| MicrobiologyBytes: Virology: Viruses & gene therapy | Updated: October 19, 2004 | Search |
Some retroviruses contain protooncogenes, which when mutated can cause cancers, however, in the production of vectors these are removed. Retroviruses can also transform cells by integrating near to a cellular protooncogene & driving inappropriate expression from the LTR, or by disrupting a tumour suppresser gene. This event, termed insertional mutagenesis, though extremely rare could still occur when retroviruses are used as vectors.
Retroviral vectors are most frequently based upon the Moloney murine leukaemia virus (Mo-MLV), which is an amphotrophic virus, capable of infecting both mouse cells, enabling vector development in mouse models, & human cells, enabling human treatment. The viral genes (gag, pol & env) are replaced with the transgene of interest & expressed on plasmids in the packaging cell line. Because the non-essential genes lack the packaging sequence (psi) they are not included in the virion particle. To prevent recombination resulting in replication competent retroviruses, all regions of homology with the vector backbone should be removed & the non-essential genes should be expressed by at least two transcriptional units (Makowitz et al, 1988). Even so, replication competent retroviruses do occur at a low frequency.
The essential regions include the 5' & 3' LTRs & the packaging sequence lying downstream of the 5' LTR. Transgene expression can either be driven by the promoter/enhancer region in the 5' LTR, or by alternative viral (e.g. cytomegalovirus, Rous sarcoma virus) or cellular (e.g. beta actin, tyrosine) promoters. Mutational analysis has shown that up to the entire gag coding sequence & the immediate upstream region can be removed without effecting viral packaging or transgene expression (Kim et al, 1998). However the exact positioning of the transgene start codon & small alterations of the 5' LTR influence transgene expression (Rivire et al, 1995). To aid identification of transformed cells selectable markers, such as neomycin & beta galactosidase, can be included & transgenes expression can be improved with the addition of internal ribosome sites (Saleh, 1997). The available carrying capacity for retroviral vectors is approximately 7.5 kb (Verma & Somia, 1997), which is too small for some genes even if the cDNA is used.
The retroviral envelope interacts with a specific cellular protein to determine the target cell range. Altering the env gene or its product has proved a successful means of manipulating the cell range. Approaches have included direct modifications of the binding site between the envelope protein & the cellular receptor, however these approaches tend to interfere with subsequent internalisation of the viral particle (Harris & Lemoine, 1996). By replacing a portion of the env gene with 150 codons from the erythropoietin protein (EPO), Kasahara et al (1994) were able to target EPO receptor bearing cells with high affinity. Coupling an antibody to the viral particle with affinity for a second cell specific antibody via a streptovadin bridge, improves viral uptake, but internalisation tends to lead to viral degradation (Roux et al, 1989). Neda et al (1991) treated viral particles with lactose which resulted in uptake by cells, principally hepatocytes, expressing asiaglycoprotein receptors. Subsequently there was efficient viral transgene expression, possibly due to acidification of the endosome allowing fusion of the viral envelope with the endosome membrane.
Viruses differ with respect to their tropisms, therefore by replacing the env gene with that of another virus, the host range can be extended, in a technique known as pseudotyping. Vesicular stomatitis virus G protein has been included in Mo-MLV derived vectors (Burns et al, 1994), which are also more stable when purified by ultracentrifugation. Recently, Qing et al (1997) improved transduction into numerous cell lines by first treating the recipient cells with an adeno-associated vector (discussed below) expressing the cellular receptor for retroviral envelope protein.
A requirement for retroviral integration & expression of viral genes is that the target cells should be dividing. This limits gene therapy to proliferating cells in vivo or ex vivo, whereby cells are removed from the body, treated to stimulate replication & then transduced with the retroviral vector, before being returned to the patient. When treating cancers in vivo, tumour cells are preferentially targeted (Roth et al, 1996, Tait et al, 1997). However, ex vivo cells can be more efficiently transduced, due to exposure to higher virus titres & growth factors (Glimm et al, 1997). Furthermore ex vivo treated tumour cells will associate with the tumour mass & can direct tumouricidal effects (Oldfield & Ram, 1995, Abdel-Wahab et al, 1997).
Though transgene expression is usually adequate in vitro & initially in vivo, prolonged expression is difficult to attain. Retroviruses are inactivated by c1 complement protein & an anti-alpha galactosyl epitope antibody, both present in human sera (Rother et al, 1995, Rollins et al, 1996). Transgene expression is also reduced by inflammatory interferons, specifically IFN-alpha & IFN-gamma acting on viral LTRs (Ghazizadeh et al, 1997). As the retroviral genome integrates into the host genome it is most likely that the viral LTR promoters are being inactivated, therefore one approach has been to use promoters for host cell genes, such as tyrosine (Diaz et al, 1998). Clearly this is an area where continued research is needed.
The lentiviral vectors used are derived from the human immunodeficiency virus (HIV) & are being evaluated for safety, with a view to removing some of the non-essential regulatory genes. Mutants of vpr & vif are able to infect neurones with reduced efficiency, but not muscle or liver cells (Blmer et al, 1997, Kafri et al, 1997).
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