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(Received for publication, September 19, 1995; and in revised form, November 22, 1995) From the
A murine model of ornithine transcarbamylase (OTC) deficiency
was used in this study to evaluate the efficacy of recombinant
adenoviruses for correcting the metabolic defect in liver. Recombinant
adenoviruses deleted in E1 and containing a human OTC cDNA expressed
little functional OTC enzyme in vivo and had no observable
impact on the underlying metabolic abnormalities of the OTC-deficient
mouse (i.e. elevated urinary orotate and serum glutamine).
E1-deleted vectors were improved through the use of the strong
constitutive promoter from cytomegalovirus driving the normal murine
homolog of OTC cDNA and the ablation of E2a with a
temperature-sensitive mutation. Infusion of this improved vector into
the mouse model was associated with a complete normalization of liver
OTC enzyme activity that persisted for at least 2 months with complete
but transient correction in serum glutamine and urine orotic acid.
These studies illustrate the utility of improved adenoviral vectors in
the treatment of liver metabolic disease. A deficiency of ornithine transcarbamylase (OTC) ( Murine models of OTC
deficiency are available for the development and evaluation of in
vivo liver-directed gene therapies. The best characterized model
is the sparse fur (spf) mouse, in which a missense mutation in
codon 117 of the OTC gene leads to a functionally defective enzyme with
hepatic OTC activity reduced to approximately 5-20% of wild-type
levels at physiologic pH(7, 8) . The other mutant is
the spf Recombinant adenoviruses have been evaluated as vectors for
liver-directed gene therapy in a variety of metabolic disorders
including OTC
deficiency(12, 13, 14, 15) .
Adenovirus is rendered defective for use as a vector by deleting the
immediate early genes E1a and E1b and incorporating a minigene
expressing the therapeutic protein. Adenovirus is efficiently targeted
to hepatocytes in vivo following intravenous infusion; high
level transgene expression can be achieved in virtually 100% of
hepatocytes, most of which are fully differentiated and not dividing.
The first use of E1-deleted viruses for gene therapy was in newborn spf Experiments of adenovirus-mediated gene transfer to liver
have not been as encouraging when performed in other species or in
adult mice. Gene transfer to liver is similarly efficient in these
experimental models; however, transgene expression is transient, often
lasting less than 14-21 days, and associated with substantial
hepatitis(13, 16, 17, 18) . We
believe this is due, in part, to destructive cellular immune responses
to the genetically corrected hepatocytes of the adult animal. This does
not occur in the newborn mouse, who is exposed to gene therapy prior to
immunologic maturity when tolerance can be induced. Deletion of E1a and
E1b is insufficient to prevent either expression of other viral genes
or actual replication at high multiplicities of
infection(18, 19) . A concerted immune response to
exogenous and endogenously produced viral protein (or transgene
product) ensues. This results in activation of both CD4 (T helper) and
CD8 (cytotoxic T) lymphocytes against the virus-infected cell and
extinction of transgene expression by either destruction of the cell or
through other indirect mechanisms(18, 20) . This is
particularly problematic for treatment of humans when the window for
inducing tolerance is in the prenatal period of 14-18 weeks
gestation(21) . We describe in this report the use of
adenoviral vectors for treatment of OTC deficiency in the previously
described murine models of the human disease. Use of a sufficiently
strong promoter with a species homologous OTC cDNA was important in
achieving curative therapeutic gene expression. An E1-deleted
adenoviral vector, made temperature-sensitive in the E2a gene,
normalized hepatic activity of OTC in the spf/y mouse and
completely corrected metabolic abnormalities in urinary orotate and
serum glutamine.
Figure 1:
Recombinant adenoviral
vectors. Diagrammatic vector map was not drawn to scale. Nomenclature
of adenoviral vectors is described in (32) .
In Vivo
Delivery of Recombinant Adenoviruses to Mouse Liver-spf/y, spf
An
E1-deleted adenoviral vector was constructed that contains human OTC
cDNA (referred to as a first generation virus) expressed from a
CMV-enhanced
Figure 2:
Cytochemical demonstration of OTC activity
in liver tissues from spf mice infused with recombinant
adenoviruses. spf/y mice infused with 5
Figure 3:
Evaluation of pathological responses of
the recipient mouse liver to recombinant adenovirus. Pathological
response in mice receiving human OTC viruses (A) or mouse OTC
viruses (B). Liver tissues were harvested at indicated time
points following infusion of first generation (H5.010CBhOTC or
H5.010CMVmOTC, light hatched boxes) or second
generation (H5.110CBhOTC or H5.110CMVmOTC, heavy
hatched boxes) recombinant adenovirus (5
Figure 4:
Urinary orotate excretion and plasma
glutamine levels in spf mice infused with recombinant
adenoviruses carrying human OTC cDNA. A and B, urine
orotate in mice infused with first and second generation recombinant
virus. C, plasma glutamine in mice infused with the second
generation virus. spf/y mice at 6-8 weeks of age were
infused with 5
The
relatively poor performance of the E1-deleted vector was felt to be, in
part, due to the inherent immunogenicity of first generation
constructs. We described in other systems that E1-deleted viruses
express viral genes whose proteins are targets for destructive cellular
immune responses(13, 18, 20, 31) .
Our first attempt to improve E1-deleted adenoviral vectors, by
inactivating the essential gene product of E2a with a
temperature-sensitive mutation, has shown promise in mouse liver, and
mouse, rat, and primate lung, using lacZ-containing
constructs(19, 31, 32, 33) . In each
case, expression of the transgene is prolonged for a variable period of
time and associated with diminished inflammation. A second generation
vector was constructed that was deleted in E1, defective in E2a due to
the ts125 mutation, and contained a human OTC cDNA minigene. As
expected, the levels of viral late gene RNA (Fig. 5A, lanes 1 and 2) and protein (Fig. 6, A and B) are diminished over that observed with the
E1-deleted virus; the associated hepatitis is also decreased (Fig. 3A). Expression of the human OTC cDNA is still
low; however, it appears slightly more stable (Fig. 2, G-I) from what is observed with the E1-deleted virus (Fig. 2, D-F). A nonspecific decrease (
Figure 5:
RNA blot analysis of liver tissues from spf mice infused with recombinant adenoviruses. spf/y
mice were infused with 2
Figure 6:
Evaluation of viral late gene expression
in liver tissue from spf mice infused with recombinant
adenoviruses. Liver tissues of spf/y mice infused with 2
First
generation vectors that differed by the promoter, the OTC cDNA, or both
were constructed and infused into spf mice. Liver tissue was
harvested 3 days later and analyzed for OTC activity by lysate enzyme
analysis (Fig. 7). The original vector expressing human OTC from
the CMV/
Figure 7:
Liver OTC activity in spf mice
infused with recombinant adenovirus. spf/y mice were infused
with 2
Figure 8:
Urinary orotate excretion and plasma
glutamine levels in spf and spf
Experiments were repeated with the second
generation virus in which the ts125 mutation in E2a was introduced into
the E1-deleted vector containing the mouse OTC minigene. This vector
performed substantially better in all categories in comparison to the
corresponding first generation construct. Expression of OTC enzyme as
measured by the histochemical stain was higher (Fig. 2M) and prolonged (Fig. 2, N and O). Inflammation was reduced (Fig. 3B), as was
expression of late viral genes at the level of RNA (Fig. 5A, lanes 3 and 4) and protein (Fig. 6, C and D). Abnormalities in urine
orotate and glutamine were completely normalized within 2-3 weeks
of gene transfer; both metabolic parameters gradually returned to
baseline levels but remained significantly improved as compared to
animals infused with lacZ virus for 2-3 months (Fig. 8A and B). A similar correction of urine
orotate and serum glutamine was achieved in the other murine model of
OTC deficiency, the spf
Our study demonstrates complete correction of orotic acid
overproduction following hepatic reconstitution of OTC in both the spf and spf Despite
normalizing serum glutamine and urine orotic acid, adenovirus-mediated
gene transfer did not significantly impact on depleted serum citrulline
(data not shown). This is consistent with the experience of orthotopic
liver transplantation in carbamyl-phosphate synthetase and OTC
deficiencies that all serum amino acids are corrected except citrulline
which remains low(6, 36) . One explanation is that
serum citrulline is primarily contributed to by extrahepatic nitrogen
ureagenesis which is not corrected using liver-directed approaches.
Through the action of a partial urea cycle in gut, including OTC,
glutamine nitrogen is converted to citrulline which is transported via
the blood to kidney, where it is converted to arginine. The role of
this extrahepatic pathway in humans appears minor as suggested by the
observation that transplant recipients realize substantially improved
tolerance to nitrogen challenges despite maintaining low serum
citrulline levels(36) .
Previous studies have clearly
implicated cellular immunity in the loss of transgene expression and
associated inflammation that has characterized E1-deleted
adenoviruses(18, 19, 20, 37) . Both
T helper cells of the T Characterization of the performance of adenoviral vectors containing
normal mouse OTC cDNA provides an opportunity to evaluate directly the
relative contribution of viral protein versus transgene
product in eliciting destabilizing cellular immunity. This is the first
example in which adenovirus-mediated gene transfer has been performed
with a transgene whose product should not be viewed as a neoantigen;
transgene-derived OTC differs by only one amino acid from the spf protein and is identical with the product of the spf Our
data in the authentic animal models of OTC deficiency support the
utility of recombinant adenoviruses for treating liver metabolic
diseases. Complete and prolonged correction of the metabolic defect has
been demonstrated following a single infusion of purified virus.
Current limitations of the technology are the potential of CTL
responses to residual expressed viral protein and possibly to OTC in
patients with null mutations. In addition, previous studies have
described the development of neutralizing antibodies to input viral
proteins that block gene transfer upon second
administration(13) . Improved vectors may diminish CTL to viral
protein but will have no effect on neutralizing antibody to capsid
protein or CTL to OTC. Activation of CD4 T helper cells to input viral
proteins is necessary for both B cell and CTL activation(37) .
Transient blockade of the initial CD4 T cell activation at the time of
virus instillation has been shown to both prolong transgene expression
and prevent formation of neutralizing antibody(39) . This
suggests a strategy for treating OTC deficiency based on
coadministration of an immune modulator together with an improved
recombinant adenovirus.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3639-3646
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS and DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is
associated with derangements in nitrogen metabolism leading to
hyperammonemic encephalopathy in humans. This X-linked recessive
disorder is the most common inborn error of urea synthesis, with an
estimated prevalence of 1:40,000 to 1:80,000 births(1) .
Approximately one-half of affected males develop marked elevations of
ammonia leading to coma in the 1st week of life. These episodes are
associated with 50% mortality(2) . Survivors of the neonatal
crisis often experience recurrent episodes of potentially
life-threatening hyperammonemia that are precipitated by excessive
protein intake or catabolic stress(3) . Since the urea cycle is
principally localized to the liver, gene therapy directed to
hepatocytes has the potential to correct the underlying metabolic
derangements. The success of orthotopic liver transplantation in this
disease indicates that hepatocyte-directed gene transfer should be
sufficient for metabolic
correction(4, 5, 6) . (abnormal skin and hair)
mouse, in which a point mutation in the final base of exon 4 of the OTC
gene leads to aberrant splicing with markedly reduced levels of OTC
mRNA and only 5% of normal OTC activity(9, 10) . In
these two mouse strains, hemizygous male and homozygous female pups are
runted and have wrinkled skin with little to no fur early in
development. On a normal diet, adult spf or spf
hemizygotes develop symptomatic
hyperammonemia, glutaminemia and severe orotic
aciduria(8, 9) , essentially identical with the
findings in affected humans. They also have a markedly shortened
lifespan and behavioral and learning abnormalities(11) .
mice(14) . Infusion of a
vector containing a rat OTC cDNA into the newborn animals led to an
increase in hepatic OTC activity in 4/15 mice which persisted for
1-2 months and was associated with decreased urinary orotic acid
excretion.
Animals
The mouse strains spf, spf, and C3HeB/J were purchased from The Jackson
Laboratory (Bar Harbor, ME) and maintained in the Wistar animal
facility. Female heterozygous spf/x or spf
/x mice were bred with normal male (x/y) C3HeB/J
mice to generate experimental spf/y or spf
/y mice. All animals used in the experiment were
between 6 and 10 weeks of age.
Construction and Propagation of the First and the Second
Generation Recombinant Adenoviruses
A summary of recombinant
adenoviruses used in this study is provided in Fig. 1.
Nomenclature used for naming the vectors was described in (32) . E1-deleted first generation recombinant adenovirus,
designated H5.010CBhOTC, was derived from a retroviral vector
containing a human OTC minigene. The coding sequence for human OTC was
removed from plasmid pHO-731 on a 1.0-kilobase HinfI
fragment(22) , blunted with Klenow, ligated with BclI
linkers, and cloned in direct orientation into the BamHI site
of the retroviral vector pgagBA(23) . A XhoI to NheI restriction fragment from this vector containing the
chicken 
-actin promoter, human OTC cDNA, 3`-untranslated sequences
including 130 base pairs of retroviral sequence was cloned in place of lacZ in the adenoviral vector
pAd.CMVlacZ(13) . This new plasmid, called
pAd.CBhOTC, expresses human OTC cDNA from a CMV-enhanced
-actin promoter and is deleted of adenoviral sequences spanning 1
to 9.6 map units. In preparation for production of virus,
pAd.CBhOTC was linearized with NheI and transfected
into 293 cells with ClaI/XbaI-restricted sub360
genomic DNA, which contains a substitution of sequence in
E3b(24) . The resulting recombinants (H5.010CBhOTC)
were grown and purified through three rounds of plaque isolations.
H5.110CBhOTC differs from H5.010CBhOTC only by a
single base pair substitution in the E2a gene which generates a
temperature-sensitive viral DNA-binding protein capable of growth at 32
°C but not 39 °C. The mutation contained within this
recombinant adenovirus was generated from the wild-type Ad5 mutant
strain H5.ts125(25) . Mouse OTC cDNA was generated by reverse
transcription-polymerase chain reaction, cloned into pGEM-T vector
(Promega), and restricted with SpeI and SacII. A
1.5-kilobase fragment containing mouse cDNA was isolated, blunted, and
cloned into the EcoRV site of an adenoviral vector
pAd.CMV-link (A CMV promoter-polylinker cassette was cloned into a
plasmid containing the adenoviral sequences 0 to 16 map units deleted
of E1a and E1b as described in the other adenovirus vectors). The new
plasmid, designated pAd.CMVmOTC, was linearized with EcoRI and cotransfected into 293 cells with ClaI/XbaI-restricted sub360 or sub360/ts125 DNA. The
resulting recombinant virus (H5.010CMVmOTC or
H5.110CMVmOTC) was purified through three rounds of plaque
isolation. The integrity of the murine OTC cDNA was confirmed by DNA
sequence analysis.
OTC Lysate Assay
Cells were harvested by scraping
into mitochondria lysis buffer (0.5% Triton, 10 mM Hepes, pH
7.4, 2 mM dithiothreitol), and total protein was extracted by
three freeze-thaw cycles. Liver tissue was homogenized in mitochondria
lysis buffer with a Polytron homogenizer. The homogenate was
centrifuged in a Microfuge at the maximum speed for 5 min, the
supernatant was transferred to a new tube, and OTC enzyme activity was
measured as described by Lee and Nussbaum (26) with
modifications. Briefly, 2-10 µg of total cellular protein was
added to 700 µl of reaction mixture (5 mM ornithine, 15
mM carbamyl phosphate, and 270 mM triethanolamine, pH
7.7) which was incubated at 37 °C for 30 min. Reactions were
stopped by adding 250 µl of 3:1 phosphoric acid/sulfuric acid (by
volume). Citrulline production was then determined by adding 50 µl
of 3% 2,3-butanedione monoxime, incubating at 95-100 °C in
the dark for 15 min, and measuring absorbance at 490 nm.OTC Histochemistry
Slides of liver tissue, less
than 3 mm thick, were fixed in 4% paraformaldehyde in PBS for 4-6
h at room temperature and subsequently washed with PBS containing 10%
sucrose for 2 h, PBS containing 20% sucrose for 2 h, and finally PBS
containing 30% sucrose overnight. The liver slides were then embedded
in OCT and sectioned for histochemical staining as described by
Mizutani(27) . Briefly, a reaction medium was prepared first,
which contained 12 mg of carbamyl phosphate, lithium salt; 20 mg of L-ornithine dihydrochloride, 3.2 g of sucrose; 20 ml of 0.05 M triethanolamine buffer, pH 7.2; 16 ml of distilled water,
and 4 ml of 1% lead nitrate. The lead nitrate solution was added
dropwise with continuous stirring, and the solution was readjusted to
pH 7.2 with 1 N NaOH. The slightly turbid substrate mixture
was filtered and used immediately. Sections were incubated for 30 min
in reaction medium at room temperature, washed with distilled water
three times, immersed in 0.37% ammonium sulfide for 1 min, rinsed with
distilled water again, and mounted for light microscopic observations.
The dark brown deposits of lead sulfide indicated the sites of OTC
activity.RNA Hybridization Analyses
Total cellular RNA was
isolated, fractionated on formaldehyde gel, and transferred onto
Hybond-N nylon filters (Amersham). DNA fragments used as probes in RNA
hybridizations were gel-purified and labeled with
[
-P]dCTP by random priming.
/y, and male C3HeB/J mice at 6-10 weeks
of age were used in this study. Blood samples were collected by
retro-orbital bleeding the day before the experiment (day -1).
Urine samples were collected at day -3 and day -1. On day
0, virus suspended in 0.1 ml of phosphate-buffered saline (PBS) was
administered to animals via the tail vein. Urine and plasma samples
were collected at weekly intervals after viral infusion. The animals
were sacrificed at day 4, 7, 14, or 28, depending on the experimental
protocol. Liver tissues were prepared for histochemical, biochemical,
and molecular biological analysis.
Immunocytochemical Analysis
Immunofluorescence
staining of adenoviral late gene products was performed as described by
Kozarsky et al.(13) . The primary antibody was a
polyclonal rabbit antibody specific to Ad5 late gene products (produced
in Dr. Wilson's laboratory). The secondary antibody was a
fluorescein isothiocyanate-labeled goat anti-rabbit IgG (Chemicon,
Temecula, CA).Determination of Urinary Orotate
Mouse urine was
collected at day -3, day -1, and at different time points
after virus infusion by leaving the animals in a metabolic cage
overnight. Urinary orotic acid levels were measured in duplicate for
each sample according to Brusilow et al.(28) .Determination of Plasma Amino Acids
Plasma amino
acids were analyzed by precolumn derivatization with o-phthaldialdehyde as described previously by Robinson et
al.(29) . After centrifugation of heparinized blood, an
aliquot of plasma was immediately precipitated with an equal volume of
0.8 N perchloric acid which contained the internal standards L--aminoadipate and L--amino-n-butyric acid. After centrifugation,
an aliquot of the supernatants was neutralized with 2 M KHCO
. These samples were derivatized using an
Autosampler. External standards were injected after every fifth
specimen.Evaluation of Liver Pathology
Histopathology was
evaluated using the criteria developed by Knodell et
al.(30) . The three criteria scored were: I, periportal
and bridging necrosis; II, intralobular degeneration and focal
necrosis; and III, portal inflammation. Analyses were performed on
three to four animals per time point.
Ineffective Genetic Reconstitution in spf Mice Using
Adenoviral Vectors Containing Human OTC
The spf and spf mice are well described animal models for
human OTC deficiency we have used to develop and evaluate adenoviral
vectors for liver-directed gene
therapy(7, 8, 9, 10) . In the two
available models, point mutations lead to dysfunctional or reduced OTC
enzyme protein resulting in metabolic and clinical abnormalities
consistent with a partial deficiency in humans. The model used
primarily in this study, the spf mouse, has an 85% reduction
in liver OTC activity which leads to an activation of de novo pyrimidine synthesis as evidenced by a 13-fold increase in
excretion of orotic acid in the urine(11) . Nitrogen
accumulates in these animals, as evidenced by a 60% increase in serum
glutamine. Conversion of
NH
to urea is
decreased to 50% of control, and the level of serum citrulline, a urea
cycle intermediate, is decreased to 25% of control(11) .-actin promoter. Pilot experiments were performed to
determine the dose of virus necessary to increase OTC enzyme activity
significantly in liver when vector was infused into spf mice.
We found that the maximally tolerated dose of a first generation virus
containing human OTC (i.e. 5 
10 particles) resulted in only a modest increase in OTC activity
over baseline. This represents 5-fold more virus than what is necessary
to transduce >80% of hepatocytes based on experiments with similar
vectors expressing a variety of reporter
genes(13, 15, 31) . A histochemical stain for
OTC activity was used to better characterize the distribution and level
of OTC expression (Fig. 2). The specificity of this assay was
demonstrated in analyses of C3H animals (Fig. 2A),
which show a dark brown reaction product in 100% of cells that was
absent in spf hemizygotes (Fig. 2C);
heterozygotes show two populations of OTC-expressing cells consistent
with lyonization of the x-chromosome (Fig. 2B).
Histochemical analysis of spf liver removed 4 days after
infusion of 5
10 particles of first generation
human OTC vector was underwhelming with expression detected at low
levels in most cells (Fig. 2D), diminishing to baseline
by days 7-14 (Fig. 2, E and F),
concurrent with the development of substantial but self-limited
hepatitis (Fig. 3A). Not surprising, there was no
significant change in either urinary orotate (Fig. 4A)
or serum glutamine (data not shown) when compared to animals that
received identical doses of lacZ virus; urinary orotate
nonspecifically decreased to approximately 50% of pretreatment levels
with both viruses, possibly due to the associated hepatitis.
10 particles of first generation H5.010CBhOTC (D, E, and F) or second generation H5.110CBhOTC (G, H, and I) human OTC-based
recombinant adenovirus were sacrificed at day 4 (D and G), 7 (E and H), and 14 (F and I) postinfusion. spf/y mice infused with 2
10 particles of first generation H5.010CMVmOTC (J, K, and L) or second generation
H5.110CMVmOTC (M, N, and O) mouse
OTC-based recombinant adenovirus were sacrificed at day 7 (J and M), 14 (K and N), and 28 (L and O). Liver tissues were analyzed for OTC activity by
histochemical staining. Liver sections from uninfected C3HeB/J (A), heterozygous spf/+ (B), and
hemizygous spf/y (C) mice were also stained as
controls. Representative photomicrographs are presented. Magnification
100.
10 particle/mouse in A and 1
10 particle/mouse in B) and evaluated for evidence of
histopathology by light microscopic inspection of paraffin sections
stained with hematoxylin and eosin. The pathological responses were
characterized in three categories: I, periportal and bridging
necrosis; II, intralobular degeneration and focal necrosis;
and III, portal inflammation. The severity in each category
was quantified by the Knodell score system. The histogram shown is the
average of at least three independent observations with S.E. shown as error bars.
10 particles of first generation
viruses (H5.010CBhOTC or H5.010CMVlacZ) or second
generation viruses (H5.110CBhOTC or H5.110CBlacZ) through tail vein. Urine and plasma samples were collected the day
before the virus infusion, and at day 4, 7, and 14 postinfusion.
Urinary orotic acid levels were measured in duplicate for each sample.
Urinary orotate/mg of creatinine are presented as a percent of
pretreatment levels and are the mean ± S.E. of at least 6
determinations. Plasma glutamine levels were determined as described
previously(29) . The levels are presented as a percent of
pretreatment levels and are the mean ± S.E. of between 4 and 10
determinations.
50% of
pretreatment levels) in urine orotate (Fig. 4B) and
nonsignificant increase in serum glutamine (Fig. 4C)
were again found in animals treated with either the lacZ or
OTC virus. We concluded from these experiments that E1-deleted
adenoviral vectors containing human OTC cDNA driven by the CMV-enhanced
-actin promoter were inadequate for gene therapy in the spf mouse, and the benefit of incorporating the ts125 mutation was
minimal.
10 particles of the first
generation (H5.010CBhOTC or H5.010CMVmOTC) or second
generation (H5.110CBhOTC or H5.110CMVmOTC)
recombinant adenovirus. At day 4 postinfusion, total liver RNA (10
µg) was isolated, fractionated in denaturing formaldehyde-agarose
gels, transferred to nylon filter, and hybridized with probes specific
to the later viral gene product hexon (A) or DNA-binding
protein (B). Lanes 1 and 2, liver RNA from spf/y mouse received first and second generation human OTC
virus. Lanes 3 and 4, liver RNA from spf/y
mouse received first and second generation mouse OTC virus. Lane
5, RNA from untreated spf liver. The intensity of
ribosomal RNA (18 S and 28 S) was similar in each lane (C),
indicating equivalent quantities of electrophoresed
RNA.
10 particles of recombinant virus were harvested 4
days later. Fresh frozen sections (6 µm) were fixed in 100%
methanol for 10 min and analyzed for viral late gene expression by
immunofluorescence using an antibody specific to viral late gene
products. Representative sections are presented. CMV/
-actin-driven
human OTC cDNA constructs, first generation (A) and second
generation (B). CMV-driven mouse OTC cDNA constructs, first
generation (C) and second generation (D).
Magnification 200.
Efficient Expression of OTC from Adenoviral Vectors with
Strong Constitutive Promoters and Species-homologous cDNAs
The
most striking aspect of the studies presented above is the paucity of
OTC expression achieved with the human OTC-based vectors and the lack
of enhanced performance in vectors containing the ts125 mutation. This
suggested problems with the human OTC cDNA functioning in the mouse
model. Direct sequence analysis of viral DNA confirmed the structure of
the human OTC minigene ruling out the trivial explanation of mutation
or rearrangement (data not shown). Analysis of RNA from liver of
treated animals revealed recombinant derived human OTC mRNA in excess
of the level of endogenous OTC transcript found in normal human liver
suggesting possible post-transcriptional inefficiencies (data not
shown). We hypothesized that greater expression of the therapeutic
protein could be achieved in a vector that contains a stronger promoter
and uses isogenic OTC cDNA. Differences in amino acid sequence between
the OTC murine and human homologs may affect the ability of the human
enzyme to fold, oligomerize, and/or traffic to the mitochondria where
it can function. In fact, 10 of the 26 amino acid differences present
between mouse and human OTC are located in the leader sequence. In
addition, the human protein may be immunogenic in the mouse. To
evaluate these hypotheses, first and second generation viruses were
constructed in which human OTC cDNA was replaced with mouse OTC cDNA,
and the CMV enhancer/chicken -actin promoter (CB) was replaced
with the strong constitutive viral enhancer/promoter from CMV.-actin promoter produced little enzyme activity above
background in spf liver. A 3-fold increase in activity was
achieved when the CMV promoter/enhancer was used to express the human
OTC cDNA. An additional 2- to 3-fold increase was realized when the
human OTC cDNA was replaced with the murine homolog in the CMV-based
vector.
10 particles of recombinant adenovirus and
sacrificed at day 3 postinfusion. Data are presented as OTC activity
(µmol of citrulline/mg of protein/h) in mice (spf or C3H)
infused with a variety of vectors. OTC activity in liver tissue was
determined as described under ``Experimental
Procedures.''
Metabolic Correction Is Complete and Prolonged in spf
Mice Treated with E2a-defective Adenoviral Vectors Containing Mouse OTC
cDNA
Gene therapy experiments were repeated with first and
second generation versions of the vector expressing mouse OTC cDNA from
the CMV promoter. The first generation mouse OTC cDNA vector was
infused into spf mice, and lysates of liver harvested 3 days
after gene transfer were analyzed for expression of OTC activity. OTC
activity was normalized with a dose of mouse OTC virus that was
2-5-fold less than the maximally tolerated dose of human OTC
virus that only partially corrected OTC deficiency. Histochemical
analysis demonstrated OTC activity in the majority of hepatocytes of spf animals treated with mouse OTC virus (Fig. 2J). Transgene expression diminished with time,
although OTC activity was detected in at least one-third of hepatocytes
at day 28 (Fig. 2L). Urinary orotate decreased to 10%
of pretreatment levels by day 7 and gradually returned to baseline by
day 42 (Fig. 8A). Correction of urinary orotate was
significantly greater than the nonspecific reduction seen with the lacZ virus, although it was not complete. A 30% decline in
serum glutamine was measured with the most substantial improvement,
realized 20 days after gene transfer, approaching normal levels,
thereafter returning to baseline within 42 days of gene transfer (Fig. 8B).
mice infused with recombinant adenoviruses carrying mouse
OTC cDNA. spf/y mice (A and B) at 6-8
weeks of age were infused with 2
10 particles of
first generation (H5.010CMVmOTC) or second generation virus
(H5.110CMVmOTC or H5.110CMVlacZ) through tail vein. spf
/y mice (C and D)
of similar age were infused with 2
10 particles of
the second generation viruses (H5.110CMVmOTC). Urine and
plasma samples were collected at day -3, day -1, and at
weekly intervals after virus infusion. Urinary orotate levels were
measured in duplicate for each sample. Urinary orotate/mg of creatinine
are presented as a percent of pretreatment levels and are the mean
± S.E. of at least 4 determinations. Plasma glutamine levels
were determined as described previously by Robinson et
al.(29) . The levels are presented as a percent of
pretreatment levels and are the mean ± S.E. of between 4 and 10
determinations.
mouse, following
infusion of second generation vector containing mouse OTC cDNA (Fig. 8, C and D).
Relationship between Genetic Complementation and
Metabolic Correction
Previous studies in mice and humans
deficient in OTC suggested reconstitution of OTC in liver should be
sufficient for some level of metabolic correction. Several lines of
transgenic mice have been established that express normal OTC cDNA in
the murine models genetically deficient in OTC. Introduction of a
minigene expressing rat OTC cDNA from an SV40 promoter into the germ
line of spf mice resulted in phenotype
conversion as evidenced by normal hair growth and diminished excretion
of orotic acid(34) . OTC activity was increased to 80-90%
of control in both liver and intestine. A similar approach was taken by
Jones et al.(35) in spf mice with a
construct carrying human OTC cDNA under control of the mouse OTC
promoter. Two transgenic hemizygous males demonstrated normal fur and
orotic acid excretion. Interestingly, these animals expressed high
levels of the transgene in intestine but low levels in liver. Both
studies implicate an important role of OTC expression in bowel and/or
liver although neither measured the impact of gene transfer on serum
amino acids which should represent more directly urea cycle function.
mouse. We also measured
the impact of gene therapy on serum amino acids and showed a
normalization of serum glutamine reflecting a decrease in nitrogen
stores. It was interesting that in the case of the first generation,
murine OTC vector, correction of serum glutamine lagged behind
biochemical correction of enzyme activity and the peak reduction in
urine orotate. The reason for this is unclear; however, we speculate it
may be due to the combined effects of increased OTC enzyme expression
with the superimposed but transient decline in liver function that
occurs because of adenovirus-induced hepatitis. The relative impact of
gene therapy on various metabolic parameters will be determined by the
balance of gene correction versus liver injury.
Implications for Gene Therapy of Liver Metabolic
Diseases
This study systematically evaluated the impact of both
the transgene and vector on the safety and efficiency of
adenovirus-mediated gene transfer to liver. Vectors containing human
OTC cDNA did not function as well in mouse liver as did the
corresponding murine OTC constructs. One explanation is that the human
OTC protein is not properly processed in a mouse cell to form
catalytically active, mitochondrial-localized enzyme. Another
explanation for the apparent dysfunction of the human OTC cDNA is that
its protein is viewed as a neoantigen in the spf mouse
eliciting confounding and destructive immune responses. Analysis of
mice infected with human OTC adenovirus detected cytotoxic T cells but
not antibodies to human OTC protein (data not shown). While primary
antigen-specific cellular immune responses may decrease stability of
transgene expression at day 7 and beyond, they cannot limit efficiency
at early time points such as day 3. subset and CTLs are necessary for
the observed destabilization of transgene expression. T
cells are activated to the input viral capsid proteins in a class
II-dependent manner, while CTLs are activated to newly synthesized
peptides presented by class I major histocompatibility complex. Viral
proteins expressed from intact open reading frames in the recombinant
virus as well as the transgene product itself are potential targets of
CTL. It has been difficult quantifying the relative contribution of
viral versus transgene protein to activation of destructive
cellular immune responses. Most experiments have used transgenes whose
protein products are easily distinguished from endogenous protein but
which run the risk of being neoantigens (e.g. lacZ,
luciferase, chloramphenicol acetyltransferase, etc.) Previous
experiments with E1-deleted viruses containing lacZ demonstrated CTLs to both viral proteins and Escherichia coli
-galactosidase; however, adaptive transfer experiments
indicated immune responses to viral antigens from 1-deleted
viruses are sufficient to ablate transgene expression (38) . allele. Transgene expression with the mouse OTC
cDNA vectors persisted longer than what we have consistently observed
with vectors expressing non-self transgenes such as
-galactosidase. Incorporating the ts125 mutation into this vector
diminished late viral gene expression and further prolonged transgene
expression. However, even under optimal conditions of isogenic OTC cDNA
in a second generation virus, the expression of transgene eventually
diminished to undetectable levels within 3-4 months of gene
transfer. This is less stable than what has been observed when
E1-deleted lacZ viruses are infused into athymic or RAG2
knockout mice (data not shown and (18) and (20) ).
Eliminating antigenicity of the transgene product will not be
sufficient to prevent all destructive cellular immune responses.
)
We thank Antoneta Radu for preparing tissue sections
and helping us with the histochemical staining of OTC activity. We also
thank Carol Pabin and Rebecca Brown for their technical assistance with
the analysis of urinary orotate and plasma amino acid.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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