This guidance applies to retro and lentiviral vectors and not to the handling of modified full length HIV which is always classified at activity class 3.
The Contained Use regulations require that all activity involving genetically modified microorganisms is classified according to the risk created by carrying out that particular activity. General guidance on classification of research projects is given on the UCL website. In most cases it is sensible and practical for the classification to be determined by the highest level of risk associated with any particular part of the project. In the case of integrating viral vector systems this is not always the case; this guidance document is intended to help people apply the classification that addresses the real risk and not merely to “tick boxes just to be on the safe side”.
The biohazard
An authoritative and readable summary of the nature of the biohazard and the risk associated with these viral vectors is given in the Compendium of Guidance produced on behalf of the HSE by the Scientific Advisory
Committee on Genetic Modification (SACGM), part of the Health and Safety Commission’s formal advisory structure. A link to this multi-part document is given on the UCL GM website, but the relevant section, 2.11
(pages 116-126), is available here SACGM Compendium Part 2 . The Compendium summarises this guidance as follows.
Activity Based Classification Summary
The degree of control needed, and therefore classification, should be determined by the risk assessment on a case-by-case basis. The risk of insertional mutagenesis, an inherent hazard associated with lentiviral/retroviral vectors, is difficult to quantify given the current available data. However, the potential likelihood of this hazard, along with others conferred by the transgene, being realised is increased where work involves the use of sharps to deliver the viral vector. Whilst ‘control of sharps’ is not one of the specified control measures in the containment tables, other CL2 measures are deemed necessary to facilitate their control (e.g. restricted access to authorised and trained personnel; written training records; and the use of gloves) therefore necessitating a classification of Class 2.
In certain circumstances, where large volumes/titres of virus and/or aerosol generating procedures are used, it may also be necessary to use additional CL2 measures to control exposure of the operator via mucosal or inhalation routes (e.g. microbiological safety cabinet; restricted access; other specific measures to minimise aerosols) therefore necessitating a classification of Class 2. This does not apply to the use of gloves and microbiological safety cabinet used solely to protect the sterility of the product.
After consultation with the HSE, the GM Safety Committee recommends that UCL should take the approach described in Part 2 section 2.11. This is summarised in the UCL context below.
Biohazard group of the vector itself
Wild-type viruses of this group vary in hazard, as you would expect, but “third generation” replication-defective viral vectors are believed to be very low in hazard and may be treated as biohazard group 1 organisms. The same applies to animal viruses even with a human tropism. Accidental inoculation of the vector is highly unlikely to cause harm. If your vector is less disabled than this, then you may need a higher level of containment as determined by the risk assessment.
Hazards of inserted sequences
In current practice, certain sequences carry a presumption that a classification above level 1 will be needed.
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Typically, these are
•
oncogenes and proto-oncogenes,
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growth factors and GF receptors
•
and other sequences coding for significant biological activity that might be harmful were these to be expressed in people accidentally exposed to the vector.
The final classification will depend on whether techniques will be used that create a likelihood of such exposure occurring, the insertion of the sequences and the likelihood of harm resulting from this insertion. Uncertainty may surround some of these factors but a balance must be struck on the basis of currently available knowledge.
The risk of insertional mutagenesis
These vectors are valuable research tool because of their capacity to insert a sequence into the genome of differentiated cells. Because the site of insertion is random, there exists a small chance that insertion might interfere with normal cellular function and give rise to uncontrolled cell proliferation. The size of this risk may be very small but must be taken into account regardless of the nature of the insert. At present the GMSC advises that there is only a very low risk to human operators when handling viral titres of less that 5x10
6 pfu per ml of viral suspension.
The likelihood of exposure
Where your experimental procedures are such that there is effectively no risk of exposure then the activity may be classified a level 1.
As the Compendium notes, the risk of exposure is greatly increased in the presence of sharps. Where this is unavoidable, as in the injection of vectors into experimental animals, then that activity will be class 2 (see also below). The risk in that situation is increased by the fact that large quantities of the vector are usually needed in order to ensure that sequences effectively insert into the target cells. Care must also be taken to avoid the use of sharps such as cover slips, glass pasteur pipettes and sharp-pointed forceps, etc.
Precautions should be taken to protect broken skin and to protect the eyes and mouth from splashes. This is effectively achieved when virus suspensions are handled in microbiological safety cabinets. You should distinguish between your use of a cabinet to protect the product and when you are using it to protect the operator from contamination. This is likely to be the case where the vector is carrying sequences whose accidental insertion may be harmful. In the latter case, the activity will be a class 2 activity.
The distinction between “activity class” and laboratory “containment level”
As stated earlier, it is usual to set as a classification level for the whole project, the highest classification applied to any of its component procedures. In certain cases this is not necessary. A current example is the preparation of a viral vector in a laboratory for administration to animals. Unless the insert is hazardous in its own right, the point of risk is the preparation of the syringe and its use and subsequent disposal.
This part of the project should be classified at 2 for the reasons quoted above from the Compendium: “Whilst
‘control of sharps’ is not one of the specified control measures in the containment tables, other CL2 measures are deemed necessary to facilitate their control (e.g. restricted access to authorised and trained personnel [for the duration of this process] ; written training records; and the use of gloves) therefore necessitating a classification of Class 2 .”
This activity will usually take place in a procedures room in a Biological Services Unit. It is unlikely that any improvement in safety will be achieved were you to carry out the injection in a microbiological safety cabinet because the main risk is from a needlestick accident. (Furthermore, it could be argued that attempting to carry out the injection in a cabinet actually increases the risk of accidental inoculation through the restriction of movement and it may raise animal welfare issues.) As the vector will be replication defective, there is very little or no chance of virus shedding from the treated animals and so the environmental risk is also low / negligible, as is the risk to BSU staff and others.
From this, it can be seen that you only need to apply the containment level 2 precautions that specifically
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protect against a risk that you recognise as belonging to that part of the project. It does not mean that you have to carry out the whole of the BSU phase of the project at animal containment level 2 and in most cases, inoculation may be the only GM activity in that project that should be classified at 2.
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Preparation and handling of vectors without inserts.
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Handling of vectors with inserted sequences that do not have harmful properties and are at titres of less that 5x10
6
/ml, including the use of needles
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Culture of cells treated with replication-defective vectors and with sequences that may or may not be harmful.
•
Handling of vectors with inserted sequences that do not have harmful properties at titres greater than
5x10
6
/ml where sharps are used.
•
Handling of vectors with inserted sequences that have harmful properties and therefore where precautions from CL2 must be taken to avoid percutaneous transmission and contact with mucosal surfaces.
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GMM notification instructions July 09