Biosafety office

February 2014

Viral Vectors – Assessing Risks

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Biosafety Office

University of Cincinnati

51 Goodman Dr.

Cincinnati, OH 45221-0767

email: inbiocom@ucmail.uc.edu

http://researchcompliance.uc.edu/Biosafety

The use of virus as vectors for genetic material delivery into cells has become very common

among the molecular biology community.

RISK ASSESSMENT

The risk assessment process is designed to assist personnel in the proper selection of appropriate

biosafety levels, training, procedural protocols, microbiological practices, safety equipment, and facilities

to prevent occupationally acquired infections.

While assessing the risk of experiments

involving viral vectors, the following factors

must be taken in consideration:

Agents Risk Group

Cell Tropism

Nature of the transgene

Mutagenesis

Safety Features – Reversion Prevention

Environmental Stability of the Vector

As genetic engineering research increases, viral

vectors are becoming an important safety issue.

It is important for users to understand the origins of

these tools and potential implications of their use.

Viruses are obligate intra-cellular parasites,

designed through the course of evolution to infect

cells, often with great specificity to a particular cell

type (tropism). They tend to be very efficient at

transfecting their own nucleic acid into the host

cell.Therefore, they have been selected as one of

the major vehicles of gene delivery.

VIRAL VECTORS

Risk Group *

(RG)

Agent Risk Description

Examples

RG-1 Agents that are not associated with disease in

healthy adult humans Bacillus subtilis, Escherichia coli K12,

adeno-associated virus (AAV)

RG-2

Agents that are associated with human

disease which is rarely serious and for which

preventive or therapeutic interventions are

often available

Staphylococcus aureus, Salmonella sp,

Herpes simplex viruses, Adenovirus

RG-3 Agents that are associated with serious or

lethal human disease for which preventive or

therapeutic interventions may be available

Mycobacterium tuberculosis, Bacillus

anthracis, HIV

RG-4 Agents that are likely to cause serious or

lethal human disease for which preventive or

therapeutic interventions are not usually

available

Ebola virus, Marburg virus, Lassa virus

Agents Risk Group

A viral vector particle has genes removed from the genome of

the wild-type parent virus to create space for the gene of

interest (transgene) and to increase safety. Although unlikely,

a viral vector may regain the deleted genes and revert to its

original form (details are further provided in Safety Features –

Reversion Prevention section). Therefore, it is important to be

aware of the risk group classification of the parent virus from

which the vector originated. Infectious agents are categorized

in different risk groups (1-4) based on their relative risk to

healthy adult humans. In the U.S., this classification system

takes the following factors into consideration:

Pathogenicity of the organism to humans

Mode of transmission and host range

Availability of effective preventive measures (e.g. vaccines)

Availability of effective treatment

* Classification systems do not address circumstances in which an individual may have increased susceptibility because

of preexisting diseases, medications, compromised immunity, or pregnancy. Determination of additional risk due to

immune status must be made in consultation with a health professional.

The Risk Group of the parent virus from which a viral vector was derived may be the first,

but not the only factor to consider while assessing the risks.

“ Risk Group is not a synonym for Biosafety Level ”

While the Risk Group classification is based on the microbiology

and epidemiology of agents, Biosafety Level corresponds to the

facilities, equipment, practices and procedures for safe conduct of

work with an agent. The biosafety levels can range from 1 to 4.

Normally agents are handled under equivalent biosafety level of

their risk group classification. But in reality, this determination

should be driven by professional judgment based on a risk

assessment. For example, experiments with a viral vector may

have to be performed adopting a biosafety level higher, or even

lower, than its parent virus risk group classification. Factors such as

cell tropism and nature of the transgene inserted into the vector

should be considered.

“ A Risk Group 1 agent may need to be handled at BSL2 ”

Viral vectors have natural host cell

populations that they can infect most

efficiently. Attachment to and entry

into a susceptible cell is mediated by

the interaction between viral surface

structures and receptors present on

the surface of the host cell. Some

viruses have limited natural host cell

range, while others are able to infect

a relatively broader range of cells

efficiently.

In some instances researchers may need to limit or, more frequently, expand the

range of cells susceptible to transduction by a viral vector. To this end, many

vectors have been developed in which the endogenous surface proteins have

been replaced by proteins from other viruses. This procedure is called

pseudotyping. Viruses in which the surface proteins have been replaced are

referred to as pseudotyped viruses.

According to the cell tropism viral vectors

receive the following classification: • Ecotropic

infect murine cells (mouse and rats)

• Amphotropic – infect mammalian cells,

including human cells

• Pantropic - infect any type of cells of any

species (e.g. VSV glycoprotein G)

“ Special care should be taken while working with pantropic or

amphotropic viruses which can infect human cells ! ”

The HIV glycoprotein (GP120) only binds to cells presenting CD4 receptors. To increase the range of cell

tropism, HIV based viral vectors usually have their envelope pseudotyped. The Vesicular Stomatitis Virus

glycoprotein G (VSV-G) has been frequently used since it binds to receptors that are present in all type of cells.

Cell Tropism

Nature of Transgene

As a safety measure, viral vectors normally have

deletion of the genes involved in replication. This

renders vectors replication incompetent, but does not

remove their ability to infect cells. In other words,

following an accidental exposure, even if a viral vector

particle does not replicate, it may infect the individual’s

cells and have its transgene expressed.

Basically, any gene which can significantly alter the cell

cycle when over-expressed is a gene of concern. These

would include kinases, growth factors, certain transcription

factors and, more importantly, oncogenes. An extensive

list of genes involved in cancer can be found at

http://atlasgeneticsoncology.org/Genes/Geneliste.html

Sometimes, a viral vector does not contain a gene to be further expressed. Instead, it may carry

oligonucleotides (e.g. micro RNA, shRNA) intended to inhibit target gene(s) of the host.

Depending on the nature of the gene to be inhibited, this may be also a concern. For example,

the inhibition a tumor suppressor may favor oncogenesis.

Replication-incompetent viral vectors still present risks !

The nature of the transgene (or of the gene to be inhibited) is the most important factor to consider while

performing the risk assessment of experiments involving viral vectors.

Mutagenesis

Some viruses, such as lentiviruses and

gamma-retroviruses, have the ability to

integrate into the host chromosome. The

new gene might be inserted in an

undesirable location in the host DNA with

harmful consequences.

Integration of the viral genome may disrupt

endogenous host genes. Activation of

proto-oncogenes or inactivation of a tumor

suppressor gene, which can lead to

increased cancer risk, are of special

concern.

Although viral vectors are occasionally created from

pathogenic viruses, they are modified in such a way as to

minimize the risk of handling them. This usually involves

the deletion of a part of the viral genome critical for viral

replication.

Genes necessary for replication of the virus are removed

from the vector, but need to be supplied somehow in order

to produce the viral vector particle. Those genes may be

supplied separately through plasmids, helper virus, or

packaging cell lines.

Replication-incompetent viral vectors can gain back the

deleted genes required for replication (become replication-

competent) through recombination – referred to as

replication-competent virus (RCV) breakthroughs. This is a

particular concern with lentiviral systems.

Split genomes of viral replication genes, providing

them in separate plasmids. Having replication

genes on different constructs means that more

recombination events would need to occur in order

to get a RCV breakthrough.

Remove viral regulatory regions. This decreases the

chance of homologous recombination occurring.

Produce virus as a transient single batch

(simultaneous transfection of plasmids) rather than

as continuous culture (use of a packaging cell line

with replication genes integrated into the genome

of the cell line). There is an increased risk of RCV

breakthroughs with the use of packaging cell lines,

especially with large-scale production.

In order to decrease the chances of RCV breakthroughs the following strategies are used:

Safety Features – Reversion Prevention

Environmental Stability of the Vector

Viruses depend on a cell to complete their cycle. Depending on their type, viruses exit a cell in different

ways. It can occur through “budding” or through cell lysis. Viruses which exit the cells through lysis are

called “naked” or non-enveloped viruses while those that exit the cells by budding are enveloped viruses.

Viral Exit from Infected Cell

HIV particle leaving a host cell by “budding”

Adenovirus particles leaving a

host cell by cell lysis

Enveloped virus

Non- enveloped virus

The envelope typically is derived from

portions of the host cell membranes

(phospholipids and proteins), but also

includes viral glycoproteins.

The lipid bilayer envelope of these viruses is

relatively sensitive to desiccation, heat and

detergents, therefore these viruses are easier to

inactivate than non-enveloped viruses, which

have longer survival outside host environments.

Resources

Biosafety in Microbiological and Biomedical Laboratories

(BMBL)

http://www.cdc.gov/biosafety/publications/bmbl5/

Genes in Cancer

http://atlasgeneticsoncology.org/Genes/Geneliste.html

NIH Guidelines

http://osp.od.nih.gov/office-biotechnology-activities/biosafety/nih-guidelines