Systemic gene therapy using an AAV44.9 vector rescues a neonatal lethal mouse model of propionic acidemia

Abstract

Propionic acidemia (PA) is rare autosomal recessive metabolic disorder caused by defects in the mitochondrially localized enzyme propionyl-coenzyme A (CoA) carboxylase. Patients with PA can suffer from lethal metabolic decompensation and cardiomyopathy despite current medical management, which has led to the pursuit of gene therapy as a new treatment option for patients. Here we assess the therapeutic efficacy of a recently described adeno-associated virus (AAV) capsid, AAV44.9, to deliver a therapeutic PCCA transgene in a new mouse model of propionyl-CoA carboxylase α (PCCA) deficiency generated by genome editing. Pcca^-/- mice recapitulate the severe neonatal presentation of PA and manifest uniform neonatal lethality, absent PCCA expression, and increased 2-methylcitrate. A single injection of the AAV44.9 PCCA vector in the immediate newborn period, systemically delivered at a dose of 1e11 vector genome (vg)/pup but not 1e10 vg/pup, increased survival, reduced plasma methylcitrate, and resulted in high levels of transgene expression in the liver and heart in treated Pcca^-/- mice. Our studies not only establish a versatile and accurate new mouse model of PA but further demonstrate that the AAV44.9 vectors may be suitable for treatment of many metabolic disorders where hepato-cardiac transduction following systemic delivery is desired, such as PA, and, by extension, fatty acid oxidation defects and glycogen storage disorders.

Keywords: AAV44.9; PCCA; cardiac tropism; genome editing; mouse model; neonatal gene therapy; organic acidemia; propionic acidemia; propionylcoa carboxylase deficiency; systemic gene therapy.

Graphical abstract

Figure 1

Mitochondrial catabolism of propionyl-CoA to succinyl-CoA Propionic acidemia (PA) is caused by mutations in the PCCA or PCCB gene, which encode for the respective subunits of propionyl-CoA carboxylase (PCC). Enzymes downstream of PCC in the metabolism of propionyl-CoA to succinyl-CoA, a Krebs cycle intermediate. Other enzymes include D-methylmalonyl-CoA epimerase (MCEE) and methylmalonyl-CoA mutase (MMUT). The figure was created with BioRender.

Figure 2

Schematic of the Pcca^{c.398_401delAAGC} allele Exon 5 of the Pcca was targeted with a single-guide RNA (sgRNA) resulting in a 4-bp deletion after Cas9 cleavage and repair by non-homologous end joining (NEHJ). Shown are Sanger sequencing results of genomic DNA from a mouse with a WT Pcca genotype compared with a mouse homozygous for the Pcca^{c.398_401delAAGC} mutation (bottom). The figure was created with BioRender.

Figure 3

Clinical and biochemical characterization of Pcca^−/− mice (A) Survival of mice with Pcca^+/+,Pcca^+/−, and a Pcca^−/− genotypes. (B) Plasma 2-methylcitrate levels from Pcca^+/+ and Pcca^+/− newborn pups (n = 13) in comparison with Pcca^−/− newborn pups (n = 6). (C) Pcca mRNA levels in the liver of Pcca^+/+ (n = 3), Pcca^+/− (n = 3), and Pcca^−/− (n = 3) mice, shown as a percentage of WT Pcca mRNA expression normalized to Actb. (D) Immunoblot of 50 μg hepatic protein from Pcca^+/+, Pcca^+/−, and Pcca^−/− newborn pups for the PCCA protein using the mitochondrial protein ubiquinol-cytochrome c reductase core protein 2 (UQCRC2) as a loading control. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 4

AAV44.9 gene delivery of PCCA rescues Pcca^−/− mice from neonatal lethality (A) Schematic of the recombinant AAV vector genome (pAAV-CBA-PCCA) packaged with the AAV44.9 capsid (CMV, cytomegalovirus; CBA, chicken β-actin). Pcca^−/− pups received a retro-orbital injection of 1e10 vg or 1e11 vg of AAV44.9-CBA-PCCA on day of life 1 (DOL 1). (B) Survival curve comparing the viability of Pcca^−/− mice treated with AAV44.9 at doses of 1e10 vg or 1e11 vg with untreated Pcca^−/− mice. (C) Plasma 2-methylcitrate in untreated neonatal Pcca^−/− mice and Pcca^−/− mice 4 days and 1–3 months after treatment with AAV44.9 at 1e11 vg. (D) Vector biodistribution in liver, heart, brain skeletal muscle, and kidneys from Pcca^−/− treated mice (n = 3) 4 days and 1 month post treatment, as determined by ddPCR. (E) PCCA and Pcca mRNA expression in the liver and heart (WT, n = 3, 3; PA, n = 2, 1; AAV44.9, n = 3, 3), shown as a percentage of WT Pcca mRNA expression normalized to Actb (liver) or Gapdh (heart). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant; ND, not detected.

Figure 5

Expression of PCCA protein in the liver and heart (A) Immunoblot of hepatic and cardiac PCCA protein from untreated Pcca^+/+ (n = 1), Pcca^−/− (n = 1), and Pcca^+/− (n = 1) and AAV44.9-treated Pcca^+/− (n = 1) and Pcca^−/− (n = 3) mice 4 days post treatment. (B) Immunoblot of hepatic and cardiac PCCA protein from untreated Pcca^+/+ (n = 2) and Pcca^−/− (n = 2) and AAV44.9-treated Pcca^−/− mice (n = 3) 30 days post treatment. (C) Quantification of PCCA protein expression relative to untreated Pcca^+/+ mice from the blots depicted in (A) and (B). ∗p < 0.05, ∗∗p < 0.003, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 6

RNA in situ hybridization in tissues 30 days after AAV44.9 gene delivery to detect PCCA expression PCCA transgene mRNA (punctate red staining) in situ hybridization (magnification, 10× with 20× inset) of liver, heart, skeletal muscle, brain, and kidney tissue from three Pcca^−/− AAV44.9-CBA-PCCA-treated mice, labeled A, B, and C.