Tracking expression of virally mediated BMP-2 in gene therapy for bone repair

Abstract

Ex vivo gene therapy using stem cells transduced with viral vectors is a useful method for delivering a therapeutic protein to augment bone repair in animal models. However, the duration of cell-mediated protein production and the fate of the transduced cells are unknown. We constructed an adenoviral vector encoding Myc epitope tagged bone morphogenetic protein (BMP)-2 gene (AdBMP-2). Rat bone marrow cells transduced with this vector produced biologically active BMP-2 protein, which was confirmed by Western blot analysis and alkaline phosphatase assay. Implantation of bone marrow cells infected ex vivo with AdBMP-2 caused orthotopic bone formation in mouse hindlimbs and bony union of critical-sized mouse radial defects. Immunohistochemical analysis revealed that rBMCs expressed Myc epitope-tagged BMP-2 protein for 14 days in vivo and became incorporated in the endochondral fracture callus. This novel adenovirus encoding for epitope-tagged BMP-2 can be used for immunohistochemical tracking of transduced cells in ex vivo gene therapy for bone repair.

Fig 1

A chart shows that the biologic activity of ALP was significantly increased in the AdBMP-2^Myc-transduced W-20-17 cells when compared with nontransduced control cells (p = 0.01).

Fig 2

(A) A Western blot is shown using BMP-2 antibody-assessed protein production of rBMCs transduced with AdBMP-2^Myc. Lane A represents recombinant human BMP-2 (60 ng). Lanes B and C contained the conditioned medium from rBMCs infected with Adβ-gal and AdBMP-2^Myc, respectively. The 30-kDa band was detected on Lanes A and C by the BMP-2 antibody under nonreducing conditions (black arrow). No signal was detected from Lane B. (B) The same membrane also was analyzed with the Myc antibody after pretreated BMP-2 antibodies were completely stripped. In Lane C the same band, which was detected by BMP-2 antibodies, also was detected by the Myc antibody confirming production of virally mediated, epitope-tagged BMP-2.

Fig 3

Radiographic examinations of orthotopic bone in SCID mice hindlimbs are shown. Orthotopic bone formation was seen after implantation of rBMCs transduced with AdBMP-2^Myc at (A) At Day 7, orthotopic bone is beginning to form. (B) At Day 14, bone formation has peaked. At (C) Day 21, and (D) Day 56, additional maturation of the orthotopic bone has occurred. Radiographs taken on Day 56 of mice receiving implants containing (E) rBMCs transduced with Adβ-gal or (F) rBMCs alone show no mineralization or orthotopic bone formation.

Fig 4

Radiographs obtained 21 days postoperatively show SCID mice with radial defects treated with: (A) collagen sponge alone, (B) AdBMP-2^Myc-transduced cells, or (C) AdBMP-2^Control-transduced cells. Arrowheads in each figure identify location of persistent radial defects (A) and area of bony union (B) and (C).

Fig 5

Immunohistochemical analyses show the defect site for radial defects treated with AdBMP-2^Myc or AdBMP-2^control-transduced cells on a collagen sponge. (A). Myc staining of the AdBMP-2^Myc cell group at Day 3 revealed positively stained cells (arrow) in the collagen sponge (arrowhead) (Stain, Myc immunostaining; original magnification, ×20). (B) Myc staining was negative in the control group at Day 3 (Stain, Myc immunostaining; original magnification, ×20). (C) Myc staining at Day 14 in the AdBMP-2^Myc group revealed positive staining of cells in the area of the bone defect (Stain, Myc immunostaining; original magnification, ×20). Positive cells were visible in areas of endochondral ossification in the defect. (D) Myc-positive staining was not observed in the AdBMP-2^Control group at Day 14 (Stain, Myc immunostaining; original magnification, ×20). (E) In the AdBMP-2^Myc group at Day 3, BMP-2 immunostaining was strongly positive in cells (arrowhead) in the collagen sponge (arrow) (Stain, BMP-2 immunostaining; original magnification, ×40). (F) Bone morphogenetic protein-2 immunostaining was similarly positive in the AdBMP-2^Control group at Day 3 (Stain, BMP-2 immunostaining; original magnification, ×40). In the (G) AdBMP-2^Myc and (H) AdBMP-2^control group at 14 days, BMP-2 immunostaining was positive in hypertrophic chondrocytes (arrowheads) in endochondral fracture callus (arrows) (scale bars, 50 μmol/L) (Stain, BMP-2 immunostaining; original magnification, ×40).