Kari J. Airenne

Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery.

Recombinant Autographa californica multiple nuclear polyhedrosis viruses (AcMNPV) have recently been shown to transduce mammalian cells in vitro. Since baculoviruses offer many advantages over viruses currently used in gene therapy, we have tested them for in vivo gene transfer by constructing a baculovirus bearing a nuclear targeted β-galactosidase marker gene (LacZ) under a CMV promoter. Both rabbit aortic smooth muscle cells (RAASMC) and human ECV-304 cells were susceptible to LacZ-baculovirus transduction. Transgene expression was evaluated in vivo by applying 1 × 109 p.f.u. of LacZ-baculoviruses or LacZ-adenoviruses in a silastic collar placed around rabbit carotid arteries in the absence of contact with blood components. As a result, baculoviruses led to transgene expression in adventitial cells in rabbit carotid arteries with efficiency comparable to adenoviruses. The β-galactosidase gene expression was transient staying at a high level for 1 week but disappearing at the 14 day time-point. The arterial structure and endothelium remained intact in the baculovirus-transduced arteries, but macrophage-specific immunostaining detected signs of inflammation comparable to adenoviruses. Baculoviruses are thus able to mediate transient gene transfer in vivo and may become useful tools for gene therapy.

Local Hypomethylation in Atherosclerosis Found in Rabbit ec-sod Gene

Extracellular superoxide dismutase (EC-SOD) protects arteries against deleterious effects of superoxide anions and the development of atherosclerosis. In this study, we cloned and characterized rabbit ec-sod gene. We identified 6 rabbit C-elements and 5 CpG clusters in the cloned sequence. One of the CpG clusters is located on the coding sequence. Because CpG clusters are potential sites for methylation and may explain the occurrence of mutations, methylation status of each of the CpG dimers located in the coding sequence CpG cluster was characterized using direct genomic sequencing. Unexpectedly, a marked reduction in the amount of methylated CpG dinucleotides in ec-sod gene was detected in atherosclerotic aortas as compared with normal aortic intima-media. Although alterations in DNA methylation are well characterized in malignant tumors, the presence of methylation changes in atherosclerosis has not been studied even though both diseases are characterized by excess cellular proliferation and alterations in gene expression. Further analysis of the whole genomic methylation by high-pressure liquid chromatography in normal and atherosclerotic aortas revealed a tendency for a decreased 5-methylcytosine (5-mC) content in atherosclerotic aortas as compared with normal arteries. Hypomethylation in atherosclerotic aortas occurred at the same level as has been reported from malignant tumors. Although a causal relationship between the methylation level and expression of EC-SOD cannot be proven, our results show that ec-sod hypomethylation is associated with the development of atherosclerosis and suggest that it may affect structure and function of ec-sod and other genes possibly involved in the development of atherosclerotic lesions.

Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered.

Generation of lentivirus vectors using recombinant baculoviruses.

In spite of advances in conventional four-plasmid transient transfection methods and development of inducible stable production cell lines, production of replication-defective lentiviral vectors in clinical scale has been challenging. Baculovirus technology offers an alternative to scalable virus production as a result of fast and easy production of baculoviruses, efficient transduction of mammalian cells and safety of the baculoviruses. As a first step toward scalable lentiviral production system, we have constructed four recombinant baculoviruses: the BAC-transfer virus expresses green fluorescent protein (GFP) as a transgene and BAC-gag-pol, BAC-vesicular stomatitis virus glycoprotein G and BAC-rev express all elements required for a safe lentivirus vector generation. Following 293T cell transduction with recombinant baculoviruses functional lentiviruses were produced. Different baculovirus concentrations, mediums and transduction times were used to find optimal conditions for lentivirus production. The unconcentrated lentiviral titers in cell culture mediums were on average 2.5 × 106u2009TUu2009ml−1, which are comparable to titers of the lentiviruses produced by conventional four-plasmid methods. Lentiviruses produced by baculovirus method transduced HeLa cells and showed sustained GFP expression. No evidence of the formation of replication competent lentiviruses was detected by p24 enzyme-linked immunosorbent assay. Our results show that baculoviruses are an attractive alternative for the production of lentiviruses in mammalian cells.

Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors

Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.

Screening of complement inhibitors: shielded baculoviruses increase the safety and efficacy of gene delivery.

One of the major obstacles in the use of baculovirus vectors for in vivo gene transfer is the virus inactivation by serum complement. In this study, we investigated the effect of decay-accelerating factor (DAF), factor H (FH)-like protein-1 (FHL-1), C4b-binding protein (C4BP), and membrane cofactor protein (MCP) on protection of baculovirus vectors from the complement-mediated inactivation. Complement regulatory proteins were displayed on baculovirus surface as fusions to membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein. This strategy resulted in abundant expression of recombinant proteins on the viral envelope while viral titers comparable to control virus were reached. The surface-modified vectors exhibited complement resistance in vitro, DAF showing the highest level of protection. Intraportal delivery of DAF-displaying baculovirus resulted in increased survival and enhanced gene expression in immunocompetent mice. Mice receiving DAF-displaying baculovirus also exhibited lower level of liver inflammation as evidenced by aspartate aminotransferase (AST). In line with this, macrophages treated with DAF baculovirus produced lower levels of inflammatory cytokines IL-1beta, IL-6, and IL-12p40 compared to control virus. These results suggest that DAF-display can protect the vector against complement inactivation but also reduce complement-mediated inflammation injury. In conclusion, complement shielded baculovirus vectors represent attractive tools for effective in vivo gene delivery.

6-O- and N-Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells

ABSTRACT Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.

How to avoid complement attack in baculovirus-mediated gene delivery

Serum inactivation of baculovirus vectors is a significant barrier to the development of these highly efficient vectors for therapeutic gene delivery. In this review we will describe the efforts taken to avoid complement attack by passive or active measures. Evidently good targets for baculovirus-mediated gene delivery include immunoprivileged tissues, such as eye, brain and testis. Similarly baculovirus vectors have also proven their efficacy in an ex vivo setting for tissue engineering. Active measures to inhibit complement include the use of pharmacological inhibitors of complement as well as surface engineering of the baculoviral vectors through the use of synthetic polymers, pseudotyping or display of complement inhibitors. Lessons learned from these studies will significantly increase the possibility of using baculovirus vectors for therapeutic applications.

Requirements for baculoviruses for clinical gene therapy applications

Baculoviruses have proven capacity for the production of recombinant proteins including virus-like particles and as viral vectors. Recent progress in preclinical studies suggest that baculoviruses have potential as new vectors for gene therapy but so far no clinical trials have been performed. To date, no specific guidelines for the use of baculoviruses as human gene therapy vectors exist but researchers can utilize existing guidelines made for other biological products. Because of the long history of research on baculoviruses, a lot of knowledge has been obtained that forms a good basis for the gene therapy development process. This article gives an overview of the current status of the application of baculovirus vectors in gene therapy and summarizes some of the challenges to overcome before the first clinical trials with baculoviruses can be accomplished.

Sleeping Beauty–baculovirus hybrid vectors for long‐term gene expression in the eye

A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid‐based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors.