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 2 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 3 Common Eukaryotic Viral Vectors Adeno-associated Virus Adenovirus Retrovirus Includes Lentivirus, MMLV, HIV or SIV replication, incompetent viruses Herpes Virus Vaccinia Virus 4
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. HEK293 cells are human cells (requiring BSL-2 practices), and require Hepatitis B vaccination or proof of immunity 5 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 6 Risk Assessment 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!. 7 Risk Assessment Summary Biosafety 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+) plasmids
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) 8 Risks Associated with Viral Vectors: Rescue of Replication Deficient Viruses
by Superinfection with Wild Viruses Viral DNA Gene of Interest Target Cell Virus Wild Virus Cells DNA Complementation The 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.
9 Risks Associated with Viral Vectors: Rescue of Replication Deficient Viruses by Superinfection with Wild Viruses Viral DNA Gene of Interest Target Cell Recombination Virus Wild Virus
Cells DNA 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. 10 Risks Associated with Viral Vectors: Insertional Mutagenesis Viral DNA Gene of Interest Target Cell Virus
Host Cell DNA Proto-Oncogene Oncogene Random integration of viral genome may disrupt endogenous host genes. Of special concern is disruption of proto-oncogenes, which can lead to increased cancer risk. 11 Viral Pseudotyping: A Double-Edged Sword 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 12 Viral Pseudotyping: A Double-Edged Sword Tropism Host Range Viral Envelope Protein Receptor for
Ram-1 / GALV VSV-G Phosphotidyl serine Phosphotidyl inositol GM3 ganglioside Pantropic All Animals Special care should be used when working with pantropic or amphotropic viruses which can infect humans! 13
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 14 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 when working with human cells 15 Specific Risks for AAV Vectors Insertional mutagenesis Increased risk when using helper virus Increased risk when gene of interest is an oncogene Latent infection 16 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 17 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 http://cshprotocols.cshlp.org/content/2009/5/pdb.prot5011.full
18 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 19 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 20 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) 21 2 Plasmid System 2 plasmid systems present safety concerns due to the increased risk of recombination from homologous recombination resulting in a replication competent virus 22 3 & 4 Plasmid Systems Spread the genomes of the helper plasmid into multiple plasmids which would require multiple replication events to form a replicative competent virus More plasmids= less risk HIV an upgrade in retroviral vectors
23 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 24 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 in post-mitotic neurons Useful for nervous system applications http://www.sciencephoto.com/media/200779/enlarge# 25 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 26 Vaccinia Virus
dsDNA virus - member of poxviridae family Wild type virus can replicate in enucleated cells Vaccinia is a human pathogen, causing severe disease in immunocompromised 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 27 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 28 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 29
Ways to Minimize Exposure Use of available technology and devices to isolate hazards from the worker e.g., Biosafety cabinets (BSC), safer needle devices, puncture-resistant sharps containers Engineering Controls: 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 30 Ways to Minimize Exposure 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 31 Engineering Controls The 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 32
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 cabinet Click to view video on this topic 33 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 Work Clean to Dirty
34 Working Inside a BSC (Contd) 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 Work Clean to Dirty 35 Certification Required annually! Contracted outside vendors certify biosafety cabinets annually
Filters are tested for leaks Air flow is verified Vibration, lighting, etc. The pressure readings on the sticker MUST match the gauge! Also should be certified when moved or repaired
36 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 The
pressure readings on the sticker MUST match the gauge! Some repairs will require decontamination of the cabinet 37 More on BSCs Absorbent Pad covering the grill. Nothing should be placed on or covering the grill
Items placed on the grill. Again, nothing should be placed on the grill 38 Filtered Vacuum Lines for Liquid Waste 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
39 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 40 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 41 Work Practices (Contd) 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 42 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!! 43 Work Practice Controls (Contd) 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 44 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 45
Animal Studies (Contd) 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 46 Personal Protective Equipment Use 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 47 Disinfection & Waste Disposal Disinfection 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) 48 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 121C (250F) for a minimum of 30 minutes 49 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 50 Work Practices Autoclaves (Contd) Autoclave Indicators used to validate decontamination Chemical indicators change color after being exposed to 121C (250F), 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 autoclave is capable of killing microorganisms A load test using Geobacillus stearothermophilus should be performed monthly 51
Testing for Replication Competent Viruses (RCV) Test producer cells and vector stocks periodically for the presence of RCV If obtaining the viral vector from a commercial source, please check the manufacturers 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 52 Testing for Replication Competent Viruses (RCV) (Contd) 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 56C, test for RCV by plaque assay or cytopathic effect 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. 53 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
Zhang WW, Kock, PE, Roth, JA. 1995. Detection of wild-type contamination in a recombinant adenoviral preparation by PCR. Biotechniques. 18:444-447. 54 Potential for Replication Competent Viruses 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 55 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 Strathdee CA, McLeod, MR. 2000. A modular set of helper dependent simplex virus expression vectors.
Mol Ther. 5: 479-485. 56 Potential for Replication Competent Viruses Vaccinia virus Testing is not required since replicating viruses are used 57 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 58 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 59
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 Investigator Adenoassociated Viruses http://www.umdnj.edu/eohssweb/documents/AdenoassociatedvirusSOPFinal5.2011.pdf Adenovirus http://www.umdnj.edu/eohssweb/documents/Adenovirus_AdenoviralVectorsSOPFinal5.2011.pdf Retroviruses http://www.umdnj.edu/eohssweb/documents/RetroviralVectorsSOPFinal5.2011.pdf Herpes Virus http://www.umdnj.edu/eohssweb/documents/HerpesVirusSOPFinal5.2011.pdf Vaccinia Virus http://www.umdnj.edu/eohssweb/documents/VacciniaVirusVectorSOPFinal5.2011.pdf 60
EOHSS Contact Information Marta Figueroa, MS, CIH 973-972-5901 [email protected] Director: Newark/ Scotch Plains Biosafety: Jessica McCormick, Ph.D. RBP 973-972-8424 Jessica.mccormick @umdnj.edu Tamara McNair, MS
Brian Eggert, MPH 973-972-3820 [email protected] 61 Were on the Web! ready.umdnj.edu http://emergency.umdnj.edu http://www.umdnj.edu/eohssweb http://www.umdnj.edu/orssweb 62
http://umdnjalerts.posterous.com/ rss.xml http://www.facebook.com /umdnjalerts http://twitter.com/ umdnjoem 63 References 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 AdenoAssociated 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. 70:84598467. MSDS Health Canada http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php 64 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. 65
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