Figure 3. Preexisting maternal immunity to Cas9 endonuclease does not impair fetal liver gene editing.

(A) Experimental design. (B) Stereomicroscopy. Numerous mG⁺ cells were observed in the livers of unsensitized and SpCas9-sensitized offspring at both 2 and 12 weeks after injection. (C) Quantification of liver editing by flow cytometry. Shown is percent mG⁺mT^–/(mG⁺mT^– + mG^–mT⁺) among live CD45^–TER119^–CD31^–EpCAM^–E-cadherin⁺ offspring hepatocytes compared by 2-tailed t test. Comparable editing was observed between the groups in the short and long term. (D) Anti-SpCas9 IgG BAbs. SpCas9-sensitized offspring demonstrated increased levels of anti-SpCas9 IgG BAbs on day of life 10 (2 weeks after injection) by 2-tailed t test, but these fell to levels comparable to those of unsensitized offspring by 12 weeks after injection. (E) T cell analysis. The prevalence of various T cell populations in the liver was assessed by flow cytometry and compared between groups by 2-tailed t test. No maternal T cells were detected in livers of SpCas9-sensitized offspring. Cytotoxic T cells were not present at increased frequency in livers of SpCas9-sensitized offspring, nor was a marker of activation (CD154) increased among them. (F) Liver histology and IHC. H&E slides were prepared and imaged at ×10 original magnification. Normal hepatocyte morphology without lymphocytic infiltration was observed in both groups in the short and long term. To assess for tissue-infiltrating T cells, IHC for CD4 and CD8 was performed and imaged at ×10 original magnification. Scant CD4⁺ helper T cells and CD8⁺ cytotoxic T cells were detected in equal frequency between the groups. Scale bars: 200 μm. (G) Serum transaminases. Serum aspartate transaminase (AST) and alanine transaminase (ALT) were measured 2 and 12 weeks after injection and compared between groups by 2-tailed t test. No elevation of AST or ALT was observed among SpCas9-sensitized offspring compared with unsensitized offspring, confirming the absence of hepatocellular injury. *P < 0.05.