Gene Therapy Platform for Bone Regeneration Using an Exogenously Regulated, AAV-2-Based Gene Expression System

Abstract

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.

Roles of exogenously regulated bFGF expression in angiogenesis and bone regeneration in rat calvarial defects

“…In addition, the expression of Cbfa1, ALP, and OC was also inhibited. Therefore, this dosage was sufficient to minimize the expression of foreign genes, consistent with the in vitro results of other researchers (11,21).…”

“…rAAV2 can infect both mitotic and post-mitotic cells of various types, producing stable gene expression without inducing toxic and immune responses (11). Considering these advantages and its tissue-specific features, rAAV2-mediated gene transfer is widely used for bone and cartilage studies (11,12). Transgenic regulation is particularly important in gene therapy.…”

“…Therefore, it is necessary to regulate transgene expression, in particular, to suppress transgene expression. The tetracycline (Tet) regulatory system (11,13,14), which has high efficiency and low toxicity, contains two components: the transactivator, tTA, and the Tet-regulated element (TRE). In the Tet-off system (14), tTA binds TRE and activates transgene expression in the absence of tet or the tet analog, doxyccyline (Dox).…”

How to use BMP-2 for clinical applications? A review on pros and cons of existing delivery strategies

“…A major concern of gene therapy is the stable and controllable overexpression of the delivered gene. In order to prevent extreme expression levels, which in terms of BMP-2 delivery might result in unwanted off-target effects, constructs with inducible promoters -such as tetracycline-sensitive promoters (TetON) -might be favorable [46].…”

Realizing the potential of gene-based molecular therapies in bone repair

“…Bone induction via endochondral ossification was observed in hindlimb muscle of immunocompetent rats (101,102) Viral particles delivered on hydroxyapatite scaffold to the back muscles of immunocompetent rats led to bone formation (103) Bone formation in thigh muscle after delivery of a tetracycline-sensitive expression system was observed in mice only when a tetracycline analogue was administered (153) Orthotopic AV Increased regeneration in a mandibular distraction osteogenesis model in rats (154) Increased bone regeneration in an osteoporotic fracture model in tibia of sheep (155) Bone formation or increased regeneration was observed in several defect models, including a critical-size mandibular defect, (33) critical-size nasal defect in athymic nude mice, (156) rib defect in horses, (157) metatarsal defect in horses, (158) and femoral critical-size defect in rats (159) Injected AVs led to partial regeneration of critical-size calvarial defects in rats, with a more vigorous response when particles were delivered in a gelatin scaffold (117) Healing of iliac crest critical-size defects in sheep was delayed compared with no treatment when viral particles were injected to injury site (160) Bone formation was observed in a dental model in immunocompetent dogs (107) Enhanced cartilage and subchondral bone was observed in femur condyle defect in immunocompetent ponies (161) Delaying administration of viral particles improved healing of femur critical-size defects in rats (162) …”