The first year plan to establish the platform of estrous synchronization and superovulation of puberty LeeSung pigs for collection of pronuclear (PN) stage embryos and prepare recipients for embryo transfer. The Cas9 endonuclease and gRNA designed from subproject 1 will be used to create triple knockout (TKO) LeeSung embryos/pigs lacking GGTA1/CMAH/β4GalNT2 genes. Some of Cas9/ gRNA injected PN eggs are going to conduct subsequent in vitro culture to blastocyst for advance TKO assay of gene targeting. The second year plan to Knock in ROSA26-PAD into genome of LeeSung pig by Combi-CRISPR strategy for tool pig generation, as well as continuously inject Cas9/gRNA into PN eggs then transfer to oviducts of recipients for development to term, and aim to generate triple knockout (TKO) LeeSung pigs lacking GGTA1/CMAH/β4GalNT2 genes., injected embryos are going to transfer into oviducts of recipient to term. The genomic DNA of newborn TKO piglets will be assay for targeting genes. The third year aim to generate ROSA26-PAD tool pig and ROSA26-PAD pig embryos will be used for replacement of EGFP or longer DNA CARGO related to human immunomodulation by recombinase-mediated cassette exchange (RMCE) manner.