Bio-ARROW - SmartForm - Viral Vectors
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 Institutional Biosafety Committee Policy UW-6102.
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) |
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*Form |
Use plasmids to generate viral particles |
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*Type |
Retrovirus (includes lentivirus) |
|
Sub-selection for Type |
Lentivirus, HIV based |
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*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. |
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*Drug Resistance |
No |
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Drug resistance explain: |
Click here to enter text. |
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*Entry into human cells |
Yes |
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*Viral vector usage: |
Administered to cells or cell cultures |
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*Nature of experimental effect |
Click here to enter text. Expression of reporter (e.g., GFP, Luciferase, photoreactive) |
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*Integration |
Yes |
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*Titer |
Titer varies. We anticipate the range for the titer to be between 108-1012 . |
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*Biosafety level |
BSL2 |
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*Enhanced practices |
Standard microbiological practices (i.e., no enhanced practices utilized) |
Recombinant viral vector: (Common viral vector with an oncogene) |
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*Form |
Use plasmids to generate viral particles |
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*Type |
Adenovirus |
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Sub-selection for Type |
Choose an item. |
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*Replication Competence |
Replication Incompetent |
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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. |
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*Drug Resistance |
No |
|
Drug resistance explain: |
Click here to enter text. |
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*Entry into human cells |
Yes |
|
*Viral vector usage: |
Administered to animals |
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*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) |
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*Integration |
Yes |
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*Titer |
1x10 12 |
|
*Biosafety level |
BSL2 |
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*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) |
|
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*Form |
Use plasmids to generate viral particles |
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*Type |
Retrovirus (includes lentivirus) |
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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) |
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*Integration |
Yes |
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*Titer |
1x10 13 |
|
*Biosafety level |
BSL2 |
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*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) |
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*Form |
Use pre-packaged viral particles |
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*Type |
Adeno-associated virus |
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Sub-selection for Type |
Choose an item. Click here to enter text. |
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*Replication Competence |
Replication Incompetent |
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Replication Deficient Explain |
Removal of cap and rep genes |
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*Drug Resistance |
No |
|
Drug resistance explain: |
Click here to enter text. |
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*Entry into human cells |
Yes |
|
*Viral vector usage: |
Administered to animals |
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*Nature of experimental effect |
Membrane proteins and ion channels Delete-and add text |
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*Integration |
No |
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*Titer |
1 x 1013 |
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*Biosafety level |
BSL2 |
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*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:
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Recombinant viral vector: (viral vector with oncogene and enhanced practices) |
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*Form |
Use plasmids to generate viral particles |
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*Type |
Retrovirus (includes lentivirus) |
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Sub-selection for Type |
Retrovirus, MMLV based VSV-G pseudotyped |
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*Replication Competence |
Replication Incompetent |
|
Replication Deficient Explain |
Removal of gag, pol and env gene |
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*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 |
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*Titer |
1 x 1013 |
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*Biosafety level |
BSL2 |
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*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 |
|
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*Form |
Use plasmids to generate viral particles |
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*Type |
Baculovirus |
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Sub-selection for Type |
Choose an item. |
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*Replication Competence |
Replication Competent |
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Replication Deficient Explain |
Click here to enter text. |
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*Drug Resistance |
No |
|
Drug resistance explain: |
Click here to enter text. |
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*Entry into human cells |
No |
|
*Viral vector usage: |
Administered to cells or cell cultures |
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*Nature of experimental effect |
Baculovirus will be used to deliver CRISPR/Cas9 to modify cellular membrane proteins. Delete-and add text |
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*Integration |
No Virus can persist but not integrate |
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*Titer |
Unknown. Will need to optimize titer. |
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*Biosafety level |
BSL1 |
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*Enhanced practices |
Standard microbiological practices (i.e., no enhanced practices utilized) Choose an item. Choose an item. |