Preexisting maternal immunity to AAV but not Cas9 impairs in utero gene editing in mice
John S. Riley, … , Philip W. Zoltick, William H. Peranteau
John S. Riley, … , Philip W. Zoltick, William H. Peranteau
Published June 17, 2024
Citation Information: J Clin Invest. 2024;134(12):e179848. https://doi.org/10.1172/JCI179848.
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Research Article Immunology

Preexisting maternal immunity to AAV but not Cas9 impairs in utero gene editing in mice

Abstract

In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer–dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.

Authors

John S. Riley, Valerie L. Luks, Cara L. Berkowitz, Ana Maria Dumitru, Nicole J. Kus, Apeksha Dave, Pallavi Menon, Monique E. De Paepe, Rajan Jain, Li Li, Lorraine Dugoff, Christina Paidas Teefey, Mohamad-Gabriel Alameh, Philip W. Zoltick, William H. Peranteau

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Figure 2

Maternal humoral immunity to Cas9 endonuclease is transferred to the fetus, while maternal T cell immunity to Cas9 endonuclease is not.

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Maternal humoral immunity to Cas9 endonuclease is transferred to the fet...
(A) Anti-SpCas9 IgG BAb ELISA. Serum concentrations of anti-SpCas9 IgG BAb were compared among the groups 28 days after primary sensitization using 1-way ANOVA. (B) Maternal-fetal transmission of anti-SpCas9 IgG BAb. Shown are the absolute and relative concentrations of anti-SpCas9 IgG BAb among three SpCas9-sensitized dams and their respective newborn fetuses. The mean fetal/maternal ratio for each pregnancy was compared by 1-way ANOVA. (C) Anti-Cas9 delayed-type hypersensitivity (DTH) reaction gross histology. Shown are images of the bilateral ears 48 hours after intradermal injection with Cas9 protein. Red arrows point to erythema and swelling consistent with a positive DTH reaction. (D) Ear skin microscopic histology. Shown are cross-sectional H&E-stained slides of the ears 72 hours after intradermal injection imaged at ×10 original magnification with ×25 original magnification inset images. Green arrows point to infiltration with small blue lymphocytes and associated edema consistent with a positive DTH reaction. Scale bars: 200 μm. (E) Measurement of ear thickness. Ear thickness was measured using microcalipers at 24, 48, and 72 hours after injection and compared among the groups using 2-way ANOVA. Significantly increased thickness was observed in the ears of SpCas9-sensitized and SaCas9-sensitized dams injected with the same Cas9 subtype to which they had been sensitized. (F–H) Anti-Cas9 DTH reaction among offspring. Unsensitized, SpCas9-sensitized, and SaCas9-sensitized BALB/c dams were mated with mTmG+/+ males and allowed to deliver. Pups were fostered and tested at 6 weeks of age. Gross histology (F), microscopic histology (G), and change in ear thickness compared by 2-way ANOVA (H) all demonstrated a negative anti-Cas9 DTH reaction, showing that maternal T cell immunity was not efficiently transferred from dam to offspring. Scale bars: 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.