Long-term Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using Adeno associated Viral Gene Therapy

Article (PDF Available)inMolecular Therapy 18(1):11-6 · October 2009with30 Reads
DOI: 10.1038/mt.2009.247 · Source: PubMed
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.
original article
© The American Society of Gene & Cell Therapy
Molecular Therapy 1
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 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.
Received 26 June 2009; accepted 21 September 2009; advance online
publication 27 October 2009. doi:10.1038/mt.2009.247
INTRODUCTION
Methylmalonic acidemia (MMA) is a severe autosomal reces-
sive inborn error of intermediary metabolism characterized by
intermittent metabolic instability, multiorgan pathology, growth
retardation, and a poor prognosis for long-term survival.
1–6
e
disorder exhibits genetic heterogeneity and can be caused by de-
cient enzymatic activity of methylmalonyl-CoA mutase (MUT)
or defective intracellular transport, processing, and metabolism
of cobalamin.
7
MUT is an important mitochondrial enzyme in
propionyl-CoA metabolism and converts -methylmalonyl-CoA
into succinyl-CoA, a Krebs cycle intermediate. A block at this
enzymatic step results in elevated plasma levels of methylmalonic
acid as well the accumulation of other propionyl-CoA-derived
metabolites such as 2-methylcitrate.
8
e etiology of the many
medical problems that patients with MMA suer is not well under-
stood. However, the wide spectrum of severity that can be seen in
patients who harbor missense mutations suggests that restoring a
very low level of enzyme activity would provide substantial clini-
cal benet.
Currently, the main treatment for aected patients is dietary
restriction of propiogenic amino acids to reduce circulating
metabolites. Liver
9–15
and/or combined liver/kidney
16,17
transplan-
tation has been performed in a limited fashion in an attempt to
improve metabolic stability through the provision of organ-spe-
cic enzymatic activity. Although this approach has been eective,
and even curative, for other metabolic disorders,
18
the clinical util-
ity of solid organ transplantation as a standard treatment for MMA
is unclear given the small number of patients that have undergone
the procedure.
14,15,19
e need for new and widely available thera-
pies for MMA is underscored by a recent multicenter European
long-term patient study, which described an overall 46% (n = 52)
mortality for patients with MMA resulting from MUT deciency
by 30 years of age.
6
We have previously developed a murine model of MMA
that replicates the severest clinical phenotype of MMA seen
in patients.
20
Aected animals display a 100–200-fold increase
in plasma methylmalonic acid concentrations and most per-
ish within the rst few days of life.
20,21
Recently, we reported
that direct hepatic, but not intramuscular, injection of an
adenovirus that expressed the Mut gene under the control of
a cytomegalovirus promoter could only partially rescue the
Mut
−/−
mice.
22
A liver-directed approach was initially selected
because the hepatocytes of Mut
−/−
mice and MMA patients
manifest morphological changes and display a severe secondary
electron transport chain defect that is likely contributory to the
pathology of the disease.
21
Additionally, we had demonstrated
that robust correction of human MUT
−/−
hepatocytes was fea-
sible with this vector.
23
However, in the surviving adenoviral-
treated Mut
−/−
mice, the Mut levels steadily declined, and most
animals died between 1 and 3 months aer treatment. ese
results suggested that persistent expression would be needed to
provide long-term amelioration of the lethal phenotype in this
murine model of MMA.
Correspondence: 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, MSC 4442, Bethesda, Maryland, 20893, USA. E-mail: venditti@mail.nih.gov
Long-term Rescue of a Lethal Murine
Model of Methylmalonic Acidemia Using
Adeno-associated Viral Gene Therapy
Randy J Chandler
1,2
and Charles P Venditti
1
1
Organic Acid Research Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health,
Bethesda, Maryland, USA;
2
Institute for Biomedical Sciences, The George Washington University, Washington, DC, USA
MT
Open
2 www.moleculartherapy.org
© The American Society of Gene & Cell Therapy
Gene Therapy for Methylmalonic Acidemia
Recombinant adeno-associated viruses (rAAVs) have been
successfully used as gene delivery vehicles in numerous animal
models of human disease and have yielded long-term transgene
expression without vector-related toxicity. Furthermore, clinical
trials using rAAV have involved hundreds of subjects, supporting
the safety of these vectors for use in humans.
24
Over 100 dier-
ent natural AAV serotypes have been isolated from a variety of
species and some display striking tissue tropism.
25
Several stud-
ies have demonstrated that rAAV serotype 8 (rAAV8) vectors can
transduce mouse hepatocytes with high eciency when delivered
via the portal vein or intraperitoneal injection in the neonatal
period.
26–28
Similarly, rAAV8 vectors can eciently transduce
skeletal muscle,
29,30
a tissue that makes a signicant contribution
to the circulating metabolite pool in patients with MMA.
20
In this report, we describe the therapeutic ecacy of a rAAV
vector as a new gene therapy treatment for MMA and apply a novel
stable isotope metabolic method to monitor the function of Mut
aer gene therapy. e treated Mut
−/−
mice have reduced circu-
lating metabolites, are phenotypically indistinguishable from their
unaected Mut
+/−
mice littermates and show sustained enzymatic
activity 1 year aer treatment with the rAAV8 vector. Our results
provide the rst evidence that systemic gene delivery using rAAV
should be useful as a treatment for patients with MMA and other
organic acidemias, disorders that currently lack denitive therapy.
is gene delivery platform and metabolic monitoring technique
should be immediately translatable to a human gene therapy trial
for MMA.
RESULTS
Gene therapy rescues the lethal Mut
−/−
phenotype
Mut
−/−
newborn mice received a direct hepatic injection with
either 1 or 2 × 10
11
vector genome copies (GCs) of rAAV8-mMut.
All Mut
−/−
mice (n = 27) injected in the neonatal period with
rAAV8-mMut survived until day of life (DOL) 90 (Figure 1).
A single Mut
−/−
mouse from the rAAV8-mMut 1 × 10
11
GC group
perished at DOL 92 following a blood collecting procedure; a full
necropsy was performed, and no abnormalities were observed.
Ninety-six percent (26/27) of the Mut
−/−
mice treated with
rAAV8-mMut have survived beyond a year. In contrast, 100% of
the untreated mutants (n = 58) perished by DOL 72, with >90% of
this group dying by DOL 24. In another control group, newborn
Mut
−/−
mice (n = 18) received a direct hepatic injection with
either 1 or 2 × 10
11
GC of rAAV8 containing green uorescent
protein (GFP) complementary DNA driven by the same chicken
β-actin promoter/enhancer (Figure 1). None of the rAAV8-GFP-
treated Mut
−/−
mice survived beyond DOL 3. Untreated Mut
−/−
mice and rAAV8-GFP-treated Mut
−/−
animals were found dead
or cannibalized; the exact causes of death were undetermined.
To determine whether direct hepatic injection was necessary to
rescue Mut
−/−
mice, newborn Mut
−/−
mice (n = 4) received an
intraperitoneal injection of 3 × 10
11
GC rAAV8-mMut. ree out
of the four (75%) of these mice survived and are still alive at DOL
120 (Figure 1). Other than mild hepatomegaly, the Mut
−/−
mice
0
050 100 150 200 250 300
Days
350 400
20
40
60
80
100
Mut
/
Mut
/
rAAV8-mMut 2 × 10
11
GC
Mut
/
rAAV8-mMut 1 × 10
11
GC
Mut
/
rAAV8-GFP
Mut
/
I.P. rAAV8-mMut 3 × 10
11
GC
Percent Mut
/
alive
*
**
Figure 1 Rescue of Mut
−/−
mice. Survival in days between the
untreated Mut
−/−
mice (n = 58), intrahepatic-injected rAAV8-GFP 1 or
2 × 10
11
GC Mut
−/−
mice (n = 18), intrahepatic-injected rAAV8-mMut
1 × 10
11
GC (n = 10) or 2 × 10
11
GC (n = 17) Mut
−/−
mice, and i.p.
rAAV8-mMut 3 × 10
11
GC injected (n = 4) Mut
−/−
mice. All three groups
of rAAV8-mMut-treated Mut
−/−
mice exhibited a significant improve-
ment 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 × 10
11
GC and **P < 10
−22
for rAAV8-mMut
2 × 10
11
GC at the 100-day time point). GC, genome copy; GFP, green
fluorescent protein; i.p., intraperitoneal.
a
b
c
Mut
/
rAAV8
Mut
/
0
20
40
60
80
100
Day 24
Day 60
*
*
**
**
Percent weight of Mut
+/
littermates
120
Mut
+/
Mut
+/
Mut
+/
Mut
/
Mut
/
rAAV8-mMut
1 × 10
11
GC
Mut
/
rAAV8-mMut
2 × 10
11
GC
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 intrahe-
patic injection of rAAV8-mMut 2 × 10
11
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) com-
pared to untreated Mut
−/−
mice (n = 6, 3) or Mut
−/−
mice treated via
an intrahepatic injection of rAAV8-mMut 1 × 10
11
GC (n = 10, 10) or
rAAV8-mMut 2 × 10
11
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.
Molecular erapy 3
© The American Society of Gene & Cell Therapy
Gene Therapy for Methylmalonic Acidemia
(n = 4) treated by rAAV8-mMut gene therapy and sacriced for
tissue harvesting on DOL 90 or at 1 year had no gross pathologic
changes noted on dissection.
Correction of growth retardation following gene
therapy
Mut
−/−
mice were indistinguishable from their Mut
+/−
lit-
termates at birth, but those rare mutants that escaped neonatal
lethality were grossly abnormal. e small number of untreated
Mut
−/−
mice that survived to DOL 24 (6/58) and 60 (3/58) were
severely growth retarded, weighing <40% of sex-matched Mut
+/−
littermates (Figure 2a). Mut
−/−
mice treated with an intrahepatic
injection of either 1 or 2 × 10
11
GC of rAAV8-mMut were grossly
indistinguishable in size and behavior compared to controls
(Figure 2b) and achieved body weights that were similar to their
Mut
+/−
sex-matched littermates on DOL 24 and 60 (Figure 2c).
Expression of Mut after treatment with rAAV8-mMut
Mut expression in liver and muscle samples from treated Mut
−/−
mice at DOL 90 and 1 year of life was analyzed using quantitative
PCR (qPCR) to measure mRNA levels and by western blotting to
examine protein content. e Mut
−/−
mice at DOL 90 injected
with either 1 or 2 × 10
11
GC of rAAV8 expressed 38 and 72% of
the endogenous Mut mRNA levels found in the liver of untreated
Mut
+/−
animals (Figure 3a), whereas the level of Mut mRNA in
the lower limb skeletal muscle of treated animals exceeded the
endogenous Mut transcript levels measured to control heterozy-
gotes (Figure 3a). Even greater expression was noted in the hearts
of the treated Mut
−/−
mice compared to untreated Mut
+/−
mice, consistent with previous observations of highly ecient
transduction of cardiac and skeletal myocytes by rAAV8 vectors.
30
Mut mRNA was variably detected in whole brain extracts and not
detected in the kidney or spleen. Western blotting showed Mut
protein in both the liver and the skeletal muscle of the treated
Mut
−/−
mice at levels that paralleled those observed in the qPCR
experiments on DOL 90 (Figure 3b). Mice studied at longer times
showed persistent expression of the Mut transgene. e levels of
Mut mRNA in the liver and skeletal muscle of treated Mut
−/−
mice 1 year aer injection diminished but was still readily detect-
able by qPCR (Figure 3a), but not by western analysis.
Gene therapy restores Mut function and activity
Several parameters reective of Mut enzymatic function were
examined in the treated Mut
−/−
mice. Circulating metabolites were
measured and taken to reect whole-body Mut enzymatic activity
because all the mice ingested a precursor unrestricted diet. e
plasma methylmalonic acid concentrations in the treated Mut
−/−
mice were signicantly lower than in untreated Mut
−/−
mice at
both time points measured (Figure 4a). e untreated Mut
−/−
mice had mean plasma methylmalonic acid concentrations of
1,342 and 1,120 µmol/l on days 24 and 60. No untreated Mut
−/−
mice survived beyond day 72; therefore, plasma methylmalonic
acid levels aer day 60 from this group could not be obtained.
Both the 1 and 2 × 10
11
GC groups of treated Mut
−/−
mice had
mean plasma methylmalonic acid concentrations between 440
and 540 µmol/l at the day 24 and 60 time points. Metabolites in
these two groups of treated Mut
−/−
mice were also measured at 90,
120, 180, and 360 days with the mean methylmalonic acid levels
at these time points ranging from 365 to 596 µmol/l (Figure 4a).
As observed in humans with MMA whom have received liver or
b
Liver
Mut
Complex III
Mut
+/
Mut
/
rAAV8
1 × 10
11
GC
Mut
/
rAAV8
2 × 10
11
GC
Mut
/
c
Skeletal muscle
Mut
Complex III
Mut
+/
Mut
/
rAAV8
1 × 10
11
GC
Mut
/
rAAV8
2 × 10
11
GC
Mut
/
90 days
a
Percent Mut
+/
Methlymalonyl-CoA mutase RNA
Mut
+/
Liver
Skeletal muscle
Heart
Kidney
Brain
Spleen
Mut
/
ND
ND
N/A
N/A
ND
N/A
100 ± 8.8
100 ± 25.9
100 ± 5.0
100 ± 17.6
100 ± 2.3
100 ± 30.0
Mut
/
rAAV8 1 × 10
11
GC
37.5 ± 1.5
178.0 ± 24.0
6,183.9 ± 1,136.1
ND
5.6 ± 0.2
ND
Mut
/
rAAV8 2 × 10
11
GC
72.3 ± 3.4
461.3 ± 214.7
3,046.4 ± 1,030.4
ND
86.6 ± 4.0
ND
1 Year Mut
+/
Liver
Skeletal muscle
Brain
Mut
/
ND
ND
ND
100 ± 10.5
105 ± 38.8
100 ± 8.4
Mut
/
rAAV8 1 × 10
11
GC
15.0 ± 1.3
N/A
N/A
Mut
/
rAAV8 2 × 10
11
GC
9.9 ± 0.7
53.7 ± 2.6
59.7 ± 2.8
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 com-
parator. 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 × 10
11
GC or 2 × 10
11
GC of rAAV8-mMut and analyzed by western blotting. The same mem-
branes 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.
4 www.moleculartherapy.org
© The American Society of Gene & Cell Therapy
Gene Therapy for Methylmalonic Acidemia
combined liver–kidney transplants, plasma metabolites were not
normalized
13,16,17
and remained 50–100-fold increased over the
level seen in unaected Mut
+/−
mice (5–10 µmol/l).
To examine whether the long-term survival and ameliorated
metabolite levels observed in the treated Mut
−/−
mice corre-
sponded with increased whole-body enzyme activity, we developed
a novel in vivo propionate oxidation assay. Mut
−/−
mice at 1 year
aer treatment were injected with 1-
13
C-sodium propionate and
the subsequent metabolism of this tracer through the Mut reac-
tion, into the Krebs cycle, with eventual oxidation into
13
CO
2
was
determined. As can be seen in Figure 4b, Mut
+/−
mice metabo-
lize ~70% of 1-
13
C-propionate into
13
CO
2
in 25 minutes. Untreated
Mut
−/−
mice convert ~10% of the dose, with very at enrich-
ment kinetics. At 1 year of age, the treated Mut
−/−
mice show a
markedly increased capacity to oxidize 1-
13
C-propionate and on
average, can convert ~40% of the injected dose into
13
CO
2
.
rAAV8-mMut rescues postneonatal Mut
−/−
mice
As an extension of rAAV8-mMut gene therapy beyond the imme-
diate neonatal period, three rare untreated Mut
−/−
mice that
survived until DOL 20 received a single intraperitoneal injec-
tion of 3 × 10
11
GC of rAAV8-mMut. At the time of injection,
the animals were hypoactive, runted (Figure 5a) and showed an
impaired ability to produce
13
CO
2
from 1-
13
C-propionate. When
studied 10 days aer receiving the rAAV8-mMut, the mice had
an improved clinical appearance (Figure 5a), fully restored propi-
onate oxidation (Figure 5b) and displayed a tenfold reduction in
plasma methylmalonic acid concentrations (Figure 5c).
DISCUSSION
e experiments undertaken in the present report were designed
to test the ecacy of rAAV-mediated gene therapy in a murine
model of MMA, a prototypical organic acidemia. Our earlier stud-
ies in Mut
−/−
mice
22
and the demonstration that liver transplanta-
tion appears benecial for a subset of MMA patients
15
led to the
0
0
0510 15
Time (minutes)
20 25 30
10
20
30
40
50
60
70
Percent 1-
13
C propionate dose oxidized
80
050 100
*
*
**
150 200 250 300
Time (days)
350 400
500
1,000
Plasma methylmalonic acid (µmol/l)
1,500
Mut
/
n = 3, 12, 6
Mut
/
rAAV8 2 × 10
11
GC n = 17
Mut
/
rAAV8 1 × 10
11
GC n = 10
2,000
2,500
Mut
+/
n = 8
Mut
/
rAAV8-mMut 1 × 10
11
GC n = 3
Mut
/
n = 6
a
b
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 × 10
11
GC rAAV8-mMut, and Mut
−/−
mice treated with 2 × 10
11
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 rep-
resent 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-
13
C-propionate oxidation
1 year after rAAV8-mMut treatment. Two hundred micrograms of 1-
13
C-
sodium propionate was injected intraperitoneally into Mut
+/−
(n = 8),
1 × 10
11
GC rAAV8-mMut-treated Mut
−/−
(n = 3), or untreated Mut
−/−
(n = 6) mice.
13
C enrichment in expired CO
2
was measured and used
to determine the percent of the administered 1-
13
C-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-
13
C-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 × 10
11
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-
13
C-propionate was injected i.p. into
Mut
−/−
mice (labeled Mut
−/−
pre-rAAV, n = 3) or control Mut
+/−
litter-
mates (labeled Mut
+/−
, n = 3) on DOL 20 prior to receiving 3 × 10
11
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.
Molecular erapy 5
© The American Society of Gene & Cell Therapy
Gene Therapy for Methylmalonic Acidemia
selection of adeno-associated virus serotype 8 as a gene delivery
vector. e observed results are striking: a single intrahepatic
injection of rAAV8-mMut delivered in the neonatal period was
sucient to uniformly rescue treated Mut
−/−
mice from certain
death for over a year. e eects of gene therapy extended beyond
immediate mortality and allowed the treated Mut
−/−
mice to gain
weight, thrive, and reproduce. Limited pathological investigations
have been performed on the treated mutants at older times and will
be the subject of future studies, particularly to examine whether
renal, hepatic, central nervous system, or pancreatic changes are
present and if they have functional consequences for the treated
mice. e treated Mut
−/−
animals were also able to tolerate a
liberalized diet in the face of elevated circulating metabolites,
which were greatly diminished compared to the untreated Mut
−/−
group, but still signicantly increased compared to heterozygous
controls. e Mut
−/−
mice, both treated and untreated, did not
receive a precursor-restricted diet, commonly employed to treat
patients
31
that likely would have further decreased methylmalonic
acid levels in the treated animals. Complete restoration of plasma
metabolites to normal in the treated mice was not expected because
patients with MMA who have received replacement liver and kid-
ney combined transplantation procedures also display persistent
MMA and methylmalonic aciduria.
16,17
Also, there is no evidence
to suggest that free methylmalonic acid can be eciently metabo-
lized, even when delivered exogenously to a wild-type host.
Consistent with many previous studies, the rAAV8-mMut
vector produced persistent expression in the liver and muscle
that was readily detected at 90 days aer therapy at the mRNA
and protein level, and at 1 year through mRNA expression and
in vivo propionate oxidation. Because the mice were treated at
the time of birth, the rapid growth of the liver and subsequent
dilution in the number of transduced cells by cell division likely
explains the relative diminution of Mut expression over time.
32
e cohort of treated Mut
−/−
mice, which is >25, have survived
to 1 year and beyond, demonstrating that even low levels of Mut
expression are sucient to provide metabolic homeostasis, and
prevent morbidity and mortality. Furthermore, although formal
testing has not been performed, the mutant animals appeared
clinically well, with no obvious neurological or behavioral phe-
notypes. rAAV-based gene vectors have previously shown prom-
ising proof-of-principle correction in other mouse models of
metabolic disease
33
and now includes a pleiotropic disorder of
organic acid metabolism.
e hereditary MMAs, as well as other inborn errors of metab-
olism that lack conventional therapy, are included in routine new-
born screening panels used by many states and countries.
34
ere
has been a vigorous and public debate on the inclusion of these
disorders in the list of conditions for which screening is oered.
In this report, we have demonstrated that a single injection of
rAAV8-mMut was sucient to cure the lethal phenotype of Mut
deciency in a murine model of MMA that closely replicates the
human condition. Our studies are the rst to demonstrate that
MMA, and by extension other organic acidemias, might be treated
by gene therapy with a safe and eective vector. is conclusion
oers strong support for the continued and expanded screening of
infants for disorders of intermediary metabolism and to the appli-
cation of gene therapy to humans with MMA.
Materials and Methods
Murine model of MMA. e targeted Mut allele harbors a deletion of exon
3 in the Mut gene. is exon encodes the putative substrate-binding pocket
in the Mut enzyme. e Mut allele does not produce mature RNA, protein,
or enzymatic activity.
20
Mut
−/−
mice on a mixed (C57BL/6 × 129SV/Ev ×
FvBN) background exhibit a semipenetrant neonatal lethal phenotype with
most mice perishing in the early neonatal period.
21
Coat colors are variable
in these mice due to parental strain contributions. Mut
−/−
mice display
massively elevated methylmalonic acid concentrations in the plasma that
progressively rises to the 2 mmol/l range until death occurs. Mut
+/−
ani-
mals have biochemical parameters identical to Mut
+/+
wild-type animals
and were used as controls throughout.
rAAV8 construction, production, and delivery.e University of
Pennsylvania Vector Core provided the expression vector, p-AAV2-CI-
CB7-RBG. e vector contains transcriptional control elements from the
cytomegalovirus enhancer/chicken β-actin promoter, cloning sites for the
insertion of a complementary DNA, and the rabbit β-globin polyA signal.
35
Terminal repeats from AAV serotype 2 ank the expression cassette. Either
the murine Mut (mMut) or GFP was cloned into pAAV2/8.CI.CB7.RBG
and packaged into rAAV8, puried by cesium chloride centrifugation, and
titered by qPCR as previously described.
26
pAAV2/8.CI.CB7.EGFP.RBG
had a titer of 2.25 × 10
13
GC/ml and pAAV2/8.CI.CB7.mMut.RBG had a
titer of 4.13 × 10
13
GC/ml. Animal studies were reviewed and approved by
the National Human Genome Research Institute Animal User Committee.
Hepatic injections were performed on nonanesthetized neonatal mice,
typically within several hours aer birth. Viral particles were diluted to
a total volume of 20 microliters with phosphate-buered saline immedi-
ately before injection and were delivered into the liver parenchyma using a
32-gauge needle and transdermal approach, as previously described.
22
Quantitative real-time PCR. Total RNA was extracted using RNeasy Mini
Kit (Qiagen, Valencia, CA), and DNase digested was preformed using
DNA-free (Ambion, Austin, TX). qPCR was accomplished with TaqMan
gene expression assays [mouse GAPD (4352932E) and murine Mut
(Mm00485312_m1) from Applied Biosystems, Foster City, CA]. Samples
were analyzed in an Applied Biosystems 7500 fast real-time PCR system, in
accordance with the manufacturer’s protocol. All samples were analyzed in
triplicate. ree individual mouse tissue samples were used to determine
the 100% comparator Mut
+/−
Mut mRNA expression level.
Western blotting. Tissue samples were homogenized with a 2-ml Tenbroeck
tissue grinder (Wheaton, Millville, NJ) in T-PER (Pierce Biotechnology,
Rockford, IL) tissue protein extraction buer in the presence of Halt (Pierce
Biotechnology) protease inhibitor cocktail. Twenty micrograms of claried
extract were used in western analysis and probed with anity-puried, rab-
bit polyclonal antisera raised against the murine Mut enzyme.
23
Complex III
Core II was used as a loading control and was also detected by immunoblot-
ting [mouse monoclonal anti-OxPhos Complex III (ubiquinol- cytochrome
c oxidoreductase) Core II antibody, Invitrogen SKU# A-11143]. e anti-
mutase antibody was used at a dilution of 1:750, and the anti- Complex
III Core II antibody was used at a dilution of 1:2,000. Horseradish perox-
idase–conjugated anti-rabbit IgG (NA934; GE Healthcare Life Sciences,
Piscataway, NJ) or rabbit anti-goat IgG (sc-2768; Santa Cruz Biotechnology,
Santa Cruz, CA) was used as the secondary antibody and was visualized
with chemiluminescence detection (Pierce Biotechnology).
Metabolic studies. Plasma was isolated from blood collected by orbital
bleeding. e samples were immediately centrifuged, and the plasma
was removed, diluted in water, and stored at −80 °C in a screw-top tube
for later analysis. Methylmalonic acid was analyzed by gas chromatog-
raphy–mass spectrometry with stable isotopic internal calibration to
measure methylmalonic acid as previously described.
36,37
In vivo 1-
13
C-
propionate oxidation was determined by collecting expired gas from
6 www.moleculartherapy.org
© The American Society of Gene & Cell Therapy
Gene Therapy for Methylmalonic Acidemia
mutant, control, and treated mice aer the animals were injected by the
intraperitoneal route with 200 micrograms of 1-
13
C-sodium propionate,
using an adaptation of a method developed to study propionate oxida-
tion in patients with methylmalonic and propionic acidemia.
38
e mice
were placed into a respiratory chamber that contained a CO
2
probe to
allow the direct measurement of CO
2
generated by each animal. An ali-
quot of expired air was removed from the chamber at each time point
for analysis of
13
C enrichment in CO
2
. e isotope ratio (
13
C/
12
C) of the
expired gas was determined with a gas isotope ratio mass spectrometer
(Metabolic Solutions, Nashua, NH). e percent dose metabolized at
each time point was calculated as % dose metabolized = total
13
C excreted
(mmol/dose (mmol) × 100%).
Statistical analyses. In all instances, P values were considered signicant
if the value was <0.05. Dierences in the survival between treated groups
were analyzed using a χ
2
test. e weights between treated and untreated
mice, and dierences in metabolite levels were assessed using a two-sided,
two-tailed unpaired Student’s t- test. e Kruskal–Wallis test was used to
determine the statistical signicance in measured propionate oxidation
rates between groups.
ACKNOWLEDGMENTS
The Intramural Research Program of the National Human Genome
Research Institute (NHGRI), National Institutes of Health (NIH) supported
this research. We thank Christelle Samen; Chenghua Yang and Irene Ginty
(NHGRI/NIH) for the care and veterinary support of the mice; David M.
Bodine, IV for providing a critical reading of the manuscript; and University
of Pennsylvania, Vector Core for AAV preps and advice. Neal Oden (The
EMMES Corporation, Rockville, MD) assisted with statistical analyses.
REFERENCES
1. Fenton, WA, Gravel, RA and Rosenblatt, DS (2001). Disorders of propionate and
methylmalonate metabolism. In: Scriver, CR, Sly, WS, Childs, B, Beaudet AL, Valle, D,
Kinzler, KW et al. (eds). The Metabolic and Molecular Bases of Inherited Disease, 8th edn.
McGraw-Hill: New York. pp. 2165–2192.
2. Matsui, SM, Mahoney, MJ and Rosenberg, LE (1983). The natural history of the
inherited methylmalonic acidemias. N Engl J Med 308: 857–861.
3. van der Meer, SB, Poggi, F, Spada, M, Bonnefont, JP, Ogier, H, Hubert, P et al.
(1994). Clinical outcome of long-term management of patients with vitamin
B12-unresponsive methylmalonic acidemia. J Pediatr 125(6 Pt 1): 903–908.
4. Nicolaides, P, Leonard, J and Surtees, R (1998). Neurological outcome of
methylmalonic acidaemia. Arch Dis Child 78: 508–512.
5. de Baulny, HO, Benoist, JF, Rigal, O, Touati, G, Rabier, D and Saudubray, JM (2005).
Methylmalonic and propionic acidaemias: management and outcome. J Inherit
Metab Dis 28: 415–423.
6. Hörster, F, Baumgartner, MR, Viardot, C, Suormala, T, Burgard, P, Fowler, B et al.
(2007). Long-term outcome in methylmalonic acidurias is influenced by the
underlying defect (mut0, mut-, cblA, cblB). Pediatr Res 62: 225–230.
7. Fenton, WA and Rosenblatt, DS (2001). Inherited disorders of folate and cobalamin
transport and metabolism. In: Scriver, CR, Sly, WS, Childs, B, Beaudet AL, Valle, D,
Kinzler, KW et al. (eds). The Metabolic and Molecular Bases of Inherited Disease, 8th edn.
McGraw-Hill: New York. pp. 3897–3933.
8. Ando, T, Rasmussen, K, Wright, JM and Nyhan, WL (1972). Isolation and identification
of methylcitrate, a major metabolic product of propionate in patients with propionic
acidemia. J Biol Chem 247: 2200–2204.
9. Nyhan, WL, Gargus, JJ, Boyle, K, Selby, R and Koch, R (2002). Progressive neurologic
disability in methylmalonic acidemia despite transplantation of the liver. Eur J Pediatr
161: 377–379.
10. Chakrapani, A, Sivakumar, P, McKiernan, PJ and Leonard, JV (2002). Metabolic
stroke in methylmalonic acidemia five years after liver transplantation. J Pediatr
140: 261–263.
11. Kayler, LK, Merion, RM, Lee, S, Sung, RS, Punch, JD, Rudich, SM et al. (2002).
Long-term survival after liver transplantation in children with metabolic disorders.
Pediatr Transplant 6: 295–300.
12. Hsui, JY, Chien, YH, Chu, SY, Lu, FL, Chen, HL, Ho, MJ et al. (2003). Living-related
liver transplantation for methylmalonic acidemia: report of one case. Acta Paediatr
Taiwan 44: 171–173.
13. Kaplan, P, Ficicioglu, C, Mazur, AT, Palmieri, MJ and Berry, GT (2006). Liver
transplantation is not curative for methylmalonic acidopathy caused by
methylmalonyl-CoA mutase deficiency. Mol Genet Metab 88: 322–326.
14. Kasahara, M, Horikawa, R, Tagawa, M, Uemoto, S, Yokoyama, S, Shibata, Y et al.
(2006). Current role of liver transplantation for methylmalonic acidemia: a review
of the literature. Pediatr Transplant 10: 943–947.
15. Morioka, D, Kasahara, M, Horikawa, R, Yokoyama, S, Fukuda, A and Nakagawa, A
(2007). Efficacy of living donor liver transplantation for patients with methylmalonic
acidemia. Am J Transplant 7: 2782–2787.
16. van’t Hoff, WG, Dixon, M, Taylor, J, Mistry, P, Rolles, K, Rees, L et al. (1998).
Combined liver-kidney transplantation in methylmalonic acidemia. J Pediatr
132: 1043–1044.
17. Nagarajan, S, Enns, GM, Millan, MT, Winter, S and Sarwal, MM (2005). Management
of methylmalonic acidaemia by combined liver-kidney transplantation. J Inherit Metab
Dis 28: 517–524.
18. Meyburg, J and Hoffmann, GF (2005). Liver transplantation for inborn errors of
metabolism. Transplantation 80(1 suppl.): S135–S137.
19. Leonard, JV, Walter, JH and McKiernan, PJ (2001). The management of organic
acidaemias: the role of transplantation. J Inherit Metab Dis 24: 309–311.
20. Chandler, RJ, Sloan, J, Fu, H, Tsai, M, Stabler, S, Allen, R et al. (2007). Metabolic
phenotype of methylmalonic acidemia in mice and humans: the role of skeletal
muscle. BMC Med Genet 8: 64.
21. Chandler, RJ, Zerfas, PM, Shanske, S, Sloan, J, Hoffmann, V, DiMauro, S et al.
(2009). Mitochondrial dysfunction in mut methylmalonic acidemia. FASEB J 23:
1252–1261.
22. Chandler, RJ and Venditti, CP (2008). Adenovirus-mediated gene delivery rescues a
neonatal lethal murine model of mut(0) methylmalonic acidemia. Hum Gene Ther
19: 53–60.
23. Chandler, RJ, Tsai, MS, Dorko, K, Sloan, J, Korson, M, Freeman, R et al. (2007).
Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine
fibroblasts and human hepatocytes. BMC Med Genet 8: 24.
24. Mueller, C and Flotte, TR (2008). Clinical gene therapy using recombinant
adeno-associated virus vectors. Gene Ther 15: 858–863.
25. Wu, Z, Asokan, A and Samulski, RJ (2006). Adeno-associated virus serotypes: vector
toolkit for human gene therapy. Mol Ther 14: 316–327.
26. Gao, GP, Alvira, MR, Wang, L, Calcedo, R, Johnston, J and Wilson, JM (2002). Novel
adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.
Proc Natl Acad Sci USA 99: 11854–11859.
27. Sarkar, R, Tetreault, R, Gao, G, Wang, L, Bell, P, Chandler, R et al. (2004). Total
correction of hemophilia A mice with canine FVIII using an AAV 8 serotype. Blood
103: 1253–1260.
28. Inagaki, K, Piao, C, Kotchey, NM, Wu, X and Nakai, H (2008). Frequency and
spectrum of genomic integration of recombinant adeno-associated virus serotype 8
vector in neonatal mouse liver. J Virol 82: 9513–9524.
29. Louboutin, JP, Wang, L and Wilson, JM (2005). Gene transfer into skeletal muscle
using novel AAV serotypes. J Gene Med 7: 442–451.
30. Wang, Z, Zhu, T, Qiao, C, Zhou, L, Wang, B, Zhang, J et al. (2005). Adeno-associated
virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol 23:
321–328.
31. Touati, G, Valayannopoulos, V, Mention, K, de Lonlay, P, Jouvet, P, Depondt, E et al.
(2006). Methylmalonic and propionic acidurias: management without or with
a few supplements of specific amino acid mixture. J Inherit Metab Dis 29:
288–298.
32. Cunningham, SC, Dane, AP, Spinoulas, A, Logan, GJ and Alexander, IE (2008).
Gene delivery to the juvenile mouse liver using AAV2/8 vectors. Mol Ther 16:
1081–1088.
33. Alexander, IE, Cunningham, SC, Logan, GJ and Christodoulou, J (2008). Potential of
AAV vectors in the treatment of metabolic disease. Gene Ther 15: 831–839.
34. Green, NS, Rinaldo, P, Brower, A, Boyle, C, Dougherty, D, Lloyd-Puryear, M et al.;
Advisory Committee on Heritable Disorders and Genetic Diseases in Newborns and
Children (2007). Committee Report: advancing the current recommended panel of
conditions for newborn screening. Genet Med 9: 792–796.
35. Daly, TM, Okuyama, T, Vogler, C, Haskins, ME, Muzyczka, N and Sands, MS (1999).
Neonatal intramuscular injection with recombinant adeno-associated virus results in
prolonged beta-glucuronidase expression in situ and correction of liver pathology in
mucopolysaccharidosis type VII mice. Hum Gene Ther 10: 85–94.
36. Marcell, PD, Stabler, SP, Podell, ER and Allen, RH (1985). Quantitation of
methylmalonic acid and other dicarboxylic acids in normal serum and urine using
capillary gas chromatography-mass spectrometry. Anal Biochem 150: 58–66.
37. Allen, RH, Stabler, SP, Savage, DG and Lindenbaum, J (1993). Elevation of
2-methylcitric acid I and II levels in serum, urine, and cerebrospinal fluid of patients
with cobalamin deficiency. Metabolism 42: 978–988.
38. Barshop, BA, Yoshida, I, Ajami, A, Sweetman, L, Wolff, JA, Sweetman, FR et al. (1991).
Metabolism of 1-13C-propionate in vivo in patients with disorders of propionate
metabolism. Pediatr Res 30: 15–22.
    • "Proof of principle for a nonsense read-through therapy in PA [219] and for chaperone therapy in MMA [220] has been achieved in cellular models. Successful gene therapy has been reported for adenoassociated viral gene delivery in the lethal Mut -/-mouse model221222223. No clinical trial has been performed in humans so far. From a pathophysiological point of view the use of antioxidants to reduce oxidative stress may be indicated in MMA and PA224225226227228229230231 . "
    [Show abstract] [Hide abstract] ABSTRACT: Methylmalonic and propionic acidemia (MMA/PA) are inborn errors of metabolism characterized by accumulation of propionic acid and/or methylmalonic acid due to deficiency of methylmalonyl-CoA mutase (MUT) or propionyl-CoA carboxylase (PCC). MMA has an estimated incidence of ~ 1: 50,000 and PA of ~ 1:100'000 -150,000. Patients present either shortly after birth with acute deterioration, metabolic acidosis and hyperammonemia or later at any age with a more heterogeneous clinical picture, leading to early death or to severe neurological handicap in many survivors. Mental outcome tends to be worse in PA and late complications include chronic kidney disease almost exclusively in MMA and cardiomyopathy mainly in PA. Except for vitamin B12 responsive forms of MMA the outcome remains poor despite the existence of apparently effective therapy with a low protein diet and carnitine. This may be related to under recognition and delayed diagnosis due to nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity.
    Full-text · Article · Sep 2014
    • "Neither the elimination of transgene expression nor the impaired mitochondrial importation and processing of the human MUT protein is suggested by our studies. The fact that circulating metabolite concentrations and in vivo propionate oxidative capacity in the AAV treated Mut −/− mice is almost exactly what we previously observed in studies using AAVs that expressed the murine cDNA provide support for this claim [5,6]. While the presence of antibodies against MUT was not directly investigated, the prolonged survival after treatment suggests the absence of an immune response. "
    [Show abstract] [Hide abstract] ABSTRACT: We demonstrate that human methylmalonyl-CoA mutase (MUT), delivered using an AAV serotype 8 vector, rescues the lethal phenotype displayed by mice with MMA and provides long-term phenotypic correction. In addition to defining a lower limit of effective dosing, our studies establish that neither a species barrier to mitochondrial processing nor an apparent immune response to MUT limits the murine model as an experimental platform to test the efficacy of human gene therapy vectors for MMA. Published by Elsevier Inc.
    Full-text · Article · Sep 2012
    • "Nonintegrating vectors based on adenovirus or adenoassociated virus (AAV) have shown high transduction efficiency in neonatal gene transfer; however, both vector systems suffer significant loss of transgene expression because of dilution of vector genome during rapid cell proliferation, especially in liver (Wang et al., 2005; Cunningham et al., 2008; Hu et al., 2011 ). Despite the reduction of transgene expression levels and vector genome copies, therapeutic effects or long-term rescue of neonatal lethality have been demonstrated in several animal models (Daly et al., 2001; CarrilloCarrasco et al., 2010; Chandler and Venditti, 2010; Yiu et al., 2010; Chandler et al., 2011; Cotugno et al., 2011; Hu et al., 2011). In these cases, expression of the normal gene in a small percentage of stably transduced cells accounts for the clinical effects. "
    [Show abstract] [Hide abstract] ABSTRACT: For genetic diseases that manifest at a young age with irreversible consequences, early treatment is critical and essential. Neonatal gene therapy has the advantages of achieving therapeutic effects before disease manifestation, a low vector requirement and high vector-to-cell ratio, and a relatively immature immune system. Therapeutic effects or long-term rescue of neonatal lethality have been demonstrated in several animal models. However, vigorous cell proliferation in the newborn stage is a significant challenge for nonintegrating vectors, such as adeno-associated viral (AAV) vector. Slightly delaying the injection age, and readministration at a later time, are two of the alternative strategies to solve this problem. In this study, we demonstrated robust and efficient hepatic gene transfer by self-complementary AAV8 vector in neonatal mice. However, transduction quickly decreased over a few weeks because of vector dilution caused by fast proliferation. Delaying the injection age improved sustained expression, although it also increased neutralizing antibody (NAb) responses to AAV capsid. This approach can be used to treat genetic diseases with slow progression. For genetic diseases with early onset and severe consequences, early treatment is essential. A second injection of vector of a different serotype at a later time may overcome preexisting NAb and achieve sustained therapeutic effects.
    Full-text · Article · Nov 2011
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