Gene therapy platform for bone regeneration using an exogenously regulated, AAV-2-based gene expression system.
Viral delivery of the therapeutic gene bone morphogenetic protein-2 (BMP-2) is a promising approach for bone regeneration. The human parvovirus adeno-associated virus (AAV) type 2 is considered one of the most encouraging viral vector systems because of its high transduction rates and biosafety ratings. Bone morphogenetic protein-2 is a highly potent osteoinductive protein, which induces bone formation in vivo and osteogenic differentiation in vitro. The exogenous regulation of BMP-2 expression in bone-regenerating sites is required to control BMP-2 protein secretion, thus promoting safe and controlled bone formation and regeneration. We have therefore constructed a dual-construct vector for the recombinant AAV (rAAV)-based recombinant human BMP-2 (rhBMP-2) gene delivery system, which is regulated by the tetracycline-sensitive promoter (TetON). Each vector was encapsidated separately, yielding two recombinant viruses. We evaluated the efficiency of rAAV-hBMP-2 to induce bone formation in ectopic and orthotopic sites. Doxycycline (Dox), an analogue of tetracycline, was orally administered to mice via their drinking water to induce rhBMP-2 expression. Bone formation was measured using quantitative imaging-microcomputerized tomography and cooled charge-coupled device imaging-to detect osteogenic activity at the cellular level, detecting osteocalcin expression. The rAAV-hBMP-2-treated mice that were given Dox demonstrated bone formation in both in vivo models compared to none in mice prevented from receiving Dox. Thus, the Tet-regulated rAAV-hBMP-2 vector is an effective means of induction and regulation of bone regeneration and repair.
Gafni
Yossi
Y
Skeletal Biotechnology Laboratory, Hebrew University-Hadassah Medical Center, Ein Kerem, P.O. Box 12272, Jerusalem 91120, Israel.
Pelled
Gadi
G
Zilberman
Yoram
Y
Turgeman
Gadi
G
Apparailly
Florence
F
Yotvat
Hagit
H
Galun
Eithan
E
Gazit
Zulma
Z
Jorgensen
Christian
C
Gazit
Dan
D
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D001861:Bone Regeneration; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D004318:Doxycycline / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H; D008822:Mice, Transgenic; D009132:Muscles / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D013234:Stem Cells / Q000166:cytology; D013752:Tetracycline / Q000494:pharmacology; D014057:Tomography, X-Ray Computed; D016212:Transforming Growth Factor beta / Q000378:metabolism
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D015675:Osteocalcin; D013752:Tetracycline; D004318:Doxycycline