Indicators of Replication Competent Adenovirus (RCA)
There are a number of indicators that might suggest the presence of replication competent adenovirus (RCA) has been generated during vector production. These include the target cells showing signs of cytopathetic effects, having to increase the multiplicity of infection (moi) to maintain transgene expression level and increasing the scale of vector production to generate sufficient vector for transduction studies,. How often should testing for replication competent adenovirus be carried out? This will depend on which generation of adenoviral vector is being used, the experience of the researchers and stringency of applying standard protocols. Further details can be found on our annual subscription service.
Gutless Adenovirus Packaging System
In third generation adenovirus almost all the virus sequence has been deleted allowing a large packaging potential of the vector. To ensure that the size of the genome is sufficient it is necessary to include stiffer DNA to generate the correct sized genome. As the transfer vector contains only the psi packaging signal, all other packging functions must to be provided by an adenovirus helper construct. The helper vector is deleted of E1 and therefore the E1 product has to be provided in trans by the packaging cell line. The psi sequence is flanked by LoxP sequences which results in the deletion of the psi sequence from the adenovirus helper genome following the expression of cre recombinase in the packaging cell line which substantially reduce the packaging of the helper genome into the virion.Further details can be found on our annual subscription service.
Complementing E1A function
• Infection with wild type adenovirus (and recombination)
• E1a-like activity can be found in a number of cell lines including indicator cell lines used for RCA detection
• EBV can complement E1a mutants
• High moi leads to E2 transcription
First, Second and Third Generation Murine Retrovirus Packaging System
First generation murine leukaemia viral vectors are derived from packaging cells lines in which the integrated proviral genome consist of the entire retroviral sequence viral but with a deleted packaging signal which is intended to prevent the packaging of the proviral derived RNA genome. In this type of simple packaging system, the generation of replication competent virus through recombination can result from a single cross-over event.
Second generation retroviral vectors are similar to first generation in that all packaging functions are provided from a single genome; however, in addition to the deletion of the packaging sequence, the 3'LTR is also deleted so that two recombination events necessary to generate wild type virus.
To further reduce the likelihood of generation wild type virus further refinements have been made whereby the gag/pol and env helper functions are on separate constructs in the packaging cell line, often referred to as a split function packaging cell line, to create the third generation vector packaging system. Where split function helper constructs are used, multiple recombination events required to generate wild type virus. Further modifications have been made to third generation packaging system to improve their safety margin. For more information register with us on an annual basis.
HIV lentiviral vectors
The HIV lentivirus genome has a genetic organisation very similar to simple retroviruses, namely sequences coding for the group specific antigen (gag) protein, the reverse transcriptase (pol) and envelope (env) protein which are flanked by long terminal repeat sequences (LTRs) at the 5’ and 3’ end of the genome. A significant difference is that HIV also codes for a number of accessory proteins which are associated with virulence and regulatory proteins required for exporting of unspliced RNA transcripts and for upregulating expression from the LTR. Specific sequences within the genome are required for binding the regulatory proteins and for the movement of RNA molecules in and out of the nucleus.
HIV lentiviral vectors are characterised by their split function nature i.e. the functions required to produce the viral particle are on separate helper genetic elements. One helper construct provides the structural gag function and a second helper construct provides the envelope protein. Both constructs lack the psi encapsidation signal and therefore their transripts cannot be packaged into the final virus particle. A third construct, the gene transfer vector, provides the transcript that will be packaged into the virus.
Safety issue regarding woodchuck post-transcriptional regulatory elements (WPRE)
The UK regulators have issued a safety note following unpublished reports of animals inoculated with lentiviral vectors developing high rates of tumour. The mechanism of tumour formation is not clear but may be due to insetrional mutagenesis or expression of proteins encoded by the vector itself. The vector used in the animal study contained the an enhancer of gene expression derived from woodchuck hepatitis virus (WHV) known as woodchuck post-transcriptional regulatory element (WPRE). This element is capable of expressing the X protein which has been associated with oncogenic properties.
Self inactivating (SIN) lentiviral vectors
Self inactivating lentivirus vectors are designed so that the internal promoter within the long terminal repeat is non-functional once the virus has integrated.
The RNA genome of retroviruses is transcribed into double stranded DNA by the action of virally encoded reverse transcriptase present in the virion. Integration of the DNA form of the virus is mediated via the long terminal repeats (LTRs) found at the ends of the genome which is generated via the action of the transcriptase enzyme.
The two ends of the genomic RNA are similar in that there is a repeated region R but differ in that at the 5’ end of the genome, there is a unique U5 region at the 3’ end of the R region. At the 3’ end of the genome is a unique U3 region located 5’ to the R region. During the synthesis of the DNA strand from the RNA genome template, the U5 regions is duplicated at the 3’ end of the genome ultimately forming one of the terminal repeats with the order U3-R-U5. The long terminal repeat is duplicate at the 5’ end of the genome in the same order (U3-R-U5). The LTR contains the regulatory elements for transcription including the promoter and enhancer which are within the U3.
Foot and mouth disease virus (FMDV)
Foot-and-mouth disease virus is a member of the picornaviridae and is the causative agent of foot-and-mouth disease, which can have devastating impact on farming and the wider economy in countries free of the virus. FMDV causes disease in a range of cloven-hooved animals, and research involving the virus must be undertaken at the highest level of containment (containment level 4). In additional to the high physical containment requirements, personnel working with the virus must be trained to a very high level of competence and there must be a robust management system.
The virus is non-enveloped with a diameter of 22-30 nm diameter. It is relatively stable in the presence of cations and is not sensitive to chloroform, di-ethyl ether or non-ionic detergents. The virus contains a single molecule of positive-sense single–stranded RNA. Infection of a cell is via receptor-mediated endocytosis, and uncoating occurs in endosomes/lysosomes. Progeny virus is released through cell lysis.
Hydrogen peroxide for fumigation
The use of hydrogen peroxide either as a gas or as a vaporised form has many advantages over formaldehyde and may become the method of choice for disinfection for safety cabinets and laboratories.
Its broad spectrum activity is due to its powerful oxidising capacity which is known to damage cellular proteins, lipids and nucleic acids. However, its use requires appropriate validation and of particular importance is the choice of model micro-organism in validation studies. Micro-organisms such as Mycobacterium tend to be more resistant to hydrogen peroxide because of their lipophilic cell wall structure. Where the genetic modification alters the lipid make-up of the cell wall it is advisable to use these GMMs as the test organism in the validation rather than a model micro-organism.
Risks associated with altered tropism of animal pathogens
When assessing the risks associated with modifying the tropism of animal pathogens, the assessment should consider whether the new construct is capable of causing sub-clinical infection in humans should accidental exposure occur as inadvertent release from containment via an infected individual could have severe environmental consequences.