viral vector training
DESCRIPTION
Viral Vector Training. Viral Vectors. Can be used as gene delivery systems Can also be used for human gene therapy All work with viral vectors must be registered with the campus Institutional Biosafety Committee (IBC) Prokaryotic or Eukaryotic viruses: - PowerPoint PPT PresentationTRANSCRIPT
Viral Vector Training
Viral Vectors • Can be used as gene delivery systems
• Can also be used for human gene therapy
• All 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 IBC– Eukaryotic viruses present biohazard concerns,
which is the focus of this training
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Eukaryotic Viral Vectors(e.g., Adenovirus, Lentivirus)
• Narrow or wide host range
• Flexibility in the type of transgene that is delivered
• Easily produced in the laboratory
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Common Eukaryotic Viral Vectors
• Adeno-associated Virus• Adenovirus• Retrovirus
– Includes Lentivirus, MMLV, HIV or SIV replication, incompetent viruses
• Herpes Virus• Vaccinia Virus
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Production of Viral Vectors
• Construction of recombinant vector with transgene(s) of interest
• Transfection of plasmids (number of plasmids differ) into host cell (typically HEK293 cells) to package recombinant viral genome
• Virus collected and used for infection of animal, cell, gene therapy, etc.
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HEK293 cells are human cells (requiring BSL-2 practices), and require Hepatitis B vaccination or proof of immunity
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 cells– Insertional mutagenesis/oncogenic potential– Inappropriate expression of gene product– Germ-line transfer of genes– Rescue by other human pathogenic viruses
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Risk Assessment
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Risk Assessments include:– Hazard Characteristics of Agent– Hazard Characteristics of Laboratory Procedures– Hazard Potential associated with work practices,
safety equipment & facility safeguards– Determination of appropriate Biosafety Level (BSL)
& any extra precautions
Risks 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!.
Risk Assessment SummaryBiosafety
Considerations Higher Risk Lower Risk
Vector Replication Replication competent Replication incompetent
Vector Design
Vector packaging functions on two plasmids
Viral genes present &expressed
Vector and packaging functions separated onto multiple
(3+) plasmidsViral genes deleted
Transgene Oncogene, Toxin encoding, Tumor Suppressor Non – oncogene, structural gene
Vector Generation Large Scale Laboratory Scale
Animal HostPermissive host
Animal engrafted with human cells
Non-permissive host
Animal Manipulation Vector administration
(e.g., use of sharps during injection)
Housing and husbandry (no use of sharps)
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Risks Associated with Viral Vectors:Rescue of Replication Deficient Viruses
by Superinfection with Wild Viruses
Target CellCell’s DNA
Viral DNAGene of Interest
Virus Wild Virus
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ComplementationThe 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.
Risks Associated with Viral Vectors:Rescue of Replication Deficient Viruses
by Superinfection with Wild Viruses
Target Cell Cell’s DNA
Viral DNAGene of Interest
VirusWild Virus
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Recombination
The 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.
Risks Associated with Viral Vectors: Insertional Mutagenesis
Virus
Target Cell
Viral DNAGene of Interest
Host Cell DNA
Proto-Oncogene
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OncogeneRandom integration of viral genome may disrupt endogenous host genes.
Of special concern is disruption of proto-oncogenes, which can lead to increased cancer risk.
Tropism
• The ability of a virus to infect a particular type of host cell
Psuedotyping
• Altering the viral envelope protein to alter tropism, thus allowing the virus to infect cells it originally could not, typically VSV-G envelope is used
Viral Pseudotyping: A Double-Edged Sword
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Viral Pseudotyping: A Double-Edged Sword
Tropism Host Range Viral Envelope Protein Receptor for Viral Envelope
Ecotropic Mouse/Rat (narrow host range) Gap70 mCAT-1
Amphotropic / Dualtropic
Mammals (wider host range) 4070A / 10A1 Ram-1 / GALV
Pantropic All Animals VSV-GPhosphotidyl serine
Phosphotidyl inositolGM3 ganglioside
Special care should be used when working with pantropic or amphotropic viruses which can infect humans!
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Adeno-Associated Virus (AAV)• Icosahedral, enveloped, ssDNA virus
• Requires a helper virus to replicate– Typically Adenovirus, Herpesvirus or Vaccinia
• Able to stably insert DNA into host chromosome, and remain latent in the absence of helper virus
• Infectious to humans with no known disease association
• May be transmitted by aerosol, droplet exposure to mucous membrane, injection and ingestion
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AAV Vector Characteristics• Limited cloning capacity
• Multi-plasmid packaging system
• Ability to be produced in high titers
• Ability to infect broad range of cells
• Long term, stable expression from randomly integrated sequences
• Replication in the presence of wild type (WT) AAV or helper virus
• BSL-1 without helper virus, BSL-2 with helper virus or whenworking with human cells
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Specific Risks for AAV Vectors
• Insertional mutagenesis
• Increased risk when using helper virus
• Increased risk when gene of interest is an oncogene
• Latent infection
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Adenovirus
• Non-enveloped, icosahedral dsDNA
• 49 immunologically distinct types
• Infectious through respiratory, mucous membranes, eye & gastrointestinal routes
• Replication deficient strains can cause respiratory inflammation, corneal injury & conjunctival damage
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Adenovirus Vector Characteristics
• Vector capacity 7.5-30 kb
• Wide host range, including humans
• Most used are replication deficient, by way of E1a and E1b deletion
• Packaged using HEK293 cells
• BSL2 recommended for in vitro and in vivo use
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http://cshprotocols.cshlp.org/content/2009/5/pdb.prot5011.full
Specific Risks for Adenovirus Vectors• Formation of replication competent viruses
• Increased risk when gene of interest is an oncogene or biotoxic material
• Inflammation
• Latentcy
• Recombination with vector and natural Adenovirus
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Retroviruses • Enveloped, ssRNA virus
• Able to inject into host DNA and become latent viruses
• Host range determined by envelope proteins
• Able to infect both proliferating & non-proliferating cells
• Include ecotropic, amphotropic & pseudotyped viruses
• BSL2 recommended for in vitro and in vivo use
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Retroviral Vector Characteristics• Vector capacity: 8kb
• Most common: – Lentivirus– MMLV– HIV/ SIV (replication incompetent forms)– Often psuedotyped with VSV-G
• Multiple plasmid packaging systems– More plasmids = less risk
(e.g. a 4 plasmid systems are better than 2 plasmid systems, less recombination risk)
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2 Plasmid System
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2 plasmid systems present safety concerns due to the increased risk of recombination from homologous recombination
resulting in a replication competent virus
3 & 4 Plasmid SystemsSpread the genomes of the helper plasmid into multiple plasmids which would
require multiple replication events to form a replicative competent virusMore plasmids= less risk
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HIV – an upgrade in retroviral vectors
Specific Risks for Retroviral Vectors
• Replication competent viruses
• Recombination with WT viruses to form replication competent strains
• Insertional Mutagenesis
• Activation of endogenous sequences
• Increased risk if the gene of interest is an oncogene
• Latent infection
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Herpes Virus
• Icosehedral, ds DNA virus
• Two immunologically distinct types - HSV1 and HSV2
• Vectors are typically replication deficient due to deletions in viral genome
• Wide host range and cell tropism
• Establishes latent infection indefinitely inpost-mitotic neurons
• Useful for nervous system applications
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http://www.sciencephoto.com/media/200779/enlarge#
Specific Risks for Herpes Vectors• Insertional mutagenesis
• Recombination that will result in a replication competent/ infectious particle
• Viral infection resulting in illness for replication competent vectors
• Latent infection
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Vaccinia Virus• dsDNA virus - member of poxviridae family
• Wild type virus can replicate in enucleated cells
• Vaccinia is a human pathogen, causing severe disease inimmunocompromised and some healthy individuals
• Virus is the component of the smallpox vaccination
• Can cause infection through ingestion, parenteral injection, absorption through broken skin, droplet or aerosol exposure
• Vaccination is available for laboratory workers
• Replication competent strains available– Mutated with decreased pathogenicity
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Vaccinia Vector Characteristics• Can hold large amount (30 kb) of foreign DNA,
stably inserted into genome for efficient replication and expression in host cells
• Can infect all mammalian cells
• Most are replication competent– One variant, MVA, can grow only in avian cells and
can remain in cytoplasm
– Other variants mutated to prevent infection, targeted to specific cells within organism
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Specific Risks for Vaccinia Vectors
• Replication competent viruses
• Potential for viral infection resulting in illness, especially in immuno-compromised subjects
• A vaccination for vaccinia virus is available. – Occupational Health Services can provide additional information
& counseling regarding its safety & protection for laboratory workers
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Ways to Minimize Exposure
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Engineering Controls: Use of available technology and devices to isolatehazards from the worker
e.g., Biosafety cabinets (BSC), safer needle devices, puncture-resistant sharps containers
Administrative Controls: Standard Operating Procedures, Exposure Control Plan, Biosafety Manual
e.g., Controls to monitor compliance, provide accessibility of control methods, investigate exposures to prevent future occurrences
Ways to Minimize Exposure
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Work Practice Controls: Manner in which task is performed to reduce exposure
e.g., Wash hands after removal of gloves; disposal of needles without recapping; no lab coats outside of lab
PPE (Personal Protective Equipment): Specialized clothing or equipment used to protect workers from exposure
e.g., lab coats, gloves, face shields, eye protection, fluid resistant aprons, head and foot coverings
Engineering ControlsThe following MUST be used when working with viral vectors:
• Biological Safety Cabinet (Class II)
• Chemical disinfectant traps with vacuum line HEPA filters
• Sharps containers & “safe needle” devices
• Centrifuge safety devices
• Specimen transport containers
• Replace glass with plastic
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Engineering ControlsBiosafety Cabinets (aka BSC, Tissue Culture Hood)
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Click to view video on this topic
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 cabinet
Working Inside a BSC• Allow cabinet to run for 10-15 min before starting work
• Check 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 towel
• Place materials as far into cabinet as possible
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Work Clean to Dirty
Working Inside a BSC (Cont’d)
• Work “clean to dirty”
• Use horizontal pipette trays and interior biohazard containers
• Disinfect spills with appropriate disinfectant
• Do not place items on the front grill or block the back grill
• Prevent turbulence when working in the BSC, use slow and deliberate motions when moving hands out of cabinet
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Work Clean to Dirty
Certification
• Required annually!
• Contracted outside vendors certify biosafety cabinets annually
• Filters are tested for leaks• Air flow is verified• Vibration, lighting, etc.
• Also should be certified when moved or repaired
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The pressure readings on the sticker MUST match
the gauge!
Repairs• Do 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 department
• Some repairs will require decontamination of the cabinet
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The pressure readings on the sticker MUST match
the gauge!
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Absorbent Pad covering the grill. Nothing should be placed onor covering the grill
Items placed on the grill. Again, nothing should be placed on the grill
More on BSCs
Filtered Vacuum Lines for Liquid Waste
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• Flask for liquid waste MUST have appropriate disinfectant • No hazardous chemicals to be used with vacuum flasks• Overflow flask is recommended• All vacuum lines MUST have HEPA filters
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
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Work Practices• Decontaminate all waste (autoclave or chemical disinfectant)
• No “sharps” (needles, glass Pasteur pipettes) may be used with these cultures unless approved by the Institutional Biosafety Committee
– Use plastic aspiration pipets– Do not use “sharps” to harvest virus pellet – All sharps MUST be properly disposed in a sharps container– For experiments requiring needles- safer devices
MUST be considered and are recommended
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Work Practices (Cont’d)• Access to the laboratory should be limited or controlled
• Viral vector work is NOT permitted on the open bench
• A biosafety cabinet must be used for all manipulations including (but not limited to): – Pipetting– Harvesting infected cells– Loading and opening containers– Initial delivery of vector to animals– Handling of infected animals
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Work Practice Controls• No eating, drinking, smoking, applying cosmetics, or
handling contact lenses
• No food or drink storage in the lab
• Minimize production of droplets or aerosols
• Transport specimens in secondary containment
• Use mechanical pipetters
• Decontaminate equipment after use
• Use universal precautions:
Treat everything as if it is infectious!!
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Work Practice Controls (Cont’d)
• Biohazard labels must be placed to indicate each area where viral vectors are used / stored:
– biosafety cabinets– Incubators– Centrifuge– Refrigerators– laboratory entrance doors– Waste containers
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Animal Studies• Some 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 procedures
• The initial delivery of vector is performed under ABSL-2 containment
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Animal Studies (Cont’d)
• All infected animals are to be manipulated in a certified BSC
• Ventilated or filtered bonnet cages are required for housing
• All cages must be changed in BSC
• All carcasses and bedding must be autoclaved or chemically treated before disposal
• Signage posted on room to indicate infected animals, and the vector of infection
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Personal Protective EquipmentUse of the following personal protective equipment is required to reduce the potential for exposure:
• Gloves• Lab Coats• Safety eyewear• Disposable gowns (animal work)• Other PPE as determined by the IBC
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Disinfection & Waste DisposalDisinfection• Most effective germicides for viral vectors are:
– 1% sodium hypochlorite (bleach)– 2% glutaraldehyde– 5% phenol
• All waste generated MUST be autoclaved or chemically disinfected PRIOR TO disposal in regulated medical waste bins (red bag)
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AutoclavingAutoclaves: 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
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Work Practices - Autoclaves
• Use autoclave bags (regular plastic bags melt!)
• Do not overload bags
• Ensure bag is partially open to allow steam to penetrate the contents
• Use appropriate secondary container for autoclaving and transporting the bag:
– Plastic: Polypropylene pans preferred over:• Polyethylene• polystyrene
– Stainless steel: durable & a good conductor of heat
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Work Practices – Autoclaves (Cont’d)
Autoclave Indicators used to validate decontamination
• Chemical 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 decontamination
• Biological indicators are designed to demonstrate that an autoclaveis capable of killing microorganisms– A load test using Geobacillus stearothermophilus should be performed monthly
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Testing for Replication Competent Viruses (RCV)
• Test producer cells and vector stocks periodically forthe presence of RCV
• If obtaining the viral vector from a commercial source, please check the manufacturer’s information as to the quality control concerning replication competent viruses
• Information as to the methods and frequency for checking viral vectors for RCV should be included with the IBC application
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Adenoassociated Virus:
• No helper virus: Not required• Helper virus used: Every viral preparation
must be tested for the presence of adenovirus prior to in vitro or in vivo use
• Heat inactivate viral preparations for 15 minutes at 56⁰C, test for RCV by plaque assay or cytopathic effect
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Testing for Replication Competent Viruses (RCV) (Cont’d)
Hehir, KM, Armentano, D, Cardoaz, LM, et al. 1996. “Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence”. J. Virol. 70:8459-8467.
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 cells
• The 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 control
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Zhang WW, Kock, PE, Roth, JA. 1995. “Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.” Biotechniques. 18:444-447.
Retrovirus - Test every 6 months, for 1 infectious unit per mL
Retrovirus (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. 1996. Improved detection of replication competent
retrovirus. J Virol Methods. 60:171-178 – Wilson, CA, Ng TH, and Miller AE. 1997. Evaluation of recommendations for replication competent
retrovirus testing associated with use of retroviral vectors. Human Gene Therapy. 8(7): 869-874.
Lentivirus– Serial transfer and by ELISA for p24 antigen– Marker rescue assay– 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.
Murine Retrovirus - Marker rescue assay, PERT, PG3S+L- or infectivity RT-PCR assays
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Potential for Replication Competent Viruses
Herpesvirus
– Viral preparations should be tested every 6 months for RCV by plaque assay
– These assays should be tested at a sensitivity level of 1 infectious unit per mL
– For in vivo work, viral preparations should be tested before each use by plaque assay
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Strathdee CA, McLeod, MR. 2000. “A modular set of helper dependent simplex virus expression vectors.” Mol Ther. 5: 479-485.
Potential for Replication Competent Viruses
Vaccinia virus
Testing is not required since replicating viruses are used
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Potential for Replication Competent Viruses
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 vector– Potential pathogenic effects of expressed transgene
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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
Institutional Biosafety Committee Review of Viral Vector Protocols
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Standard Operating Procedures
Adenoassociated Viruseshttp://www.umdnj.edu/eohssweb/documents/AdenoassociatedvirusSOPFinal5.2011.pdf
Adenovirushttp://www.umdnj.edu/eohssweb/documents/Adenovirus_AdenoviralVectorsSOPFinal5.2011.pdf
Retroviruseshttp://www.umdnj.edu/eohssweb/documents/RetroviralVectorsSOPFinal5.2011.pdf
Herpes Virushttp://www.umdnj.edu/eohssweb/documents/HerpesVirusSOPFinal5.2011.pdf
Vaccinia Virushttp://www.umdnj.edu/eohssweb/documents/VacciniaVirusVectorSOPFinal5.2011.pdf
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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 Investigator
EOHSS Contact Information
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Director: Marta Figueroa, MS, CIH
973-972-5901 [email protected]
Newark/ Scotch Plains
Biosafety:
Jessica McCormick, Ph.D. RBP
973-972-8424Jessica.mccormick
@umdnj.edu
Tamara McNair, MS 973-972-8419
Brian Eggert, MPH 973-972-3820
Piscataway/ New Brunswick
Biosafety:
Tracy Pfromm, MPH 732-235-8376
Camden/ Stratford Biosafety:
Tom Boyle, MS, RBP 865-566-6189
We’re on the Web!
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ready.umdnj.edu
http://emergency.umdnj.edu http://www.umdnj.edu/eohssweb http://www.umdnj.edu/orssweb
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http://www.facebook.com/umdnjalerts
http://umdnjalerts.posterous.com/rss.xml
http://twitter.com/umdnjoem
• Braun, A. 2006. “Biosafety in Handling Gene Transfer Vectors.” Current Protocols in Human Genetics. 12.1-12.18.
• CDC-BMBL, 5th ed., www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf
• 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.
• 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.pdf
• Forestell, SP, Nando, JS, Bohnlein, E and Rigg, RJ. 1996. Improved detection of replication competent retrovirus. J Virol Methods. 60:171-178.
• Hazardous and Radioactive Waste Disposal Standard Operating Procedure, Comparative Medicine Resources http://njms.umdnj.edu/research/cmr/sop.cfm
• Hehir, KM, Armentano, D, Cardoaz, LM, et al. 1996. “Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence”. J. Virol. 70:8459-8467.
• MSDS Health Canada http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php
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References
• NCI-Fredrick Safetygram (ISM-193, April 2001): http://web.ncifcrf.gov/Campus/safety/safetygram/ism-193.pdf
• Strathdee CA, McLeod, MR. 2000. “A modular set of helper dependent simplex virus expression vectors.” Mol Ther. 5: 479-485.
• Stanford University, “Working with Viral Vectors,” http://www.stanford.edu/dept/EHS/prod/researchlab/bio/docs/Working_with_Viral_Vectors.pdf
• University of Texas Health Science Center at Houston “Guidelines for the Safe Handling of Adenoviral Vectors in Laboratory, Animal and Human Experiments” http://www.uth.tmc.edu/safety/biosafety/adenoviral.pdf
• Wilson, CA, Ng TH, and Miller AE. 1997. Evaluation of recommendations for replication competent retrovirus testing associated with use of retroviral vectors. Human Gene Therapy. 8(7): 869-874.
• Young, L.S., Searle, P.F., Onion, D., and V. Mautner. 2006. “Viral gene therapy strategies: from basic science to clinical application.” J. of Pathology. 208:299-318.
• Zhang WW, Kock, PE, Roth, JA. 1995. “Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.” Biotechniques. 18:444-447.
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References
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