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We thank Harding and Martinez for their comments regarding our recent article published in HGG Advances. In the proof-of-concept study described in the article, we assessed 10 humanized phenylketonuria (PKU) mice homozygous for the phenylalanine hydroxylase (PAH) c.1222C>T (p.Arg408Trp) variant (hereafter referred to as PKU R408W mice). Four control mice (2 males and 2 females) were treated with vehicle, three mice (all females) received a 5 mg/kg dose of lipid nanoparticles (LNPs), and three mice (1 male and 2 females) received a 2.5 mg/kg dose of LNPs. We observed no effect of sex on blood phenylalanine (Phe) concentrations before treatment, with the females having a mean level of 1,281 μmol/L and the males having a mean level of 1,231 μmol/L, although the small sample size precludes a definitive conclusion. Following LNP treatment, Phe levels were completely normalized (<125 μmol/L) in all treated mice. Given the small number of mice treated with two different LNP doses, we are unable to rule out a sex-specific effect on the magnitude of the response to treatment.
We observed a mean 29% whole-liver corrective editing in the higher-dosed mice and a mean 26% whole-liver corrective editing in the lower-dosed mice. If only 75% of bulk liver DNA is derived from hepatocytes, as cited by Harding and Martinez, and LNPs preferentially target hepatocytes, then we would estimate there was 35%–40% corrective editing of hepatocyte alleles in the treated mice. In a prior study using adeno-associated virus-based editor systems to correct the pathogenic variant in Pahenu2/enu2 mice,1 the investigators observed mean correction rates in the liver of 9.7%, 18.6%, 22.1%, and 25.1% at 4, 8, 14, and 26 weeks post-treatment, respectively. They found that Pah-mRNA correction rates were higher—16.7%, 34.4%, 38.5%, and 43.6%, respectively1—which aligns with our assumptions. Nonetheless, we acknowledge that our work represents an indirect measurement of the restoration of normal PAH enzyme activity. Assessments of liver PAH enzyme activity, anti-PAH immunohistology, and other phenotypes are currently underway to better characterize the PKU R408W mouse model and its response to editing treatments.
Reference
- 1.Villiger L., Grisch-Chan H.M., Lindsay H., Ringnalda F., Pogliano C.B., Allegri G., Fingerhut R., Häberle J., Matos J., Robinson M.D., et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat. Med. 2018;24:1519–1525. doi: 10.1038/s41591-018-0209-1. [DOI] [PubMed] [Google Scholar]