VECTOR SAFETY INFORMATION 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 ITRcontaining 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. 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
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. Lentiviral Vectors: Material Information Pseudotyped lentiviral vectors contain recombinant transgene sequences (e.g. encoding reporter or therapeutic genes) as well as viral packaging and regulatory sequences which are flanked by lentiviral long terminal repeats (LTRs). The removal of viral structural genes renders the vector replication-defective and the LTRs may or may not carry modifications rendering them selfinactivating or SIN. Lentiviral vector particles are pseudotyped with heterologous envelope proteins (e.g. VSV-G envelope glycoproteins). In the vectors, the lentiviral LTRs and remaining coding sequences typically comprise less than 30% of the wild type lentiviral genome. Lentiviral vectors are replication-defective and generated by transient transfection of HEK293 cells using three or four plasmids (the transfer LTR-containing plasmid, the helper packaging plasmid and the plasmid encoding a heterologous envelope glycoprotein gene as well as, potentially, a separate plasmid encoding Rev). Lentiviral vector preparations produced by the Penn Vector Core are filtered, concentrated by ultracentrifugation, resuspended in PBS and stored at -80 C. Routine quality control conducted for preclinical lentiviral vector preparations includes a determination of titer and yield by quantitative PCR and an endotoxin analysis. Additional assays offered include the detection of replication competent lentivirus (RCL). Preclinical lentiviral vectors fall into rdna registration category Class III-D (experiments that require institutional biosafety committee approval before initiation). Since the vectors are produced from an agent associated with human disease, they fall into risk group 2 or 3 (depending upon the generation and origin). Pseudotyped recombinant lentiviral vectors must be used under BSL-2 or enhanced BSL-2 containment. It is important to note that the 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. A detailed guidance document Biosafety Considerations for Research with Lentiviral Vectors prepared by the Recombinant DNA Advisory Committee (RAC) is also available at, http://osp.od.nih.gov/sites/default/files/resources/lenti_containment_guidance_0_0.pdf
Adenoviral Vectors: Material Information Adenoviral vectors consist of recombinant transgene sequences (e.g. encoding reporter or therapeutic genes) inserted at E1 region of an E1 or E1 E3 deleted adenoviral backbones. The E1 deletion renders the vector replication defective and dependent upon E1 and other viral sequences present in human 293 cells. Adenoviral cultures are normally provided as purified viral particles in phosphate buffered saline. The viral stock consists of particles containing the vector genome (infectious particles) and a variable number of empty viral capsids in PBS. Other trace components present include, but are not limited to, inorganic salts, vitamins and other nutrients, and human cellular proteins, carbohydrates, amino acids, and fats. The material is normally shipped and stored frozen.