Liver-Directed Recombinant Adeno-Associated
Viral Gene Delivery Rescues a Lethal Mouse
Model of Methylmalonic Acidemia and Provides
Long-Term Phenotypic Correction
Nuria Carrillo-Carrasco,1*Randy J. Chandler,1,2*Suma Chandrasekaran,1and Charles P. Venditti1
Methylmalonic acidemia is a severe metabolic disorder caused by a deficiency of the ubiquitously expressed
mitochondrial enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation has been used to treat a small
number of patients with variable success, and whether liver-directed gene therapy might be employed in such a
pleiotropic metabolic disorder is uncertain. In this study, we examined the therapeutic effects of hepatocyte-
directed delivery of the Mut gene to mice with a severe form of methylmalonic acidemia. We show that a single
intrahepatic injection of recombinant adeno-associated virus serotype 8 expressing the Mut gene under the
control of the liver-specific thyroxine-binding globulin (TBG) promoter is sufficient to rescue Mut–/–mice from
neonatal lethality and provide long-term phenotypic correction. Treated Mut–/–mice lived beyond 1 year of
age, had improved growth, lower plasma methylmalonic acid levels, and an increased capacity to oxidize
[1-13C]propionate in vivo. The older treated mice showed increased Mut transcription, presumably mediated by
upregulation of the TBG promoter during senescence. The results indicate that the stable transduction of a small
number of hepatocytes with the Mut gene can be efficacious in the phenotypic correction of an inborn error of
organic acid metabolism and support the rapid translation of liver-directed gene therapy vectors already
optimized for human subjects to patients with methylmalonic acidemia.
l-methylmalonyl-CoA into succinyl-CoA in the mitochon-
drial matrix (Fenton et al., 2001). This reaction is essential for
the metabolism of propionyl-CoA, an important intermediate
in the degradation of isoleucine, valine and odd-chained fatty
acids. Deficiency of MUT causes isolated methylmalonic
acidemia (OMIM #251000), a severe disorder of intermediary
metabolism associated with multisystemic disease and lethal
metabolic instability (Oberholzer et al., 1967; Stokke et al.,
1967). Patients with complete absence of enzyme activity are
classified as mut0and have a poor prognosis for long-term
survival despite aggressive nutritional and medical manage-
ment (Matsui et al., 1983; van der Meer et al., 1994; Nicolaides
et al., 1998; de Baulny et al., 2005; Dionisi-Vici et al., 2006;
Ho ¨rster et al., 2007).
ethylmalonyl-CoA mutase (MUT) (EC 184.108.40.206) is a
mitochondrial enzyme that catalyzes the conversion of
2003; Kaplan et al., 2006b; Kasahara et al., 2006) and/or com-
bined liver–kidney transplantation (van ‘t Hoff et al., 1998;
Nagarajan et al., 2005) has been used to treat a subset of mut0
patients. Although solid organ recipients do not experience
peripheral metabolic correction after transplantation (van ‘t
have improved growth and protection from intermittent met-
abolic crises. These observations suggest that gene and cell
therapy directed toward the liver might have therapeutic ef-
variability in the selection of candidates for liver transplanta-
tion, timing of the surgery, lack of standard monitoring pre-
genetic phenotypes of recipient patients have confounded the
a treatment for methylmalonic acidemia. Experimental studies
1Organic Acid Research Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes
of Health Bethesda, MD 20892.
2Institute for Biomedical Sciences, George Washington University, Washington, DC 20052.
*N.C.C. and R.J.C. contributed equally to this study.
HUMAN GENE THERAPY 21:1147–1154 (September 2010)
ª Mary Ann Liebert, Inc.
We have previously reported the temporal correction of a
mediated gene therapy (Chandler and Venditti, 2008) and
described long-term correction in the same model, using a
recombinant adeno-associated virus serotype 8 (rAAV8)
b-actin (CBA) promoter (Chandler and Venditti, 2010). In
wild-type mice, the Mut enzyme is present ubiquitously
(Chandler et al., 2007a) but is most highly expressed and ac-
tive in the liver (Wilkemeyer et al., 1993), an organ that plays a
central role in methylmalonyl-CoA metabolism. The rAAV8-
CBA-mMut-treated Mut–/–mice showed evidence of trans-
gene expression in numerous organs, including the liver,
skeletal muscle, and heart, with significant levels of Mut
protein detected in the liver (Chandler and Venditti, 2010).
Although the contribution of Mut enzymatic activity from
each tissue to the corrected phenotype was not discernable,
the sustained improvement in the Mut–/–mice in our earlier
rAAV experiments might largely be explained by expression
of the Mut transgene in hepatocytes.
In this study, we explore the therapeutic efficacy of liver-
directed gene therapy as a potential treatment for meth-
ylmalonic acidemia. In addition to targeting a cell type
established to be central in the pathology of methylmalonic
acidemia (Hayasaka et al., 1982; Krahenbuhl et al., 1991;
Chandler et al., 2009; de Keyzer et al., 2009), selective ex-
pression of the Mut gene in the liver might help minimize
transgene-directed immune responses (Franco et al., 2005)
and afford rapid translation to the development of an
rAAV8-MUT vector similar to those given to human subjects
with hemophilia (Manno et al., 2006) and a1-antitrypsin de-
ficiency (Brantly et al., 2009). An rAAV8 vector that expresses
the Mut gene from the liver-specific thyroxine-binding
globulin (TBG) promoter (Hayashi et al., 1993; Xiao et al.,
1998) was constructed and delivered to neonatal Mut–/–mice.
Hepatic expression of the viral Mut transgene completely
rescues Mut–/–mice from neonatal lethality, restores growth,
and affords long-term survival and yet, exactly as is ob-
served in methylmalonic acidemia patients who have re-
ceived liver transplants, it does not completely correct
biochemical abnormalities. Expression of the Mut gene in a
small percentage of stably transduced cells accounts for the
clinical effects and is higher in older mice, likely because of
senescence upregulation of the transgene from the TBG
promoter. The results of our experiments have therapeutic
implications for other disorders of intermediary metabolism,
particularly inborn errors of organic acid metabolism.
Materials and Methods
Construction and production of rAAVs
The expression construct, p-AAV8-CI-TBG-RGB, was
provided by the University of Pennsylvania Vector Core
(Philadelphia, PA). The plasmid contains transcriptional
TBG promoter, and the rabbit globin polyadenylation signal.
These elements are flanked by inverted terminal repeat
mutase and enhanced green fluorescent protein (GFP) cDNAs
were cloned into pAAV2/8.CI.TBG.RBG (see Supplementary
Fig. 1 at www.liebertonline.com/hum). The expression vec-
tor pAAV2/8.CI.CB7.mMut.RBG has been described and
contains the cytomegalovirus enhancer/chicken b-actin pro-
moter driving the expression of the murine methylmalonyl-
CoA mutase cDNA (Chandler and Venditti, 2010). The vector
genomes were packaged into an AAV8 capsid, purified by
cesium chloride gradient purification, and titered by quanti-
tative PCR as previously described (Hayashi et al., 1993; Gao
et al., 2002).
Murine experiments were approved and performed ac-
cording to the regulations and standards of the National
Human Genome Research Institute (NHGRI, Bethesda, MD)
Animal Care and Use Committee (ACUC). The mice used
harbor a deletion of exon 3 in the Mut gene. This mutation
abolishes the production of mature RNA, protein, and
detectable enzymatic activity (Chandler et al., 2007a). Mice
homozygous for this mutation (Mut–/–) display a severe me-
thylmalonic acidemia phenotype that is lethal in the new-
born period and is accompanied by progressive elevation of
methylmalonic acid (MMA) levels to 2000mM (normal
range, 5–10mM) at the time of death. Heterozygous animals
(Mutþ/?) appear normal and have biochemical profiles
identical to those of wild-type animals, and were used as
controls throughout the study. Immediately before injection,
2?1011or 4?1011vector genome copies (GC) of either the
Mut.RGB vector were diluted to a total volume of 20ml with
on nonanesthetized neonatal mice, typically within several
hours of birth, using a 32-gauge needle and transdermal ap-
Livers from untreated mice and mice injected in the neo-
natal period with pAAV2/8.CI.TBG.GFP.RGB were collected
at 60 days of life, embedded, frozen immediately on dry ice,
and stored at ?808C. Frozen liver sections (thickness, 8mm)
were placed onto glass slides, stained with 40,6-diamidino-2-
phenylindole (DAPI), and mounted with Fluoro-Gel aqueous
mounting medium (Electron Microscopy Sciences, Hatfield,
PA). Random slides from each liver section were evaluated
by confocal microscopy (510 NLO Meta; Carl Zeiss, Ober-
kochen, Germany) with fluorescein isothiocyanate (FITC)
and DAPI filters for the same microscope and camera set-
tings and using the threshold level from untreated control
mice as a reference for liver background fluorescence, as
previously described (Wang et al., 2010b). The total number
of cells was calculated with software and manually vali-
dated. GFP-labeled cells were quantified manually by careful
examination of 13 randomly selected sections by three in-
dependent observers. Each observer evaluated 680 cells and
the results were averaged as the number of GFP-positive
nuclei per total nuclei visualized. The GFP intensity in each
image was also determined.
Quantitative real-time PCR analysis
Mut RNA expression in liver was calculated for untreated
Mut–/–mice (n¼3), and for Mutþ/?mice (n¼3) and Mut–/–
mice treated with 2?1011GC of rAAV8-TBG-mMut, 90 days
(n¼3) and 270 days (n¼3) after injection. Total RNA from
1148CARRILLO-CARRASCO ET AL.
Valencia, CA). Residual DNA was digested with DNA-free
(Ambion, Austin, TX). Mut and Serpina7 (TBG) transcripts
were quantified by real-time PCR, in triplicate, using TaqMan
gene expression assays with mouse glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) (4352932E), murine
thyroxine-binding globulin (Mm00626105_m1) probes, all
from Applied Biosystems (Foster City, CA), and analyzed
with an Applied Biosystems 7500 real-time PCR system.
Expression of Mut and Serpina7 was normalized on the basis
of liver GAPDH transcript levels.
Vector genome copy number
Genome copy (GC) number was measured by quantitative
real-time PCR analysis as described previously. A standard
curve was prepared, using serial dilutions of a plasmid car-
rying the murine Mut cDNA. Genomic DNA was extracted
from murine liver samples and 100ng of DNA was used to
determine the genome copy number of rAAV at 90 days
(n¼2) and 270 days (n¼3) of life.
Tissue samples were homogenized with a 2-ml Tenbroeck
tissue grinder (Wheaton Science International, Millville, NJ) in
tissue protein extraction reagent (T-PER; Pierce Protein Re-
search Products/Thermo Scientific, Rockford, IL). Sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–
PAGE) was done with 35mg of clarified liver extract and
transferred to nylon membranes. Western blot analysis was
performed with a rabbit polyclonal antibody against murine
Mut at a 1:500 dilution (Chandler et al., 2007b) and mouse
monoclonal anti-OxPhos (oxidative phosphorylation) complex
III core II antibody (A-11143; Invitrogen, Carlsbad, CA) at a
1:4000 dilution; incubation was for 3hr. Horseradish peroxi-
dase (HRP)-conjugated goat anti-rabbit antibody (1858415;
Pierce Protein Research Products/Thermo Scientific) at a
(1858413; Pierce Protein Research Products/Thermo Scientific)
at a 1:30,000 dilution was incubated for 1hr. Signal was de-
tectedwithSuperSignal West Picochemiluminescent substrate
(34080; Pierce Protein Research Products/Thermo Scientific).
Blood samples were obtained by retro-orbital sinus plexus
sampling, using a sterile glass capillary tube at 24, 60, 90, 120,
180, 270, and 360 days of life, for measurement of plasma
methylmalonic acid (MMA) levels. Samples were immedi-
ately centrifuged and plasma was collected, diluted in water,
and frozen at ?808C until measurements were performed.
Plasma MMA levels were quantified by gas chromatography-
mass spectrometry with stable isotopic internal calibration as
previously described (Marcell et al., 1985; Allen et al., 1993).
In vivo propionate oxidation studies
Mutþ/?, untreated Mut–/–, and treated Mut–/–mice were
injected intraperitoneally with 200mg of sodium [1-13C]pro-
pionate (Cambridge Isotope Laboratories, Andover, MA).
[1-13C]propionate oxidation was measured by collecting ex-
pired gas,using anadaptation ofa method developed tostudy
propionate oxidation in patients with methylmalonic and
propionic acidemia, as previously described (Chandler and
Venditti, 2010). Mice were placed into a respiratory chamber
that contained a CO2probe to allow the direct measurement of
CO2generated by each animal. An aliquot of expired air was
removed from the chamber at each time point for analysis of
13C enrichment in CO2. The isotope ratio (13C/12C) of the col-
lected breath was measured with a gas isotope ratio mass
spectrometer (Metabolic Solutions, Nashua, NH). The percent
dose metabolized was calculated as follows: percent dose
metabolized¼total13C excreted (mmol)/dose (mmol)?100.
In all instances, p values were considered significant if the
value was less than 0.05. Differences in survival between
treated groups were analyzed by w2test. The weights be-
tween treated and untreated mice and differences in me-
tabolite levels were assessed by two-sided, two-tailed
unpaired Student t test. The Kruskal–Wallis test was used to
determine the statistical significance of differences in mea-
sured propionate oxidation rates between groups at 25min.
Twenty-one Mut–/–mice received a single intrahepatic
injection of either 2?1011GC (n¼15) or 4?1011GC (n¼6) of
rAAV8-TBG-mMut at birth. In the untreated mutant group,
94% perished before 21 days of life and only one of the un-
treated mice was alive by 72 days of life. In contrast, all but
one of the Mut–/–mice that received 2?1011GC of AAV8-
TBG-mMut (n¼15), and all that received 4?1011GC of
AAV8-TBG-mMut (n¼6), were alive at 21 days of life after
injection. After weaning on day 24, 93% (14 of 15) of the
2?1011GC-treated mutants were alive (Fig. 1). One year after
directed gene therapy. Survival (in days) of untreated Mut–/–
mice (n¼58) and those treated with either 2?1011GC
(n¼15) or 4?1011GC (n¼6) of rAAV8-TBG-mMut in the
neonatal period. Mice treated with 4?1011GC of AAV8-
TBG-mMut (n¼6) had 84% survival (5 of 6) by 360 days of
life compared with 53% (8 of 15) for those treated with
2?1011GC of AAV8-TBG-mMut. Both treated groups had
significantly increased survival at 24, 60, and 100 days and
beyond (*p<0.01) compared with the group of untreated
Long-term survival of Mut–/–mice after liver-
LIVER-DIRECTED GENE THERAPY FOR MMA1149
injection, 53% (8 of 15) of the Mut–/–mice that received
2?1011GC of rAAV8-TBG-mMut and 83% (5 of 6) of the
Mut–/–mice that received 4?1011GC were still alive. In the
group that received the dose of 2?1011GC (n¼15) one
animal died soon after birth, another was found dead at 30
days, one was killed at 90 days to harvest tissue, and another
two were reported sick and were killed at 90 days of life.
Another two Mut–/–mice from this group were found dead
after their cage accidentally flooded at about 270 days of life.
Postmortem examination of the treated Mut–/–mice that
perished during the study period revealed no evident cause
of death or gross abnormalities in any organ on necropsy.
maintained 60–80% of body weight when compared with
their Mutþ/?littermates (Fig. 2). Treated Mut–/–mice were
significantly greater in size than untreated Mut–/–mice on
days24and 60(p<0.01), andclose tothesizeofsex-matched,
mice achieved and
There were no differences between the weights of Mut–/–mice
treated with 2?1011or 4?1011GC of rAAV8-TBG-mMut
(data not presented). Although the older rAAV8-TBG-mMut-
treated Mut–/–mice were slightly smaller than heterozygous
controls, they were otherwise vigorous and appeared grossly
normal. Although formal testing has not been performed,
these animals appeared clinically well, with no obvious
neurological or behavioral phenotypes (see Supplementary
Video 1 at www.liebertonline.com/hum).
A single intrahepatic injection of rAAV8-TBG-mMut
(2?1011GC) at birth produced detectable levels of Mut
mRNA in the liver of Mut–/–mice. When compared with the
Mut RNA expression of untreated Mutþ/?mice (n¼3),
Mut–/–mice killed at 90 days had 13% of transcript levels
(n¼3) and mice killed at 270 days of life had 46% of tran-
script levels (n¼3) (Fig. 3A). An immunoblot showed
immune-reactive Mut protein in some of these liver samples
(Fig. 3C). Transgene copy numbers in genomic DNA ex-
tracted from the livers of treated mice averaged 0.05 and 0.06
per diploid genome at 90 days (n¼2) and 270 days (n¼3) of
life, respectively. Serpina7 (TBG) mRNA, derived from the
endogenous genomic locus, had a 2.4-fold increase in ex-
pression at 270 days compared with 90 days of life (Fig. 3B).
To approximate the amount of hepatic correction achieved
in our treated Mut–/–mice, we injected 2?1011GC of rAAV8-
TBG-GFP and evaluated the liver of mice 60 days after a
neonatal intrahepatic injection. Clusters of cells accounting
for approximately 9% of hepatocytes visualized were GFP
positive (Fig. 3D). Attempts to perform immunohistochem-
istry with anti-Mut antibodies were unsuccessful.
A decrease in plasma MMA concentration was used as an
indirect measure of increased Mut enzymatic activity. Plas-
ma MMA concentrations were assayed at 24, 60, 90, 120, 180,
270, and 360 days of life. Mut–/–mice treated with rAAV8-
TBG-mMut at 2?1011GC had significantly lower plasma
MMA levels compared with untreated Mut–/–mice (Fig. 4A).
Treated Mut–/–mice had mean plasma MMA levels of
709mM (n¼11) and liver Mut mRNA expression of 13%
(n¼3) at 90 days of life. At 270 days of life, the treated mice
had a mean plasma MMA concentration of 379mM (n¼8)
and liver Mut mRNA expression of 46% (n¼3). There was
no difference in MMA concentrations between the 2?1011
and 4?1011GC-treated groups (data not presented). Overall,
plasma MMA levels in treated Mut–/–mice remained
elevated 50 to a 100 times above the levels of Mutþ/?mice
Whole-animal propionate oxidation capacity, which is de-
pendent on the functional activity of methylmalonyl-CoA
posttreatment with either 2?1011GC of rAAV8-TBG-mMut
(n¼4) or 2?1011GC of rAAV8-CBA-mMut (n¼4) were in-
was included as a comparator for long-term, whole-body
correction. The in vivo metabolism of this tracer involves a
series of enzymatic reactions, including methylmalonyl-CoA
mutase, and results in oxidation of [1-13C]propionate to13CO2
via the Krebs cycle. Assuming normal activity of other enzy-
matic reactions, an increase in sodium [1-13C]propionate
oxidized to13CO2by treated mice relative to untreated Mut–/–
mice indicates an increase in methylmalonyl-CoA mutase
activity. As can be seen in Fig. 4B, Mutþ/?mice (n¼12)
rAAV8-TBG-mMut. Weights were calculated as a percentage
of sex-matched Mutþ/?littermates. Error bars represent
plus and minus 1 standard deviation. Treated Mut–/–mice
showed improved growth compared with the group of un-
treated Mut–/–mice at 24 and 60 days (*p<0.01). Results for
the treated Mut–/–group compared with the treated Mutþ/?
group were significantly different at all points (p<0.01).
Top: Appearance at 1 year of age of a Mut–/–mouse treated
with rAAV8-TBG-mMut (right) and an age- and sex-matched
treated Mutþ/?littermate (left).
Improved growth parameters for mice treated with
1150CARRILLO-CARRASCO ET AL.
metabolize approximately 69.4?4.3% of [1-13C]propionate
into13CO2in 25min whereas untreated Mut–/–mice (n¼9)
oxidize 12.6?2.2% of the dose, with flat enrichment kinet-
ics. At 1 year of age, rAAV8-TBG-mMut-treated Mut–/–
mice (n¼4) showed an increased capacity to metabolize
[1-13C]propionate and could convert approximately 32.5?
8.1% of the injected dose into13CO2in 25min, which is not
significantly different (p¼0.20) from the 39.8?9.4% range
observed in a group of mice treated with 2?1011GC of
The successful outcomes seen in some patients with me-
thylmalonic acidemia who have received liver transplants
led us to perform liver-directed gene delivery experiments
in a mouse model that faithfully replicates the severest
phenotype of methylmalonic acidemia. We selected the TBG
promoter because it has been used in rAAVs that have suc-
cessfully corrected liver-specific metabolic disorders in mice,
such as ornithine transcarbamylase deficiency (Moscioni
of AAV8-TBG-mMut (2?1011GC). (A) Liver Mut mRNA expression was determined by quantitative PCR. Treated Mut–/–
mice had significantly higher mRNA expression: 13% at 90 days of life (n¼3; p<0.01) and 46% at 270 days of life (n¼3;
*p<0.01), compared with untreated Mutþ/?mice (n¼3). Untreated Mut–/–mice had less than 0.5% of the Mut mRNA
expression of Mutþ/?mice. (B) The expression of Serpina7 (TBG) was assayed by quantitative PCR in treated Mut–/–mice. The
mice at 270 days (n¼3) had, on average, 2.4-fold increased Serpina7 RNA levels compared with younger animals (n¼3). ( C)
Immunoblotting showed that one of two treated Mut–/–mice studied at 90 days had hepatic Mut protein levels similar to that
of Mutþ/?mice. No Mut protein was detected in untreated Mut–/–mice. (D) rAAV-mediated transgene expression in the liver.
Shown is GFP expression and localization in liver sections of mice killed 60 days after injection of rAAV8-TBG-GFP at 2?1011
GC (A–C) compared with control mice (D–F). GFP expression (A and D) and nuclear DNA (B and E) images were merged (C
and F), using LSM Image Browser software (Carl Zeiss). The white arrow indicates an area where the nuclear contour is
preserved with green signal present. Color images available online at www.liebertonline.com/hum.
Increased expression of methylmalonyl-CoA mutase in the liver of mice treated with a single intrahepatic injection
LIVER-DIRECTED GENE THERAPY FOR MMA1151
et al., 2006) and apolipoprotein deficiency (Lebherz et al.,
2007), and has been extensively studied in nonhuman pri-
mates (Wang et al., 2010a). The use of a hepatotropic rAAV
serotype 8 (Gao et al., 2002) further enhanced delivery of the
transgene to the liver. Mut RNA levels in rAAV8-TBG-
mMut-treated Mut–/–mice were consistent with the GFP
expression pattern in the liver seen after treatment with
rAAV8-TBG-GFP (Fig. 3), suggesting that the correction of
approximately 9% of hepatocytes is responsible for main-
taining long-term metabolic stability and phenotypic cor-
rection in treated Mut–/–mice.
Gene therapy with either 2?1011GC of rAAV8-TBG-
mMut or 2?1011GC of rAAV8-CBA-mMut resulted in
comparable phenotypic and metabolic correction of Mut–/–
mice. For example, at 60 days the growth effects were similar
between the treated mutant groups (72?18% [Fig. 2] vs.
81?15%; p¼0.20), as were the plasma MMA levels
(528?133mM [Fig. 4B] vs. 511?160mM; p¼0.77). Even 1
year after injection, Mut–/–mice treated with 2?1011GC of
rAAV8-TBG-mMut had a propionate oxidative capacity that
was equivalent to that observed in mice treated with 2?1011
GC of rAAV8-CBA-mMut (Fig. 4B). The hepatic RNA and
protein expression seen at later times in Mut–/–mice treated
with 2?1011GC of rAAV8-TBG-mMut establish that the TBG
promoter can effectively direct long-term expression in a
small population of transduced cells, as the GFP reporter
Endogenous TBG (Serpina7) transcript levels increased
over time, suggesting there was an increase in the promoter-
driven expression of Mut from the rAAV-TBGmMut trans-
gene in our treated mice. Such an observation would be
consistent with a prior report that established TBG as a
senescence-upregulated protein in rodents (Savu et al., 1991).
We therefore quantified the expression of TBG RNA at 90
and 270 days of life and found an approximate 2.4-fold in-
crease in the levels of RNA at the later time point (Fig. 3B).
Because mice at both the 90- and 270-day time points had
similar genome copy levels of the rAAV8-TBG-mMut vector,
the most plausible explanation for the apparent increase in
Mut transcript levels at 270 days compared with 90 days in
the rAAV8-TBG-mMut-treated Mut–/–mice would be tran-
scriptional upregulation from the TBG promoter in the AAV
vector, as occurs at the endogenous locus. Further studies
will be required to evaluate more thoroughly whether pro-
moter regulation may be used to modulate the effects of gene
therapy in methylmalonic acidemia, but these data support
the use of an rAAV configured with a liver-specific pro-
moter, especially if such a vector could facilitate a physio-
logical, long-term increase in the expression of a therapeutic
Complete biochemical correction of methylmalonic acid-
emia, in humans or mice, is unlikely. Patient studies have
clearly, and repeatedly, documented failure of metabolites to
normalize in liver (Kaplan et al., 2006a) and/or combined
liver–kidney transplant recipients (van ‘t Hoff et al., 1998;
Nagarajan et al., 2005). In the gene therapy experiments
presented here, we evaluated plasma MMA levels to indi-
rectly measure Mut activity, not as a definitive parameter to
methylmalonic acid (MMA) levels at 24, 60, 90, 120, 180, 270, and 360 days of life in untreated Mut–/–mice and Mut–/–mice
treated with 2?1011GC of rAAV8-TBG-mMut. The mean plasma MMA levels of untreated Mut–/–mice were 1,361mM at 24
days (n¼13) and 1,120mM at 60 days (n¼6). The mean plasma MMA levels of treated Mut–/–mice were considerably lower
at 703mM in mice 24 days old (n¼12; *p<0.01) and 528mM in mice 60 days old (n¼11; *p<0.01). Plasma MMA levels in
older mice stabilized between 345 and 465mM after 90 days (n¼4–8 at each point). The plasma MMA levels in unaffected
Mutþ/?mice ranged from 5 to 10mM. (B) In vivo [1-13C]propionate oxidation in Mut–/–mice 1 year after treatment with 2?1011
GC of rAAV8-TBG-mMut or 2?1011GC of rAAV8-CBA-mMut. Mice were injected with 200mg of sodium [1-13C]propionate
and the percentage of dose oxidized was measured at 25min. rAAV8-TBG-mMut-treated Mut–/–mice (n¼4) oxidized
32.5?8.1% of the injected tracer, compared with 12.6?2.2% by untreated Mut–/–mice (n¼9) (*p<0.01). rAAV8-CBA-mMut-
treated Mut–/–mice (n¼4) oxidized 39.8?9.4% of the injected tracer (p value not significant vs. rAAV8-TBG-mMut-treated
mice). Untreated Mutþ/?mice (n¼12) oxidized 69.4?4.3% of the injected tracer at the same time point. Error bars surround
the 95% confidence intervals.
Increase in methylmalonyl-CoA mutase activity in mice treated with 2?1011GC of AAV8-TBG-mMut. (A) Plasma
1152 CARRILLO-CARRASCO ET AL.
establish that the disease phenotype had been corrected.
Numerous metabolic changes have been documented in
methylmalonic acidemia, but the role(s) that each plays in
the pathology of this heterogeneous disease remains unclear.
AAV8-TBG-mMut-treated Mut–/–mice display substantially
increased concentrations of MMA but appear grossly nor-
mal, suggesting that a small amount of Mut enzymatic ac-
tivity can provide long-term phenotypic correction and
demonstrating that stably transduced cells persist in the
setting of an abnormal metabolic milieu.
The data presented here examine the efficacy of liver-
directed gene therapy in a tractable experimental animal
model and help clarify some of the confounding issues that
have surrounded liver transplantation for methylmalonic
acidemia patients. Our results clearly demonstrate the effi-
cacy of this approach and are consistent with the suggestion
offered by some authors to perform a liver transplantation as
soon as possible as a treatment for this disorder (Morioka
et al., 2007). Because the stable transduction of a small
number of hepatocytes appears sufficient to obtain thera-
peutic effects in a mouse model that replicates a severe form
of the condition, liver-directed gene therapy would likely
benefit patients with methylmalonic acidemia and, possibly,
other inborn errors of organic acid metabolism. Last, our
vector has been harmonized in design with those currently
being used in human clinical trials and is similar to one that
has been tested in nonhuman primates (Wang et al., 2010a).
This should facilitate the translation of our murine studies to
a clinical gene therapy trial for methylmalonic acidemia.
The authors thank Stephen Wincovitch for confocal mi-
croscopy guidance, Denise Larson for performing frozen
sections of liver, Cherry Yang and Irene Ginty for mouse care
and technical assistance, David M. Bodine IV for providing a
critical reading of the manuscript, and the University of
Pennsylvania Vector Core for AAV preparations and advice.
N.C.C., R.J.C., S.C., and C.P.V. were supported, in part, by
the Intramural Research Program of the National Human
Genome Research Institute, National Institutes of Health.
Author Disclosure Statement
No competing financial interests exist.
Allen, R.H., Stabler, S.P., Savage, D.G., and Lindenbaum, J.
(1993). Elevation of 2-methylcitric acid I and II levels in serum,
urine, and cerebrospinal fluid of patients with cobalamin de-
ficiency. Metabolism 42, 978–988.
Brantly, M.L., Chulay, J.D., Wang, L., Mueller, C., Humphries,
M., Spencer, L.T., Rouhani, F., Conlon, T.J., Calcedo, R., Betts,
M.R., Spencer, C., Byrne, B.J., Wilson, J.M., and Flotte, T.R.
(2009). Sustained transgene expression despite T lymphocyte
responses in a clinical trial of rAAV1-AAT gene therapy. Proc.
Natl. Acad. Sci. U.S.A. 106, 16363–16368.
Chandler, R.J., and Venditti, C.P. (2008). Adenovirus-mediated
gene delivery rescues a neonatal lethal murine model of mut0
methylmalonic acidemia. Hum. Gene Ther. 19, 53–60.
Chandler, R.J., and Venditti, C.P. (2010). Long-term rescue of a
lethal murine model of methylmalonic acidemia using adeno
associated viral gene therapy. Mol. Ther. 18, 11–16.
Chandler, R.J., Sloan, J., Fu, H., Tsai, M., Stabler, S., Allen, R.,
Kaestner, K.H., Kazazian, H.H., and Venditti, C.P. (2007a).
Metabolic phenotype of methylmalonic acidemia in mice and
humans: The role of skeletal muscle. BMC Med. Genet. 8, 64.
Chandler, R.J., Tsai, M.S., Dorko, K., Sloan, J., Korson, M.,
Freeman, R., Strom, S., and Venditti, C.P. (2007b). Adenoviral-
mediated correction of methylmalonyl-CoA mutase deficiency
in murine fibroblasts and human hepatocytes. BMC Med.
Genet. 8, 24.
Chandler, R.J., Zerfas, P.M., Shanske, S., Sloan, J., Hoffmann, V.,
dysfunction in mut methylmalonic acidemia. FASEB J. 23,
de Baulny, H.O., Benoist, J.F., Rigal, O., Touati, G., Rabier, D.,
and Saudubray, J.M. (2005). Methylmalonic and propionic
acidaemias: Management and outcome. J. Inherit. Metab. Dis.
de Keyzer, Y., Valayannopoulos, V., Benoist, J.F., Batteux, F.,
Lacaille, F., Hubert, L., Chretien, D., Chadefeaux-Vekemans,
B., Niaudet, P., Touati, G., Munnich, A., and de Lonlay, P.
(2009). Multiple OXPHOS deficiency in the liver, kidney,
heart, and skeletal muscle of patients with methylmalonic
aciduria and propionic aciduria. Pediatr. Res. 66, 91–95.
Dionisi-Vici, C., Deodato, F., Roschinger, W., Rhead, W., and
Wilcken, B. (2006). ‘‘Classical’’ organic acidurias, propionic
aciduria, methylmalonic aciduria and isovaleric aciduria:
Long-term outcome and effects of expanded newborn screen-
ing using tandem mass spectrometry. J. Inherit. Metab. Dis. 29,
Fenton, W.A., Gravel, R.A., and Rosenblatt, D.S. (2001). Dis-
orders of propionate and methylmalonate metabolism. In The
Metabolic & Molecular Bases for Inherited Disease (McGraw-Hill,
New York) pp. 2165–2194.
Franco, L.M., Sun, B., Yang, X., Bird, A., Zhang, H., Schneider,
A., Brown, T., Young, S.P., Clay, T.M., Amalfitano, A., Chen,
Y.T., and Koeberl, D.D. (2005). Evasion of immune responses
to introduced human acid a-glucosidase by liver-restricted
expression in glycogen storage disease type II. Mol. Ther. 12,
Gao, G.P., Alvira, M.R., Wang, L., Calcedo, R., Johnston, J., and
Wilson, J.M. (2002). Novel adeno-associated viruses from
rhesus monkeys as vectors for human gene therapy. Proc.
Natl. Acad. Sci. U.S.A. 99, 11854–11859.
Hayasaka, K., Metoki, K., Satoh, T., Narisawa, K., Tada, K.,
Kawakami, T., Matsuo, N., and Aoki, T. (1982). Comparison of
cytosolic and mitochondrial enzyme alterations in the livers of
propionic or methylmalonic acidemia: A reduction of cyto-
chrome oxidase activity. Tohoku J. Exp. Med. 137, 329–334.
Hayashi, Y., Mori, Y., Janssen, O.E., Sunthornthepvarakul, T.,
Weiss, R.E., Takeda, K., Weinberg, M., Seo, H., Bell, G.I., and
Refetoff, S. (1993). Human thyroxine-binding globulin gene:
Complete sequence and transcriptional regulation. Mol.
Endocrinol. 7, 1049–1060.
Ho ¨rster, F., Baumgartner, M.R., Viardot, C., Suormala, T.,
Burgard, P., Fowler, B., Hoffmann, G.F., Garbade, S.F., Ko ¨lker,
S., and Baumgartner, E.R. (2007). Long-term outcome in me-
thylmalonic acidurias is influenced by the underlying defect
(mut0, mut–, cblA, cblB). Pediatr. Res. 62, 225–230.
Hsui, J.Y., Chien, Y.H., Chu, S.Y., Lu, F.L., Chen, H.L., Ho, M.J.,
Lee, P.H., and Hwu, W.L. (2003). Living-related liver trans-
plantation for methylmalonic acidemia: Report of one case.
Acta Paediatr. Taiwan 44, 171–173.
Kaplan, P., Ficicioglu, C., Mazur, A.T., Palmieri, M.J., and
Berry, G.T. (2006a). Liver transplantation is not curative for
LIVER-DIRECTED GENE THERAPY FOR MMA 1153
methylmalonic acidopathy caused by methylmalonyl-CoA Download full-text
mutase deficiency. Mol. Genet. Metab. 88, 322–326.
Kaplan, P., Ficicioglu, C., Mazur, A.T., Palmieri, M.J., and
Berry, G.T. (2006b). Liver transplantation is not curative for
methylmalonic acidopathy caused by methylmalonyl-CoA
mutase deficiency. Mol. Genet. Metab. 88, 322–326.
Kasahara, M., Horikawa, R., Tagawa, M., Uemoto, S., Yokoya-
ma, S., Shibata, Y., Kawano, T., Kuroda, T., Honna, T., Tanaka,
K., and Saeki, M. (2006). Current role of liver transplantation
for methylmalonic acidemia: A review of the literature.
Pediatr. Transplant. 10, 943–947.
Kayler, L.K., Merion, R.M., Lee, S., Sung, R.S., Punch, J.D.,
Rudich, S.M., Turcotte, J.G., Campbell, D.A., Jr., Holmes, R.,
and Magee, J.C. (2002). Long-term survival after liver trans-
plantation in children with metabolic disorders. Pediatr.
Transplant. 6, 295–300.
Krahenbuhl, S., Chang, M., Brass, E.P., and Hoppel, C.L. (1991).
Decreased activities of ubiquinol:ferricytochrome c oxidore-
ductase (complex III) and ferrocytochrome c:oxygen oxidore-
ductase (complex IV) in liver mitochondria from rats with
hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.
J. Biol. Chem. 266, 20998–21003.
Lebherz, C., Sanmiguel, J., Wilson, J.M., and Rader, D.J. (2007).
Gene transfer of wild-type apoA-I and apoA-I Milano re-
duce atherosclerosis to a similar extent. Cardiovasc. Diabetol.
Manno, C.S., Pierce, G.F., Arruda, V.R., Glader, B., Ragni, M.,
Rasko, J.J., Ozelo, M.C., Hoots, K., Blatt, P., Konkle, B., Dake,
M., Kaye, R., Razavi, M., Zajko, A., Zehnder, J., Rustagi, P.K.,
Nakai, H., Chew, A., Leonard, D., Wright, J.F., Lessard, R.R.,
Sommer, J.M., Tigges, M., Sabatino, D., Luk, A., Jiang, H.,
Mingozzi, F., Couto, L., Ertl, H.C., High, K.A., and Kay, M.A.
(2006). Successful transduction of liver in hemophilia by
AAV-Factor IX and limitations imposed by the host immune
response. Nat. Med. 12, 342–347.
Marcell, P.D., Stabler, S.P., Podell, E.R., and Allen, R.H. (1985).
Quantitation of methylmalonic acid and other dicarboxylic
acids in normal serum and urine using capillary gas chro-
matography-mass spectrometry. Anal. Biochem. 150, 58–66.
Matsui, S.M., Mahoney, M.J., and Rosenberg, L.E. (1983). The
natural history of the inherited methylmalonic acidemias. N.
Engl. J. Med. 308, 857–861.
Morioka, D., Kasahara, M., Horikawa, R., Yokoyama, S., Fuku-
da, A., and Nakagawa, A. (2007). Efficacy of living donor liver
transplantation for patients with methylmalonic acidemia.
Am. J. Transplant. 7, 2782–2787.
Moscioni, D., Morizono, H., McCarter, R.J., Stern, A., Cabrera-
Luque, J., Hoang, A., Sanmiguel, J., Wu, D., Bell, P., Gao, G.P.,
Raper, S.E., Wilson, J.M., and Batshaw, M.L. (2006). Long-term
correction of ammonia metabolism and prolonged survival in
ornithine transcarbamylase-deficient mice following liver-
directed treatment with adeno-associated viral vectors. Mol.
Ther. 14, 25–33.
Nagarajan, S., Enns, G.M., Millan, M.T., Winter, S., and Sarwal,
M.M. (2005). Management of methylmalonic acidaemia by
combined liver–kidney transplantation. J. Inherit. Metab. Dis.
Nicolaides, P., Leonard, J., and Surtees, R. (1998). Neurological
outcome of methylmalonic acidaemia. Arch. Dis. Child. 78,
Nyhan, W.L., Gargus, J.J., Boyle, K., Selby, R., and Koch, R.
(2002). Progressive neurologic disability in methylmalonic
acidemia despite transplantation of the liver. Eur. J. Pediatr.
Oberholzer, V.G., Levin, B., Burgess, E.A., and Young, W.F.
(1967). Methylmalonic aciduria: An inborn error of metabo-
lism leading to chronic metabolic acidosis. Arch. Dis. Child.
Savu, L., Vranckx, R., Rouaze-Romet, M., Maya, M., Nunez,
E.A., Treton, J., and Flink, I.L. (1991). A senescence up-
regulated protein: The rat thyroxine-binding globulin (TBG).
Biochim. Biophys. Acta 1097, 19–22.
Stokke, O., Eldjarn, L., Norum, K.R., Steen-Johnsen, J., and
Halovorsen, S. (1967). Methylmalonic acidemia: A newborn
error of metabolism which may cause fatal acidosis in the
neonatal period. Scand. J. Clin. Lab. Invest. 20, 313–328.
van der Meer, S.B., Poggi, F., Spada, M., Bonnefont, J.P., Ogier,
H., Hubert, P., Depondt, E., Rapoport, D., Rabier, D., Char-
pentier, C., Parvy, P., Bardet, J., Kamoun, P., and Saudubray,
J.M. (1994). Clinical outcome of long-term management of
patients with vitamin B12-unresponsive methylmalonic aci-
demia. J. Pediatr. 125, 903–908.
van ‘t Hoff, W.G., Dixon, M., Taylor, J., Mistry, P., Rolles, K.,
Rees, L., and Leonard, J.V. (1998). Combined liver–kidney
transplantation in methylmalonic acidemia. J. Pediatr. 132,
Wang, L., Calcedo, R., Wang, H., Bell, P., Grant, R., Vanden-
berghe, L.H., Sanmiguel, J., Morizono, H., Batshaw, M.L., and
Wilson, J.M.. (2010a). The pleiotropic effects of natural AAV
infections on liver-directed gene transfer in macaques. Mol.
Ther. 18, 126–134.
Wang, L., Wang, H., Bell, P., McCarter, R.J., He, J., Calcedo, R.,
Vandenberghe, L.H., Morizono, H., Batshaw, M.L., and
Wilson, J.M. (2010b). Systematic evaluation of AAV vectors for
liver directed gene transfer in murine models. Mol. Ther. 18,
Wilkemeyer, M.F., Andrews, E.R., and Ledley, F.D. (1993).
Genomic structure of murine methylmalonyl-CoA mutase:
Evidence for genetic and epigenetic mechanisms determining
enzyme activity. Biochem. J. 296, 663–670.
Xiao, W., Berta, S.C., Lu, M.M., Moscioni, A.D., Tazelaar, J., and
Wilson, J.M. (1998). Adeno-associated virus as a vector for
liver-directed gene therapy. J. Virol. 72, 10222–10226.
Address correspondence to:
Dr. Charles P. Venditti
Organic Acid Research Section
Genetics and Molecular Biology Branch
National Human Genome Research Institute
National Institutes of Health
Building 49, Room 4A18
Bethesda, MD 20892
Received for publication January 15, 2010;
accepted after revision May 4, 2010.
Published online: July 30, 2010.
1154 CARRILLO-CARRASCO ET AL.