Characterization Of Bioprosthetic Heart Valves Of GGTA1/CMAH/β4GalNT2 Gene Targeted Pigs

Background:Surgical heart valve replacement is an established lifesaving treatment for diseased heart valve.Bioprosthetic heart valves(BHVs)made from glutaraldehyde-fixed porcine or bovine tissues are widely used in clinics but exhibit age-dependent structural valve degeneration(SVD)which is associated with the immune response against BHVs.Three major xenoantigens present on commercial BHVs,galactosea-α1,3-galactose(αGal),N-glycolylneuraminic acid(Neu5Gc)and glycan products of β-1,4-N-acetyl-galactosaminyl transferase 2(β4Gal NT2)are eliminated through CRISPR/Cas9-mediated gene targeting in the present study.The genetically modified porcine pericardium showed reduced immunogenicity but comparable collagen composition and physical characteristics of the pericardium from wild-type pigs.Our data suggested that BHVs from TKO pigs is a promising alternative for currently available BHVs from wild-type pigs.Objective: Galactose-α1,3-galactose(αGal),N-glycolylneuraminic acid(Neu5Gc)whose synthesis are catalyzed by α(1,3)galactosyltransferase(encoded by GGTA1gene)and CMP-Neu5 Ac hydroxylase(encoded by CMAH gene),Sd(a)xenoantigen produced by β-1,4-N-acetyl-galactosaminyl transferase 2(β4Gal NT2)are widely expressed in pigs,which cause immune responses.In order to get porcine bioprosthetic heart valves of GGTA1/CMAH/β4Gal NT2 gene targeted pigs,can be an ideal source of BHVs in clinic cardiac replacement therapy.Methods:1.To target the porcine GGTA1,CMAH and β4Gal NT2 genes,single guide RNAs(sg RNAs)were designed using online tools(http://crispr.mit.edu/),ligated into digested p X330,Cas9-sg RNA targeting plasmids were constructed successfully.Then plasmids were transfected into PFF,after G418 selection,single colonies were collected,and sequenced,GGTA1/CMAH/β4Gal NT2 biallelic mutation cells were obtained.2.Pigs were obtained using somatic cell nuclear transfer(SCNT)technique.Genomics were extracted from tissues,PCR products were insert into p MD18-T vector,single clones was analysed.Peripheral blood mononuclear cells(PBMCs)were separated from piglets used for detecting antigen epitopes by flow cytometry.porcine pericardium were analyzed by immunofluorescence for antigen epitopes.3.After incubating with human serum,The extent of binding human immunoglobulins including Ig G,Ig M were tested.The content of collagen were tested by hydroxyproline quantification assay.Pericardium were tested by Uniaxial mechanical test.Results: 27 individual male cell clones and 38 individual female cell clones were screened,and finally identified only 1 male clone and two female clones with double allele mutations.After using somatic cell nuclear transfer techniques,8 piglets were obtained,Expression of αGal,Neu5 G and Sd(a)on TKO pigs was negative and the human Ig G/Ig M binding to pericardium was minimal.The analysis of collagen composition and physical characteristics of porcine pericardium from the TKO pigs indicated that elimination of the three xenoantigens had no significant impact on the physical proprieties of porcine pericardium.Conclusions:GGTA1/CMAH/β4Gal NT2 KO pigs were successfully produced by CRISPR/Cas9,indicating that CRISPR/Cas9 was high efficiency tools in gene targeting.GGTA1/CMAH/β4Gal NT2 deficient pigs could be optimized sources of GBHVS.Therefore,these three gene modified pigs could be useful for future clinical cardiac replacement therapy.