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Penn Vector Core: Biosafety Information
Biosafety Information
AAV Vectors |
Lentiviral Vectors |
AAV Vectors: General Information
AAV vectors contain recombinant transgene sequences (e.g., reporter or therapeutic genes) flanked by the AAV inverted terminal repeats (ITRs) which consist of only 6% of the wild type AAV genome. The removal of the viral structural genes renders the vector replication-defective and dependent on adenovirus helper functions provided in trans (note: At UPenn, AAV vectors are generated in the presence of a helper plasmid, not helper virus). AAV vectors are generated by transient transfection of HEK293 cells using three plasmids (the cis ITR-containing plasmid, the trans plasmid encoding AAV replicase and capsid genes and the adenoviral helper plasmid) which result in the pseudotyping of vector genomes with different serotype capsid proteins. The recombinant vectors are purified by tangential flow filtration followed by iodixanol gradient purification and buffer exchange. Routine quality control conducted for preclinical vector preparations includes titer and yield determination by qPCR (expressed in genome copy units), detection of endotoxin, and purity assessment by SDS-PAGE/densitometry; additional assays may include infectious titer determination by TCID50 analysis. Assays for the detection of replication competent (RCL) AAV particles are not currently available for serotypes other than AAV2 and not conducted for preclinical grade vectors.
AAV vectors are based on AAV viruses which are non-pathogenic in humans and the vectors themselves are not known to cause any diseases in humans or animals. Although wild type AAV can enter mammalian cells in the presence of adenovirus and their genomes integrate into host cell DNA, AAV vector genomes remain primarily episomal in target cells and have a low (if any) frequency of integration. According to NIH guidelines, AAV vectors typically fall into Class III-D-3 (Experiments that require institutional biosafety committee approval before initiation). If the vectors are designed to express cDNA from higher risk group organisms (e.g HIV) , they can move to a Class III-D-2. In terms of biosafety containment level, at UPenn, we stipulate whether we are registering vectors for generation or use or both. BSL2 conditions must be used for the generation of AAV vectors only because of the transformed HEK293 cells that are used for production. Purified AAV vectors may be subsequently used under BLS1 conditions. Additional information is provided below – please keep in mind that the vectors are different from the viruses from which they were derived but some of the information you need for your registration may not be known for the vectors and may need to be taken from the information provided below for the wild type virus.
Recombinant Vector Nomenclature – AAV vectors from UPenn are named as follows:
Vector Platform/Serotype. Promoter. Intron (if applicable). Transgene.Polyadenylation Signal eg. AAV2/9.CB7.CI.eGFP.rBG. (Other elements might include IRES, WPRE). In general, AAV vectors are either named according to capsid serotype alone eg. AAV9 or named according to replicase and capsid serotype eg. AAV2/9 (with the capsid serotype always appearing after the backslash) Recombinant AAV vectors generated by transient transfection using AAV2 replicase proteins (which are not packaged so not present in the purified vector) typically consist of AAV2 ITR genomes pseudotyped with various serotype capsid proteins. Thus the nomenclature AAV9 and AAV2/9 is interchangeable. AAV2 vectors (ie. with AAV2 ITR genomes pseudotyped with AAV2 capsid proteins) are generally referred to only as AAV2 (as opposed to AAV2/2).
Sample Plasmid Maps of AAV cis and trans plasmids
Example: pAAV.CB7.CI.PI.eGFP.rBG cis plasmid
Example: pAAV2/9 trans plasmid
Adeno-associated virus (AAV viruses and AAV vectors)
These are infectious human viruses with no known disease association. Some AAV types are common in the general population, and these viruses have the ability to integrate into the host chromosome. The NIH Guidelines state that adeno-associated virus (AAV) types 1 through 4, and all recombinant AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus (which in the case at UPenn) can in most cases be handled at biosafety level 1 (BSL1). This level of containment made is modified by other considerations (e.g transgene.)
Adeno-associated virus (Wild type)
Virology: Adeno-associated virus gets its name because it is often found in cells that are simultaneously infected with adenovirus. Parvoviridae; icosahedral, 20-25 nm in diameter; single stranded DNA genome with protein capsid. AAV is dependent for replication on presence of wild type adenovirus or herpesvirus; in the absence of helper virus, AAV will stably integrate into the host cell genome. Co-infection with helper virus triggers lytic cycle as do some agents which appropriately perturb host cells. Wild type AAV integrates preferentially into human chromosome 19q13.3-qter; recombinant vectors lose this specificity and appear to integrate randomly, thereby posing a theroretical risk of insertional mutagenesis.
- Clinical features: No known pathology for wild type AAV serotype 2.
- Epidemiology: Not documented definitively. Infection apparently via mouth, esophageal or intestinal mucosa.
- Treatment: No specific treatment.
- Laboratory hazards: Ingestion, droplet exposure of the mucous membrane, direct injection.
Laboratory Hazards PPE
- Exposure of mucus membrane eyes, nose, mouth
- Use of safety goggles or full face shields.
- Use of appropriate face mask
- Injection Use of safety needles; NEVER re-cap needle or remove needle from syringe
- Aerosol inhalation- Use of appropriate respiratory protection
- Direct contact with skin gloves, lab coat, closed shoes
*The above PPE are often required IN ADDITION to working in a certified Biosafety Cabinet.
- Use with Animals: BL1 housing.
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