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Penn Vector Core: Biosafety Information

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Biosafety Information

AAV Vectors

Lentiviral Vectors
Adenoviral Vectors
Material Safety Data Sheet (to come)

 

AAV Vectors: Material Information

AAV vectors contain recombinant transgene sequences (e.g. encoding reporter or therapeutic genes)
flanked by the AAV inverted terminal repeats (ITRs). The AAV ITRs, consisting of only 6% of the wild
type AAV genome, are the only AAV specific sequences packaged into the vector particles. The
removal of the viral structural genes renders the vector replication-defective and dependent on
adenovirus helper functions provided in trans. AAV vectors produced by the Penn Vector Core are
generated in the presence of a helper plasmid, not helper virus. The 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 a
determination of titer and yield by quantitative PCR and endotoxin analysis. Additional assays may
include a purity assessment by SDS-PAGE/densitometry and/or an infectious titer determination by
TCID50 analysis. Assays for the detection of replication competent (RCAAV) AAV particles are not
currently available for serotypes other than AAV2 and not conducted for preclinical 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 virus
is dependent for replication on the presence of adenovirus or herpesvirus and will, in the absence of
helper virus, stably integrate into the host cell genome, AAV vector genomes remain primarily
episomal in target cells and have a low (if any) frequency of integration. Cultures of replication
defective AAV vectors are non-infectious and are not hazardous materials as defined by OSHA
1919.1200. 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 is the case at UPenn) can in most cases be handled at biosafety level 1 (BSL-1). This level of
containment made is modified by other considerations.


AAV vectors typically fall into rDNA registration category Class III-D (experiments that require
institutional biosafety committee approval before initiation). If the vectors are to be used for in vivo
studies, registration Class III-D-3 (experiments involving whole animals) could be appropriate. If the
vectors are designed to express cDNA from higher risk group organisms (e.g HIV), they would move
to a Class III-D-2. In terms of biosafety containment level, UPenn requests investigators to state if the
vectors are to be registered for generation, for use, or both. BSL-2 conditions must be used for the
generation of AAV vectors due to the transformed HEK293 cells that are used for production. Purified
AAV vectors may be subsequently used under BLS-1 conditions. Additional information is provided in
the attached MSDS. It is important to note that vectors are different from the viruses from which they
were derived but some of the safety information may be the same as required 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

Map of pENN AAV CB7 CI-eGFP(P1046)

 

 

Example: pAAV2/9 trans plasmid

Map of pAAV 2/9 (P0008) Q

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.

Laboratory Hazards PPE

*The above PPE are often required IN ADDITION to working in a certified Biosafety Cabinet.

 

Lentiviral Vectors: Material Information (to come)

Adenoviral Vectors: Material Information (to come)

 

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