Viral Vectors for Gene Transfer

The following table lists the most common viral vectors used for gene transfer/gene therapy studies and the required biosafety levels for producing vector stocks and/or producer lines. The currently recommended tests for replication competent virus (RCV) are also given. Select the links below for additional information about biosafety levels and tests for replication competent virus.


VirusBiosafety LevelTest for Replication Competent Virus
Adeno-associated virus (with adenovirus) BSL-2 Replication-competent adenovirus
Adeno-associated virus (adenovirus-free) BSL-1 Not required
Adenovirus BSL-2 Polymerase chain reaction (PCR) for E1a
Foamy virus (replication competent) BSL-2 Not applicable
Foamy virus (replication defective) BSL-1 Not required
Gamma retrovirus (ecotropic pseudotyped) BSL-1 Not required
Gamma retrovirus (other pseudotypes) BSL-2 Marker rescue assay
Herpes virus amplicons BSL-2 Plaque assay
Lentivirus (non-HIV pseudotyped) BSL-2 Serial transfer and ELISA for p24 antigen
Example Lentiviral RCV Protocol (Word)
Vaccinia virus BSL-2 Not applicable

Adeno-associated virus vectors (adenovirus free) Adeno-associated virus vector stocks generated with adenovirus-free packaging systems can be handled at a BSL-1 level, and no further testing is needed for studies in mice. Reference for adenovirus free packaging system: Allen JM, Halbert CL, Miller AD. 2000. Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6. Mol Ther 1:88-95.

Adeno-associated virus vectors (with adenovirus) Adeno-associated virus vector stocks generated with adenovirus must be tested for the presence of replication-competent adenovirus after heat-inactivation. Reference for a RCV assay: Hehir KM, Armentano D, Cardoza LM, et a1. 1996. Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence. J Virol 70:8459-8467.

Adenovirus vectors The following conditions must be met before adenovirus vector stocks can be used in animals and handled at a BSL-1 level. First, the vector must contain less than 2/3 of the wild-type genome; currently this only includes what are known as "gutless" vectors. Second, the vector stocks must be tested for RCV by PCR for E1a prior to use. The vector stock should be tested at a limit of sensitivity of 1 in less than 106 virus particles compared to a known positive control and the results of the test must be available upon request. For murine studies, if only the second condition is met, the vector must be administered at BSL-2 and the animals held for at least one hour prior to their return to standard ABSL-1 housing. Reference for E1a PCR assay: Zhang WW, Kock PE, Roth JA. 1995. Detection of wild-type contamination in a recombinant adenoviral preparation by PCR. Biotechniques 18: 444-447.

Foamy virus vectors Foamy virus vector stocks generated with packaging systems shown to be free of RCV by a marker rescue assay can be used in mice and handled at a BSL-1 level without further testing. Foamy virus vectors which are replication-competent must be handled at a BSL-2 level. Reference for a marker rescue assay: Trobridge GD, Russell DW. 1998. Helper-free foamy virus vectors. Hum Gene Ther 1998 9:2517-2525. Results should be <1 infectious units/milliliter.

Gamma retrovirus vectors (ecotropic pseudotyped) Gamma retrovirus vectors pseudotyped with an ecotropic envelope (one which allows for the transduction of mouse cells but not human cells) and generated by direct plasmid transfection can be handled at a BSL-1 level. There is no requirement that the vector stocks and/or producer lines be tested prior to use in mice; however, a marker rescue assay for RCV is recommended as part of any experimental design. Ecotropic producer cells generated by transduction with non ecotropic pseudotyped producer cells must be handled at a BSL-2 level until demonstrated to be free of RCV by a marker rescue assay.

Gamma retrovirus vectors (other pseudotyped) Gamma retrovirus vectors pseudotyped with envelopes other than ecotropic must be tested for RCV by a marker rescue assay prior to being approved for use at BSL-1/ABSL-1. The vector stock or producer line should be tested at a limit of sensitivity of 1 infectious unit per milliliter and the test should include a known positive control. Reference for a marker rescue assay: Miller AD, Buttimore C. 1986. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 6:2895-2902.

Herpes virus vectors Herpes virus generated using amplicons must be must be tested for RCV by a plaque assay prior to approval for use at BSL-1 and ABSL-1. The vector stock should be tested at a limit of sensitivity of 1 infectious unit per milliliter and the test should include a known positive control. Herpes virus vectors based on attenuated herpes virus must always be handled at a BSL-2 level. Reference for a plaque assay: Strathdee CA, McLeod MR. 2000. A modular set of helper-dependent herpes simplex virus expression vectors. Mol Ther 5:479-485.

Lentivirus vectors Lentivector systems with HIV envelope gene sequences require BSL-2 with BSL-3 practices at a minimum.

Lentivirus vectors Lentivirus vector stocks generated with packaging systems devoid of the HIV envelope gene must be tested for RCV by serial transfer in a cell line documented to be capable of supporting wild type HIV and ELISA assay for p24 antigen prior to approval for use at BSL-1 and ABSL-1. The vector stock should be tested at a limit of sensitivity of 1 infectious unit per milliliter. A volume of at least 1 milliliter (or equivalent if viral stock is concentrated) must be tested. Lentivector systems with HIV envelope gene sequences require BSL-2 with BSL-3 practices at a minimum. Strictly adhere to the published protocol: Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. 1998. A third-generation lentivirus vector with a conditional packaging system. J Virol 72: 8463-8471. For questions contact the Research & Biological Safety Office at 206.221.7770. Example Lentiviral RCV Protocol (Word)

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RCV Testing to Lower Containment

In general, vector stocks and/or producer lines must be tested for RCV prior to approval for use at a lowered containment level. The following steps are required to lower containment for viral vector use.

  1. Contact EH&S Research and Occupational Safety (ROS) at ehsbio@uw.edu or 206-221-7770 to determine if lowering containment is possible for the desired work.
  2. Complete the referenced test for RCV.
  3. Submit a completed Request for Change to Biological Use Authorization (BUA) with the testing results attached to EH&S ROS. Include the following information with the submission.
    • Test protocol or reference to published protocol
    • Vector name
    • Batch number
    • Results for samples, including controls
  4. EH&S and the IBC Chair confirm results that meet the testing requirements and lower the containment level accordingly. The BUA letter is updated to reflect the change and all necessary parties are notified.
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Oncogenes in Viral Vectors

Inclusion of oncogenes in integrating viral vectors typically requires a heightened containment level. Register any oncogenes with EH&S. Genes listed in the Cancer Gene Census database are known oncogenes. To see if a gene is listed as an oncogene, search using the RefSeq gene name. Use the search box in the Census table, not the search box on the site's toolbar.

See Third Generation Lentiviral Vectors: Lowering Biocontainment for Oncogenic Inserts for the IBC's working definition of third generation lentiviral vectors. If you would like to request lower biocontainment for your work in a third generation lentiviral vector system, contact EH&S Research and Occupational Safety at ehsbio@uw.edu or 206.221.7770.

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