Development of RAG2 -/- IL2Rγ -/Y immune deficient FAH-knockout miniature pig

Abstract

Human hepatocyte transplantation for liver disease treatment have been hampered by the lack of quality human hepatocytes. Pigs with their large body size, longevity and physiological similarities with human are appropriate animal models for the in vivo expansion of human hepatocytes. Here we report on the generation of RAG2^-/-IL2Rγ^-/YFAH^-/- (RGFKO) pigs via CRISPR/Cas9 system and somatic cell nuclear transfer. We showed that thymic and splenic development in RGFKO pigs was impaired. V(D)J recombination processes were also inactivated. Consequently, RGFKO pigs had significantly reduced numbers of porcine T, B and NK cells. Moreover, due to the loss of FAH, porcine hepatocytes continuously undergo apoptosis and consequently suffer hepatic damage. Thus, RGFKO pigs are both immune deficient and constantly suffer liver injury in the absence of NTBC supplementation. These results suggest that RGFKO pigs have the potential to be engrafted with human hepatocytes without immune rejection, thereby allowing for large scale expansion of human hepatocytes.

Keywords: FAH; IL2Rγ; RAG2; immunodeficient; liver damage; pig.

Figure 1

Targeted disruption of RAG2 by CRISPR/Cas9. (A) Schematic representation of the workflow to generate RGFKO pigs. RAG2^-/-IL2Rγ^-/YFAH^-/- (RGFKO) pigs were generated through sequential mutation of the RAG2 gene to generate RAG2^-/- fetus first followed by mutations of IL2Rγ and FAH genes to generate triple knockout RGFKO pigs. To mutate the targeted gene(s), single guide RNA (sgRNA) targeting the respective gene(s) was first designed. Day 33 fetal fibroblasts were then electroporated with sgRNA and Cas9 plasmids, followed by drug selection. Positive clones were used as donor cell for somatic cell nuclear transfer (SCNT). SCNT embryos were then transferred into surrogate sows to establish pregnancy. (B) Endogenous RAG2 locus and the sgRNA targeting site are shown. (C) Genomic DNA was obtained from nine clones (C1-C9) after puromycin selection. RAG2 was amplified by PCR for further Sanger sequencing. Ctrl: no template control. (D) Alignment of Sanger sequencing results of clone C9 with wildtype RAG2 sequence demonstrating a one nucleotide insertion (⁺1bp) and a four nucleotides deletion (Δ4bp) mutation. (E) Six fetuses were obtained from surrogate sows transplanted with SCNT embryos using clone C9 as donor nuclei. (F) Representative results of T7-endonuclease I assays, and gel shift assays performed on the genomic DNA of fetuses F1- F5. Wildtype genomic DNA was used as control (Ctrl).

Figure 2

Targeted disruption of IL2Rγ and FAH by CRISPR/Cas9. (A) Endogenous IL2Rγ and FAH loci and their sgRNA targeting site are shown. (B) Genomic DNA was obtained from 16 clones (C24-C39) after puromycin selection. IL2Rγ and FAH were amplified by PCR for further Sanger sequencing. Ctrl: no template control. (C) Alignment of Sanger sequencing results of clone C25 with wildtype IL2Rγ and FAH sequences. 16 and 137 nucleotide deletion (Δ16bp, Δ137bp), and one nucleotide insertion (⁺1bp) mutations were observed in IL2Rγ. Deletion mutations of 1, 31, 297 and 415 nucleotides were observed in FAH (Δ1bp, Δ31bp, Δ297bp, Δ415bp). (D) Two live RGFKO piglets (P1 and P2) were obtained after 113 days of pregnancy from surrogate sows transplanted with SCNT embryos using clone C25 as donor nuclei. (E) Representative results of T7-endonuclease I assays and gel shift assays performed on the genomic DNA of RGFKO piglet (P1). WT: Wildtype. (F) Alignment of Sanger sequencing results of RGFKO piglets (P1 and P2) in the target fragments in RAG2, IL2Rγ, and FAH genes. One nucleotide insertion (⁺1bp) and 4 nucleotide deletional (Δ4bp) mutations were observed in RAG2. 137 nucleotide deletion mutation (Δ137bp) was observed in IL2Rγ. 1 nucleotide deletion mutation (Δ1bp) was observed in FAH.

Figure 3

Immunological characterization of RGFKO pigs. (A) Representative images of the thymus of wildtype (WT) and RGFKO pigs. (B) Representative images of the spleen of WT and RGFKO piglets. (C) Representative spleen sections of WT and RGFKO piglets stained with hematoxylin and eosin. Left: normal spleen architecture in WT pig; Right: lacked lymphoid follicles and germinal centers, as well as reduced lymphoid aggregation in the white pulp in RGFKO pig. Boxed region indicating the white pulp region was further magnified. (D–F) Flow cytometric analysis of the peripheral blood (D, E) and splenocytes (F) of WT and RGFKO piglets. (D) Shown are representative images of histograms of macrophage/granulocyte (M/G) marker staining. The boxed region, representing M/G negative population, is further gated upon for T cells (M/G-CD3⁺CD16^-) and NK cells (M/G-CD3-CD16⁺) analysis. (E, F) Shown are representative plots of CD45RA against CD3. B cells are identified as CD3^-CD45RA⁺. Numbers indicate the proportion of the indicated population as a percentage of total peripheral blood mononuclear cells (D, E) or splenocytes (F). (G) qPCR analysis of the expression of the indicated genes in splenocytes of RGFKO and WT piglets. Shown are fold change of the indicated genes over housekeeping gene (GAPDH). Data shown are mean ± standard error (n = 2 for RGFKO, n = 3 for WT). ns: not significant. ***p < 0.001. (H) Western blot analysis of the expression of IL2Rγ in splenocytes of RGFKO and WT piglets. β-actin was used as a loading control. (I) PCR analysis of germline TRB-V, TRD-V and IGH-V genes, and V(D)J-recombined TRB-VDJ, TRD-VDJ and IGH-VDJ genes in splenocytes of RGFKO and WT pigs. Shown are representative images of PCR amplification products visualized by DNA gel electrophoresis.

Figure 4

RGFKO pigs suffer hepatic damage when taken off NTBC. (A) Schematic of the NTBC dosing regimen for RGFKO pigs. Surrogate sows were supplemented with NTBC (5g/L) at a dose of 10 ml NTBC per 100 kg body weight for the first 94 days of gestation. NBTC dose was increased to 12 ml NTBC per 100 kg body weight thereafter until delivery of RGFKO piglets. RGFKO piglets were supplemented with 12 ml NTBC per 100 kg body weight for the first 3 days after birth. NTBC was then withdrew after that. (B) FAH immunohistochemistry analysis of the liver of RGFKO and wildtype (WT) pigs using anti-FAH. (C, D) Western blot analysis of the expression of FAH in hepatocytes (C) and testes (D) of RGFKO and WT piglets. β-actin was used as a loading control. (E) Representative liver sections of WT and RGFKO piglets stained with haematoxylin and eosin. Boxed region was magnified to show diffuse hepatocellular injury and cytoplasmic ballooning degeneration. (F, G) Evaluation of hepatocyte apoptosis via TUNEL assay was performed on liver sections of RGFKO and WT pigs. DAPI (blue) was used to stain the nuclei of hepatocytes, while TUNEL positive cells were stained red (F). TUNEL positive cells were quantified through image analysis (G). (H) qPCR analysis of the expression of the indicated genes in hepatocytes of RGFKO and WT piglets. Shown are fold change of the indicated genes over housekeeping genes (GAPDH). Data shown are mean ± standard error (n = 2 for RGFKO, n = 3 for WT). ns, not significant. ***p < 0.001.