Current Gene Therapy - Volume 10, Issue 3, 2010

The widely used viral gene therapy vectors include retrovirus, lentivirus, adenovirus and adeno-associated virus, which have proven to be clinically valuable in numerous trials. These vectors, however, possess respective drawbacks and may raise safety concerns [1] although recent clinical trials have shown encouraging results [2]. Moreover, the production of these vectors has posed challenges in large scale production. In contrast to the commonly employed viral vectors, baculovirus is an insect virus and has been extensively employed for recombinant protein production by infecting its natural host insect cells since 1980s. However, two groups in the mid-1990s uncovered that baculovirus can enter mammalian cells and mediate transgene expression as long as the transgene is driven by a promoter active in mammalian cells [3,4]. Since then, following studies have demonstrated that baculovirus can efficiently transduce cells originating from human, rodent, porcine, bovine, rabbit, fish and avian species [5]. Baculovirus also transduces dividing and nondividing cells as well as primary cells including mesenchymal stem cells [6] and embryonic stem cells [7]. In mammalian cells, baculovirus neither replicates nor imposes apparent cytotoxicity, hence minimizing the possible side effects. Also baculovirus is non-pathogenic, hence the handling and production can be carried out in biosafety level 1 laboratories. Baculovirus is easy to construct and produced to high titers and can accommodate a single large insert or multiple genes encompassing the regulatory elements [5,8,9]. These findings and attributes have fueled the interests to explore baculovirus as a vector for gene therapy and a plethora of emerging applications. In this special issue, 3 papers review the development of baculovirus vectors for in vitro applications including cell-based assay development for drug screening [10], display of eucaryotic proteins [11] and recombinant protein production [12]. Baculovirus has been used for in vivo gene delivery, hence its in vivo applications and monitoring are crucial and reviewed in this issue [13]. Inspired by the accumulation of more fundamental knowledge and discovery of new permissive cell types, baculovirus has also been exploited in emerging fields such as cancer therapy [14], vaccine development [15] and tissue regeneration [16], which are reviewed by Madhan et al., Wang and Balasundaram, and Lin et al., respectively. Due to the mounting interests and applications, whether baculovirus is truly safe and what immune responses are provoked after baculovirus transduction of cells or administration into animals have emerged as an important issue, which is reviewed by Abe and Matsuura [17]. Finally, the translation of baculovirus technology from bench to bedside hinges critically on whether the baculoviral vectors can be produced and purified in large scale at reasonable cost, therefore, the bioprocessing of baculoviral vectors is reviewed by Aucoin et al. [18]. Compared to other well-known viral vectors, the development of baculoviral vector for gene delivery is still in its infancy. To date baculovirus-mediated gene therapy remains unknown to a large population of scientific community and has yet to advance to clinical trials. Nevertheless, the potential of baculovirus has attracted a rapidly increasing number of research groups to enter this field, with research topics covering the fundamental virology studies, viral vector development, new applications, to downstream production and purification. Although more needs to be done to elucidate and ameliorate the safety profile and efficacy, it is anticipated that this technology will yield dividends in coming years.

Modern drug discovery programs utilize a wide variety of technologies to aid in identification of potential drug targets, and progress them through the often long and winding path of finding novel drug-like molecules. Recombinant cell-based assays are an important tool in the drug discovery process for investigating the biological mechanisms of potential drug targets and conducting screening campaigns in the hunt for biologically active molecules. Historically, stable cell lines expressing the target protein(s) of interest have been used for these assays. Although such cell lines can be useful, their development can be laborious and the resulting cell line affords little experimental flexibility. Transient gene expression approaches provide an alternative to the often tedious task of developing and maintaining numerous stable cell lines. Recently the unique properties of modified baculoviruses, containing mammalian expression cassettes and referred to as BacMam viruses, have been exploited to facilitate rapid and reproducible transient cell-based assay development. This review will focus on the many features of BacMam virus gene delivery that make it a powerful system for cell-based assay development and screening.

The baculovirus/insect cell expression system is best known, and used, as a research tool for the production of recombinant proteins often requiring post-translational modifications. Although studies on the larger scale use of the system have been reported on for three decades, the recognition of this system as an industrially relevant platform for the production of biologics has only been recently achieved with the approvals of baculovirus-derived human and veterinary vaccines for commercialization. The full utility of baculoviruses, however, does not end with the production of recombinant proteins; baculoviruses are increasingly being studied for their ability to transduce mammalian cells and use as a therapeutic themselves. There is, therefore, a need to revisit the current state of the art in the bioprocessing of baculoviruses as the product. This paper is an extensive review of what is currently known about the amplification and recovery of baculoviruses and highlights the gaps that have not been focused on in the midst of optimizing this system for protein production.

Baculoviruses are safe and high-capacity vectors for gene delivery which have matured from the initial successful experiments performed in liver cells into convenient tools to transduce almost any cell from any origin in vitro and ex vivo. This is a result of 15 years of intensive vector development as well as studies performed in vertebrate cells to reveal important factors affecting the transduction efficacy. Now, at the stage when the first evidence of meaningful use of baculoviruses for therapeutic applications has been reported, there is no doubt that the technology will meet the expectations as highly useful platform for many applications of gene delivery. This review summarizes the pre-clinical in vivo work carried out with baculoviruses and discusses remaining challenges which still need to be solved.

The baculovirus Autographa californica nuclear polyhedrosis virus is an attractive candidate for eukaryotic virus display. A variety of strategies exists to incorporate and present target proteins on the surface of infected insect cells as well as on budded virions. Native baculovirus proteins such as the major envelope protein and the capsid protein, but also foreign scaffolds such as the vesicular stomatitis virus G-1 protein or the influenza A virus hemagglutinin serve as fusion partners for surface presentation of target proteins. The purposes of surface display are manifold; efficient presentation of antigenic epitopes on budded virions serves to induce specific immune response, designed surface modifications alter the binding properties and host specificities of the baculovirus to e.g. mammalian cells and help to enhance mammalian cell transduction. Eukaryotic surface display libraries based on the baculovirus system allow selecting for specific binding proteins while providing post translational modifications. Here we describe the different possibilities and strategies to accessibly present foreign proteins and peptides on the surface of insect cells and baculoviruses, as well as the applications thereof, including vector design, cloning strategies and construction and screening of surface expression libraries.

Application of viral vectors derived from human viruses to mediate immune response in animals and humans has been greatly hampered by the problems associated with pre-existing immunity and associated toxicities. Among few non-human viral vectors, baculovirus has now evolved as a novel tool for vaccine vector development. With broad tissue tropism and expanded bio-safety profile suitably supplemented with intrinsic immunostimulatory properties, baculovirus has now attained a niche position in the arena of vaccine development. Recombinant envelope-modified baculovirus equipped with novel shuttle promoters for in vivo transduction has shown promising results in several animal models. Baculovirus mediated induction of systemic and mucosal immune responses through intranasal or oral administration has now opened an entirely new way for the development of new generation vaccines. Gaining additional insight into the baculovirus biology and its interaction with non-native hosts will certainly promote this human-friendly virus as a potential vector for clinical applications.

Many different types of therapeutic genes, ranging from suicide genes, tumor suppressor genes, to genes encoding tumor-specific antigens, have been successfully delivered by insect baculoviral vectors to treat tumours in animal models. These encouraging results observed to date underscore the potential for using the non-human baculovirus to combat human cancer. The present review outlines the advances in this area and highlights the challenges behind translating the findings from research with baculoviral vectors into clinical practice.

The baculovirus Autographa californica nuclear polyhedrosis virus has been widely used not only to achieve a high level of foreign gene expression in insect cells but also for efficient gene transduction into mammalian cells without any replication. In addition to the efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. The baculovirus has abundant CpG motifs in the viral genome and is capable of inducing pro-inflammatory cytokines and interferons through Toll-like receptor-dependent and -independent signaling pathways in a cell-type-specific manner. The baculovirus also has a strong adjuvant activity, and recombinant baculoviruses encoding neutralization epitopes elicit protective immunity in mice. This review deals with the current status of our knowledge of the induction of host innate immune responses by baculovirus and discusses the future prospects for baculovirus vectors.

Since the discovery of mammalian cells susceptible to baculovirus transduction in 1995, baculovirus has emerged as a safe, economical and convenient tool for introducing foreign genetic material into mammalian systems. The feasibility of baculovirus as a novel gene transfer vehicle for large scale mammalian protein expression was not obvious at first, as the baculovirus genome is unable to amplify within mammalian cells and expression of the transferred foreign gene does not usually last more than 20 days. However, several recent articles have demonstrated that baculovirus contains both the elements - hr sequences, and the factor - IE2 protein, which can function in mammalian cells to dramatically boost baculovirus-transduced gene expression. The longevity of genes transferred by baculovirus can also be extended by inserting a replication ori with the necessary factors and induce self-replicating episomes. Lastly, peptide-based baculovirus surface modification and two groups of drugs: histone deacetylase inhibitors and microtubule depolymerizing agents, which improve baculovirus gene expression were described. In this article we summarized ways to activate, enhance, and prolong baculovirus-mediated foreign protein expression. The mechanism behind IE2 activation through its unique nuclear body structure was given special emphasis, including our most current data which suggested PML NBs could be disrupted by IE2. Finally, strategies to maximizing mammalian protein production through baculovirus gene transfer are proposed in prospect to current breakthroughs.

Baculovirus is an effective vector for gene delivery into various mammalian cells, including chondrocytes and mesenchymal stem cells, and has been employed for diverse applications. By gene delivery and expression of the growth factor, recombinant baculovirus has been shown to modulate the differentiation state of the cells and stimulates the production of extracellular matrix and tissue formation, hence repairing the damaged cartilage and bone in vivo. This article reviews the studies pertaining to the applications of baculovirus-mediated gene delivery in cartilage and bone tissue engineering and discusses recent progress, future applications and potential hurdles.