Short-term rescue of neonatal lethality in a mouse model of propionic acidemia by gene therapy

Abstract

Propionic acidemia (PA) is a metabolic disorder that causes mental retardation and that can be fatal if untreated. PA is inherited in an autosomal recessive fashion involving mutations in PCCA or PCCB encoding the alpha and beta subunits of propionyl-CoA carboxylase (PCC). Current treatment is based on dietary restriction of substrate amino acids, which attenuates symptoms. However, patients still experience episodes of hyperammonemia that can cause progressive neurologic damage. In this paper, we have tested gene therapy approaches to PA in a stringent mouse model of PCCA deficiency, in which homozygous knockout mice are born but die within 36 hr. In this work, we have delivered first-generation and helper-dependent adenovirus serotype 5 (Ad5) vectors expressing the human PCCA cDNA by intraperitoneal injection into newborn mice. Unmodified Ad5 vectors mediated extensive transduction of the peritoneum with weak liver transduction as determined by luciferase imaging and dsRed expression. In contrast, modification of Ad5 with polyethylene glycol detargeted the virus from the peritoneum and retargeted it for transduction in the liver. When vectors expressing PCCA were injected, significant increases in life span were observed for both the unmodified and polyethylene glycol (PEG)-modified Ad5 vectors. However, this rescue was transient. Similarly, adeno-associated virus serotype 8-mediated transduction also produced only transient rescue. These data show first proof of principle for gene therapy of PA and demonstrate the potential utility of PEG to modify viral tropism in an actual gene therapy application.

FIG. 1.

Metabolic pathway for propionyl-CoA carboxylase (PCC). The pathways in a normal patient (A) and in a propionic acidemia patient (B).

FIG. 2.

Luciferase imaging of gene delivery in neonatal mice. ICR mice were injected within 6 hr of birth intraperitoneally with 2.5×10⁹ VP of unmodified or PEGylated AdLucIREShrGFP and luciferase activity was imaged 24, 48, and 72 hr after injection, using a Lumazone imager. Shown are images of a representative pup with luciferase expression in pseudo-color overlaid on the white light image “0” represents uninjected mice.

FIG. 3.

DsRed microscopy of gene delivery in neonatal mice. ICR mice were injected intraperitoneally within 6 hr of birth with 2.5×10⁹ VP of unmodified or PEGylated Ad-DsRed. Seventy-two hours after injection the pups were frozen embedded in O.C.T. and sectioned (thickness of sections, 20 μm). Confocal images were taken after DAPI staining. Blue represents DAPI-stained nuclei and red represents DsRed fluorescence.

FIG. 4.

Survival of PCCA^−/− neonatal mice. The litters from PCCA^+/− crosses were injected with the indicated vectors within 6 hr of birth and they were monitored for survival time. At 1 week, the remaining pups were killed and all were genotyped. Kaplan–Meier survival curves are shown only for PCCA^−/− mice, because PCCA^+/+ and PCCA^+/− mice all survived. (Note: Uninjected animals survived at a rate indistinguishable from that of Ad-LacZ-injected mice and are not shown on the graph for clarity.) (A) Survival of mice receiving unmodified, PEGylated, and 50% unmodified plus 50% PEGylated first-generation (Ad-CMV-hPCCA) vector at a total vector dose of 2.5×10⁹ viral particles (VP) injected intraperitoneally. (B) Survival of mice receiving unmodified and PEGylated first-generation (Ad-CMV-hPCCA) vector at a vector dose of 5×10¹⁰ VP injected intraperitoneally. (C) Survival of mice receiving unmodified helper-dependent vectors expressing the hPCCA transgene under the control of the CMV promoter (HD-Ad-CMV-hPCCA) or the PEPCK promoter (HD-Ad-CMV-hPCCA), and PEGylated HD-Ad-PEPCK-hPCCA (PEG-HD-Ad-PEPCK-hPCCA), all injected intraperitoneally at a dose of 2.5×10⁹ VP. (D) Survival of mice injected with PEG-HD-Ad-PEPCK-hPCCA either intraperitoneally (i.p.) or intrahepatically (i.h.) at a dose of 2.5×10⁹ VP. (E) Survival of mice receiving adeno-associated virus serotype 8 (AAV8-hPCCA) intraperitoneally injected at a vector dose of 1.2×10¹⁰ VP. (A) through (E) all present the same group intraperitoneally injected with 2.5×10⁹ VP of Ad-CMV-LacZ, which serves as a nontherapeutic control vector. This group was indistinguishable in terms of survival from the uninjected group (data not shown).

FIG. 4.

Survival of PCCA^−/− neonatal mice. The litters from PCCA^+/− crosses were injected with the indicated vectors within 6 hr of birth and they were monitored for survival time. At 1 week, the remaining pups were killed and all were genotyped. Kaplan–Meier survival curves are shown only for PCCA^−/− mice, because PCCA^+/+ and PCCA^+/− mice all survived. (Note: Uninjected animals survived at a rate indistinguishable from that of Ad-LacZ-injected mice and are not shown on the graph for clarity.) (A) Survival of mice receiving unmodified, PEGylated, and 50% unmodified plus 50% PEGylated first-generation (Ad-CMV-hPCCA) vector at a total vector dose of 2.5×10⁹ viral particles (VP) injected intraperitoneally. (B) Survival of mice receiving unmodified and PEGylated first-generation (Ad-CMV-hPCCA) vector at a vector dose of 5×10¹⁰ VP injected intraperitoneally. (C) Survival of mice receiving unmodified helper-dependent vectors expressing the hPCCA transgene under the control of the CMV promoter (HD-Ad-CMV-hPCCA) or the PEPCK promoter (HD-Ad-CMV-hPCCA), and PEGylated HD-Ad-PEPCK-hPCCA (PEG-HD-Ad-PEPCK-hPCCA), all injected intraperitoneally at a dose of 2.5×10⁹ VP. (D) Survival of mice injected with PEG-HD-Ad-PEPCK-hPCCA either intraperitoneally (i.p.) or intrahepatically (i.h.) at a dose of 2.5×10⁹ VP. (E) Survival of mice receiving adeno-associated virus serotype 8 (AAV8-hPCCA) intraperitoneally injected at a vector dose of 1.2×10¹⁰ VP. (A) through (E) all present the same group intraperitoneally injected with 2.5×10⁹ VP of Ad-CMV-LacZ, which serves as a nontherapeutic control vector. This group was indistinguishable in terms of survival from the uninjected group (data not shown).