2 Viral Vectors Can be used as gene delivery systems Can also be used for human gene therapyAll work with viral vectors must be registered with the campus Institutional Biosafety Committee (IBC)Prokaryotic or Eukaryotic viruses:Recombinant prokaryotic viruses (bacteriophages) must be registered with IBCEukaryotic viruses present biohazard concerns, which is the focus of this training
3 Eukaryotic Viral Vectors (e.g., Adenovirus, Lentivirus) Narrow or wide host rangeFlexibility in the type of transgene that is deliveredEasily produced in the laboratory
4 Common Eukaryotic Viral Vectors Adeno-associated VirusAdenovirusRetrovirusIncludes Lentivirus, MMLV, HIV or SIV replication, incompetent virusesHerpes VirusVaccinia Virus
5 Production of Viral Vectors Construction of recombinant vector with transgene(s) of interestTransfection of plasmids (number of plasmids differ) into host cell (typically HEK293 cells) to package recombinant viral genomeVirus collected and used for infection of animal, cell, gene therapy, etc.HEK293 cells are human cells (requiring BSL-2 practices), and require Hepatitis B vaccination or proof of immunity
6 Biosafety Concerns Require Risk Assessment Risk Assessment considers the potential for the following risks which pose a hazard to laboratory staff which include:Generation of Replication Competent Viruses (RCV)Infection of unintended target cellsInsertional mutagenesis/oncogenic potentialInappropriate expression of gene productGerm-line transfer of genesRescue by other human pathogenic viruses
7 Risk Assessment Risk Assessments include: Hazard Characteristics of AgentHazard Characteristics of Laboratory ProceduresHazard Potential associated with work practices, safety equipment & facility safeguardsDetermination of appropriate Biosafety Level (BSL) & any extra precautionsRisks for infection are DIMINISHED by the nature of the vector system (and its safety features)OR;EXACERBATED by the nature of the transgene insert encoded by the vector!.
8 Risk Assessment Summary Biosafety ConsiderationsHigher RiskLower RiskVector ReplicationReplication competentReplication incompetentVector DesignVector packaging functions on two plasmidsViral genes present & expressedVector and packaging functions separated onto multiple (3+) plasmidsViral genes deletedTransgeneOncogene, Toxin encoding, Tumor SuppressorNon – oncogene, structural geneVector GenerationLarge ScaleLaboratory ScaleAnimal HostPermissive hostAnimal engrafted with human cellsNon-permissive hostAnimal ManipulationVector administration (e.g., use of sharps during injection)Housing and husbandry (no use of sharps)
9 Risks Associated with Viral Vectors: Rescue of Replication Deficient Virusesby Superinfection with Wild VirusesViral DNAGene of InterestVirusWildVirusCell’s DNATarget CellComplementationThe genome from the wild virus provides the missing proteins needed for the viral vector to replicate. The superinfected cell functions similarly to a packaging line.
10 Risks Associated with Viral Vectors: Rescue of Replication Deficient Virusesby Superinfection with Wild VirusesVirusWildVirusViral DNAGene of InterestCell’s DNATarget CellUnlikely to be HIV (due to gene deletions); theoretical risk is pseudo-typed VSV-g replication-competent virus bearing gene of interest and able to infect dividing and non-dividing cells.RecombinationThe genome from the wild virus randomly recombines with the viral vector, providing sufficient genetic material for the viral vector to replicate. The resulting rescued virus may possess pieces of the original insert gene. The viral genome is impossible to predict due to random recombination. The virus may exhibit altered virulence.
11 to increased cancer risk. Risks Associated with Viral Vectors:Insertional MutagenesisViral DNAGene of InterestVirusHost Cell DNATarget CellProto-OncogeneOncogeneRandom integration of viral genome may disrupt endogenous host genes. Of special concern is disruption of proto-oncogenes, which can leadto increased cancer risk.
12 Viral Pseudotyping: A Double-Edged Sword TropismThe ability of a virus to infect a particular type of host cellPsuedotypingAltering the viral envelope protein to alter tropism, thus allowing the virus to infect cells it originally could not, typically VSV-G envelope is used
13 Viral Envelope Protein Receptor for Viral Envelope Viral Pseudotyping: A Double-Edged SwordTropismHost RangeViral Envelope ProteinReceptor for Viral EnvelopeEcotropicMouse/Rat (narrow host range)Gap70mCAT-1Amphotropic / DualtropicMammals (wider host range)4070A / 10A1Ram-1 / GALVPantropicAll AnimalsVSV-GPhosphotidyl serinePhosphotidyl inositolGM3 gangliosideSpecial care should be used when working with pantropicor amphotropic viruses which can infect humans!
14 Adeno-Associated Virus (AAV) Icosahedral, enveloped, ssDNA virusRequires a helper virus to replicateTypically Adenovirus, Herpesvirus or VacciniaAble to stably insert DNA into host chromosome, and remain latent in the absence of helper virusInfectious to humans with no known disease associationMay be transmitted by aerosol, droplet exposure to mucous membrane, injection and ingestion
15 AAV Vector Characteristics Limited cloning capacityMulti-plasmid packaging systemAbility to be produced in high titersAbility to infect broad range of cellsLong term, stable expression from randomly integrated sequencesReplication in the presence of wild type (WT) AAV or helper virusBSL-1 without helper virus, BSL-2 with helper virus or when working with human cells
16 Specific Risks for AAV Vectors Insertional mutagenesisIncreased risk when using helper virusIncreased risk when gene of interest is an oncogeneLatent infection
17 Adenovirus Non-enveloped, icosahedral dsDNA 49 immunologically distinct typesInfectious through respiratory, mucous membranes, eye & gastrointestinal routesReplication deficient strains can cause respiratory inflammation, corneal injury & conjunctival damage
18 Adenovirus Vector Characteristics Vector capacity kbWide host range, including humansMost used are replication deficient, by way of E1a and E1b deletionPackaged using HEK293 cellsBSL2 recommended for in vitro and in vivo use
19 Specific Risks for Adenovirus Vectors Formation of replication competent virusesIncreased risk when gene of interest is an oncogene or biotoxic materialInflammationLatentcyRecombination with vector and natural Adenovirus
20 Retroviruses Enveloped, ssRNA virus Able to inject into host DNA and become latent virusesHost range determined by envelope proteinsAble to infect both proliferating & non-proliferating cellsInclude ecotropic, amphotropic & pseudotyped virusesBSL2 recommended for in vitro and in vivo use
21 Retroviral Vector Characteristics Vector capacity: 8kbMost common:LentivirusMMLVHIV/ SIV (replication incompetent forms)Often psuedotyped with VSV-GMultiple plasmid packaging systemsMore plasmids = less risk (e.g. a 4 plasmid systems are better than 2 plasmid systems, less recombination risk)
22 2 Plasmid System2 plasmid systems present safety concerns due to the increased risk of recombination from homologous recombination resulting in a replication competent virusHelper: all structural proteins needed to package new virus within the packaging cell line; packaging sequence deletedVector: gene of interest and packaging sequence – lacks structural genes needed to form a replicative competent virus in the host cell.Construct efficient at delivery of the transgene but was shown to be replication competent via several recombination events in the packaging cell – double crossover event resulted in packaging sequence in vector plasmid to join helper plasmid (containing structural proteins).Earlier generation vector systems (MMLV) used a 2 plasmid system:Helper: all structural proteins needed to package new virus within the packaging cell line and the packaging sequence deleted
23 HIV – an upgrade in retroviral vectors 3 & 4 Plasmid SystemsSpread the genomes of the helper plasmid into multiple plasmids which would require multiple replication events to form a replicative competent virus More plasmids= less riskHIV – an upgrade in retroviral vectorsFurther separating the structural proteins of the helper plasmid.HIV has many accessory genes required for replication – removal makes recombination event less likely
24 Specific Risks for Retroviral Vectors Replication competent virusesRecombination with WT viruses to form replication competent strainsInsertional MutagenesisActivation of endogenous sequencesIncreased risk if the gene of interest is an oncogeneLatent infection
25 Herpes Virus Icosehedral, ds DNA virus Two immunologically distinct types - HSV1 and HSV2Vectors are typically replication deficient due to deletions in viral genomeWide host range and cell tropismEstablishes latent infection indefinitely in post-mitotic neuronsUseful for nervous system applications
26 Specific Risks for Herpes Vectors Insertional mutagenesisRecombination that will result in a replication competent/ infectious particleViral infection resulting in illness for replication competent vectorsLatent infection
27 Vaccinia Virus dsDNA virus - member of poxviridae family Wild type virus can replicate in enucleated cellsVaccinia is a human pathogen, causing severe disease in immunocompromised and some healthy individualsVirus is the component of the smallpox vaccinationCan cause infection through ingestion, parenteral injection, absorption through broken skin, droplet or aerosol exposureVaccination is available for laboratory workersReplication competent strains availableMutated with decreased pathogenicity
28 Vaccinia Vector Characteristics Can hold large amount (30 kb) of foreign DNA, stably inserted into genome for efficient replication and expression in host cellsCan infect all mammalian cellsMost are replication competentOne variant, MVA, can grow only in avian cells and can remain in cytoplasmOther variants mutated to prevent infection, targeted to specific cells within organism
29 Specific Risks for Vaccinia Vectors Replication competent virusesPotential for viral infection resulting in illness, especially in immuno-compromised subjectsA vaccination for vaccinia virus is available.Occupational Health Services can provide additional information & counseling regarding its safety & protection for laboratory workers
30 Ways to Minimize Exposure Engineering Controls:Use of available technology and devices to isolate hazards from the workere.g., Biosafety cabinets (BSC), safer needle devices, puncture-resistant sharps containersAdministrative Controls:Standard Operating Procedures, Exposure Control Plan, Biosafety Manuale.g., Controls to monitor compliance, provide accessibility of control methods, investigate exposures to prevent future occurrences
31 Ways to Minimize Exposure Work Practice Controls:Manner in which task is performed to reduce exposuree.g., Wash hands after removal of gloves; disposal of needles without recapping; no lab coats outside of labPPE (Personal Protective Equipment):Specialized clothing or equipment used to protect workers from exposuree.g., lab coats, gloves, face shields, eye protection, fluid resistant aprons, head and foot coverings
32 Engineering ControlsThe following MUST be used when working with viral vectors:Biological Safety Cabinet (Class II)Chemical disinfectant traps with vacuum line HEPA filtersSharps containers & “safe needle” devicesCentrifuge safety devicesSpecimen transport containersReplace glass with plastic
33 Engineering Controls Biosafety Cabinets (aka BSC, Tissue Culture Hood) All work with viral vectors, infection of animals, handling infected animals, animal necropsy, cage changing, etc. MUST be performed inside a certified, Class II, biosafety cabinetClick to view video on this topic
34 Working Inside a BSCAllow cabinet to run for min before starting workCheck magnahelic gauge to be sure hood is functioning properly (compare with number on annual certification sticker)Disinfect surfaces (including equipment)Cover work surface with disinfectant-soaked towelPlace materials as far into cabinet as possibleWork Clean to Dirty
35 Working Inside a BSC (Cont’d) Work “clean to dirty”Use horizontal pipette trays and interior biohazard containersDisinfect spills with appropriate disinfectantDo not place items on the front grill or block the back grillPrevent turbulence when working in the BSC, use slow and deliberate motions when moving hands out of cabinetWork Clean to Dirty
36 The pressure readings on the sticker MUST match the gauge! CertificationRequired annually!Contracted outside vendors certify biosafety cabinets annuallyFilters are tested for leaksAir flow is verifiedVibration, lighting, etc.Also should be certified when moved or repairedThe pressure readings on the sticker MUST match the gauge!
37 The pressure readings on the sticker MUST match the gauge! RepairsDo NOT use the cabinet if it is malfunctioning (e.g.: noise, vibration, or the pressure gauge reads no pressure/ too much pressure)Physical Plant does NOT perform repairs. Certified vendors must be contacted by your departmentSome repairs will require decontamination of the cabinetThe pressure readings on the sticker MUST match the gauge!
38 More on BSCsAbsorbent Pad covering the grill. Nothing should be placed on or covering the grillItems placed on the grill. Again, nothing should be placed on the grill
39 Filtered Vacuum Lines for Liquid Waste Flask for liquid waste MUST have appropriate disinfectantNo hazardous chemicals to be used with vacuum flasksOverflow flask is recommendedAll vacuum lines MUST have HEPA filters
40 Centrifuge Safety Cups Centrifuge safety cups or sealed rotors must be used when working with viral vectors They are to be loaded and unloaded in the biological safety cabinet
41 Work PracticesDecontaminate all waste (autoclave or chemical disinfectant)No “sharps” (needles, glass Pasteur pipettes) may be used with these cultures unless approved by the Institutional Biosafety CommitteeUse plastic aspiration pipetsDo not use “sharps” to harvest virus pelletAll sharps MUST be properly disposed in a sharps containerFor experiments requiring needles- safer devices MUST be considered and are recommended
42 Work Practices (Cont’d) Access to the laboratory should be limited or controlledViral vector work is NOT permitted on the open benchA biosafety cabinet must be used for all manipulations including (but not limited to):PipettingHarvesting infected cellsLoading and opening containersInitial delivery of vector to animalsHandling of infected animals
43 Work Practice Controls No eating, drinking, smoking, applying cosmetics, or handling contact lensesNo food or drink storage in the labMinimize production of droplets or aerosolsTransport specimens in secondary containmentUse mechanical pipettersDecontaminate equipment after useUse universal precautions:Treat everything as if it is infectious!!
44 Work Practice Controls (Cont’d) Biohazard labels must be placed to indicate each area where viral vectors are used / stored:biosafety cabinetsIncubatorsCentrifugeRefrigeratorslaboratory entrance doorsWaste containers
45 Animal StudiesSome animal systems are not permissive hosts and do not support replication competent viruses. These are safer systems, but all animals infected with viral vectors should be handled using ABSL-2 proceduresThe initial delivery of vector is performed under ABSL-2 containment
46 Animal Studies (Cont’d) All infected animals are to be manipulated in a certified BSCVentilated or filtered bonnet cages are required for housingAll cages must be changed in BSCAll carcasses and bedding must be autoclaved or chemically treated before disposalSignage posted on room to indicate infected animals, and the vector of infection
47 Personal Protective Equipment Use of the following personal protective equipment is required to reduce the potential for exposure:GlovesLab CoatsSafety eyewearDisposable gowns (animal work)Other PPE as determined by the IBC
48 Disinfection & Waste Disposal Most effective germicides for viral vectors are:1% sodium hypochlorite (bleach)2% glutaraldehyde5% phenolAll waste generated MUST be autoclaved or chemically disinfected PRIOR TO disposal in regulated medical waste bins (red bag)
49 Autoclaving Autoclaves: Time, Pressure, Heat Pressure vessels that use saturated steam under a pressure of approximately 15 psi to achieve a chamber temperature of a least 121°C (250°F) for a minimum of 30 minutes
50 Work Practices - Autoclaves Use autoclave bags (regular plastic bags melt!)Do not overload bagsEnsure bag is partially open to allow steam to penetrate the contentsUse appropriate secondary container for autoclaving and transporting the bag:Plastic: Polypropylene pans preferred over:PolyethylenepolystyreneStainless steel: durable & a good conductor of heat
51 Work Practices – Autoclaves (Cont’d) Autoclave Indicators used to validate decontaminationChemical indicators change color after being exposed to 121°C (250°F), but they have no time factor!Tape indicators can ONLY be used to verify that the autoclave has reached normal operating temperatures for decontaminationBiological indicators are designed to demonstrate that an autoclave is capable of killing microorganismsA load test using Geobacillus stearothermophilus should be performed monthly
52 Testing for Replication Competent Viruses (RCV) Test producer cells and vector stocks periodically for the presence of RCVIf obtaining the viral vector from a commercial source, please check the manufacturer’s information as to the quality control concerning replication competent virusesInformation as to the methods and frequency for checking viral vectors for RCV should be included with the IBC application
53 Testing for Replication Competent Viruses (RCV) (Cont’d) Adenoassociated Virus:No helper virus: Not requiredHelper virus used: Every viral preparation must be tested for the presence of adenovirus prior to in vitro or in vivo useHeat inactivate viral preparations for 15 minutes at 56⁰C, test for RCV by plaque assay or cytopathic effectHehir, KM, Armentano, D, Cardoaz, LM, et al “Molecular characterization of replication-competentvariants of adenovirus vectors and genome modifications to prevent their occurrence”. J. Virol. 70:
54 Potential for Replication Competent Viruses Adenovirus:Replication competent viruses can be produced upon successive amplification. These viruses are produced when adenoviral DNA recombines with E1-containing DNA in HEK293 cellsThe E1a assay can be used to check for RCV and must be done before in vitro or in vivo use. The vector stock should be tested at a limit of sensitivity of 1 in 106 virus particles compared to known positive controlZhang WW, Kock, PE, Roth, JA “Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.” Biotechniques. 18:
55 Potential for Replication Competent Viruses Retrovirus - Test every 6 months, for 1 infectious unit per mLRetrovirus (ecotropic & amphotropic)Amplification in permissive cell lines, and screening by appropriate assay (i.e. PG-4S+L- or marker rescue)Forestell, SP, Nando, JS, Bohnlein, E and Rigg, RJ Improved detection of replication competent retrovirus. J Virol Methods. 60:Wilson, CA, Ng TH, and Miller AE Evaluation of recommendations for replication competent retrovirus testing associated with use of retroviral vectors. Human Gene Therapy. 8(7):LentivirusSerial transfer and by ELISA for p24 antigenMarker rescue assayDull, T, Zufferey, R, Kelly M, mandel, RJ, Nguyen M, Trono D, Naldini L A third generation lentivirus vector with a conditional packaging system. J Virol. 72:Murine Retrovirus - Marker rescue assay, PERT, PG3S+L- or infectivity RT-PCR assays
56 Potential for Replication Competent Viruses HerpesvirusViral preparations should be tested every 6 months for RCV by plaque assayThese assays should be tested at a sensitivity level of 1 infectious unit per mLFor in vivo work, viral preparations should be tested before each use by plaque assayStrathdee CA, McLeod, MR “A modular set of helper dependent simplex virus expression vectors.”Mol Ther. 5:
57 Potential for Replication Competent Viruses Vaccinia virusTesting is not required since replicating viruses are used
58 Institutional Biosafety Committee Review of Viral Vector Protocols The NIH rDNA guidelines indicate that the IBC is responsible for performing a risk assessment of rDNA work and will determine the appropriate biosafety level (BSL)Major considerations to the BSL for viral vector work:Potential human tropism of the vectorPotential pathogenic effects of expressed transgene
59 Institutional Biosafety Committee Review of Viral Vector Protocols It is the responsibility of the protocol applicant to provide enough information to the IBC to justify WHY a particular vector should be used at BSL-2 and not BSL-3, particularly in the cases in which the transgene is potentially oncogenic or immunosuppressive to humans
60 Standard Operating Procedures EOHSS has developed standard operating procedures for working with viral vectors, which includes the information in this training The signature page must be signed by all those working with the viruses in the lab AND the Principal InvestigatorAdenoassociated Viruses Adenovirus Retroviruses Herpes Virus Vaccinia Virus
64 ReferencesBraun, A “Biosafety in Handling Gene Transfer Vectors.” Current Protocols in Human GeneticsCDC-BMBL, 5th ed.,Dull, T, Zufferey, R, Kelly M, mandel, RJ, Nguyen M, Trono D, Naldini L A third generation lentivirus vector with a conditional packaging system. J Virol. 72:Environmental Health and Safety. The University of Iowa, “ Adeno-Associated Virus and Adeno-Associated Viral Vectors” https://research.uiowa.edu/ehs/files/documents/biosafety/AAV.pdfForestell, SP, Nando, JS, Bohnlein, E and Rigg, RJ Improved detection of replication competent retrovirus. J Virol Methods. 60: Hazardous and Radioactive Waste Disposal Standard Operating Procedure, Comparative Medicine ResourcesHehir, KM, Armentano, D, Cardoaz, LM, et al “Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence”. J. Virol. 70:MSDS Health Canada
65 ReferencesNCI-Fredrick Safetygram (ISM-193, April 2001):Strathdee CA, McLeod, MR “A modular set of helper dependent simplex virus expression vectors.” Mol Ther. 5:Stanford University, “Working with Viral Vectors,”University of Texas Health Science Center at Houston “Guidelines for the Safe Handling of Adenoviral Vectors in Laboratory, Animal and Human Experiments”Wilson, CA, Ng TH, and Miller AE Evaluation of recommendations for replication competent retrovirus testing associated with use of retroviral vectors. Human Gene Therapy. 8(7):Young, L.S., Searle, P.F., Onion, D., and V. Mautner “Viral gene therapy strategies: from basic science to clinical application.” J. of Pathology. 208: Zhang WW, Kock, PE, Roth, JA “Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.” Biotechniques. 18:
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