Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.Recombinant adenoviruses currently are used for a variety of purposes, including gene transfer in vitro, vaccination in vivo, and gene therapy (1-4). Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained.Decades of study of adenovirus biology have resulted in a detailed picture of the viral life cycle and the functions of the majority of viral proteins (5, 6). The genome of the most commonly used human adenovirus (serotype 5) consists of a linear, 36-kb, double-stranded DNA molecule. Both strands are transcribed and nearly all transcripts are heavily spliced. Viral transcription units are conventionally referred to as early (E1, E2, E3, and E4) and late, depending on their temporal expression relative to the onset of viral DNA replication (6). The high density and complexity of the viral transcription units poses problems for recombinant manipulation, which therefore is usually restricted to specific regions, particularly E1, E2A, E3, and E4. In most recombinant vectors, transgenes are introduced in place of E1 or E3, the former supplied exogenously. The E1 deletion renders the viruses defective for replication and incapable of producing infectious viral particles in target cells; the E3 region encodes proteins involved in evading host immunity and is dispensable for viral production per se.Two approaches traditionally have been used to generate recombinant adenoviruses. The first involves direct ligation of DNA fragments of the adenoviral genome to restriction endonuclease fragments containing a transgene (7,8). The low efficiency of large fragment ligations and the scarcity of unique restriction sites have made this approach technically challenging. The second and more widely used method involves homologous recombination in mammalian cells capable of complementing defective adenoviruses (''packaging lines'') (9, 10). Homologous recombination results in a defective adenovirus that can replicate in the packaging line (e.g., 293 or 911 ce...

Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.

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Osteosarcoma is the most common nonhematologic malignancy of bone in children and adults. The peak incidence occurs in the second decade of life, with a smaller peak after age 50. Osteosarcoma typically arises around the growth plate of long bones. Most osteosarcoma tumors are of high grade and tend to develop pulmonary metastases. Despite clinical improvements, patients with metastatic or recurrent diseases have a poor prognosis. Here, we reviewed the current understanding of human osteosarcoma, with an emphasis on potential links between defective osteogenic differentiation and bone tumorigenesis. Existing data indicate osteosarcoma tumors display a broad range of genetic and molecular alterations, including the gains, losses, or arrangements of chromosomal regions, inactivation of tumor suppressor genes, and the deregulation of major signaling pathways. However, except for p53 and/or RB mutations, most alterations are not constantly detected in the majority of osteosarcoma tumors. With a rapid expansion of our knowledge about stem cell biology, emerging evidence suggests osteosarcoma should be regarded as a differentiation disease caused by genetic and epigenetic changes that interrupt osteoblast differentiation from mesenchymal stem cells. Understanding the molecular pathogenesis of human osteosarcoma could ultimately lead to the development of diagnostic and prognostic markers, as well as targeted therapeutics for osteosarcoma patients.

Bone morphogenetic protein 9 (BMP‐9) is a member of the transforming growth factor (TGF)‐β/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/β‐catenin signalling plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. Wnt3A and BMP‐9 enhanced each other’s ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP‐9‐induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR‐5 or low‐density lipoprotein receptor‐related protein (LRP)‐6 exerted no inhibitory effect on BMP‐9‐induced ALP activity. β‐Catenin knockdown in MSCs and MEFs diminished BMP‐9‐induced ALP activity, and led to a decrease in BMP‐9‐induced osteocalcin reporter activity and BMP‐9‐induced expression of late osteogenic markers. Furthermore, β‐catenin knockdown or FrzB overexpression inhibited BMP‐9‐induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP‐9 induced recruitment of both Runx2 and β‐catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between β‐catenin and Runx2, plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs.

With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.