Replication-competent virus

From a bio-safety point of view, it is necessary to remove as much of the HIV viral sequences as possible. Rev removal would permit elimination of additional viral sequences from the vector. This removal minimizes the possibilities for recombination events that could lead to a replication-competent virus. With Rev removed, as-acting elements, not requiring viral proteins for function, must be introduced in the vector. RNA export elements derived from simple retroviruses, such as CTE, can be used to replace Rev in... 

DNA amplified as bacterial artificial chromosome in Escherichia coli Rescue of replication-competent virus progeny and packaging of amplicon vectors. Hum Gene Ther 9 2787-2794. 

Saeki Y, Ichikawa T, Saeki A, Chiocca EA, Tobler K, et al. 1998. Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coir. rescue of replication-competent virus progeny and packaging of amplicon vectors. Hum. Gene Ther. 9 2787-94... 

The second system induces a rapid death of the cell by apoptosis if viral proteins are being synthesized in that cell. This second system permits the destruction of any HIV-1 infected cell, no matter whether the provirus was formerly active or silent The active provirus is detected by the presence of Rev active proteins. As this protein is involved in the rapid transport of the transcripts to the cytoplasm, avoiding or reducing the rate of splicing of the introns, its activity is essential for a correct viral gene expression, so no replication-competent viruses can escape from its detection. An apoptotic gene, p53, is inside an intron that can only be over-expressed through the 5 -LTR promoter of the vector and the presence of the Rev protein otherwise, it is... 

In the case of biopharmaceuticals, several additional tests are common which reflect their production processes. Examples include the absence of replication-competent virus in the case of viral products, and an... 

Being replication competent, virus rapidly spreads horizontally throughout the culture of primary CEF or immortalized DFl cells, and therefore, almost any transfection protocol suffices. The following protocol (i.e., sections 3.3.1-33.3) works well and minimizes cell death. 

The presence of replication- competent virus in the vector preparation is problematic, since it could be associated with increased toxicity and may lacilitate the spread of the recombinant in vivo (18-20). Replication-competent adenovirus occurs by either cross contamination of the initial plaque with replication-competent vims from an adj acent plaque or reversion of the E1 deletion during the propagation of the recombinant by homologous recombination between sequences in the vector flanking the El deletion and the transfected El sequences in 293 cells. 

Gao GP, Engdahl RK, Wilson JM. A cell line for high-yield production of El -deleted adenovirus vectors without the emergence of replication-competent virus. Hum Gene Ther 2000 11 213-219. 

AAV vectors have not been studied to the same extent as adenoviral or retroviral systems, however they appear to be associated with fewer safety risks than the other viral systems. This is due to the elimination of all sequences coding for viral proteins, thereby greatly reducing the risk of an immune reaction against the vector. There remain, however, the potential problems of insertional mutagenesis and the generation of replication competent virus. 

An important safety issue of viral vectors is whether or not the recombinant viruses are able to replicate in the infected cells. Replication of viral vectors is unwanted in most gene-therapy approaches. Therefore, replication-defective vectors have been designed, which are able to perform only one initial infectious cycle within the target cell. In addition, replication-competent vectors have been designed, which are able to productively infect the target cell and to spread in the target tissue. 

Yu DC, Sakamoto GT, Henderson DR. Identification of the transcriptional regulatory sequences of human kallikrein 2 and their use in the construction of calydon virus 764, an attenuated replication competent adenovirus for prostate cancer therapy. Cancer Res 1999 59 1498-1504. 

Finally, replication-competent viral vector systems have been combined with standard cancer therapy in animal models, resulting in synergistic effects. This has, for example, been shown for ONYX-015 plus cisplatin or ionizing radiation [108,112] and for an AdElB 55k-deleted virus expressing HSV-tk (plus ganciclovir) in combination with the topoisomerase inhibitor topotecan [114]. 

Numerous transcriptionally targeted replication-competent adenovimses and herpes viruses have been shown to be highly specific (up to 10,000-fold relative to nontarget cells) in cell culture experiments. In addition, animal models have demonstrated their safety and efficacy. It thus appears that the transcriptional targeting of viral replication holds some promise as a strategy for cancer gene therapy. 

Allen, J. M., Debelak, D. J., Reynolds, T. C. and Miller, A. D. (1997). Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by nonhomologous recombination during AAV vector production. J. Virol. 71, 6816-6822. 

Kimpton, J. and Emerman, M. (1992) Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene../. Virol. 66, 2232-2239. 

The oncolytic viruses include adenovirus, measles, reovirus, vesicular stomatitis virus (VSV),HSV,poxvirus, and vaccinia. Specific examples include (1) ONYX-015, which is an adenoviral oncolytic virus, administered to patients with liver metastases of colorectal cancer and pancreatic cancer [29], (2) Reolysin, which is an oncolytic reovirus administered to patients with glioma [30], and (3) MV-CEA, which is an oncolytic measles virus expressing carcinoembryonic antigen, administered to patients with ovarian cancer [31]. Some oncolytic viruses are wild type and are apparently not pathogenic in humans, such as the Newcastle disease virus (NDV), which is an RNA avian paramyxovirus. PV701, a naturally attenuated, replication-competent strain of NDV, has been administered to patients with advanced solid tumors [32], The applicability of oncolytic viruses as a therapy for clinical oncology trials is due to their potential selectivity the ability to kill tumor cells but not normal cells. However, the level of attenuation of viral replication in normal cells is limited for most oncolytic vectors. 

Subramanian, T. and G. Chinnadurai. 2006. Temperature-sensitive replication-competent adenovirus shRNA vectors to study cellular genes in virus-induced apoptosis. Meth. Mol. Med. 130, 125-34. 

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