Sendai virus (SeV), or murine parainfluenza virus type 1, is an enveloped,150-200nm in diameter, single strand, negative-sense RNA virus belonging to the Paramyxoviridae family. It was first isolated from mice in Sendai city of Japan in the 1950s and its typical infected hosts are rodents and swine, resulting in a highly transmissible respiratory tract infection amongst the hosts. Sendai virus replication occurs in the cytoplasm of infected cells and is used as a model virus in virology research.
Why Sendai virus is used as a gene delivery or expression vector? • No Potential Pathogenicity for Humans
Safety is one of the great concerns associated with medical use of any viral vectors. No link between SeV and any human diseases has been reported for the nearly six decades since the discovery of this virus. It has been used in laboratories for a long period of time and no accidental infections in humans occurred. In a clinical study, intranasal inoculation of SeV in adult volunteers produced no harmful outcomes (Slobod et al. 2004). Similarly, intramuscular inoculations as great as 5x109 CIU (cell infectious units)/60 kg produced no adverse events in a clinical trial of gene therapy for peripheral arterial disease (PAD) using the F gene-deleted Sendai virus.
• Nonintegrating Nature
SeV and all other nonsegmented negative strand RNA viruses (the Mononegavirales) have no nuclear phase throughout their life cycles, which are completed entirely in the cytoplasm in an RNA replicon form (reviewed in Conzelmann 1998; Lamb and Parks 2007; Nagaiet al. 2011). Therefore, these viruses replicate in enucleated cells or in the presence of the inhibitors of DNA-dependent RNA polymerase such as actinomycin D anda-amanitin (Armeanu et al. 2003). In theory, these features of the Mononegavirales eliminate the possibility of genotoxicity caused by integrationinto and any other interactions with chromosomes, representing the first-line advantage of SeV and related viruses in vector generation.
Integration of the vector genome (i.e, its DNA copied via reverse transcription) into chromosomes of target cells has been a precondition for trans-gene expression from these retroviral vectors, raising fear or safety concerns such as proto-oncogene activation and other disorders in cellular gene expression, which are comprehensively regarded as a genotoxicity concern.
• Lack of Homologous/Heterologous Recombination and Genome Mixing
Systematic phylogenetic analyses suggested that virtually no recombination events occurred in the evolutionary process of the Mononegavirales (Chare et al. 2003). No homologous or heterologous recombination has been reported over a long period of SeV research, eliminating the possibility of the emergence of unforeseen recombinant viruses. This situation may be partly explained by the fact that the SeV RNA genome is tightly bound with N proteins to form a ribonucleoprotein (RNP) complex and does not become naked RNA throughout the life cycle; hence, it has minimal opportunities for base pairing. The SeV polymerase can sometimes jump from one position to another during replication and continue to copy a distal region of thegenome template to yield a deletion-type defective interfering (DI) particle or copy back the nascent chain to yield a copy back DI particle (Re et al. 1985). This change may leave open the possibility that homologous recombination may occur, albeit with low efficiency, when two distinct RNP strands are replicating in a single cell. However, there have been no reports to date that support this possibility.
An additional advantage of using SeV and the Mononegavirales as expression vectors is that they possess a single, nonsegmented RNA genome and, hence, do not undergo the gene reassortment seen in RNA viruses with segmented genomes such as orthomyxo-, arena-, bunya-, and reoviruses (reviewed in Simon-loriere and Holmes 2011).
• Very High Performance in Foreign Gene Expression
After entering into host cells, SeV initiates transcription and replication in the cytoplasm by its RNA-dependent RNA polymerase. As the SeV accessory C protein encodes an anti-apoptosis function, its cytopathic effects are not very extensive, allowing a fairly long time span of infected cell survival. Because the amplification of genomic RNA and mRNAs is vigorous, SeV replicates to high titers along with a high level of viral protein expression in cells. Consequently, the continued robust expression of inserted foreign genes is expected. However, the robust expression of trans-genes is not always advantageous and can sometimes be hazardous. In this context, down regulating the expression of foreign genes to various extents is possible by selecting the site of their insertion in the SeV genome.
As SeV reaches as high as 1010 CIU (cell infectious units)/ml in the allantoic fluid of embryonated chicken eggs and 108-9 CIU/ml in the culture supernatant of cell lines or primary cells, the production of vector virus stocks with high titers is guaranteed, which represents a critically important justification for its practical use.
• Very Broad Target Cell Range
Conventional gammaretrovirus vectors frequently derived from Moloney murine leukemia virus cannot infect nondividing cells such as neurons and muscle cells. The adenovirus hardly infects blood cells and hardly enters through the apical surface of epithelial cells. The main site of virus entry in epithelial tissues appears to be the basolateral surface because of the polarized localization on the basolateral surface of the specific receptor for the fiber proteins, CAR (the coxsackie virus and adenovirus receptor) (Walters et al. 1999). Therefore, gene transfer through epithelial tissues by the adenovirus vector is not easy.
SeV uses as its receptor the terminal sialic acids of glycosylated molecules ubiquitously present on the apical surface of epithelial cells as well as other cell surfaces. SeV can readily infect both dividing and nondividing cells and exhibits a very broad target cell range. Thus, a wide variety of diseases can potentially be the therapeutic targets of the SeV vector. Importantly, SeV tropism is restricted because the tryptic endoprotease required for activating the viral fusion protein is expressed in limited tissue types only (Nagai 1993). Thus, SeV vector stocks have to be activated before use by cleaving the inactive precursor fusion protein with a low concentration of trypsin (Homma and Ohuchi 1973). This is another key to the safety of the SeV vector because the progeny produced from initially infected cells does not gain infectivity in the absence of such enzymes.
• Additional Advantageous Aspects
In addition to the advantages described of the characteristics of SeV as a gene transfer vector, the following properties of SeV are also listed as additional advantages. SeV can attach to target cells and transfer its genome into these cells instantly within seconds (Masaki et al. 2001; Ikeda et al. 2002). This ability is of particular merit in a situation where, for example, stopping the bloodstream is required during gene delivery. SeV is also able to traverse the mucous layer of the nasal cavity and airway in vivo, readily accessing the epithelial cell surface (Yonemitsu et al. 2000 Griesenbach et al. 2002)
