Recombinant adeno-associated viral vector types 4 and 5. Preparation and application for CNS gene transfer.
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Animals↗
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Viral vectors for gene delivery in tissue engineering.
The goal of tissue engineering is the production of functional, biocompatible tissues by seeding cells within biological or synthetic scaffolds. One tissue engineering approach involves the genetic modification of cells that are seeded onto (or into) scaffolds prior to implantation. The genetic modification is achieved through gene delivery, with can utilize viral transduction or non-viral transfection systems. Although novel non-viral systems have continued to emerge as innovative vehicles for controlled gene delivery, viruses remain the most efficient means by which exogenous genes can be introduced into and expressed by mammalian cells. Retrovirus, adenovirus, adeno-associated virus and herpes virus are widely studied viral gene transfer systems and have attracted the most attention in the field of transduction. This review thoroughly discusses the genomic structures of each virus type, along with the advantages and disadvantages of their use in tissue engineering applications.
Adenoviridae↗
Concentration of viral vectors by co-precipitation with calcium phosphate.
BACKGROUND: The envelope glycoproteins, surface unit (SU) and transmembrane (TM) of the murine leukemia virus (MLV) are not covalently linked and tend to dissociate upon high-speed centrifugation, leading to loss of vector infectivity. This study describes a gentle and simple method to concentrate MLV vectors or HIV vectors pseudotyped with MLV envelopes. Having a fast and inexpensive method to concentrate large volumes of vector supernatant will facilitate in vivo experiments and clinical trials that require high titer vector stocks. METHODS: The methods employed in the study were co-precipitation of viral supernatant with calcium phosphate, low-speed centrifugation, dialysis, and infection assays with Lac-Z transducing vectors. RESULTS: Murine leukemia virus vectors and HIV vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV.G) or MLV envelopes were concentrated successfully using the calcium phosphate co-precipitation method. Parameters that influence virus yield and the reproducibility of the method were investigated. The optimized protocol involves virus harvest in serum-free media, co-precipitation using 60mM calcium chloride, pelleting at 2,000 g, resuspending the pellet in a small volume of 0.1M EDTA-saline, and dialysis against saline to remove EDTA. Volumes were decreased from 300 ml to 10 ml, with 50-100% recovery, and titers can be concentrated up to 1,000-fold. CONCLUSIONS: The calcium phosphate co-precipitation method to concentrate virus is applicable to retrovirus and lentivirus preparations. It uses simple techniques and does not require expensive equipment. Multiple rounds of co-precipitation can be carried out if required.
Animals↗
Non-viral vectors in cancer gene therapy: principles and progress.
This review focuses on the use of synthetic (non-viral) delivery systems for cancer gene therapy. Therapeutic strategies such as gene replacement/mutation correction, immune modulation and molecular therapy/'suicide' gene therapy type approaches potentially offer unique and novel ways of fighting cancer, some of which have already shown promise in early clinical trials. However, the specific and efficient delivery of the genetic material to remote tumors/metastases remains a challenge, which is being addressed using a variety of viral and non-viral systems. Each of these disparate systems has distinct advantages and disadvantages, which need to be taken into account when a specific therapeutic gene is being used. The review concentrates on particulate gene delivery systems, which are formed through non-covalent complexation of cationic carrier molecules (e.g. lipids or polymers) and the negatively charged plasmid DNA. Such systems tend to be comparatively less efficient than viral systems, but have the inherent advantage of flexibility and safety. The DNA-carrier complex acts as a protective package, and needs to be inert and stable while in circulation. Once the remote site has been reached the complex needs to efficiently transfect the targeted (tumor) cells. In order to improve overall transfection specificity and efficiency it is necessary to optimize intracellular trafficking of the DNA complex as well as the performance after systemic administration. Common principles and specific advantages or disadvantages of the individual synthetic gene delivery systems are discussed, and their interaction with tumor-specific and generic biological barriers are examined in order to identify potential strategies to overcome them.
Animals↗
Highly efficient regulation of gene expression by tetracycline in a replication-defective herpes simplex viral vector.
Employing the tetracycline repressor tetR and the wild-type hCMV major immediate-early promoter, we have developed a highly sensitive tetracycline-inducible transcription switch in mammalian cells (T-REx; Invitrogen, Carlsbad, CA, USA). In view of the previous difficulty in achieving regulatable gene expression in recombinant HSV vector systems, we constructed a T-REx-encoding replication-defective HSV-1 recombinant, QR9TO-lacZ, that encodes two copies of the tetR gene controlled by the HSV-1 immediate-early ICP0 promoter and a reporter, the LacZ gene, under the control of the tetO-bearing hCMV major immediate-early promoter. Infection of cells, such as Vero, PC12, and NGF-differentiated PC12 cells, with QR9TO-lacZ led to 300- to 1000-fold tetracycline-regulated gene expression. Moreover, the expression of the LacZ gene by QR9TO-lacZ can be finely controlled by tetracycline in a dose-dependent fashion. Efficiently regulated gene expression can also be achieved in vivo following intracerebral and footpad inoculations in mice. The demonstrated capability of T-REx for achieving high levels of sensitively regulated gene expression in the context of the HSV-1 genome will significantly expand the utility of HSV-based vector systems for studying gene function in the nervous system and delivering regulated gene expression in therapeutic applications, particularly in the treatment of CNS diseases.
Animals↗
Adeno-associated viral vectors for gene transfer and gene therapy.
Adeno-associated virus (AAV) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as vectors for gene therapy. In contrast to other gene delivery systems, rAAVs lack all viral genes and show long-term gene expression in vivo without immune response or toxicity. Over the past few years, many applications of rAAVs as therapeutic agents have demonstrated the utility of this vector system for long-lasting genetic modification and gene therapy in preclinical models of human disease. New production methods have increased rAAV vector titers and eliminated contamination by adenovirus. In addition, vectors for regulatable gene expression and vectors retargeted to different cells have been engineered. These advancements are expected to accelerate and facilitate further animal model studies, providing validation for use of rAAVs in human clinical trials.
Animals↗
Nonreplicating DNA viral vectors for suicide gene therapy: the adenoviral vectors.
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Animals↗
Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies.
The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene6, and compared it with two other transfection reagents, Tfx-50 and LipoTaxi. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.
Adult↗
In vitro selection of viral vectors with modified tropism: the adeno-associated virus display.
Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor-specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.
Base Sequence↗
Polycistronic viral vectors.
Traditionally, vectors for gene transfer/therapy experiments were mono- or bicistronic. In the latter case, vectors express the gene of interest coupled with a marker gene. An increasing demand for more complex polycistronic vectors has arisen in recent years to obtain complex gene transfer/therapy effects. In particular, this demand is stimulated by the hope of a more powerful effect from combined gene therapy than from single gene therapy in a process whose parallels lie in the multi-drug combined therapies for cancer or AIDS. In the 1980's we had only splicing signals and internal promoters to construct such vectors: now a new set of biotechnological tools enables us to design new and more reliable bicistronic and polycistronic vectors. This article focuses on the description and comparison of the strategies for co-expression of two genes in bicistronic vectors, from the oldest to the more recently described: internal promoters, splicing, reinitiation, IRES, self-processing peptides (e.g. foot-and-mouth disease virus 2A), proteolytic cleavable sites (e.g. fusagen) and fusion of genes. I propose a classification of these strategies based upon either the use of multiple transcripts (with transcriptional mechanisms), or single transcripts (using translational/post-translational mechanisms). I also examine the different attempts to utilize these strategies in the construction of polycistronic vectors and the main problems encountered. Several potential uses of these polycistronic vectors, both in basic research and in therapy-focused applications, are discussed. The importance of the study of viral gene expression strategies and the need to transfer this knowledge to vector design is highlighted.
Artificial Gene Fusion↗
Characterization of wild-type adeno-associated virus type 2-like particles generated during recombinant viral vector production and strategies for their elimination.
The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077-3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.
Base Sequence↗
An integrin-targeted non-viral vector for pulmonary gene therapy.
Gene therapy offers potential for the treatment of severe respiratory diseases. However, the vectors which are currently available have drawbacks limiting their therapeutic application. Here we report on an integrin-targeted, non-viral gene delivery system for pulmonary gene transfer. We demonstrate that this vector can deliver the lacZ reporter gene to the lung, transfecting bronchial epithelium and parenchymal cells with similar efficiency to an adenoviral vector and with greater efficiency than a cationic liposome. In addition, vector administration can be repeated leading to further gene expression without inducing inflammation. The advantages of this novel gene delivery system provide considerable potential for targeted gene therapy in vivo. Gene Therapy (2000) 7, 393-400.
Animals↗
Use of recombinant modified vaccinia Ankara viral vectors for equine influenza vaccination.
Recombinant modified vaccinia Ankara (MVA) vectors expressing equine influenza virus genes were constructed and evaluated for use in equine vaccination. Two strains of recombinant MVA, expressing either hemagglutinin (HA) or nucleoprotein (NP) genes were constructed. Each influenza virus gene was cloned from A/equine/Kentucky/1/81 (Eq/Ky) into an MVA construction plasmid, and was introduced to the deletion III locus of the wild type MVA genome by homologous recombination. Recombinant viruses were plaque purified, and antigen expression was confirmed by immunostaining. Two ponies were primed by vaccination with 50 microg HA-DNA and two ponies were vaccinated with 50 microg NP-DNA using the PowderJect XR research device. Six and 10 weeks later, ponies were immunized with 2 x 10(9) infectious units of recombinant MVA encoding the homologous influenza antigen, equally divided between intramuscular and intradermal sites in the neck. A marked rise in influenza virus-specific IgGa and IgGb serum antibody titers was detected following administration of MVA boosters with both HA and NP antigens. Influenza virus-specific lymphoproliferative responses and IFN-gamma mRNA production were also strongly elicited by both antigens. This study demonstrates the facility with which recombinant MVA viruses expressing defined pathogen genes can be constructed, and provides preliminary evidence of the immunogenicity and safety of these vectors in the horse.
Animals↗
Seasonal abundance and parity of stock-associated Culicoides species (Diptera: Ceratopogonidae) in different climatic regions in southern Africa in relation to their viral vector potential.
Seasonal abundance and parity in Culicoides populations, in the vicinity of livestock, were determined at seven sites in five different climatic regions with 220 V down-draught blacklight-traps. In 418 collections made between October 1983 and December 1986, a total of 2,134,171 Culicoides, of which 342,571 were identified to species level and sexed, were collected; 267 of these collections (182,321 Culicoides) were graded for parity. In the frost-free summer rainfall area, Culicoides were collected in large numbers in light-traps throughout the year; this implies breeding and possible virus transmission throughout the winter in certain parts of South Africa. However, where frost occurred, Culicoides numbers usually peaked in late summer and dropped sharply after the first frost. In the latter areas, small Culicoides collections during winter may be due to low winter temperatures and rainfall; low temperatures negatively affect adult activity and reduce the rate of development of larvae and pupae; low rainfall would lead to a reduction of available larval habitats. Relatively large numbers of Culicoides were collected in winter in the temperature frost-free winter rainfall area. In each of the four summer rainfall areas, one Culicoides species remained dominant throughout the year: at two of these areas this species was C. imicola. Other abundant species in some of these summer rainfall areas were C. schultzei s.l. and C. zuluensis. In the winter rainfall area, C. zuluensis, C. magnus, C. gulbenkiani and C. imicola shared abundance. It was established that abdominal pigmentation is an indicator of parity in C. imicola in South Africa. With the increase in Culicoides numbers towards the end of summer, there was also a rise in the proportion of parous (pigmented) females in most Culicoides species, which signifies a higher vector potential for African horsesickness and bluetongue towards the end of summer. This coincides with the seasonal occurrence of viral diseases transmitted by Culicoides species. Nulliparous (unpigmented) females of all Culicoides species were present throughout the year at all sites where Culicoides were continuously collected, confirming uninterrupted breeding in these areas.
African Horse Sickness↗
Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors.
In polarized epithelial cells, maturation sites of enveloped viruses that form by budding at cell surfaces are restricted to particular membrane domains. Recombinant vaccinia viruses were used to investigate the sites of surface expression in the Madin-Darby canine kidney (MDCK) cell line of the hemagglutinin (HA) of influenza virus, the G glycoprotein of vesicular stomatitis virus (VSV), and gp70/p15E of Friend murine leukemia virus (MuLV). These glycoproteins could be demonstrated by immunofluorescence on the surfaces of MDCK cells as early as 4 h post-infection. In intact MDCK monolayers, vaccinia recombinants expressing HA produced a pattern of surface fluorescence typical of an apically expressed glycoprotein. In contrast, cells infected with vaccinia recombinants expressing VSV-G or MuLV gp70/p15E exhibited surface fluorescence only when monolayers were treated with EGTA to disrupt tight junctions, as expected of glycoproteins expressed on basolateral surfaces. Immunoferritin labeling in conjunction with electron microscopy confirmed that MDCK cells infected with the HA recombinant exhibited specific labeling of the apical surfaces whereas the VSV-G and MuLV recombinants exhibited the respective antigens predominantly on the basolateral membranes. Quantitation of surface expression by [125I]protein A binding assays on intact and EGTA-treated monolayers confirmed the apical localization of the vaccinia-expressed HA and demonstrated that 95% of the VSV-G and 97% of the MuLV gp70/p15E glycoproteins were localized on the basolateral surfaces. These results demonstrate that glycoproteins of viruses that normally mature at basolateral surfaces of polarized epithelial cells contain all of the structural information required for their directional transport to basolateral plasma membranes.
Animals↗
Adeno-associated viral vector gene transfer into leptomeningeal xenografts.
Leptomeningeal carcinomatosis is a painful and debilitating complication of cancer. Indwelling reservoirs provide continuous assess to the subarachnoid space, making leptomeningeal cancer potentially amenable to gene therapy. Adeno-associated virus (AAV) is a defective virus not associated with any human disease. We used an AAV vector to transduce medulloblastoma (DAOY) cells in a nude rat model of leptomeningeal disease. After intraventricular injection of vector carrying the bacterial lacZ gene, beta-galactosidase positive cells were found in the implanted tumor and in ependymal and subependymal cells but not in underlying normal brain parenchyma. No evidence of virally-mediated toxicity was noted in the animals. The results of this pilot study demonstrate that AAV vectors may be used to transfer and express foreign genes in established leptomeningeal tumors.
Adenovirus E1A Proteins↗
Recombinant adeno-associated viral vectors mediate long-term transgene expression in muscle.
Gene transfer to muscle holds overt promise for the treatment of inherited myopathies, lysosomal storage disorders, and serum protein deficiencies. In addition, muscle could provide a reservoir for delivery of therapeutic molecules like blood clotting factors, erythropoietin, or insulin. To date, successful gene transfer to muscle has been limited by the inefficiency of the vector delivery systems and the transient nature of gene expression. In this paper, we show that a vector based on recombinant adeno-associated virus (rAAV) can efficiently transduce adult mouse skeletal muscle. Transduced myofibers escape immune elimination and transgene expression is robust beyond 5 months. Importantly, input vector DNA appears to undergo conversion from single-stranded genomes to high-molecular-weight concatameric forms. These data suggest that rAAV might have a significant advantage over many other viral and nonviral gene delivery methods, and holds significant promise as a vector for gene transfer to mature muscle.
Animals↗
Transductional and transcriptional targeting of cancer cells using genetically engineered viral vectors.
Gene delivery vectors, including adenovirus (Ad) and adeno-associated virus (AAV), are inefficient and non-selective for cancer due to low levels of viral receptors with high levels on other tissues, including liver. We tested Ads and AAVs with the SIGYPLP-targeting peptide inserted into virus capsids for transduction in a panel of cancer cells. Six of twelve lines (C8161, PC-3, G-CCM, MKN-45, LnCAP and A549) were transduced, independently of native viral tropism. Furthermore the candidate cancer gene therapy promoter FLT-1 was active in three of these six cell lines. This offers the potential for dual targeting of selected cancer cells.
Adenoviridae↗