Gene therapy has received much attention over the past decade.
The adenovirus has advantages as a vector system in gene therapy
because of its ease of producing a high titer recombinant virus
and high transduction efficiency, as well as its ability to transfer
the gene of interest, even into non-dividing cells. Gene therapy
with the adenoviral vector has been examined extensively and used
in clinical trials10,13.
Bone morphogenetic proteins (BMPs) have a unique ability to alter
the differentiation pathway of mesenchymal cells toward chondrogenic
and osteogenic lineages with the ultimate induction of endochondral
bone at ectopic or orthotopic sites. Ectopic bone formation in
vivo is performed by the implantation of recombinant human
(rh) BMP-2 protein with a foreign body carrier. Various types of
carriers, such as collagen and poly-lactic acid, have been evaluated in
vivo studies. However, the disadvantage with this approach
is that a surgical procedure must be carried out.
In the present study, we evaluated the osteoinductive activity
of an adenoviral vector carrying BMP-2 gene, in vitro and in
vivo.
In Vitro Study
Construction of the BMP-2-expressing recombinant
adenovirus
The details of construction were described previously?8. A replication-deficient
type 5 adenoviral vector-carrying BMP-2 gene, AxCAOBMP-2, was constructed
according to the COS/TPC method as previously described5. This vector system with deletion
of the E1 and E3 regions4 contained
the human BMP-2 gene under the control of a CAG promoter (chicken
b-actin promoter and cytomegalovirus enhancer). The control vector was
AxCALacZ4 (a gift from I. Saito
and Y. Kanegae), which carries the Escherichia coli lacZ
cDNA in the same expression unit as AxCAOBMP-2.
Cell culture
C2C12 myoblasts (American Type Culture Collection) were grown
in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO,
Grand Island, NY, U.S.A.) containing 10% heat-inactivated
fetal bovine serum (FBS; GIBCO), penicillin G (100 IU/ml),
and streptomycin (100 mg/ml). C2C12 cells were cultured
in an incubator at 37°C in a humidified atmosphere containing 5% CO2
and 95% air.
Indirect immunofluorescence
C2C12 cells were seeded at 2 104 cells
per well on four-well Lab-Tek Chamber Slides (Nalgen Nunc International,
Naperville, IL, U.S.A.) in DMEM containing 5% FBS. After
24 hours, the cells were exposed to AxCAOBMP-2 or AxCALacZ at a
multiplicity of infection (MOI) of 100, for 1 hour. Then, the cells
were rinsed with phosphate buffered saline (PBS) and returned to fresh
medium. Twenty-four hours after infection, the cells were fixed
with 3.7% formaldehyde in PBS for 10 minutes. The cells
were incubated with a monoclonal antibody against BMP-2 (h3b2/17.8.1),
provided by Genetics Institute (Cambridge, MA, U.S.A.), for 1 hour
at a 1:100 dilution. The cells were rinsed with PBS and stained
with fluorescein isothiocyanate-conjugated goat anti-mouse IgG,
F (ab’)2 fragment (1:50 dilution; Organon Teknika, Duhn,
NC, U.S.A.) for 20 minutes. They were then examined by fluorescence
microscopy.
Alkaline phosphatase activity and osteocalcin
production
The cells were seeded at 2 105 cells/well
on six-well plates (each group consisting of three wells) in DMEM
containing 5% FBS. Twenty-four hours later, the cells were
exposed to AxCAOBMP-2 at MOIs of 20, 4, and 0 (mock: uninfected)
and AxCALacZ at a MOI of 20 for 1 hour. Then, the cells were rinsed
with PBS, and the growth medium was replaced with fresh medium.
Two days after infection, the cells were rinsed with PBS and
the growth medium was replaced, as already described. On day 5 after
infection, the cells were washed with PBS and lysed in 50 mM Tris-HCl,
0.5% Nodient P-40, pH 7.5. On day 5 after treatment, the
growth medium was removed and the cells were lysed. Alkaline phosphatase (ALP)
activity and total protein in the cell lysates were determined by
the 4-nitrophenylphosphate method. The amount of osteocalcin that
was secreted into the culture medium on day 5 after the infection
was determined by radioimmunoassay with use of a mouse osteocalcin
assay kit (Biomedical Technologies, MA, U.S.A.).
In Vivo Study
Animals
Fifteen Wistar rats (male; 9 weeks old; weight: 230-250 g) were
randomly assigned to groups of five animals each. They were given
25 ml of AxCAOBMP-2 (8.75 108 pfu, Group
I), 5 ml of AxCAOBMP-2 (1.75 108 pfu,
Group II), or 25 ml of AxCALacZ (1.75 108 pfu,
control group). All procedures and virus inoculum were approved
by the Institute of Laboratory Animals, Graduate School of Medicine,
Kyoto University.
Surgical procedure
Cyclophosphamide, provided by Shionogi and Co. (Osaka, Japan),
was given at a dose of 125 mg/kg injected intraperitoneally
the day before the vector injection. On the day of the injection,
each rat was anaesthetized by an intraperitoneal administration of
sodium pentobarbital (5.0 mg per 100 g of body weight). Following
sterile preparation of the operative region, 25 ml of AxCAOBMP-2
and AxCALacZ and 5 ml of AxCAOBMP-2 suspended in 20 ml of Hanks’ balanced
salt solution (HBSS; GIBCO) were injected with a microsyringe into
a right calf muscle.
Radiographic evaluation
Twenty-one days after the vector injection, the rats were sacrificed
with an overdose of sodium pentobarbital. The region that had received
the injection was excised together with the surrounding tissue, and
soft radiographs were prepared (SOFRON, SRO-M50; Sofron, Tokyo,
Japan).
Histological analysis
The specimens with peripheral tissue were fixed in 10% formalin
neutral buffer solution (pH 7.4), demineralized in EDTA, and embedded
in paraffin. They were cut into 4-mm-thick sections and stained with
hematoxylin and eosin.
Statistical analysis
The results are presented as mean ± standard
error of mean (S.E.M.). The statistical analysis of differences
in the value of ALP activity and osteocalcin was performed by an
analysis of variance (ANOVA), followed by Fisher’s comparison
test.
Indirect Immunofluorescence
In the C2C12 cells infected with AxCAOBMP-2 at an MOI of 100,
BMP-2-positive cells were detected (Fig. 1-A). BMP-2 protein accumulated mainly
in the cytoplasm of gene-transferred cells. In the cells infected by
AxCALacZ, few BMP-2-positive cells were seen and only faint background
staining was observed (Fig. 1-B).
ALP Activity and Osteocalcin Production
The ALP activity and osteocalcin production on day 5 after the
treatment are shown in Fig. 2. In the uninfected cells and cells
infected with AxCALacZ, ALP activity and osteocalcin production
were only slightly elevated on day 5 after the treatment. Cells
infected with AxCAOBMP-2 expressed ALP activity and osteocalcin
production in a dose-dependent fashion.
Radiographic Findings
On day 21, radiographs revealed opaque shadows in muscles in
Groups I and II (Figs. 3-A and 3-B). The oval shadows in muscles in
Group I were larger, with higher radiopacity than those in Group II.
Radiopaque images were not observed in the control virus-treated
group (Fig. 3-C)
or in the AxCAOBMP-2 group treated without immunosuppression (data
not shown).
Histological Findings
No inflammatory responses were seen in any group. Light-microscopic
examination disclosed new bone formation in Groups I and II (Figs. 4-A and 4-B). However, there
was no evidence of osteoinduction in the control virus-treated group
(Fig. 4-C).
Around the trabecular bone, osteoclasts and osteoblasts were observed
in both AxCAOBMP-2-treated groups. The area of trabecular bone was greater
in Group I than in Group II.
We constructed a BMP-2 expressing replication-deficient adenoviral
vector, AxCAOBMP-2, and evaluated the activity of this vector in
vitro and in vivo. It was demonstrated
that myoblastic cell line C2C12 cells infected with this vector
produced BMP-2 protein. Furthermore, these cells were converted
to osteoblast lineage cells in vitro. The expression
of BMP-2 protein was demonstrated by immunoflourescence in C2C12
cells infected with AxCAOBMP-2. BMP-2 accumulated predominantly
in the cytosol. These results demonstrated that the BMP-2 gene can
be transferred into C2C12 cells via AxCAOBMP-2 and the protein can also
be produced efficiently in the cells after only 24 hours of infection.
In vivo study demonstrated osteoinduction in
both AxCAOBMP-2-treated groups, with immunosuppression seen radiologically
and histologically. In both AxCAOBMP-2-treated groups, new bone
formation, including a considerable amount of trabecular bone, was
detected on day 21 after infection. The trabecular area was greater
in Group I than in Group II.
Various types of carriers, such as collagen, have been evaluated in
vivo studies. However, the disadvantage of this surgical procedure
is that intervention is necessary. In clinical applications, it
can be difficult to obtain sufficient bone formation induced by
rhBMP-2 protein. Furthermore, these carriers may induce an immune response.
Several gene delivery methods for osteoinduction by BMPs have been
developed. Adenovirus has advantages as a vector system in gene
therapy because of its ease of producing a high titer recombinant
virus and high transduction efficiency, as well as its ability to
transfer the gene of interest, even into non-dividing cells. However, the
utility of such vectors may be limited because of the immune response
elicited by adenoviral proteins. It was reported that bone formation
by an adenoviral vector expressing BMP-2 was detected in nude rats
but not in immunocompetent rats1.
We confirmed that there was very little difference in the expression
of LacZ between immunosuppressed rats and normal rats on day 3 after
injection in X-gal staining; however, osteoinduction was not seen
in normal rats9. To overcome T-cell
responses, several methods have been used, such as inhibiting the
CD28/B7 pathway3, blocking
NF-k B activation6, inhibiting
tumor necrosis factor a activity14,
administering an anti-CD4 antibody5,
and immunosuppression2,12. Cyclophosphamide
is used clinically as an anti-cancer agent in the treatment of Hodgkin’s
disease and other leukemias. Cyclophosphamide, administered at a
dose of 300 mg/kg the day before the vector injection in
mice, was effective in blocking the humoral response and enhanced
the effectiveness of the second injection with the adenoviral vector12. Therefore, bone formation may be
elicited over a period of time by readministration of AxCAOBMP-2.
The results of this study suggest that gene therapy with AxCAOBMP-2
under transient immunosuppression may be useful for bone reconstruction.