Murine models have been generated for two of the common human organic acidemias, propionic acidemia and methylmalonic acidemia, but have been difficult to manipulate. Both models feature almost immediate neonatal lethality in homozygote mutants, which has made basic investigations challenging (Miyazaki et al., 2001
; Peters et al., 2003
). Our initial experimental approach was to examine, in a proof-of-principle fashion, whether virus-mediated gene delivery could rescue the affected mice from uniform fatality in the neonatal period and extend the life span. We developed and tested an E1,E3-deleted adenovirus that expressed the murine methylmalonyl-CoA mutase gene in the E1 region under the control of the CMV promoter and, independently, eGFP from the E3 region under the control of the RSV promoter (Chandler et al., 2007
). Intrahepatic delivery was rapid, taking only minutes to inject an average litter of mice, minimally stressful to the neonatal pups, and biologically effective. However, the nature of the intrahepatic injection procedure was variable and associated with general mortality because treated heterozygotes and wild-type animals also perished. Pathological investigations of the liver at a time when inflammation might be expected to be observed were unrevealing in the treated animals. The exact cause of mortality is unknown but was suspected to be related to the stress of the procedure, because survival on day 15 () among the Ad-Mut-GFP-treated animals (54 of 77; 70.0%) was similar to that seen among the Ad-GFP-treated animals (19 of 26; 73%). Mechanistic explanations for the increased mortality will be important to examine in future studies.
A strong viral promoter driving the expression of the target gene was selected because we hypothesized that an immediate burst of methylmalonyl-CoA mutase expressed in the liver might provide enough enzyme to allow the neonatal pups to survive until weaning. The overall success of this approach was most obvious from the survival statistics, showing that >50% of the mutant mice that were treated by direct hepatic injection were alive at weaning, whereas uninjected controls (), mice that received the same virus injected in the skeletal muscle, and Ad-GFP-treated animals uniformly perished ().
During the period between injection and weaning, hepatically treated mutants were indistinguishable from heterozygote and wild-type littermates and had normal body weights, habitus, and behaviors. However, when the surviving Mut−/−
mice were weaned and placed on regular chow, they slowly stopped growing, failed to gain weight, and eventually perished. One animal survived 8 months after a single viral treatment but most died shortly after weaning. The reason for the death is uncertain but was likely related to the expected diminution of enzymatic activity from dilution effects, immune responses, or silencing effects on viral gene expression (Coude et al., 1990
; Guo et al., 1996
; Connelly, 1999
). Because adenovirus is a nonintegrating vector and the liver rapidly expands between the neonatal period and the time at weaning, dilution effects may be the most significant factor contributing to the diminution of MUT
expression in the treated mice. Other variables, such as the change in diet when the animals were transitioned from milk to chow, were also likely contributory to the deaths seen after weaning.
To assess the efficacy of virus-mediated delivery, time course studies were conducted, using a validated qPCR assay to examine RNA expression; and Western analysis, using highly specific anti-mutase antibodies, to demonstrate virus-directed protein production. As might be expected for a low-volume, direct injection method, some variability in the degree of expression at the RNA and protein levels was observed (). However, mRNA and protein levels exceeding those seen in wild-type animals were present in some treated mice (, column A; and , lane 3), whereas other injected mutants showed lower level expression but definite evidence of transcripts. As final support for the restoration of enzymatic activity in the livers of treated mice, plasma MMA levels were measured and showed a statistically significant decrease between treated and untreated mutants (). The lower methylmalonic acid levels seen in these mice indicate that significant hepatic methylmalonyl-CoA mutase activity was restored and was responsible for the lowered metabolite levels. It further suggests that the survival observed in the treated Mut−/− cohort likely correlates with more efficient adenoviral transduction and lower MMA levels, because roughly the same proportion of treated mutant mice in this study had lower metabolite levels when compared with survival at weaning. The fact that other regimens, including intramuscular injections, failed to produce survivors indicates that successful rescue gene delivery should be targeted to the liver in neonatal animals.
The demonstration of survival-based, adenovirus-mediated gene delivery to treat neonatal Mut−/−
mice has implications for future treatment strategies. The studies described here prove that virus-mediated rescue of neonatal lethality in Mut−/−
mice, and by extension, other lethal organic acidemia murine models, can be successfully accomplished. These results are surprising when the pleiotropic nature of the enzyme defect in MMA is considered (Wilkemeyer et al., 1993
) and suggest that metabolic cooperativity of propionate metabolism (Wilkemeyer et al., 1992
; Stankovics and Ledley, 1993
) may contribute to the biological effects observed by transient restoration of metabolism in the hepatocyte. The rapidity and severity of symptom onset in this model differ when compared with other inborn error of metabolism models that have been treated successfully with integration viruses and transposons as gene delivery agents to achieve permanent correction, such as lysosomal storage disorders (Ponder et al., 2002
), aminoacidopathies (Chen and Woo, 2005
), and bilirubin metabolic defects (Nguyen et al., 2005
). Such considerations led to the selection of an adenovirus in our initial proof-of-principle correction experiments because these vectors can be highly concentrated, have been demonstrated to have biological effects in other intermediary metabolic mouse models, such as ornithine transcarbamylase (OTC) deficiency (Stratford-Perricaudet et al., 1990
; Ye et al., 1996
), citrullinemia (Patejunas et al., 1998
; Ye et al., 2000
), and glycogen storage disorder type I (Nguyen et al., 2005
), and can be easily engineered with strong heterologous expression cassettes. Our results encourage the application of other vector systems, if efficient in early transduction of hepatocytes, to treat Mut−/−
mice. Other promoters and viral delivery systems, in particular adeno-associated viral serotype 8 vectors, might be especially effective in this application and will be the subject of future gene delivery studies in this murine model.
Adenovirus-mediated correction to extend the life span of Mut−/− mice has additional utility to generate older mice with severe methylmalonic acidemia and diminished or absent viral gene expression. Such mice have been used in pilot experiments to study the effects of dietary manipulations and provide an animal model of childhood methylmalonic acidemia that is analogous to the state of severely affected patients who have survived a neonatal crisis. Furthermore, rescued Mut−/− mice may also be useful to study the unusual pathology that is seen in MMA patients, such as pancreatitis, renal disease, and stroke syndrome, which have not been detected in neonatal animals.