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Viral Vectors are often designed so that they can enter human cells and deliver genes of interest. Viral vectors are usually replication-deficient – genes necessary for replication of the virus are removed from the vector and supplied separately through plasmids, helper virus, or packaging cell lines.

Viral vectors frequently used are:

  • Retrovirus/ lentivirus
  • Adenovirus
  • Baculovirus
  • Poxvirus
  • Herpes virus
  • Alphavirus
  • Adeno-associated Virus (AAV)

There are several biosafety concerns that arise with the use of viral vectors.

  • Tropism (natural or modified)
  • Recombination to make a replication competent virus (RCV)
  • Delivery/expression of oncogenes/potential oncogenes or biological toxin genes

The host range of the virus is called tropism. Modifications made to the virus can change the host range for the virus. A virus or viral vector is considered amphotropic when it can recognize receptors and can replicate in a broad range of hosts or broad range of cell types. In contrast, a virus or viral vector is considered ecotropic when it can recognize receptors and can replicate only in its original host species or cell type. It is important to understand the host range of the viral vector as it may have important implications in the event of an exposure or release. Some viral vectors can be modified to be able to enter human cells. Conversely, vector modifications can lower the risk by eliminating the ability to enter human cells. 

Examples of how glycoprotein changes modify viral vector tropism:

  • Changing the host range to include human cells by adding VSV-G
  • Creating pseudotyped viruses that have the structural and enzymatic core from the parent virus and the envelope glycoprotein from another virus (e.g., avian pseudotyped removes ability to enter mammalian cells and enables entry into avian cells)
  • Engineering viral vector to enter only specific cell types (e.g., able to enter only neuron cells, able to enter specific tissues)
  • Modifying to create an amphotropic vector (e.g., Murine Leukemia Virus)

In addition to recombinant modifications made to the viral vector, assess the possibility of events that could occur to create a replication competent virus. Replication-competent virus (RCV) breakthroughs occur when replication-deficient viral vectors gain back the deleted genes required for replication through recombination. Recombination can occur during cell culture production, with existing competent virus or a virus latent in the recipient. Adenoviral vectors are known to have a relatively high rate of RCV breakthroughs compared to other viral vectors. It is important to understand the factors in the experimental design that would reduce or increase the possibility of an RCV. RCVs are of concern because if present, they would create progeny and go through all stages of their life cycle. In the event of an exposure or release, this could pose additional risk.

Because a RCV could pose an additional risk, it is important to try to reduce the probability of one occurring. Several strategies may be used to decrease chances of RCV breakthroughs:

  • Separate genomes of viral replication genes (e.g., Gag, Pol). Having replication genes on different constructs means that more recombination events would need to occur to get a RCV breakthrough.
  • Remove viral regulatory regions (e.g., Rev). This decreases the chance of homologous recombination occurring.
  • Produce virus as a transient single batch (i.e., simultaneous transfection of plasmids) rather than as continuous culture using 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 during large-scale production.

Risk assessment must also include what genes are expressed or targeted. Special attention is needed when there is oncogenic potential or when expressing a biological toxin gene. (e.g., tumor suppressors, oncogenes, potential oncogenes, partial or full biological toxin genes) In these instances, enhanced practices may be needed.

Information on containment of Adeno-associated virus (AAV):

UW-Madison Policy 6089 Appropriate Containment for Select Opportunistic and Borderline Pathogens

Additional guidance documents available:

Use of Lentivirus & Lentiviral Vectors

Use of Adeno-associated virus (AAV) & Adeno-associated viral vectors

Use of Adenovirus & Adenoviral vectors

 

OBS would like to assist in improving safety whenever possible.

Please contact the Office of Biological Safety at 263-9013 or biosafety@fpm.wisc.edu for further information or if you have any questions.

 

Things to consider when filling out this table include:

  • PPE differences
  • Containment strategies
  • Signage considerations
  • Description of activities and biosafety precautions in place

The overall risk assessment for the use of viral vectors will involve assessing information found in various sections of the biosafety protocol. Please also check other sections such as PPE, Containment, Signage as well as the Research Description to see if information needs to be added.

Table notes:
In addition to filling out the table, there would be things to note in other sections for viral vectors. There may be PPE, containment and signage considerations. Additionally, activities and biosafety precautions are described in the Research Description.

 Recombinant viral vector: (Common viral vector)

*Form

Use plasmids to generate viral particles

*Type

Retrovirus (includes lentivirus)

Sub-selection for Type

Lentivirus, HIV based

*Replication Competence

Replication Incompetent

Replication Deficient Explain

The third-generation packaging system is split into two plasmids: one encoding Rev and one encoding Gag and Pol.

Tat is eliminated from the 3rd generation system through the addition of a chimeric 5' LTR fused to a heterologous promoter on the transfer plasmid. Expression of the transgene from this promoter is no longer dependent on Tat transactivation.

*Drug Resistance

No

Drug resistance explain:

Click here to enter text.

*Entry into human cells

Yes

*Viral vector usage:

Administered to cells or cell cultures

*Nature of experimental effect

Click here to enter text.                  

Expression of reporter (e.g. GFP, Luciferase, photoreactive)

*Integration

Yes

*Titer

Titer varies. We anticipate the range for the titer to be between 108-1012 .

*Biosafety level

BSL2

*Enhanced practices

Standard microbiological practices (i.e. no enhanced practices utilized)

 

Recombinant viral vector: (Common viral vector with an oncogene)

*Form

Use plasmids to generate viral particles

*Type

Adenovirus

Sub-selection for Type

Choose an item.

*Replication Competence

Replication Incompetent

Replication Deficient Explain

Replication-defective (RD) vector has the essential E1A and E1B genes deleted and replaced by an expression cassette with a high activity cytomegalovirus immediate early (CMV) promoter which drives expression of the foreign transgene

Adenoviral vector lacks the E3 genes which in general prevents infected cells from elimination by the immune systems.

*Drug Resistance

No

Drug resistance explain:

Click here to enter text.

*Entry into human cells

Yes

*Viral vector usage:

Administered to animals

*Nature of experimental effect

Click here to enter text.                  

Oncogenic potential (e.g. expression or upregulation of an oncogene, knock out or repression of a tumor suppressor, impact cell cycle, transformation to create cell line, repression of senescence, inhibition of programmed cell death)

*Integration

Yes

*Titer

1x10 12

*Biosafety level

BSL2

*Enhanced practices

Changes in animal practices as described in other sections (e.g. disinfection/inactivation, containment, research description)          

Use of safety sharps

 

Recombinant viral vector: (Common viral vector with a toxin gene)

*Form

Use plasmids to generate viral particles

*Type

Retrovirus (includes lentivirus)

Sub-selection for Type

Lentivirus, HIV based

*Replication Competence

Replication Incompetent

Replication Deficient Explain

The third-generation packaging system is split into two plasmids: one encoding Rev and one encoding Gag and Pol.

Tat is eliminated from the 3rd generation system through the addition of a chimeric 5' LTR fused to a heterologous promoter on the transfer plasmid. Expression of the transgene from this promoter is no longer dependent on Tat transactivation.

*Drug Resistance

No

Drug resistance explain:

Click here to enter text.

*Entry into human cells

Yes

*Viral vector usage:

Administered to cells or cell cultures

*Nature of experimental effect

Click here to enter text.

Apitoxin gene                

Expression of biological toxin genes (full length or active subunits)

*Integration

Yes

*Titer

1x10 13                                          

*Biosafety level

BSL2

*Enhanced practices

Changes in PPE as described in the PPE section      

More frequent disposal of biohazardous waste    

Elimination of glass/substitution for glass

 

Recombinant viral vector: (rAAV administered to an animal outside of containment)

*Form

Use pre-packaged viral particles

*Type

Adeno-associated virus

Sub-selection for Type

Choose an item.

Click here to enter text.

*Replication Competence

Replication Incompetent

Replication Deficient Explain

Removal of cap and rep genes

*Drug Resistance

No

Drug resistance explain:

Click here to enter text.

*Entry into human cells

Yes

*Viral vector usage:

Administered to animals

*Nature of experimental effect

Membrane proteins and ion channels

Delete-and add text

*Integration

No

*Titer

1 x 1013

*Biosafety level

BSL2

*Enhanced practices

Restricted access with signage during work with viral vector          

Changes in PPE as described in the PPE section      

Changes in animal practices as described in other sections (e.g. disinfection/inactivation, containment, research description)

Note: 
For this example, BSL2 was selected because the titer is  1 X 10 13 and enhanced practices were added because the administration of the viral vector is performed outside of containment.  In addition to filling out this table, there would be things to note in other sections for viral vectors. There may be PPE, containment and signage considerations. Additionally, activities and biosafety precautions are described in the Research Description. For high titer rAAV, please see UW Policy 6089 Appropriate Containment for Select Opportunistic and Borderline Pathogens.

 

Recombinant viral vector: (viral vector with oncogene and enhanced practices)

*Form

Use plasmids to generate viral particles

*Type

Retrovirus (includes lentivirus)

Sub-selection for Type

Retrovirus, MMLV based

VSV-G pseudotyped

*Replication Competence

Replication Incompetent

Replication Deficient Explain

Removal of gag, pol and env gene

*Drug Resistance

No

Drug resistance explain:

Click here to enter text.

*Entry into human cells

Yes

*Viral vector usage:

Administered to animals

Administered to cells or cell culture

*Nature of experimental effect

Click here to enter text.                  

Oncogenic potential (e.g. expression or upregulation of an oncogene, knock out or repression of a tumor suppressor, impact cell cycle, transformation to create cell line, repression of senescence, inhibition of programmed cell death)

*Integration

No

*Titer

1 x 1013

*Biosafety level

BSL2

*Enhanced practices

Use of safety sharps 

Elimination of glass/substitution for glass

Changes in animal practices as described in other sections (e.g. disinfection/inactivation, containment, research description)

 

Replication competent viral vector

*Form

Use plasmids to generate viral particles

*Type

Baculovirus                  

Sub-selection for Type

Choose an item.

*Replication Competence

Replication Competent

Replication Deficient Explain

Click here to enter text.

*Drug Resistance

No

Drug resistance explain:

Click here to enter text.

*Entry into human cells

No

*Viral vector usage:

Administered to cells or cell cultures

*Nature of experimental effect

Baculovirus will be used to deliver CRISPR/Cas9 to modify cellular membrane proteins.         

Delete-and add text

*Integration

No                                                                                                       

Virus can persist but not integrate

*Titer

Unknown.  Will need to optimize titer.

*Biosafety level

BSL1

*Enhanced practices

Standard microbiological practices (i.e. no enhanced practices utilized)                    

Choose an item.

Choose an item.

 



Keywordsviral, vector, vectors, viral vectors, microbes   Doc ID127582
OwnerTara S.GroupARROW - Institutional Biosafety Committee
Created2023-05-02 15:24:02Updated2023-08-14 14:46:30
SitesARROW - Institutional Biosafety Committee
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