Long-term rescue of a lethal murine model of methylmalonic acidemia using adeno-associated viral gene therapy

Abstract

Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating metabolites, transgene expression, and whole animal propionate oxidation were examined as outcome parameters after gene therapy. One-hundred percent of the untreated Mut(-/-) mice (n = 58) died by day of life (DOL) 72, whereas >95% of the adeno-associated virus-treated Mut(-/-) mice (n = 27) have survived for > or = 1 year. Despite a gradual loss of transgene expression and elevated circulating metabolites in the treated Mut(-/-) mice, the animals are indistinguishable from unaffected control littermates in size and activity levels. These experiments provide the first definitive evidence that gene therapy will have clinical utility in the treatment of MMA and support the development of gene therapy for other organic acidemias.

Figure 1

Rescue of Mut^−/− mice. Survival in days between the untreated Mut^−/− mice (n = 58), intrahepatic-injected rAAV8-GFP 1 or 2 × 10¹¹ GC Mut^−/− mice (n = 18), intrahepatic-injected rAAV8-mMut 1 × 10¹¹ GC (n = 10) or 2 × 10¹¹ GC (n = 17) Mut^−/− mice, and i.p. rAAV8-mMut 3 × 10¹¹ GC injected (n = 4) Mut^−/− mice. All three groups of rAAV8-mMut-treated Mut^−/− mice exhibited a significant improvement in survival relative to untreated and rAAV8-GFP-treated Mut^−/− mice. The intrahepatic-injected rAAV8-mMut-treated Mut^−/− mice show significantly improved survival at 24, 60, and 100 days and beyond compared to the untreated and rAAV8-GFP-treated Mut^−/− mice (*P < 10⁻²² for rAAV8-mMut 1 × 10¹¹ GC and **P < 10⁻²² for rAAV8-mMut 2 × 10¹¹ GC at the 100-day time point). GC, genome copy; GFP, green fluorescent protein; i.p., intraperitoneal.

Figure 2

Growth and phenotypic correction after rAAV8-mMut gene therapy. (a) An untreated Mut^−/− mouse (left) compared to a Mut^+/− littermate at day of life 55 to illustrate the relative difference in size and appearance. The Mut^−/− mouse is severely growth retarded and has achieved only 40% of the Mut^+/− littermate weight. Shortly after this photo was taken, the Mut^−/− mouse became lethargic and perished. (b) A Mut^−/− mouse (left, neck flexed) that received a single intrahepatic injection of rAAV8-mMut 2 × 10¹¹ GC aside a Mut^+/− littermate (right, neck extended) at day of life 120. The treated Mut^−/− animal is similar in size to a control Mut^+/− littermate. (c) Growth correction between untreated Mut^−/− mice, intrahepatic rAAV8-mMut-treated Mut^−/− mice, and untreated age-, diet-, and gender-matched Mut^+/− littermates. The graph depicts the percent weight at day of life 24 and 60 of Mut^+/− diet- and gender-matched littermates (n = 25, 25) compared to untreated Mut^−/− mice (n = 6, 3) or Mut^−/− mice treated via an intrahepatic injection of rAAV8-mMut 1 × 10¹¹ GC (n = 10, 10) or rAAV8-mMut 2 × 10¹¹ GC (n = 17, 17) at birth. The rAAV8-mMut-treated Mut^−/− mice at both doses showed significant growth improvement compared to the untreated Mut^−/− mice (*P < 10⁻⁶ for day 24, **P < 0.01 for day 60). Error bars represent plus and minus one standard deviation. GC, genome copy.

Figure 3

Methylmalonyl-CoA mutase expression after rAAV8-mMut treatment. (a) Quantitative PCR analysis of Mut expression in tissues. The level of Mut mRNA (plus or minus one standard deviation) detected in the various tissues from Mut^+/− controls was set at 100% and used as a comparator. GAPDH was independently examined for normalization. ND equals none detected (<1%), N/A equals not analyzed. The treated Mut^−/− mice show significant expression in the liver, muscle, heart, and brain after neonatal gene delivery at 90 days that diminished after 1 year. (b) Western analysis of liver (top) and lower limb skeletal muscle (bottom) total extracts were prepared from Mut^+/− (day 90), untreated Mut^−/− (day 45), and Mut^−/− mice (day 90) that had received either 1 × 10¹¹ GC or 2 × 10¹¹ GC of rAAV8-mMut and analyzed by western blotting. The same membranes were probed with either anti-methylmalonyl-CoA mutase antibody (labeled Mut) or an anti-ubiquinol-cytochrome c oxidoreductase antibody (labeled complex III) to control for loading and mitochondrial content. Immunoreactive Mut enzyme is present in all lanes, except those from the untreated Mut^−/− mice. The mitochondrial loading control shows approximately the same intensity in each sample. GC, genome copy.

Figure 4

Metabolic improvements after rAAV8-mMut treatment. (a) Plasma methylmalonic acid levels (µmol/l) were measured at time points of 24, 90, 120, 180, and 360 days after birth in the rAAV8-mMut-treated Mut^−/− mice as an indication of Mut activity. Three groups are presented: untreated Mut^−/− mice, Mut^−/− mice treated with 1 × 10¹¹ GC rAAV8-mMut, and Mut^−/− mice treated with 2 × 10¹¹ GC rAAV8-mMut. Untreated and treated Mut^+/− mice have plasma methylmalonic acid levels between 5 and 10 µmol/l, and are not depicted in this graph. The numbers in each group are presented in the graph. Error bars represent plus and minus one standard deviation. The rAAV8-mMut-treated mutant mice show a significant reduction in plasma methylmalonic acid levels compared to the untreated Mut^−/− mice at all time points (*P < 0.001 on day 24, **P < 0.01 on day 90). (b) 1-¹³C-propionate oxidation 1 year after rAAV8-mMut treatment. Two hundred micrograms of 1-¹³C-sodium propionate was injected intraperitoneally into Mut^+/− (n = 8), 1 × 10¹¹ GC rAAV8-mMut-treated Mut^−/− (n = 3), or untreated Mut^−/− (n = 6) mice. ¹³C enrichment in expired CO₂ was measured and used to determine the percent of the administered 1-¹³C-propionate dose that was oxidized. Error bars surround the 95% confidence intervals. The rAAV8-mMut-treated Mut^−/− mice show a significant increase in the ability to oxidize 1-¹³C-propionate compared to the untreated Mut^−/− mice at 25 minutes (*P < 0.01). GC, genome copy.

Figure 5

rAAV8-mMut treatment in 20-day-old Mut^−/− mice. (a) A Mut^−/− mouse was treated on day of life (DOL) 20 with 3 × 10¹¹ GC rAAV8-mMut delivered intraperitoneally (i.p.), and serial weights were measured in parallel with a control Mut^+/− littermate. The treated Mut^−/− mouse achieved the control weight 40 days after treatment. (b) Two hundred micrograms of 1-¹³C-propionate was injected i.p. into Mut^−/− mice (labeled Mut^−/− pre-rAAV, n = 3) or control Mut^+/− littermates (labeled Mut^+/−, n = 3) on DOL 20 prior to receiving 3 × 10¹¹ GC rAAV8-mMut. The study was repeated 10 days after the viral injection and propionate oxidation at 20 minutes in the treated Mut^−/− mice (labeled Mut^−/− post-rAAV, n = 3) achieved levels that were significantly greater than the untreated mutants (*P < 0.01) and equivalent to the controls. Error bars surround the standard deviation. (c) Plasma methylmalonic acid levels in response to rAAV8 treatment on DOL 20 in Mut^−/− mice (n = 3). Before (pre-rAAV; mean 1,011 µmol/l) was significantly more than after (post-rAAV; mean 118 µmol/l). *P < 0.05. Error bars surround the standard deviation. GC, genome copy.