Skip to main content
. 2024 May 9;134(12):e179848. doi: 10.1172/JCI179848

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.

Figure 4

(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.