Development of a Cell-Based Gene Therapy Approach to Selectively Turn Off Bone Formation

Abstract

Cell and gene therapy approaches are safer when they possess a system that enables the therapy to be rapidly halted. Human mesenchymal stem cells were transduced with an adenoviral vector containing the cDNA for bone morphogenetic protein 2 (AdBMP2) to induce bone formation. To make this method safer, a system to quickly kill these virally transduced cells was designed and evaluated. Cells were encapsulated inside poly(ethylene glycol) diacrylate (PEG-Da) hydrogels that are able to shield the cells from immunological destruction. The system involves an inducible caspase-9 (iCasp9) activated using a specific chemical inducer of dimerization (CID). Delivering AdBMP2-transduced human mesenchymal stem cells encapsulated in PEG-Da hydrogel promoted ectopic ossification in vivo, and the iCasp9 system allowed direct control of the timing of apoptosis of the injected cells. The iCasp9-CID system enhances the safety of delivering AdBMP2-transduced cells, making it a more compelling therapeutic for bone repair and spine fusion. J. Cell. Biochem. 118: 3627-3634, 2017. © 2017 Wiley Periodicals, Inc.

Keywords: BIOMATERIAL; BONE FORMATION; CELL THERAPY; DELIVERY SYSTEM; IN VIVO; INDUCIBLE SUICIDE GENE.

Fig. 1

(A) Cell viability of human mesenchymal stem cells possessing a stably integrated inducible caspase 9 (iCasp9) when treated with a chemical inducer of dimerization (CID) or vehicle. All data are reported as the mean ± standard deviation for n = 3. (B) Quantification of alkaline phosphatase (ALP) in W20-17 cells. Media collected from the AdBMP2-transduced hMSCs-iCasp9 cells cultured in the presence of CID or vehicle was added to culture media of W20-17 cells, and 72 h later alkaline phosphatase activity was measured by a chemiluminescent assay. Alkaline phosphatase activity was reported in relative luminescence units (RLUs). All data are reported as the mean ± standard error of the mean for n = 3. * Represents significant difference between groups (P <0.05). (C) LIVE/DEAD staining cultured in complete medium after 24 h. Cells in culture medium (A–C), in the presence of 50 μg of a chemical inducer of dimerization (CID) and 100 ng/ml polyethylenimine (PEI). Maximum intensity projection of green (FITC) channel (A,D,G), red (rhodamine) channel (B,E,H), and merge of all channels (C,F,I). Dead cells appear red and live cells appear green; 20× mag.

Fig. 2

(A) Far-red fluorescence emission after (B) direct injection or (C) encapsulation in PEGDA hydrogel of AdBMP2-AdiRFP transduced hMSC-iCasp9 cells into NOD/SCID mice in the presence or absence of CID. Quantification of fluorescence intensity in mice receiving these cells (Day 0) in the absence and presence of CID was reported as the mean ± standard error of the mean for n = 4. * Represents significant difference of fluorescence intensity between groups (P <0.05).

Fig. 3

MicroCT analysis of tissues 2 weeks after intramuscular delivery of AdBMP2-transduced hMSCs possessing the inducible caspase 9 (iCasp9). (A) Representative three-dimensional reconstruction of bone formation in mice treated with chemical inducer of dimerization (CID, 50 μg) compared to (B) mice treated with vehicle. (C–F) Representative photomicrographs of hematoxylin and eosin stained section of the newly forming bone.

Fig. 4

Mice exposed to CID or vehicle at 2, 4, 6, and 8 days after injection of AdBMP2-AdiRFP transduced hMSCs-iCasp9 cells during the formation of heterotopic bone formation. (A) Far-red fluorescence expression in the mice. (B) Quantification of far-red fluorescence in the mice. (C) Representative three-dimensional reconstructions of the microCT data collected from imaging the mice. (D) Quantification of bone volume in the mice. All data are reported as the mean ± standard error of the mean for n = 3. * Represents significant difference between groups (P <0.05).