Guidelines

Working with Retroviral Vectors - Guidelines

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1. Purpose and Objectives

These guidelines explain the minimum requirements that should be met when working with or using retroviral vectors, including lentiviral vectors.

2. Definitions, Terms, Acronyms

Dealing - as defined by the OGTR – conduct experiments with GMOs; make, develop, produce, manufacture GMOs; breed; propagate; use GMOs in the manufacturing of non GM products; grow, raise, culture GMOs; import GMOs; transport or store GMOs

DIR - Dealings involving Intentional Release. This is a licence issued to an Accredited Organisation to undertake high risk dealings outside the containment facilities such as laboratory or glasshouse.

DoH - Commonwealth Department of Health (formerly Department of Health and Ageing)

DNIR - Dealings Not Involving Intentional Release. This is a licence issued to an Accredited Organisation to undertake high risk dealings within a certified facility such as laboratory or glasshouses.

Exempt Dealing - a type of GM dealing, one that poses a very low risk

GM/GMO/GMMO - Genetic Modification/Genetically Modified Organism/Genetically Modified Microorganism

IBC - Institutional Biosafety Committee, a Committee that is established by an Accredited Organisation, as required by the OGTR

IBSC - Institutional Biosafety Sub-Committee

Lentiviruses - are a subclass of Retroviruses. They have been adapted as gene delivery vehicles (vectors) due to their ability to integrate into the genome of non-dividing cells, which is the unique feature of Lentiviruses as other Retroviruses can infect only dividing cells

OGTR - Office of the Gene Technology Regulator

PC1, PC2, PC3 certified facilities/Certified facilities - Physical containment facilities certified by the OGTR.

PPE - Personal Protective Equipment

Retroviral Vectors - are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro).

SOP - Safe Operating Procedure

Workers - Workers include staff, students (undergraduate and postgraduate), visitors and contractors

3. Guideline Scope/Coverage

These guidelines apply to all persons undertaking research on genetically modified organisms, in particular, when working with retroviral vectors, including lentiviral vectors.

4. Guideline Statement

These guidelines assist workers in ensuring that they are compliant with the requirements as set out in the Gene Technology Regulations 2001 (C'th), and in ensuring safe working practices are followed when conducting work with or using retroviral vectors, including lentiviral vectors.

5. Understanding Retroviral Vectors

Retroviral vectors, including lentiviral vectors, are a common tool used by researchers to introduce a gene into the genome of a target cell or organism thanks to their ability to transduce a wide range of cell types and the ability of some lentiviral vectors in particular to infect both dividing and non-dividing cells.

The efficiency of those vectors which are able to transduce human cells comes with some inherent risk, namely the possibility for the generation of replication competent retroviral particles, and also the accidental delivery of genes which may confer an oncogenic modification. Progressive generations of vector systems have become available that minimize these possibilities by splitting the components of the packaged retro/lentivirus into multiple plasmids. As part of this process, retrovirus particles are produced that are replication defective. That is, they are designed to be unable to continue to infect their host after they deliver their contents.

These new systems are less efficient but also provide a considerably reduced risk of the generation of a replication competent virus. The following guideline outlines ways in which risks associated with working with retroviral vectors can be further minimised.

6. Considerations Required when Using Retroviral Vectors

The primary consideration when using retroviral vectors must be the specific nature of the work being undertaken. 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 Office of the Gene Technology Regulator (OGTR) has determined that in vitro use of replication defective retroviral vectors capable of transducing human cells may be carried out within PC2 containment under Notifiable Low Risk Dealing (NLRD) conditions as long as they have the following safety features:

  • All viral genes removed from the vector, AND
  • Packaging proteins are supplied in trans from multiple unlinked loci with minimum sequence overlap, AND

EITHER

  • Self inactivating deletion in 3’UTR (SIN), OR
  • Essential packaging proteins only – gag, pol, rev and an envelope protein. See Gene Technology Regulations 2001 (C'th)

For the majority of NLRD lentiviral work, routine PC2 containment is adequate, given the work practices discussed below. Isolation of such work e.g. in a dedicated room or on a dedicated bench, is at the discretion of the workplace.

The level of risk associated with such a vector is also dependent upon the type of gene it is being used to transfect. Nucleic acid which is known or suspected to be oncogenic or capable of complementing the defective viral vector will require a consideration of handling techniques above those already in place with, for example, standard marker genes. Details of these gene classes and their categorisation can be found under Schedule 3 Parts 2 & 3 of the Gene Technology Regulations 2001 (C'th).

Most in vivo use of replication defective retroviral vectors (including lentiviral vectors) must be performed under Licence (Dealing for Non-Intentional Release) conditions; this is primarily a reflection of the biosafety issues involved in the handling of live animals. The delivery of the vector must be done in a way to stringently minimize the risk of autoinoculation by the researcher. Following inoculation of the animals, the animals and their waste and bedding must be handled in such a way to further minimise the risk to animal house staff posed by the potential shedding of replication defective retroviral vector or (unlikely) replication competent retrovirus.

However, in vivo use of replication defective retroviral vectors (including lentiviral vectors) that are unable to transduce human cells or where the gene is not a toxin, not an oncogenic modification and not immunomodulatory, may be conducted under an NLRD.

6.1 Work practices

The University of Queensland 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. They do not apply to the initial construction of the vectors in E. coli.

6.1.1 Personal Protective Equipment (PPE) and safety 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.
  • Waterproof covering over any break in skin integrity.
  • 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.
  • 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.

6.1.2 Specific work procedures

  • 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.
  • Manipulations such as shaking, mixing and ultrasonic disruption must also be carried out in a Class II Biosafety cabinet.

6.1.3 Dedicated work space

  • 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.

6.1.4 Sharps

  • Sharps must be eliminated from experimental procedures wherever possible.
  • Sharps must be disposed of appropriately.

6.1.5 Clean-up and waste disposal

  • Waste disposal must be in accordance with the OGTR Guidelines for Transport, Storage and Disposal of GMOs (2011), and any other requirements specified in the relevant OGTR dealing.
  • 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 (eg. 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).

6.1.6 Animal work

  • The animal facility manager(s) must be notified of work involving the inoculation of animals with retroviral vectors.
  • Animal waste and bedding material containing virus particles must be autoclaved prior to disposal in clinical or animal waste stream.
  • Animals should be kept in an isolated room post inoculation while still shedding virus.

6.1.7 Storage requirements

The OGTR details specific storage requirements for GMOs in their Guideline for Transport, Storage and Disposal of GMOs (2011). These requirements include records of material being stored to be kept for easy identification of samples stored.

Please consult PPL 2.40.07 Storage and Labelling Requirements for GMOs for further details.

6.2 Downgrading work

There is no standard method recognised by the OGTR to determine if work or material is no longer infectious and can be downgraded (i.e. no standard washing protocol for cells in culture). Therefore, it is recommended that the researchers establish and verify their own protocol prior to considering downgrading work.

For example, the testing of supernatants by ELISA type assay could be used to assess the effectiveness of the final washes and would be performed especially when trialling new protocols.

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 or Faculty.

6.3 Occupational health

Health surveillance may be required for some work involving retroviral vectors (eg: some work with animals, any PC3 work). Consideration should be given to this requirement when completing the initial risk assessment.

The Occupational Health Nurse Advisor will be able to provide advice on precautions that you may or must undertake.

Vaccinations and immunisations may also be required for some work (e.g. animal work). Please consult PPL 2.60.08 Vaccinations and Immunisations.

7. Contact for Additional Information

Biosafety Advisor
UQ OHS Division
Phone: 336-52365
Email: biosafety@uq.edu.au

Custodians
Director, Occupational Health and Safety
Mr Jim Carmichael
Custodians
Director, Occupational Health and Safety
Mr Jim Carmichael