Viral vectors replication-competent

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. 

Galanis E, Vile R, Russell SJ. 2001. Delivery systems intended for in vivo gene therapy of cancer Targeting and replication competent viral vectors. Crit Rev Oncol Hematol. 38 177-192. 

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... 

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]. 

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. 

One major concern in the use of Ad recombinant vectors is the emergence of replication competent Ad vims as a result of recombination events between the viral sequences. Ad protein expression by replication-competent Ad vims, but also from the vector itself, results in the in vivo induction of a potent immune response. 

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... 

Fig. 3 Delivery systems used in clinical gene transfer. During gene therapy, an expression vector (therapeutic gene) is transferred to somatic cells via a delivery system, for example, a viral or nonviral vector (replication-incompetent), a naked nucleic acid, or a recombinant, mostly conditionally replication-competent microorganism. The gene...

The retroviral vectors in clinical use have mainly been derived from murine leukemia virus (MLV) [14]. MLV causes leukemia in mice and replication-competent retrovirus (RCR) in a contaminated vector preparation was shown to cause leukemia in severely immunosuppressed monkeys. RCR absence therefore has to be verified before human use of retroviraUy modified cells MLV vector use in vivo has been very rare. The genome of the retroviral vectors consists of two copies of single-stranded RNA, which contains one or more coding regions flanked by the viral control elements, the so-called long terminal repeat (LTR)... 

Murine retroviral vectors to be used forhuman transfer experiments (less than 10 liters)that contain less than 50% oftheir respective parental viral genome and that have been demonstrated to be free of detectable replication competent retrovirus can be maintained, handled, and administered, under BLl containment. 

Fekete DM, Cepko CL (1993) Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo. Mol Cell Biol 13 2604-2613. 

Assessment of the immimogenic consequences of gene delivery should also be possible in the chosen species this is particularly important for the evaluation of viral vectors. There has been much discussion on the value of using a permissive host to assess potential human risk from these vectors. The natural route of exposure - i.e. respiratory for adenoviruses - traditionally used to assess permissivity, seems unnecessary to assess the safety of gene therapy products, particularly with the core study approach discussed above. Systemically-administered replication-competent adenovirus will infect (i.e. gain entry into the cell) and be pathogenic in numerous tissues (Duncan et al., 1978). Gene expression and viral replication depend on the species, route of exposure, and individual tissue susceptibility (Torres et al., 1996 Bett et al., 1962). Thus i.v. administration of viral vectors to mammalian test species should permit the evaluation of potential toxicity of widely-distributed vectors. 

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. 

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