Biosafety considerations for working with Retroviral Vectors

Under Gene Technology regulations the classification of work using these vectors in tissue culture and in vivo depends on the DNA insert subcloned into the vector backbone and their ability to transduce human cells. The OGTR provide the following flow chart to assist in classifying dealings involving the use of replication defective retroviruses.

One of the benefits of using these retroviral vector systems is that they integrate the genes they carry into the chromosome of the target cell. The virus itself can't replicate and so, after a period of time, the resulting transfected cell line or animal will no longer express the viral vector but will continue to express the integrated gene. The safety features incorporated into the replication defective, retroviral vectors prevent the virus from remobilizing. Therefore, stably transfected cell lines or animals infected with these viral vectors may be reclassified to a lower level of dealing (and ergo containment) after sufficient time has elapsed that infectious viral particles are no longer present.

In the case of transduced cell lines, after the virus is no longer active, dealings may be considered exempt dealings. In the case of animals, the dealing can be considered as either an exempt dealing for wild type animals or PC1-NLRD if the animals are transgenic or knockout mice, rats, rabbits or guinea pigs.

The OGTR do not provide a specified timeframe between viral transduction and reclassification of a given dealing but instead leave it to the IBC to satisfy itself that no infectious virus remains.

After considering the literature on virus stability and clearance rates the University of Western Australia IBC has concluded that:

• For cell culture work – a dealing involving the use of a replication defective, retroviral vector with a PC2-NLRD initial classification, can be reclassified as an Exempt dealing after a period of at least 72 hours AND the three or more total (100 %) media changes.
• For animal work – a dealing involving inoculation with a replication defective, retroviral vector, originally classified as a PC2-NLRD, can be treated as a PC1-NLRD (if used in transgenic or knockout, mice, rats, guinea pigs or rabbits) or an Exempt dealing (if used in wild type animals) after a period of 48 hours.

The IBC may require PCR validation of viral vector absence in certain circumstances.

First take into account the specific nature of the work. These considerations only apply when working with media containing virus particles or cell lines which contain and may be producing these particles. Note that they do not apply to the initial construction of the vectors in E. coli.

It is essential that a risk assessment be done prior to all work, and that personnel working on this material be required to read and sign off that they understand what work practices are to be followed. The level of containment of work involving retroviral vectors will depend upon several factors:

• the vector system (notably its ability to transduce human cells) and its ability to regenerate vector-competent retrovirus, and the amount (titre) of vector to be used.
• the nature of the inserted transgene.
• the host organism.

The University of Western Australia Institutional Biosafety Committee recommends the following work practices when using replication defective retroviral vectors:

NOTE: These considerations only apply when working with media containing virus particles or cell lines which contain and may be producing these particles. Note that they do not apply to the initial construction of the vectors in E. coli.

• Personal protective equipment (PPE) must always be worn when working with these vectors.
  • gloves must be pulled over the elasticised wrist cuffs of a long sleeved lab gown which is rear fastening. Safety glasses must also be worn.
  • additional PPE may apply depending on the nature of the work involved.
  • Consult relevant OGTR Licence dealings that may require this as a Condition of the Licence. For example face shields and double gloving could be considered depending on the level of risk and procedures being performed.
• For centrifugation of this material, centrifuge rotor cups should have an aerosol tight seal. Loading and unloading of centrifuge rotors should be done in the Class II Biosafety cabinet.
• Use of dedicated work space, storage space and incubators is recommended but not essential, depending upon the risk assessment. Such dedicated work space is particularly important when working with live animals, owing to the increased risks involved (see above). If space permits, a separate room is ideal.
• All work involving such vectors must be performed in a Class II Biosafety cabinet, unless specified otherwise in the relevant OGTR dealing. This includes the inoculation of animals.
• Sharps must be eliminated from experimental procedures wherever possible.
• The testing of supernatants could be considered for Licenced (DNIR) work. This is used to determine the effectiveness of the final washes and would be performed, especially when trialling new protocols.
• Clean-up procedures should be documented and displayed in a prominent position such as next to the Biosafety cabinet. Disinfectants (and their concentrations) must be effective against retroviral vectors. The expiry date for disinfectants that can lose their activity (such as bleach)must be clearly labelled on the bottle.
• Waste disposal procedures should also be documented and displayed. Provision should be made to ensure that there is no risk of perforation of the biohazard bag prior to autoclaving (e.g. placing pipette tips etc. into a solid container which is then placed into an autoclave bag along with other biohazardous waste – note this container must be vented to allow penetration of steam during autoclaving).
• The animal facility manager(s) must be notified of work involving the inoculation of animals with retroviral vectors.
• All workers must be familiar with and closely follow any additional documentation on working with replication defective retroviral vectors that may be provided by their Institute/Faculty.