人骨形态发生蛋白-2重组腺病毒的构建与制备

将人BMP-2的编码区cDNA克隆至穿梭载体pShuttle,以PI-SceI和I-CeuI切下含BMP-2编码区cDNA的片断,在体外与PI-SceI/I-CeuI切开的腺病毒DNA连接,构建重组有BMP-2全长编码区基因的腺病毒DNA,PCR鉴定正确后,经PacI酶切线性化,在脂质体介导下转染HEK293细胞,反复冻融制备重组腺病毒,空斑形成试验测定病毒滴度约为7.5×106～1.5×107pfu/ml。以BMP-2重组腺病毒感染体外培养的小鼠成肌细胞C2C12,Westernblot检测证实有BMP-2表达。     

Combination of adeno-associated virus and adenovirus vectors expressing bone morphogenetic protein-2 produces enhanced osteogenic activity in immunocompetent rats

We have previously shown that gene therapy using adeno-associated virus (AAV) carrying bone morphogenetic proteins (BMPs) is a promising strategy for new bone formation in vivo in SD rats. However, it had a relatively low transduction efficiency. We investigate here whether enhanced osteogenic activity can be achieved without eliciting a severe immune response, using a cocktail of AAV-BMP2 and adenovirus (Ad)-BMP2 as a vector system. The muscles of SD rats were injected with either AAV-BMP2, Ad-BMP2, or an AAV-BMP2/Ad-BMP2 cocktail, and the in vivo bone formation was determined at eight weeks post-injection. Radiographic examination demonstrated that the addition of a low level of Ad-BMP2 to AAV-BMP2 produced significantly higher new bone formation than the use of AAV-BMP2 alone. Histological and immunohistological analysis revealed an enlarged bone-forming area and a long-term BMP2 expression, without pronounced infiltration of lymphocytes. Our results provide the first evidence that the introduction of a low level of adenovirus in vivo in immunocompetent subjects can greatly enhance AAV-mediated gene transfer, without inducing severe immune responses. This cocktail vector system may offer an attractive way of improving the efficiency of AAV-based gene delivery.     

Structural and functional healing of critical-size segmental bone defects by transduced muscle-derived cells expressing BMP4

BACKGROUND: Our previous studies have shown that muscle-derived cells, including a population of muscle stem cells, transduced with a retroviral vector expressing bone morphogenetic protein 4 (BMP4) can improve the healing of critical-size calvarial defects. However, we did not evaluate the functionality of the healed bone. The purpose of this study was to determine whether primary muscle-derived cells transduced with retroBMP4 can heal a long bone defect both structurally and functionally. METHODS: Primary muscle-derived cells were genetically engineered to express BMP4 and were implanted into 7-mm femoral defects created in syngeneic rats. Muscle-derived cells transduced with retroLacZ were used in the control group. Bone healing was monitored by radiography, histology, and biomechanical testing at designated time points. RESULTS: Most of the defects treated with muscle-derived cells expressing BMP4 formed bridging callous by 6 weeks after surgery, and exhibited radiographically evident union at 12 weeks after cell implantation. Histological analysis at 12 weeks revealed that the medullary canal of the femur was restored and the cortex was remodeled between the proximal and distal ends of each BMP4-treated defect. In contrast, the defects treated with muscle-derived cells expressing beta-galactosidase displayed nonunion at all tested time points. An evaluation of the maximum torque-to-failure in the treatment group indicated that the healed bones possessed 77 +/- 28% of the strength of the contralateral intact femora. Torsional stiffness and energy-to-failure were not significantly different between the treated and intact limbs. CONCLUSIONS: This study demonstrated that primary muscle-derived cells transduced with retroBMP4 can elicit both structural and functional healing of critical-size segmental long bone defects created in rats.     

Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation

Adeno-associated virus (AAV) is so far the most valuable vehicle for gene therapy because it has no association with immune response and human disease. The present study was conducted to investigate the feasibility of AAV-mediated BMP4 gene transfer for bone formation. In vitro study suggested that AAV-BMP4 vectors could transduce myoblast C2C12 cells and produce osteogenic BMP4. In vivo study demonstrated that new bone formation could be induced by direct injection of AAV-BMP4 into the skeletal muscle of immunocompetent rats. Histological analysis revealed that the newly formed bone was induced through endochondral mechanism. Immunohistochemical staining further demonstrated that AAV-BMP4 gene delivery could mediate long-term transduction, and the involvement of BMP4 expression was responsible for the endochondral ossification. This study is, to our knowledge, the first report in the field of AAV-based BMP gene transfer and should be promising for clinical orthopaedic applications.     

Peptide drugs accelerate BMP‐2‐induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling

Both W9 and OP3‐4 were known to bind the receptor activator of NF‐κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide‐induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3‐4 accelerated BMP‐2‐induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL‐binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP‐2‐induced bone regeneration by the RANKL‐binding peptides.     

Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7

Bone morphogenetic proteins (BMPs) are well-established agents for inducing orthotopic and ectopic bone formation. However, their clinical usefulness as regenerative agents may be limited by a short in vivo half-life and low specific activity. BMP gene therapy is an alternative route for exploiting the bone-inductive activity of this class of molecules. To test the feasibility of this approach, we examined the osteogenic activity of AdCMV-BMP7, an adenovirus containing BMP7 cDNA under control of the CMV promoter that was constructed using Cre/lox recombination (Hardy et al. [1997] J. Virol. 71:1842-1849). Adenovirus vectors were shown to readily infect a wide variety of cell types in vitro including osteoblasts, fibroblasts, and myoblasts. COS7 cells transduced with AdCMV-BMP7 produced high levels of BMP-7 (approximately 0.5 microg/10(6) cells). Furthermore, transduction of C2C12 murine myoblast cells with AdCMVBMP-7 suppressed the muscle phenotype and induced in vitro osteoblast differentiation. To test its in vivo biological activity, AdCMV-BMP7 was mixed with a bovine bone-derived collagen carrier (10(8) plaque-forming units virus/site) and was implanted into mouse muscle and dermal pouches. In both cases, an ossicle containing cortical and trabecular bone and a clearly defined marrow cavity formed at the site of virus implantation within 4 weeks. These data demonstrate that AdCMV-BMP7 transduced cells produce biologically active BMP-7 both in vitro and in vivo and show that gene therapy by direct viral transduction using a virus/matrix implant may be a viable route for stimulating bone regeneration.     

Growth and differentiation of a long bone in limb development,repair and regeneration

Repair from traumatic bone fracture is a complex process that includes mechanisms of bone development and bone homeostasis. Thus, elucidation of the cellular/molecular basis of bone formation in skeletal development would provide valuable information on fracture repair and would lead to successful skeletal regeneration after limb amputation, which never occurs in mammals. Elucidation of the basis of epimorphic limb regeneration in amphibians would also provide insights into skeletal regeneration in mammals, since the epimorphic regeneration enables an amputated limb to re‐develop the three‐dimensional structure of bones. In the processes of bone development, repair and regeneration, growth of the bone is achieved through several events including not only cell proliferation but also aggregation of mesenchymal cells, enlargement of cells, deposition and accumulation of extracellular matrix, and bone remodeling.     

Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy.

Recombinant adenoviruses with E1 sequences deleted efficiently transfer genes into a wide variety of target cells. Antigen- and nonantigen-specific responses to the therapy lead to toxicity, loss of transgene expression, and difficulties with vector readministration. We have created new cell lines that allowed the isolation of more disabled adenovirus vectors that have both E1 and E4 deletions. Studies with murine models of liver-directed gene therapy indicated that the E1- and E4-deleted vector expresses fewer virus proteins and induces less apoptosis, leading to blunted host responses and an improved safety profile. The impact of the E4 deletion on the stability of vector expression was confounded by immune responses to the transgene product, which in this study was beta-galactosidase. When transgene responses were eliminated, the doubly deleted vector was substantially more stable in mouse liver than was the E1-deleted construct. These studies indicate that adenovirus vectors with both E1 and E4 deletions may have advantages in terms of safety and efficacy over first-generation constructs for liver-directed gene therapy.     

Development of the vertebral morphogenetic field in the mouse: interactions between Crossveinless-2 and Twisted Gastrulation

Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2; Tsg and Cv2; Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.