(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) ()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) .
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 (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) .
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 spfmice(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.
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/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.
An
E1-deleted adenoviral vector was constructed that contains human OTC
cDNA (referred to as a first generation virus) expressed from a
CMV-enhanced -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.
Figure 2:
Cytochemical demonstration of OTC activity
in liver tissues from spf mice infused with recombinant
adenoviruses. spf/y mice infused with 5 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.
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 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.
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 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.
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 (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.
Figure 5:
RNA blot analysis of liver tissues from spf mice infused with recombinant adenoviruses. spf/y
mice were infused with 2 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.
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
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.
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/-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.
Figure 7:
Liver OTC activity in spf mice
infused with recombinant adenovirus. spf/y mice were infused
with 2 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.''
Figure 8:
Urinary orotate excretion and plasma
glutamine levels in spf and spf 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.
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 mouse, following
infusion of second generation vector containing mouse OTC cDNA (Fig. 8, C and D).
Our study demonstrates complete correction of orotic acid
overproduction following hepatic reconstitution of OTC in both the spf and spf 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.
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 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) .
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 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.
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.