Rescue of glutaric aciduria type I in mice by liver-directed therapies

Abstract

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase (Aass) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.

Figure 1.. Gcdh^−/− knockout mice: phenotype and rescue by hepatocyte transplantation.

(A) Known lysine catabolism pathway in peroxisomes, cytosol, and mitochondria. (B) Brain hemorrhage (arrow) of Gcdh^−/− mice after 4 days on high-protein diet exposure. (C) Representative H&E staining of the hippocampus with vacuolation (arrow). Boxed area shown with higher magnification on the right. (D) Kaplan-Meier survival curves of Gcdh^−/− knockout mice transplanted with wild-type hepatocytes (Gcdh^+/+) on high-protein diet. Glutaric acid (E) and 3-OH-glutaric acid (F) concentrations in liver and brain of groups after 10 (non-transplanted) or 160 (transplanted) days on high-protein diet. (G) H&E staining of hippocampal brain sections. Boxed area shown with higher magnification on the right. Quantification of hippocampal vacuolation (H) and meningeal hemorrhage (I) (Arbitrary Units [AU]: 0 = absence; 1 = low; 2 = intermediate; 3 = high; 4 = very high). (J) RFP immunohistochemistry detecting healthy hepatocytes (Gcdh+/+, transgenic mTmG) and (K) Western blot (GCDH and beta actin) of liver lysates from low and high repopulated transplanted animals (n=4 per group). Data is presented as means ± SD. Significance was validated with Student’s t test (D, E) or Mann-Whitney U test (H, I); *p≤0.05, **p≤0.01 and *** p≤0.005. All mice were transplanted at the age of 2 months and experiments were performed at age of 8 months. Non-transplanted controls were age-matched. GCDH: Glutaryl-Co-A Dehydrogenase; AASS: Alpha Aminoadipate-Semialdehyde Synthase; RFP: Red Fluorescent Protein; mTmG: membrane Tomato membrane GFP.

Figure 2.. Phenotype and transplantation experiments of double knockout (Gcdh^−/−/Aass^-/)^- mice.

(A) Kaplan-Meier survival curves of single (Gcdh^−/−) and double (Gcdh^−/−Aass^−/−) knockout mice on high-protein diet. (B-C) Glutaric acid (B) and 3-OH-glutaric acid (C) concentrations in liver and brain tissue of single (Gcdh^-/) and double (Gcdh^−/−/Aass^−/−) knockout mice after 5 and 60 days on high-protein diet, respectively. (D) Kaplan-Meier survival curves of double (Gcdh^−/−/Aass^−/−) knockout mice transplanted with Gcdh^−/− hepatocytes. Glutaric acid (E) and 3-OH-glutaric acid (F) concentrations in liver and brain of double knockout (Gcdh^−/−/Aass^−/−) groups after 5 days (transplanted) and 60 days (non-transplanted), on high-protein diet. Representative AASS immunostaining (G) and Western blot (H) of livers from transplanted double (Gcdh^−/−/Aass^−/−) knockout mice. (I) H&E staining of hippocampal brain sections with vacuolation (arrows). Boxed area shown with higher magnification on the right. (J-K) Quantification of hippocampal vacuolation (J) and brain meningeal hemorrhage (K). Arbitrary Units (AU): 0 = absence; 1 = low; 2 = intermediate; 3 = high; 4 = very high. Data is presented as means ± SD. Significance was validated with t test (B, D) or with Mann-Whitney U test (H, J); *p≤0.05, **p≤0.01 and *** p≤0.005. All mice were transplanted at the age of 2 months and experiments were performed at age of 8 months. Non-transplanted controls were age-matched.

Figure 3.. Phenotype of liver-specific GA-1 model.

Gcdh^−/− hepatocytes were transplanted into TIRF (transgene free Il2rg^−/−/Rag2^−/−/Fah^−/−) mice, which have a normal lysine catabolism (Gcdh^+/+ /Aass^+/+). (A) Kaplan Meier survival curve of TIRF mice transplanted with Gcdh^−/− hepatocytes (B) Representative FAH immunohistochemistry of TIRF liver (FAH negative) transplanted with Gcdh^−/− hepatocytes (FAH positive). Glutaric acid (C) and 3-OH-glutaric acid (D) concentrations in liver and brain of transplanted and non-transplanted mice on high protein diet. (E) Summary of hepatocyte transplantation models and their outcomes. Color codes for whole body and/or liver (transplanted hepatocytes) of mice in the diagram correspond to: Blue: Gcdh^−/−/Aass^+/+ (single knockout); Yellow: Gcdh^−/−/Aass^−/− (double knockout); Grey: Gcdh^+/+/Aass^+/+(wild-type). Significance was validated with Mann-Whitney U test (*p≤0.05 and **p≤0.01). All mice were transplanted at the age of 2 months and experiments were performed at age of 8 months (see methods). Non-transplanted controls are age matched. Il2rg: Il-2 receptor gamma; Rag2: recombination activating gene 2; fah: fumarylacetoacetate hydrolase.

Figure 4.. Liver-directed AAV gene therapy in Gcdh^−/− mice.

(A-I) Five-week-old Gcdh^−/− mice were intravenously injected with AAV-Gcdh or AAV-GFP at a dose of 1.5×10¹² vg/mouse. (A) Kaplan Meier survival curves of Gcdh^−/− mice on high-protein diet. (B) GCDH Western blot analysis of liver and brain lysates from AAV -treated mice after harvesting or expiration (controls). (C) Representative GCDH immunostaining of liver in treatment group. (D) C5-DC metabolite levels in whole blood of all experimental groups before and 4 days after high protein diet. Glutaric acid (E) and 3-OH-glutaric acid (F) concentrations in liver and brain tissue of Gcdh^−/− mice at 140 days (AAV-Gcdh) or upon expiration (AAV-GFP and untreated). (G) H&E staining of hippocampal brain sections showing vacuolation (arrows) and meningeal hemorrhage (arrowheads). Boxed area shown with higher magnification on the right. (H to I) Quantification of hippocampal vacuolation (H) and brain meningeal hemorrhage (I). Arbitrary Units (AU): 0 = absence; 1 = low; 2 = intermediate; 3 = high; 4 = very high. (J-K) Neonatal Gcdh^−/− pups treated with low (3×10¹¹ vg/mouse), intermediate (7.5×10¹¹ vg/mouse) and high (1.5×10¹² vg/mouse) dose of AAV. (J) Kaplan Meier survival curves of treated Gcdh^−/− mice on high protein after weaning. Western blot (K) and GCDH immunostaining (L) of treated (high dose) Gcdh^−/− mice after expiration. Data is presented as means ± SD. Significance was validated with t-test (D,E,F) or Mann-Whitney U test (G,H); *p≤0.05, **p≤0.01 and ***p≤0.005. All mice were transplanted at the age of 2 months and experiments were performed at age of 8 months (see methods for details). Non-transplanted controls were age matched. AAV: Adeno-Associated Virus; C5-DC: glutarylcarnitine; GCDH: Glutaryl-Co-A Dehydrogenase.

Figure 5.. Liver-specific deletion of Aass in Gcdh^−/− mice using AAV-CRISPR.

Neonatal Gcdh^−/− mice were injected with a low (2.4×10¹¹ vg/mouse), intermediate (6×10¹¹ vg/mouse), or high (1×10¹² vg/mouse) dose of AAV expressing Cas9 under a liver specific promoter and sgRNA targeting the Aass gene. (A) Kaplan Meier survival curves of experimental groups on high protein diet. Glutaric acid (B) and 3-OH-glutaric acid (C) concentrations in liver and brain of wild-type C57BL/6 mice and treated (AAV-CRISPR, high dose) Gcdh^−/− mice after 60 days on high protein diet and upon expiration (day 4, AAV-GFP). (D) AASS immunostaining of livers of experimental groups (E) Representative hippocampal sections of mice injected with AAV-CRISPR or AAV-GFP showing vacuolation (arrows) and hemorrhage (arrowheads). Quantification of hippocampal vacuolation (F) and meningeal hemorrhage (G). Data is presented as means ± SD. Significance was validated with t test (B) and with Mann-Whitney U test (D) (**p≤0.01, and ***p≤0.005 AAV: Adeno-Associated Virus; Gcdh: Glutaryl-Co-A Dehydrogenase. AASS: Alpha Aminoadipate-Semialdehyde Synthase.