Endocrinology Vol. 140, No. 1 260-265
Copyright © 1999 by The Endocrine Society
Construction and in Vivo Efficacy of a Replication-Deficient Recombinant Adenovirus Encoding Murine Growth Hormone
Yitzhak Marmary,
Albert F. Parlow,
Corinne M. Goldsmith,
Xinjun He,
Robert B. Wellner,
Kazuhito Satomura,
Martin F. Kriete,
Pamela Gehron Robey,
Lynnette K. Nieman and
Bruce J. Baum
Gene Therapy and Therapeutics Branch, National Institute of Dental
and Craniofacial Research (Y.M., C.M.G., X.H., R.B.W., B.J.B.),
Bethesda, Maryland 20892; National Hormone and Pituitary Program,
Harbor-UCLA Medical Center (A.F.P.), Torrance, California 90509;
Craniofacial and Skeletal Diseases Branch, National Institute of Dental
and Craniofacial Research (K.S., P.G.R.); Animal Care Unit, National
Institute of Dental and Craniofacial Research (M.F.K.); and
Developmental Endocrinology Branch, National Institute of Child Health
and Human Development (L.K.N.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Bruce J. Baum, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 10 Center Drive, MSC-1190, Building 10, Room 1N-113, Bethesda, Maryland 20892. E-mail: bruce_j_baum{at}nih.gov
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Abstract
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We have constructed a recombinant, replication-deficient,
first-generation adenovirus-encoding mouse GH (mGH), AdCMVmGH. This
virus directed mGH production from an epithelial cell line in
vitro in a dose-dependent manner. When injected into the
quadriceps muscle or submandibular ducts of mGH-deficient Snell dwarf
mice, AdCMVmGH resulted in the production of significantly elevated
serum mGH levels. Furthermore, after im injection, dwarf mice increased
in weight by 8% over 4 days and close to 100% by 30 days. When
AdCMVmGH was administered to 3- to 4-week-old rats by iv injection to
assess general metabolic responses, serum mGH, insulin-like growth
factor 1, triglycerides and cholesterol levels were
significantly elevated. AdCMVmGH should be a valuable experimental tool
for the controlled, directed expression of mGH in preclinical mouse
model studies.
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Introduction
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THE MANAGEMENT of numerous systemic protein
deficiencies (e.g. diabetes, hemophilia A, GH deficiency)
has been greatly facilitated by applications of molecular biological
methods for the production of large amounts of pure, recombinant
hormones. Though the repetitive injection of recombinant hormones
circumvents concerns over coadministration of pathologic contaminants
of biochemically-purified proteins from natural sources, this treatment
approach is still viewed as inconvenient and is certainly not ideal (1, 2). Among the many approaches being attempted to improve the management
of patients with systemic protein deficiencies is direct in
vivo gene transfer to both somatic and germ line cells (3, 4, 5). The
stable transfer of a gene encoding the deficient protein could lead, in
principle, to the correction of the clinical defect (6).
Thus far, no in vivo gene transfer approach has replaced the
conventional repetitive injection mode of clinical management. However,
considerable progress has been made, and the results of several recent
animal model studies are quite encouraging (4, 5). The most frequently
used recombinant viruses in such studies are replication-deficient
adenoviruses. In their studies of models of erythropoietin-responsive
anemias using recombinant adenoviruses, Leiden and colleagues have
noted the importance of using a transgene that encodes a protein
identical to the test species’ native protein (7, 8). For example, in
immunocompetent mice injected with an adenovirus encoding murine
erythropoietin, transgene expression dramatically improved over that
seen when mice were injected with an adenovirus encoding human
erythropoietin (7).
We have made a substantial effort to explore the therapeutic potential
of reengineering exocrine salivary glands to secrete proteins in an
endocrine fashion (9). Salivary glands are a convenient, if not often
considered, site for in vivo gene transfer, because their
excretory ducts can be readily cannulated in the mouth. One target
disease that we have chosen to study is GH deficiency (10). The present
series of experiments represents an extension of these efforts. The
most widely used animal model for GH deficiency is the Snell dwarf
mouse (11, 12, 13). These mice have a mutation on chromosome 16 and are
unable to express Pit-1, a transcription factor necessary for the
production of several anterior pituitary hormones and for the
GH-releasing factor receptor (11, 12). Herein, we describe the
construction of a replication-deficient, recombinant adenovirus
encoding murine GH and initial demonstration of its efficacy and
potential utility, through in vivo experiments.
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Materials and Methods
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Adenovirus construction
From the plasmid pKL-mGH (which was a generous gift of Dr. P.L.
Chang, McMaster University, Hamilton, Ontario, Canada), a 750-bp
fragment containing the mouse GH (mGH) complementary DNA (cDNA) was
obtained by digestion with SalI and HindIII (13).
This fragment was then cloned into the SalI and
HindIII sites of the plasmid pACCMV-pLpA (a generous gift of
Dr. C. Newgard, University of Texas-Southwestern Medical Center,
Dallas, TX). The resultant vector, pACCMVmGH (Fig. 1
), contains the cytomegalovirus
(CMV) promoter-enhancer, and the SV40 polyadenylation sequence, and was
subjected to DNA sequencing to confirm nucleotide sequences in the two
joining regions. Thereafter, pACCMVmGH was cotransfected with pJM17
(a generous gift of Dr. F. Graham, McMaster University)
into 293 (human embryonic kidney) cells, as previously
described (10, 14). The recombinant virus so generated was
plaque-purified and named AdCMVmGH.
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Figure 1. Shuttle vector constructed with mGH cDNA. As
described in Materials and Methods, pACCMVmGH was
constructed using pACCMV-pLpA and the mGH cDNA. CMV indicates the
cytomegalovirus promoter/enhancer; mGH indicates the transgene cDNA;
and SV40, the SV40 polyadenylation sequence; AMPr, the ampicillin
resistance gene; and Ad5, type 5 adenovirus sequences.
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In vitro infection of cells
AdCMVmGH was used to infect SMIE cells, an epithelial cell line
derived from adult rat submandibular gland (15). SMIE cells were grown
in DMEM, supplemented with 10% FBS, 2 mM glutamine, 100
U/ml penicillin, and 100 µg/ml streptomycin. Cells were infected at
various multiplicities of infection (MOIs) for up to 72 h. Culture
media samples were assayed for mGH using the RIA procedure described
below for serum samples. To assess the electrophoretic characteristics
of mGH secreted into serum-free culture media, aliquots were
precipitated with ice-cold ethanol, subjected to SDS-PAGE (10, 14),
transferred to nitrocellulose membranes (Hybond ECL, Amersham,
Arlington Heights, IL), and incubated with polyclonal antirat GH serum
(lot no. AFP-411S, generously provided by the National Hormone and
Pituitary Program, Harbor-UCLA Medical Center).
In vivo gene transfer
All animals were treated according to procedures approved by the
National Institute of Dental and Craniofacial Research Animal Care and
Use Committee. Anesthesia for all gene transfer procedures was obtained
by im injection of ketamine chloride (60 mg/kg) and xylazine (5 mg/kg),
as previously described (9, 10). Dwarf mice (6–7 wks old) and their
normal litter mates were obtained from Jackson Laboratories (Bar
Harbor, ME). Wistar-derived rats (3–4 wks old) were obtained from
Harlan Sprague Dawley, Inc. (Indianapolis, IN). Animals
were allowed to acclimate to our facility for approximately 1 week.
Thereafter, each animal received 5 x 109
plaque-forming units (pfu) of either AdCMVmGH or the control
virus, Addl312 (a generous gift of Dr. T. Shenk, Princeton University,
Princeton, NJ). Virus typically was suspended in 25–50 µl of vehicle
[10 mM Tris (pH 7.4), 0.1 mM
MgCl2, 10% glycerol] and administered either by im
injection (quadriceps), by iv infusion (femoral vein), or by
submandibular duct cannulation, as described previously (10). For all
animals studied at the 4-day time point, an injection of dexamethasone
(4 mg/kg) was administered at the time of gene transfer and the
following 3 days, to limit the host immune response (16). At the
conclusion of these experiments, animals were euthanized by
CO2 gas, and blood samples were obtained from the tail
vein. For all animals followed for 70 days, dexamethasone was
administered as above, but blood was obtained from the thoracic cavity
after euthanasia.
RIA of sera and serum chemistry analyses
Mouse and rat sera were prepared from blood samples obtained as
above. For mGH determination with sera and culture media (above), a
double-antibody RIA was used, including highly purified mouse pituitary
GH (lot no. AFP-10783B) as tracer and reference, and antiserum to rat
GH (lot no. AFP-411S) at a dilution of 1:3,000,000. This RIA is
specific for mGH and unreactive with other hormones of the mouse
pituitary gland. The within-assay coefficient of variation was 4.7%,
and the between-assay coefficient of variation was 7.4%. For assay of
rat GH, purified rat pituitary GH (lot no. AFP 3190B) was used as
tracer and reference, as was the above antiserum to rat GH (lot no.
AFP-411S), also at 1:3,000,000. In our assay for mouse GH, both rat and
mouse GH are nearly equally reactive. These immunoreagents are
distributed to all requestors by the National Hormone and Pituitary
Program, Harbor-UCLA Medical Center, Torrance, CA (FAX: 310-222-3432).
Insulin-like growth factor 1 (IGF-1) levels in rat sera were determined
by RIA (Endocrine Sciences, Inc., Calabasas Hills, CA).
Routine rat serum chemistry parameters also were assessed (ANTEC
Diagnostics, Farmingdale, NY).
Histological analyzes of growth plates
Bones were obtained from dwarf animals and normal litter mates,
fixed for 16–24 h in 4% paraformaldehyde, and then demineralized for
7–10 days in 10% EDTA. After dehydration and embedding in paraffin,
the bones were sectioned at 5 µm and stained with
hematoxylin/eosin.
Statistical analyses
Results obtained from in vivo experiments were tested
for statistical significance using a t test (for parametric
data) or a Mann-Whitney U test (for nonparametric data).
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Results
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Characterization of mGH expression in vitro
As described in Materials and Methods, we constructed a
replication-deficient, recombinant adenovirus-encoding mGH, AdCMVmGH.
To test the ability of this virus to direct the expression of mGH, we
infected an epithelial cell line (SMIE), which normally does not make
GH. As shown in Fig. 2, we analyzed the
culture media using a quantitative RIA procedure. Cells were infected
with different MOIs, from 0–300, and the culture media was assayed for
mGH at different time points up to 72 h (Fig. 2). For all positive
media samples, the slopes of the generated RIA dilution curves were
parallel to those determined with authentic mGH standards (data not
shown). Production of mGH by SMIE cells was dependent on AdCMVmGH dose
and increased with time. Little-to-no mGH was expressed at MOIs less
than 10. At higher viral doses, substantial levels of mGH were
detected. For example, at 72-h postviral infection, the culture media
contained 6.7 ± 0.3, 46.2 ± 1.8, and 183.7 ± 6.5
ng/ml mGH for cells infected at MOIs equal to 10, 100, and 300,
respectively. The presence of mGH in the culture media was also
demonstrated by Western blot analysis (not shown).
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Figure 2. mGH production in SMIE cells in
vitro. SMIE cells were infected with different doses of
AdCMVmGH (MOI = 0–300) for up to 72 h. Medium was assayed
for mGH as described in Materials and Methods. Data
shown are the mean ± SE for three separate
determinations. Where error bars are not shown, they are
smaller than the symbol.
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Efficacy of mGH expression in vivo
To test the in vivo efficacy (and thus, the potential
experimental utility) of AdCMVmGH, we administered the virus either via
a single im injection (quadriceps) or into the submandibular duct of
Snell dwarf mice. As a control for virus administration, we injected
some animals with Addl312, a similar replication-deficient adenovirus
but without any transgene. As shown in Fig. 3, which presents serum levels of mGH,
the dwarf mice used in these experiments endogenously expressed very
low levels of GH (1.08 ± 0.06 ng/ml), whereas their normal litter
mates express considerably higher levels (20.35 ± 22.9 ng/ml).
Intramuscular administration of AdCMVmGH to eight dwarf mice resulted
in an elevation of serum mGH levels, in all animals, to values
significantly greater then those receiving the control vector, (range:
3.3–249.2 ng/ml; mean ± SE, 42 ± 29.7;
Mann-Whitney test, P < 0.002) when measured 4 days
post injection. Conversely, GH values were not significantly elevated
after im AdCMVmGH administration to normal animals, compared with that
observed after Addl312 administration. Submandibular administration of
AdCMVmGH was attempted in four dwarf mice. Despite the small size of
these animals, submandibular glands were successfully cannulated in
three of four mice, resulting in serum mGH levels (64.1, 3.4, 11.0
ng/ml) well above that found in control dwarf mice (see above).
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Figure 3. Effect of im AdCMVmGH administration on mGH levels
in the serum of dwarf mice. Dwarf mice or their normal litter mates
were injected with either AdCMVmGH or Addl 312 (control virus), at
5 x 109 pfu/animal. After 4 days, sera were obtained
and mGH measured, as described in Materials and Methods.
Data shown are the mean ± SE of results from four to
eight animals. Differences seen in dwarf mice are significant, by
Mann-Whitney test (P < 0.002), whereas differences
between normal litter mates are not significant.
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Biological responses to mGH
As an initial test of the biological activity of the mGH directed
by AdCMVmGH, we used a global measurement parameter, animal body weight
gain (Fig. 4). Normal mouse litter mates
weighed approximately 25–30 g, and im injection of either Addl312 or
AdCMVmGH had no effect on their body weight over the 4-day experimental
period. Dwarf mice typically weighed about one fourth to one third the
size of their normal litter mates (
6–9 g). Injection of AdCMVmGH,
however, resulted in a highly significant (P = 0.01)
increase in the weight of the dwarf mice (range, 2.5–15.1%; mean
± SE, 8.15 ± 1.6%), compared with that seen when
the mice were injected with Addl312 (-1.55 ± 3.0). In a second
experiment, dwarf mice also received a single im dose of either Addl312
or AdCMVmGH (5 x 109 pfu) but were followed for more
than 2 months. As shown in Fig. 5, the
animals injected with AdCMVmGH grew in an almost linear fashion for up
to 30 days and plateaued thereafter. Conversely, animals injected with
Addl312 showed essentially no weight gain over the experimental period.
At time of sacrifice, animals injected with AdCMVmGH had 2-fold higher
serum GH levels than the control animals (4.2 ± 0.1
vs. 2.0 ± 0.4 ng/ml; t = 4.7, P
= 0.005).
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Figure 4. Effect of im AdCMVmGH administration on body
weight in dwarf mice. Animals were infected with either AdCMVmGH or
Addl 312, as described in Fig. 3 . Animals were weighed at the start of
the experiment and after 4 days. Data are the mean ±
SE of results from four to eight animals. Differences seen
in dwarf mice are significant, by Mann-Whitney test
(P = 0.01), whereas differences between normal
litter mates are not significant.
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Figure 5. Effect of im AdCMVmGH administration on the time
course of body weight gain in dwarf mice. Animals were injected with
either AdCMVmGH or Addl312, as described in Fig. 4, and were weighed
periodically for up to 70 days. Animals were killed at day 70, and
serum was obtained for mGH determination. Data are the mean ±
SE of results from three (AdCMVmGH) or four (Addl312)
animals.
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Because of blood volume limitations, we were able to obtain only enough
serum for measurement of mGH from dwarf mice. To assess several
metabolic parameters that may be altered by the biological activity of
GH, we administered AdCMVmGH to young rats (3–4 wks old) via an iv
route. This route of recombinant adenoviral administration in rats
primarily leads to transduction of hepatocytes in the liver (17) and
marked transgene expression. Table 1
provides data from these experiments in which sera were obtained 4 days
post injection. When compared with animals injected with the control
virus Addl312, injection of AdCMVmGH (on average) led to an increase
(>10-fold) in serum GH in these animals (mouse and rat GH were
indistinguishable with our RIAs). Four of the six animals had dramatic
elevations in GH (>375 ng/ml). Further, serum IGF-1 levels were
increased approximately 35%; serum cholesterol, approximately 60%;
and serum triglycerides, approximately 40%. No significant changes
were detected in serum glucose or serum protein levels or in the
BUN:creatinine ratio.
The growth plate, a primary target of GH, also was examined in dwarf
mice and their normal litter mates. Dwarf mice exhibit an expanded
growth plate, marked by an overt delay in maturation with a poorly
developed site of secondary ossification of the epiphysis, and reduced
bone formation in the metaphysis (18). We observed no consistent
changes in the growth plates of dwarf mice 4 days after im
administration of AdCMVmGH (not shown).
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Discussion
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This study demonstrates that a recombinant adenovirus can direct
the in vivo expression of biologically-active mGH. As judged
by electrophoretic and immunochemical characteristics, the transgene
product seemed identical to authentic GH. Most importantly, when the
virus was administered to dwarf mice deficient in GH, functional mGH
production was observed. Serum GH levels exceeded normal levels, and
the dwarf mice gained an average of approximately 8% of their
pretreatment body weight in just 4 days, and nearly doubled their body
weight in 30 days. We did not, however, see significant changes in the
growth plates of dwarf mice at the 4-day time point. Though this may
reflect an insufficient time period of elevated GH exposure, it also
may reflect the effects of dexamethasone administration on the
skeleton. Because adenoviral vectors elicit a potent immune response,
the administration of antiinflammatory drugs is required (16).
This study additionally shows that AdCMVmGH can be successfully and
efficiently delivered to rodents in vivo using
three different routes. The primary delivery site for AdCMVmGH in
our in vivo dwarf mouse experiments was the quadriceps
muscle. We also presented one set of experiments conducted in young
(3–4 week) rats, in which the virus was administered via the femoral
vein, a delivery route leading primarily to transduction of the liver
in rats (17). The iv administrations generally led to high serum GH
levels in animals and several significant serum chemistry changes
consistent with systemic mGH action and anabolic effects. Additionally,
we administered AdCMVmGH via retrograde intraductal instillation after
successful cannulation of the submandibular glands of dwarf mice. In
three of four dwarf mice, submandibular glands were successfully
transduced, leading to serum mGH levels comparable with those seen
after im injection.
Previously, we have shown that rat salivary epithelial cells readily
can be infected in vivo by recombinant adenoviruses using an
intraductal route of delivery (19). Furthermore, we have shown that
salivary glands can secrete adenoviral-encoded transgene products in an
endocrine manner (9, 10). Recently, Goldfine and colleagues (20)
reached similar conclusions. They have shown that rodent salivary
glands can be transduced by direct administration of plasmid DNA,
albeit at much lower levels than seen with adenoviruses. In their
studies, hGH was secreted from salivary glands into the bloodstream
(20).
The initial choice of muscle as a primary target site was, in part,
driven by practical issues of size with the dwarf mice. However,
skeletal muscle has emerged as an extremely useful target site for the
delivery of circulating proteins (21), allowing fairly stable transgene
expression after a single injection of a first-generation recombinant
adenovirus (months, to >1 yr) in rodents and nonhuman primates (7, 8).
Furthermore, in immunocompetent animals, such as those used herein, the
host immune response to recombinant adenoviruses administered in muscle
seems to be somewhat milder than that observed when the virus infects
other sites (4, 5, 7). Nonetheless, there are several examples of the
prolonged (months) expression of a circulating transgene-directed
protein after the iv administration of a first-generation adenoviral
vector in immuocompetent animals (e.g. Refs. 22, 23, 24). In the
present study, after 70 days, mice injected with AdCMVmGH had average
GH levels that were approximately 2-fold higher than mice that received
Addl312.
In summary, we have constructed an adenoviral vector that directs the
functional expression of mGH at different tissue sites in
vivo. Given the increased clinical interest in GH therapy for
adult GH deficiency, age-associated decreases in bone mass, and cardiac
performance (25, 26, 27, 28), as well as congenital GH deficiency, we
anticipate that AdCMVmGH would be a valuable experimental tool for
mouse model, preclinical studies.
Received May 15, 1998.
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Top
Abstract
Introduction
