AAV gene therapy corrects OTC deficiency and prevents liver fibrosis in aged OTC-knock out heterozygous mice

Abstract

Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder of the urea cycle. Hemizygous males and heterozygous females may experience life-threatening elevations of ammonia in blood and brain, leading to irreversible cognitive impairment, coma, and death. Recent evidence of acute liver failure and fibrosis/cirrhosis is also emerging in OTC-deficient patients. Here, we investigated the long-term consequences of abnormal ureagenesis in female mice heterozygous (Het) for a null mutation in the OTC gene. Two-month-old Het OTC knockout (KO) mice received a single dose of self-complementary adeno-associated virus (AAV) encoding a codon-optimized human OTC gene at 1×10¹⁰, 3×10¹⁰, or 1×10¹¹ vector genome copies per mouse. We compared liver pathology from 18-month-old treated Het OTC-KO mice, age-matched untreated Het OTC-KO mice, and WT littermates, and assessed urinary orotic acid levels and vector genome copies in liver at 4, 10, and 16months following vector administration. Het OTC-KO female mice showed evidence of liver inflammation and the eventual development of significant fibrosis. Treatment with AAV gene therapy not only corrected the underlying metabolic abnormalities, but also prevented the development of liver fibrosis. Our study demonstrates that early treatment of OTC deficiency with gene therapy may prevent clinically relevant consequences of chronic liver damage from developing.

Keywords: AAV; Gene therapy; Liver fibrosis; OTC deficiency.

Figure 1. Characterization of adult OTC-KO Het mice

(A) OTC enzyme activity in liver (Het, n=3; WT, n=4). (B) Plasma ammonia (Het, n=15; WT, n=10). (C) Urinary orotic acid (Het, n=34; WT, n=13). (D) Body weight of 3-month-old Het and WT mice (n=5). (E, F) OTC immunofluorescent staining on liver sections of 6-month-old Het and WT mice, respectively. (G and H) OTC histochemical staining on liver sections of 6-month-old Het and WT mice, respectively. Scale bar is 200 μm. Means ± STD are shown. * p<0.05; ****p<0.0001 (Mann-Whitney test).

Figure 2. Development of liver fibrosis in aged OTC-KO Het mice and in an OTC deficiency patient

Collagen staining with Sirius Red on liver sections from: (A) 6-month-old WT mouse, (B) 12-month-old WT mouse, (C, D) 18-month-old WT mouse, (E) 6-month-old Het mouse, (F) 12-month-old Het mouse, (G, H) 18-month-old Het mouse, (I) 11-year-old OTC deficiency patient, and (J) normal 21-year-old female. Representative pictures are shown. Scale bar is 500 μm for all except for (D) and (H), where it is 200 μm. (K) Quantification of liver fibrosis area in 6-, 12-, and 18-month-old Het and WT mice. Means ± STD are shown (n=4–19). * p<0.05 (Mann-Whitney test).

Figure 3. Abnormal fat staining and lymphocytic infiltrate in the liver of OTC-KO Het mice

Oil-Red-O staining for lipids in liver from a WT (A, C) and OTC-KO Het mouse (B, D), showing representative images at low and high magnification, respectively. (E, F) Haemotoxylin and eosin staining on liver sections from a WT and OTC Het mouse, respectively. The liver from the OTC-KO Het mouse shows a lymphocytic infiltrate, which we frequently observed in these animals. (G) Liver enzyme levels in 18-month-old Het and WT mice. Scale bar is 300 μm (A, B), 50 μm (C, D), and 150 μm (E, F). ALT, alanine aminotransferase; AST, aspartate aminotransferase. Means ± STD are shown (Het, n=8; WT, n=5). * p<0.05 (Mann-Whitney test).

Figure 4. Robust and sustained normalization of urinary orotic acid in OTC-KO Het mice by AAV gene therapy

Urinary orotic acid levels in AAV8-treated OTC-KO Het mice normalized within one week following vector treatment and remained within the normal range through the experiment. Vector doses for each group were: (A) 1×10¹¹, (B) 3×10¹⁰, and (C) 1×10¹⁰ GC/mouse. Means ± STD are shown (n=5).

Figure 5. Prevention of liver fibrosis in OTC-KO Het mice by AAV gene therapy

Collagen staining with Sirius Red on liver samples harvested from 18-month-old AAV8-treated OTC-KO Het mice (16 months post-vector administration at the doses of 1×10¹¹, 3×10¹⁰, and 1×10¹¹ GC/mouse) compared to untreated OTC-KO Het mice and WT littermates. Representative pictures taken at 4x (scale bar is 500 μm) and 10× (scale bar is 200 μm) magnifications of the groups are shown (n=5).

Figure 6. Long-term and high levels of OTC enzyme activity in the liver of OTC-KO Het mice following AAV gene therapy

Two-month-old OTC-KO Het mice received a single tail vein injection of AAV8 vector at the doses of 1×10¹¹, 3×10¹⁰, and 1×10¹¹ GC/mouse. Liver samples were harvested 16 months later and stained for OTC enzyme activity. A representative picture of each group is shown (n=5; scale bar is 200 μm).

Figure 7. Dose-correlated vector GC in the liver of AAV8-treated OTC-KO Het mice at different time points post vector administration

Two-month-old OTC-KO Het mice received a single tail vein injection of AAV8 vector at the doses of 1×10¹¹ (A), 3×10¹⁰ (B), and 1×10¹⁰ (C) GC/mouse. Liver samples were harvested 4, 10, and 16 months later for vector GC analysis by real-time PCR. Means ± STD are shown (n=5). * p<0.05 (Tukey’s test). n.s., not significant.