Download 1071 - ex vivo gene therapy to produce bone using different cell types

EX VIVO GENE THERAPY TO PRODUCE BONE USING DIFFERENT CELL TYPES
*Musgrave, D; *Bosch, P; **Ghivizzani, S; *Whalen, J; *Niyibizi, C; *Usas, A; +*Huard, J
+*Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. 4151 Rangos Research Center, 3705 Fifth Ave., Pittsburgh, PA 15213-2583, 412-6927822, Fax: 412-692-7095, [email protected]
Introduction: Recombinant human bone morphogenetic protein-2 (rhBMP2) induces in vivo bone formation4 but is hindered by one time dosing,
dilutional effects, and its short half-life. The use of gene therapy to deliver
BMP-2 may circumvent these limitations1.2. This study evaluated the
advantages and disadvantages of using a bone marrow stromal cell line,
primary bone marrow stromal cells, primary muscle derived cells, primary
articular chondrocytes, and primary skin fibroblasts in ex vivo BMP-2 gene
delivery.
Methods: Cell Populations A bone marrow stromal cell line (OIMSC) from
a mouse model of human osteogenesis imperfecta (OI) was obtained and
maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 1%
L-glutamine. Primary bone marrow stromal cells (BMSCs) were isolated
from normal adult mice littermates and maintained in DMEM supplemented
with 10% FBS, 1% P/S, and 1% L-glutamine. Primary muscle derived cells
were isolated from normal adult mice according to a previously described
technique3 and maintained in DMEM supplemented with 10% FBS and 1%
P/S. Primary articular chondrocytes were isolated from adult New Zealand
white (NZW) rabbits and maintained in Ham’s F-12 media supplemented with
10% FBS and 1% P/S. Primary skin fibroblasts were isolated from skin
biopsies of adult NZW rabbits and maintained in Ham’s F-12 media
supplemented with 10% FBS and 1% P/S. Construction of Viral Vector A
plasmid containing the BMP-2 cDNA was provided (Genetics Institute,
Cambridge, MA) for construction of a E1 and E3 genes deleted, replication
defective, adenoviral vector under the control of the CMV promoter (AdBMP2). In Vitro BMP-2 Secretion
Equal numbers of each cell type were plated
in 12 well plates, infected with AdBMP-2, and allowed to secrete BMP-2 for
72 hours. Control cells were not infected with AdBMP-2. The AdBMP-2
infection was performed in quadruplet to allow statistical analysis.
The amount of BMP-2 in the supernatants was determined by enzyme
linked immunosorbent assay (ELISA). The primary antibody consisted of a
mouse monoclonal anti-BMP-2 antibody (Genetics Institute, Cambridge, MA)
and the secondary antibody consisted of a goat antimouse antibody conjugated
to horseradish persoxidase. Included in the ELISA were negative control
wells (media) and positive control wells containing known concentrations of
rhBMP-2 to establish a standard curve used to determine BMP-2 secretion.
Alkaline Phosphatase Assay In vitro osteogenic responsiveness to rhBMP-2
was assessed by quantification of alkaline phosphatase production. Equal
numbers of cells were plated in 12 well plates and stimulated with 200 ng/ml
of rhBMP-2 (Genetics Institute, Cambridge, MA) for 5 days. Control cells
were not stimulated. Alkaline phosphatase activity was determined using a
commercially available kit (Sigma Diagnostics, St. Louis, MO). The
procedure was performed in quadruplet to allow statistical analysis (Student’s
t-test). In Vivo Bone Formation Cells were co-transduced with a retrovirus
encoding for a nuclear-localized β-galactosidase enzyme and AdBMP-2 on
successive days. The cells were then trypsinized and 5 x 105 cells were
suspended in 20 microliter aliquots of HBSS and injected into the triceps
surae musculature of adult immunodeficient (SCID) mice. Separate animals
injected with the same cells not transduced with AdBMP-2 served as controls.
Animals were sacrificed at 2, 3, 4, and 5 weeks after injection. Radiographs
and histologic sections were obtained of the hind limbs. All procedures were
approved by the animal research and care committee.
Results: All cell types secreted nanogram quantities of BMP-2 over 3 days
(Fig. 1). This amount was 1000x less than the µgram quantities of exogenous
rhBMP-2 required in previous studies4 to induce in vivo bone formation. The
OIMSCs (p=0.0001) and muscle derived cells (p=0.001) produced large
increases in alkaline phosphatase in response to rhBMP-2 stimulation (Fig. 2).
The BMSCs (p=0.07 ) and chondrocytes (p=0.03) were moderately BMP-2
responsive and the fibroblasts were not BMP-2 responsive. All BMP-2
transduced cell types induced in vivo ectopic bone formation when injected
into rodent hind limbs. The ectopic bone was radiographically detectable only
in animals injected with BMP-2 transduced OIMSCs (Fig. 3) and muscle
derived cells. Staining for β-galactosidase revealed labeled OIMSCs and
muscle derived cells (Fig. 4) lining and within ectopic bone, locations
normally occupied by osteoblasts and osteocytes, thereby confirming these
cells possessed osteocompetence. Few labeled BMSCs were found lining
bone, confirming the in vitro data suggesting only a few of these cells
possessed osteocompetence. Labeled chondrocytes were identified lining and
within ectopic bone, confirming some of these cells were osteocompetent.
Finally, no labeled fibroblasts were found lining or within ectopic bone,
confirming that the fibroblasts were not osteocompetent.
Discussion: This study established the feasibility of using primary bone
marrow stromal cells, primary articular chondrocytes, primary skin
fibroblasts, and an osteogenesis imperfecta bone marrow stromal cell line in
ex vivo gene therapy to produce bone. The in vitro ELISA data demonstrated
that, after being transduced with AdBMP-2, all the cell types secreted
nanogram quantities of BMP-2 over 3 days (Fig. 1). These quantities were
1000 fold less than the µgram quantities of exogenous rhBMP-2 required to
induce bone formation4. Therefore, BMP-2 gene delivery appears more
efficient than single dose, exogenous rhBMP-2 protein administration in
inducing bone formation. The in vitro alkaline phosphatase data indicated the
significant osteogenic potential of the OIMSCs and primary muscle derived
cells (Fig. 2). Histologic sections demonstrated labeled OIMSCs and muscle
derived cells (Fig. 4) in locations normally occupied by osteoblasts and
osteocytes, further supporting their osteocompetence. In contrast, fibroblasts
displayed no in vitro osteocompetence (Fig. 2), produced only
microscopically detectable bone, and were not found in locations normally
occupied by osteogenic cells. Primary BMSCs and articular chondrocytes
were intermediate in terms of osteocompetence, both in vitro and in vivo (Fig.
2). This data demonstrated the advantages and disadvantages of using each of
the cell types in ex vivo gene therapy to produce bone. Each cell type, with its
own distinct favorable characteristics, may have a role in musculoskeletal
gene therapy applications.
References
1. Lieberman JR, et al.: JOR 16: 330-9; 1998.2. Musgrave
DS, et al..: Bone 24(6): 541-7; 1999.3. Qu Z, et al.:JCB 142:1257-1267,
1998.4. Wang EA, et al.: PNAS USA 87: 2220-4; 1990.
Acknowledgements: We would like to thank the Genetic Institiute
**Department of Molecular Genetics & Biochemistry, University of
Pittsburgh, Pittsburgh, PA.
Poster Session - Gene Therapy - VALENCIA D
46th Annual Meeting, Orthopaedic Research Society, March 12-15, 2000, Orlando, Florida
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