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 Therapeutics

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 4

Hereditary tyrosinemia can be cured by AAV9-delivered CRISPR/Cas9 in utero gene editing in the presence of maternal preexisting immunity to Cas9 but not AAV.

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Hereditary tyrosinemia can be cured by AAV9-delivered CRISPR/Cas9 in ute...
(A) Tyrosine degradation pathway. We designed an sgRNA to induce indels in the Hpd gene, thereby replicating the effects of nitisinone (NTBC) through CRISPR/Cas9 gene editing. (B) Experimental design. (C) Kaplan-Meier survival curve. Injected offspring of unsensitized and SpCas9-sensitized dams demonstrated comparable survival off NTBC by log-rank (Mantel-Cox) test, while significantly decreased survival was observed among injected offspring of AAV9 indirectly sensitized dams. (D) Weight change from baseline. Transient weight loss was observed after NTBC withdrawal among injected offspring of unsensitized and SpCas9-sensitized dams with return to baseline after 10 days. Severe weight loss preceded death among injected offspring of AAV9 indirectly sensitized dams (similar to disease controls), and those few that did survive showed prolonged weight loss prior to eventual recovery. (E) Liver function testing. Hepatocellular injury was transient among injected offspring of unsensitized and SpCas9-sensitized dams. By contrast, injected offspring of AAV9 indirectly sensitized dams showed prolonged and, in most cases, unresolved hepatocellular injury and cholestatic liver failure. (F) Histology. Shown are H&E and IHC for HPD at ×10 original magnification. Characteristic signs of liver injury including hepatocyte ballooning (feathery) degeneration (blue arrows) were observed among injected offspring of AAV9 indirectly sensitized dams off NTBC. Loss of HPD expression by IHC was observed in a patchy pattern among injected offspring of unsensitized and SpCas9 directly sensitized dams. This was notably decreased among injected offspring of AAV9 indirectly sensitized dams. Scale bars: 200 μm. (G) Liver editing. Shown is the percentage insertions/deletions (indels) at the Hpd locus by next-generation sequencing compared by 1-way ANOVA. Significantly impaired editing was observed among AAV9 indirectly sensitized offspring but not SpCas9 directly sensitized offspring. ****P < 0.0001.