Send Orders of Reprints at reprints@benthamscience.net Current Gene Therapy, 2013, 13, 000-000 1 1566-5232/13 $58.00+.00 © 2013 Bentham Science Publishers Thymidine Kinase-Mediated Shut Down of Bone Morphogenetic Protein-4 Expression Allows Regulated Bone Production Barbara Lombardo 1,2 , Teresa Rocco 2 , Maria Teresa Esposito 2 , Bruno Cantilena 2 , Sara Gargiulo 2 , Adelaide Greco 2,3 , Donatella Montanaro 2 , Arturo Brunetti 2,3 and Lucio Pastore 1,2, * 1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli "Federico II", Via S. Pansini 5, 80131 Napoli, Italia; 2 CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italia; 3 Dipartimento di Scienze Biomorfologiche e Funzionali, Università degli Studi di Napoli "Federico II", Via S. Pansini 5, 80131 Napoli, Italia Abstract: Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previ- ously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK ex- pression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the con- tralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic ac- tivity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with gan- ciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore block- ing unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor. Keywords: FG-B4, FG-B4TK, bone production regulation. INTRODUCTION Bone morphogenetic proteins (BMPs) are extremely powerful in inducing osteoblast differentiation of bone mar- row stromal cells (BMSC) and bone formation in vitro and in vivo [1-3]. Several BMPs are currently under study to deter- mine their efficacy for the treatment of numerous bone pathologic conditions [4, 5]. The BMPs family is composed of more than 40 growth factors and belongs to the transform- ing growth factor- (TGF) superfamily. All the members of the superfamily share a high degree of homology within the C-terminal seven-cysteine region and bind to specific cell surface receptors to control morphogenetic events during tissue and organ development [6]. Many of these proteins are characterized by their ability to induce ectopic and ortho- topic bone formation when delivered in vivo [7, 8]. BMPs are able to stimulate BMSCs chemotaxis and proliferation, followed by their subsequent differentiation into chondro- cytes and osteoblasts [9]. Many family members of BMPs exhibit osteogenic activity and are promising therapeutic *Address correspondence to this author at the CEINGE-Biotecnologie A- vanzate, Via Gaetano Salvatore 486, 80145, Napoli, Italia; Tel: +39-081- 3737885; Fax: +39-081-3737808; E-mail: pastore@ceinge.unina.it tools for skeletal regeneration. Adenoviral vectors carrying cDNAs for BMP-2, -7, and -9 effectively generate bone in a variety of in vivo models [10, 11]. BMP-2, -4, and -7 stimu- late osteogenic and chondrogenic differentiation via BMP receptor (BMPR) types I and II [12] through SMADs signal- ing [13]. Recombinant BMP-2 and -7 proteins have already tested for clinical application in complicated traumas such as open and discontinuous fractures [14, 15]. Strategies of BMPs delivery are based on recombinant protein embedded within matrices or implantation of BMPs-secreting cells [16]. In our experimental work, we focused on BMP-4 be- cause its expression levels increase ten times compared to baseline during fracture repair and is able to induce en- dochondral ossification [17, 18]. BMPs overexpression may also lead to bone overproduc- tion and exostosis that can cause problems in their clinical applications [19]. BMPs secretion can be regulated using different approaches such as promoters responsive to tetra- cyclin [20], tamoxifen or rapamycine [21]. However, most of these systems shows “leakiness”: in fact, it is virtually im- possible to completely block transgene production with these promoters. In our work, we have decided to induce apoptosis of transduced cells with, as a consequence, block transgene secretion. In order to achieve apoptosis of transduces cells,