Search

Abstract Background Previous studies have identified the carbohydrate epitope Galα1–3Galβ1–4GlcNAc-R (termed the α-galactosyl epitope), known as the α-Gal antigen as the primary xenoantigen recognized by the human immune system. The α-Gal antigen is regulated by galactosyltransferase (GGTA1), and α-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop α-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system. Results The mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the α-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6 months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits. Conclusions α-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these α-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed.

Background: Previous studies have identified the carbohydrate epitope Galα1–3Galβ1–4GlcNAc-R (termed the α-galactosyl epitope), known as the α-Gal antigen as the primary xenoantigen recognized by the human immune system. The α-Gal antigen is regulated by galactosyltransferase (GGTA1), and α-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop α-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system. Results: The mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the α-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6 months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits. Conclusions: α-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these α-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed. [ABSTRACT FROM AUTHOR]

BACKGROUND: α-Galactosyl (α-Gal) is the main target antigen in hyperacute rejection resulting from animal tissues or xenotransplantation. OBJECTIVE: To develop a Gal antigen-deficient rabbit model in order to objectively evaluate the immunogenicity risk of animal-derived biomaterials and the local response to implantation in the host. METHODS: New Zealand white rabbits, SPF grade, 6-8 months old, were selected as model animals to prepare Gal antigen-deficient rabbits. Using CRISPR/Cas9-mediated gene editing technology, two complementary sgRNAs were designed and constructed for the 8th exon of GGTA1 gene to regulate Gal antigen expression in rabbits. After transcription, GGTA1 sgRNA mRNAs and Cas9 mRNA were co-microinjected into in vitro cultured rabbit fertilized eggs, which were then implanted into surrogate mother rabbits after a brief in vitro culture, and the neonatal rabbits were obtained by natural pregnancy. The success of gene editing was verified by gel electrophoresis and gene sequencing. The expression of Gal antigen was detected with reference to the method given by the industry standard (YY/T 1561-2017). The study protocol was approved by the Animal Ethics Committee of the Laboratory Animal Resources Laboratory of the National Institutes for Food and Drug Control (approval No. 2017(B)007). RESULTS AND CONCLUSION: The embryos were transferred to four surrogate rabbits after gene editing. FIfteen gene-edited pups were obtained after natural pregnancy. The appearance and feeding behavior of the pups were not abnormal. Gel electrophoresis and gene sequencing results showed that 14 out of 15 rabbits were successfully edited, but the edited bases were not the same. The Gal antigen of the main organs was detected randomly, and its expression was reduced by more than 99.96%. Therefore, Gal-antigen-deficient rabbits are expected to be used for immunogenic risk assessment of animal-derived medical devices and in situ implantation experiments of heterogeneous bone and cornea, in order to be able to more objectively and scientifically evaluate the safety and effectiveness of animal-derived medical devices. [ABSTRACT FROM AUTHOR]