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Review
. 2004;176(1-3):95-108.
doi: 10.1159/000075031.

Gene therapy approaches for bone regeneration

Renny T Franceschi  1 , Shuying Yang, R Bruce Rutherford, Paul H Krebsbach, Ming Zhao, Dian Wang
Affiliations
Review

Gene therapy approaches for bone regeneration

Renny T Franceschi et al. Cells Tissues Organs. 2004.

Abstract

Gene therapy represents a promising approach for delivering regenerative molecules to specific tissues including bone. Several laboratories have shown that virus-based BMP expression vectors can stimulate osteoblast differentiation and bone formation in vivo. Both in vivo and ex vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Adenovirus and direct DNA delivery of genes encoding regenerative molecules can heal critical-sized defects of cranial and long bones. Although osteogenic activity can be demonstrated for individual BMP vectors, substantial synergies may be achieved using combinatorial gene therapy to express complimentary osteogenic signals including specific combinations of BMPs or BMPs and transcription factors. Further control of the bone regeneration process may also be achieved through the use of inducible promoters that can be used to control the timing and magnitude of expression for a particular gene. Using these types of approaches, it should be possible to mimic natural processes of bone development and fracture repair and, in so doing, be able to precisely control both the amount and type of bone regenerated.

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Figures

Fig. 1
Fig. 1
Strategies for delivering therapeutic genes to tissue sites. In vivo transduction involves direct delivery of a viral or nonviral gene therapy vector to the target tissue of interest using a suitable carrier matrix. Ex vivo gene therapy first requires transduction of syngeneic cells in tissue culture followed by implantation at the regeneration site of the patient.
Fig. 2
Fig. 2
Induction of endochondral bone formation by BLK cells transduced ex vivo with AdCMV-BMP7; histological analysis. BLK cell cultures were transduced with AdCMV-BMP7 (m.o.i. 100). After 24 h, cells were trypsinized and resuspended in type I collagen hydrogels that were subcutaneously implanted in C57BL/6 mice. At the times indicated, implants were harvested, fixed in 4% paraformaldehyde, demineralized in 10% formic acid, dehydrated and embedded in paraffin. Sections (8 μm) were cut and stained with hematoxylin and eosin. Bone (B) and cartilage (C) areas are indicated. An ossicle containing trabecular bone, cortical bone and marrow was formed over a 4-week period.
Fig. 3
Fig. 3
Quantitation of the osteogenic activity. BLK cells were transduced with AdCMV-BMP7 and implanted into C57BL/6 mice as described in figure 2. Osteogenesis by implants was assessed by homogenizing ossicles in 15% trichloroacetic acid and measuring total calcium in supernatants. a Effect of cell number. BLK cells were transduced at an m.o.i. of 100 and the indicated numbers of cells were implanted. Osteogenesis was assessed after 3 weeks. b Time course. BLK cells (1 × 107 cells/implant) were transduced at an m.o.i. of 100, implanted and harvested at the times indicated. c, d Effect of virus titer. BLK cells were transduced with the indicated virus titers. One set of cells was implanted and harvested after 3 weeks for measurement of bone formation (c) and a second set was incubated in serum-free medium for 24 h for measurement of BMP7 secretion by Western blotting (d).
Fig. 4
Fig. 4
Retention of implanted BLK (lacZ)-AdCMV-BMP7-transduced cells during bone induction. BLK cells were stably transfected with pCMV-neo-lacZ plasmid encoding the bacterial β-galactosidase gene. Cells were transduced with empty adenovirus (control) or AdCMV-BMP7 (BMP7), implanted and harvested at the times indicated. Implants were stained for β-galactosidase activity before paraffin embedding and sectioning. Samples were stained with eosin only to allow visualization of blue β-galactosidase staining. Blue staining cells associated with osteoblast layers in 2- and 6-week samples are indicated by arrows. Only 2× magnifications of control implants are shown while 2× and 20× magnifications are shown for the AdCMV-BMP7 group. Bone formed by implanted, virally transduced cells was composed primarily of cells from the host.
Fig. 5
Fig. 5
Regeneration of a critical size cranial defect with AdCMV-BMP7-transduced fibroblasts [from Krebsbach et al., 2000]. Dermal fibroblasts isolated from Lewis rats were transduced with AdCMV-BMP7 (a) or lacZ control virus (b). Cells were adsorbed to a gelatin sponge and placed in a 9-mm-diameter cranial defect created in a Lewis rat host. Calvariae were isolated for histological analysis after 4 weeks. Black arrows designate the surgical margins. Original magnification: ×25. AdCMV-BMP7-transduced cells were able to repair a calvarial defect while control cells produced only fibrous connective tissue.
Fig. 6
Fig. 6
Regeneration of a long bone critical size defect with AdCMV-BMP7-transduced syngeneic fibroblasts [from Rutherford et al., 2002]. Dermal fibroblasts isolated from Fischer rats were transduced with lacZ control virus (a, c, e) or AdCMV-BMP7 (b, d, f). Cells were suspended in collagen hydrogel, adsorbed to gelatin sponges and placed in 3-mm surgical osteotomies in the femurs of Fischer rats. After 6 weeks, radiographs were taken (e, f) and tissues were processed for histology (a–d). Arrowheads mark margins of the defect. Double arrow identifies cartilage. Original magnification: 10× (a, b) and 50× (c, d). Partial healing of the long bone defect was only seen with AdCMV-BMP7-transduced cells.
Fig. 7
Fig. 7
Cotransduction of mesenchymal cell lines with adenoviruses expressing combinations of BMPs synergistically stimulates alkaline phosphatase activity. C3H10T1/2 and ST2 cells were transduced with the indicated adenoviruses. Cells were harvested after 8 days for measurement of alkaline phosphatase activity and DNA (for normalization). Cotransduction of cells with combinations of either AdCMV-BMP2 or AdCMV-BMP4 with AdCMV-BMP7 stimulated alkaline phosphatase 2–4 times more than would be predicted if effects of individual adenoviruses were additive. 10T1/2 = C3H10T1/2 mouse mesenchymal cell line; LacZ = AdCMV-lacZ; B2 = AdCMV-BMP2; B4 = AdCMV-BMP4; B7 = AdCMV-BMP7. Significantly different from the sum of activity in cells individually transduced with AdCMV-BMP2 and AdCMV-BMP7, * p < 0.001; significantly different from the sum of activity in cells individually transduced with AdCMV-BMP4 and AdCMV-BMP7, + p < 0.001.
Fig. 8
Fig. 8
In vivo bone formation by C3H10T1/2 cells transduced with adenoviruses expressing Runx2 and BMP2 [from Yang et al., 2003]. Cells were transduced with indicated adenoviruses and implanted into immunodeficient mice. After 4 weeks, transplants were harvested for histological examination (a), determination of total calcium (b) and morphometric analysis (c). For morphometric analysis, results are expressed as the ratio of total bone area/total implant area. Statistical analysis: significantly different from AdLacZ, ap < 0.001; significantly different from AdBmp2, bp < 0.05. Implants of cells transduced with control virus contained residual gelfoam carrier (g) and fibrous tissue (f), but no bone or cartilage. Cells transduced with AdCMV-Runx2 alone contained small areas of both bone (b) and cartilage (c) as well as a small marrow cavity (m). Both AdCMV-BMP2 alone and AdCMV-BMP2 plus AdCMV-Runx2-treated groups formed large ossicles with clearly defined cortical and trabecular bone as well as a marrow cavity. The total amount of bone as measured either by total calcium or fractional bone area was greatest in the AdCMV-BMP2 plus AdCMV-Runx2-treated group. Control = AdCMV-lacZ control virus; B2 + R2 = cells transduced with AdCMV-BMP2 plus AdCMV-Runx2.
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