Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia

Abstract

Despite the implementation of lifesaving newborn screening programs and a galactose-restricted diet, many patients with classic galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that the administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which decreased not only galactose-1 phosphate (gal-1P) in the liver but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, noninvasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of galactosemia. Although our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that targets the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety.

Keywords: adeno-associated viral vectors; breath test; classic galactosemia; galactose oxidation; gene therapy; lipid nanoparticles; mRNA-based therapy.

Graphical abstract

Figure 1

Schematic representation of breath test analysis After injection with ¹³C-galactose, the animal is quickly relocated to the air-sealed chamber. Expired air was collected using a 20-mL glass syringe every 15 min over 2 h. A tank of CO₂-free air was used to flush out the chamber for 10 s before subsequent samples. The collected air samples were stored in a Exetainer vial and the analyses of ¹³CO₂ enrichment used a published protocol.

Figure 2

Evaluation of whole-body ¹³C-galactose oxidation in WT, homozygous GalT-KO (GG), and heterozygous GalT-KO (Het) mice After i.p. administration of 5 mg [1-¹³C] galactose, the amount of ¹³CO₂ in the expelled air over time was measured and calculated as described in materials and methods.

Figure 3

Dose dependency of whole-body ¹³C-galactose oxidation in GalT-deficient mice after experimental AAVrh10 GALT gene replacement therapy (A) Four-week-old GalT KO male mice were injected with a single i.p. dose of AAVrh10 GALT at a dose of 1 × 10¹², 1 × 10¹³, and 1 × 10¹⁴ vg/kg body weight, respectively (n = 3 per group). Seven days after the injection, the amount of ¹³CO₂ in the expelled air over time was measured and calculated as described in materials and methods. Curves are interpolated using a “cubic spline.” (B and C) Specific GALT protein quantification (B) and (C) Specific GALT enzyme activity analysis in GalT-KO and AAVrh10-GALT-treated mice. (D) The comparison of the disease-relevant biomarker gal-1P in liver samples of GalT-KO and AAVrh10-GALT-treated mice. Values presented as mean ± SD. (∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001).

Figure 4

Single iv dose of LNP1-encapsulated GALT mRNA version 22 resulted in long half-lived GALT protein and normalized whole-body galactose oxidation (A) Kinetic analysis of liver GALT protein using different cohorts of GalT-KO mice after a single dose of 1 mpk GALT mRNA version 22. (B) Whole-body galactose oxidation was measured in the treated animals 3 days after treatment. (∗∗p < 0.005; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001).

Figure 5

Time course analyses of whole-body galactose oxidation in GalT KO mice treated with LNP1-encapsulated GALT mRNA version 22 therapy or AAVrh10-GALT gene therapy (A) Time course analyses of whole-body galactose oxidation with a single dose (1 mpk) of GALT mRNA version 22 in the same cohort of GalT-KO mice. (B–D) Time course analyses of whole-body galactose oxidate in the same cohorts of GalT-KO mice treated with 3 different doses (1 × 10¹², 1 × 10¹³, and 1 × 10¹⁴ vg/kg body weight) of the experimental AAVrh10 GALT gene therapy.

Figure 6

Time course analyses of whole-body galactose oxidation in GalT KO mice treated with LNP4-encapsulated GALT mRNA version 22 therapy (A–C) Time course analyses of whole-body galactose oxidation with a single, but 3 different dosages (0.5, 1, and 2 mpk) of GALT mRNA version 22 in the same cohorts of GalT-KO mice. (D) Western blot analysis of GALT expression in the animals 23 days after treatment. Values presented as mean ± SD.