Experiments using Lentiviral Vectors require DHRC IBC approvals before initiation of experiments and should be done in BSL-2 designated room.

Transcription

1 Experiments using require DHRC IBC approvals before initiation of experiments and should be done in BSL-2 designated room. 1.0 Purpose and Scope To provide guidance and describe the biological safety considerations when working with lentiviral vectors and the engineering and operational practices that are approved to mitigating these risks. Note tha there are minimum standards and that some experiments may require higher containment practices. This should be decided jointly by the Principal Investigator and the DHRC Institutional Biosafety Committee. 2.0 Requirement No one is allowed to work with lentivirus without prior training by the Principal Investigator who supervises their work, or their designated technical expert. The worker should demonstrate good laboratory safety practices and tissue culture technique and an understanding of this SOP prior to being permitted to work with lentivirus. Additionally, Training done to laboratory staff should be documented. Mandatory to all personnel working with Lentiviral vectors Read the DHRC Biological Safety Manual Read the Viral Vector Guideline Health Canada E-Learning modules 1, 2, 3, 4, 5, 6, 7, 8, 9 and Background Lentiviruses are members of the viral family Retroviridae that are characterized by their use of viral RT (reverse transcriptase) and IN (integrase) for stable insertion of viral genomic information into the host genome. Unlike other retrovirus, lentiviruses have the capability to infecting both dividing and nondividing cells. Lentiviruses include bovine lentiviruses (e.g., Bovine immunodeficient virus, Jembrana disease virus); equine lentiviruses (e.g., Equine infectious anemia virus); feline lentiviruses (e.g., Feline immunodeficient virus); Ovien/caprine lentivirus (e.g., Caprine arthritis-encephalitis virus, Ovine lentivirus, Page 1 of 11 Date: 13 June 2014

2 Visna virus); and Primate lentiviruses (e.g., Human immunodeficiency virus (HIV) types 1-3, Simian AIDS retrovirus SRV-1, Human T-cell lymphotropic virus type 4 and Simian immunodeficiency virus). Most of lentiviral vectors presently in use are HIV derived vectors 4.0 Guidance Information The major risks to be considered for research with HIV based lentivirus vectors are potential for generation of replication-competent lentivirus (RCL), and potential for oncogenesis. These risks can be mitigated by the nature of the vector system (and its safety features) or exarcerbated by the nature of the transgene insert encoded by the vector. The production of safer, more effective replication-incompetent HIV-derived vectors has been achieved through several subsequent modifications. The first generation were produced by transient transfection fo 293T cells using 3 plasmids. Second generation lentivirus vector were made by deleting the four accessory genes (vpr, vpu, vif and nef) from the packaging plasmid. The absence of the accessory genes resulted in a safer plasmid by reducing the chance of the generation replication-competent lentivirus. The third generation letivirus vector are self-inactivating vectors, which have a deletion in the U3 enhancer region resulting in a transcriptionally inactive vector that cannot be converted into full lenght RNA. The tat protein is a strong transcriptional activator of the HIV-1LTR promoter essential for viral replication. In addition to deletion and feature developed in the first three generations, in the fourth generation of lentiviral vector, number of genes from HIV-1 used in the system has been reduced to three (i.e. gag, pol, and rev), VSV-G gene from Vesicular Stomatitis Virus is used in place of the HIV-1 envelope, genes encoding the structural and other components required for packaging the viral genome are separated onto four to five plasmids and may contain inducible promotor. In fact, advanced vector systems that separated vector and packaging functions onto multiple plasmids are less likely to generate RCL and are considered as low risk. Late generation vectors in which 4 plasmids are used to produce the viral particles and which a deletion is created in the LTR upon integration have a Page 2 of 11 Date: 13 June 2014

3 lower risk of generating competent virus, so their use is encouraged when practical. Lentivirfal vectors are generally classed as RG-2. However thay may be classed as RG-2+ and require additional operational precaution if the vector system is an earlier generation, of if the transgene encodes a biological toxin, an oncogene, a cell cyl=cle regulator or an inhibito of a tumor suppresson (e.g., sirna for a tumor suppressor). If this is the case it will be indicated on the biohazard permit approved by DHRC IBC. The risk assessment will need to be evaluated based on the potential of generation of replication/competent adenovirus (RCL), aerosolizing procedures and the potential of oncogenesis from the transgene among other risk factors including the use of needles and sharps 5.0 Storage Lentiviral vector stocks must be in leak-proof secondary containers and stored in a -80 o C freezer clearly marked with a warning label to indicate that Lentiviral vector is present in that freezer. 6.0 Transportation Viral vector should be carried in a double leak-proof container. AAV is transported from the laboratory storage place to the animal facility in closed cryovials or Eppendorf tubes and placed into a 15 or 50 ml screw cap tube with absorbent material in case of a spill. The screw cap tube is place into a closed Styrofoam box containing ice packs. The Styrofoam box should be labelled with PI name, contact information, product name and a biohazard symbol. 7.0 General Laboratory Practices Centrifugation must be done in closed containers and using sealed rotors or safety cups. Safety cups are to be opened inside the biosafety cabinet. All vacuum lines must be fitted with a HEPA filter as described in the DHRC Biological Safety Manual and DHRC Cell Culture Guidelines. All laboratory staff working with or supervising work with Lentiviral vector must be made aware of the hazards associated with the work, required safety practices and procedures, and proper handling of the agent Page 3 of 11 Date: 13 June 2014

4 All laboratory staff working with or supervising work with Lentiviral vector must be up to date with laboratory safety and bloodborne pathogens/biosafety trainings. Signs and labels (including the universal biohazard symbol must be placed to indicate each area where Lentiviral vector is used or stored (including biosafety cabinets, incubators, refrigerators, laboratory entrance doors, etc). Laboratory inventory and laboratory procedure log book should be completed after the use of Lentiviral vector. All Lentiviral vector manipulation should be done in designated BSL-2 laboratory and using BSL-2 procedures Doors have to be kept closed when using the BSL-2 room and BSL-2 signage posted at the entrance. Only authorized personel should enter into BSL-2 room The laboratory should be kept clean and free of materials that are not pertinent to the work The gloves and other PPE worn during work should be taken off before leaving the BSL-2 room Avoid the use of sharps whenever possible and dispose of them immediately. If feasible use plastic disposable transfer pipettes rather that glass Paster pitettes. Common use equipment must be immediately disinfected after use and laboratory disinfection should be done at the end of the work and procedure documented. All cell culture work using Lentiviral vector must be conducted in a certified Class II biological Safety cabinet. 8.0 Personal Protective Equipment Disposable gloves Gloves should be worn during all manipulations. Double gloves are better because micro-holes may be present. Inspect gloves for obvious holes when you are putting them on. Change outer gloves at regular intervals and whenever they become obviously contaminated. Wash hands thouroughly with soap immediately after removing gloves. Page 4 of 11 Date: 13 June 2014

5 Always treat your outer gloves as contaminated. Remove them and replace with clean gloves before touching things outside of the biological safety cabinet (e.g. the stereomicroscope, the perfusion pump, the microscope, the centrifuge, or the incubator) is necessary use the one clean hand / one dirty hand technique. People often touch their face and eyes unconsciously. If you find that you are doing this with gloves on your hands then wear goggles to preent yourself from doing so since this could results in you infecting yourself. Lab Coats Gown or equivalent when introducing vector into animals or performing necropsies. Lab coats are adequate for tissues culture manipulations. Lab coat should be worn in the BSL-2 room only and removed before leaving the BSL-2 room. Lab coats with knit cuffs are recommended to ensure that no bare skin is exposed between the gloves and the lab coat. Goggles and Facial mask Goggles and or face shield is recommended, especially during the AAV infusion and necropsies procedures. N-95 respirator can be used during stereotaxic injection or infusion 9.0 Lentiviral vector administration into animals Lentiviral vectors cannot replicate (even in wild-type form) in rodents. Therefore, animal biosafety level 1 for long-term use in infected rodents is generally considered acceptable. However, at the time of transfer of lentiviral vector and/or lentivirally infected cells into the animals (e.g., by injection or surgery), the animals may still have infectious virus on their wound or body secretion that could be transmitted to research staff. The initial delivery vector is performed under BSL-2 conditions in one of the two surgery room (G0248 or G1256). After the surgery, animals are then housed in filter top cages, in a designated ABSL-2 for at least 72 hours. The following information must be posted at the entrance of ABSL-2 animal room Page 5 of 11 Date: 13 June 2014

6 A description of special housing required to ensure safety of animal facility personnel A label on the animal cage indication the hazardous materials administered to live animal. The name of individual(s) responsible for handling the materials All bedding, waste and animals shall be treated as biohazardous for at least 72 hours post exposure or until the initial cage change that occurs after the 72 hour time period Decontamination of animal waste, caging and any other contaminated equipment is required before disposal. Once the period of potential infectivity is over, the containment level is reduced to ABSL-1. The animal are then transferred to a clean cage and the ABSL-2 cage will stay in a ABSL-2 quarantine space for appropriate waste disposal and cleaning. ABSL-2 cages are autoclaved before they are cleaned and washed Waste disposal All used sharps must be placed, un-capped, immediately into a rigid sharps container and disposed as biohazardous waste as described in the DHRC Biological Safety Manual All potentially contaminated lab materials should be collected in a red biohazard bag and disposed as biohazardous waste as described in the DHRC Biological Safety Manual. Infectious liquid waste will be treated with 10% bleach for 30 min, and can then be drain disposed. Bedding waste from the 72 hour period post-injection are collected in a red biohazard bag and disposed as biohazardous waste as described in the DHRC Biological Safety Manual. Contaminated cages are autoclaved before they are cleaned and washed 11.0 Decontamination Lentiviral vector is an enveloped virus, so it is susceptible to inactivation by sufficiently long treatment with 70% ethanol and freshly diluted 10% bleach. Page 6 of 11 Date: 13 June 2014

7 Surface working area The most effective disinfectant against AAV is a 1% Sodium Hypochloride (bleach) solution that is made fresh daily. To make this solution, dilute 1 volume of Javex to 9 volume of water Ensure a 15 minutes contact time Use this disinfectant for treatment of reusable equipment, surfaces and liquid waste Disinfectant alternative include 2% glutaraldehyde, and 0.25% sodium dodecyl.sulfate (SDFS). Animal cages Autoclaving for 1 hour at 121 o C ir 250 o F (15 lbs psi of steam pressure). Use this disinfection method for reusable equipment, liquid waste or solid waste. Equipments Stereomicroscope, stereotaxic apparatus, persurion pump, microscope, centrifuge. Clean equipment use 70% ethanol 12.0 Accidental spill Immediately notify others around you Don appropriate personal protective equipment, such as gloves, labcoat, and safety glasses Use forceps to remove any broken glass or other sharp items, which should be placed into biohazard sharps containers Cover the spill with paper towels or other absorbent materials Apply 10% bleach directly onto and around the paper towels covering the spill Allow 15 minute contact time before cleaning, starting at perimeter and working inwards towards center Dispose of materials into biohazard bins For large spills, you may contact the Biosafety Officer or the Neuroscience Coordinator and the DHRC Security for assistance at Employee Exposure Page 7 of 11 Date: 13 June 2014

8 Consequences of exposure and appropriate post-exposure treatments are not well defined, and so the emphasis should be placed on prevention. Nonetheless, if an exposure should occur, personnel should wash exposed areas for several minutes with soap and water. Additionally, most lentiviral vector used are derived from human HIV, in case of exposure, if medical attention is required the person should be directed to Hospital St-Luc. Have the MSDS in your hand available to medical personnel. Exposure from splash Eye, skin and/or mucous membrane, rinse a minimum of 15 minutes with water, call the security 2444 and mention code blue and seek for medical attention at Hospital St-Luc Emergency room. Exposure from aerosols-inhalations Call the security and mention code blue and seek for medical attention at Hospital St-Luc Emergency room. Needlestick and/or sharps Exposure Contaminated skin should be wash with copious amounts of water. Call the security and mention code blue and seek for medical attention at Hospital St-Luc Emergency room. All incident and or accidents should be reported to the lab manager/principal investivator, biosafety officer, Neuroscience Coordinator or DHRC administration Precautions and Symptoms of exposure Lentiviral vector may be transmitted by aerosol, droplet exposure to the mucous membrane, ingestion and injection. Retroviruses can act as insertional mutagens, due to their ability to integrate into the host s DNA. Transcriptional activation of host genes adjacent to the site of integration may result, a process dependent on the enhancer/promoter within the viral long terminal repeat (LTR). Replication defective retroviruses can recombine with endogenous retroelements, thus reestablishing or enhancing the pathogenic potential of the virus undergoing recombination. Page 8 of 11 Date: 13 June 2014

9 While murine retroviruses are inactivated by human complement and are not capable of causing human disease, lentiviruses are not inactivated by human complement and can cause disease. Symptomatology: Fever / flu-like symptoms Possible inflammation of infected tissues 15.0 Emergency (Power failure, Fire, Building evacuation) Situations requiring evacuation include fire, hazardous material release, bomb threats and earthquakes. All fire alarms must be treated as real emergencies and building evacuation must occur. The need for evacuation in other situations will be determined by emergency personnel and you will be advised if evacuation is necessary. If evacuation is necessary: Shut down equipment and secure hazardous materials. Calmly proceed to nearest exit Close doors but let them unlock, in case of fire check doors for heat before opening. Follow instructions from emergency personnel. Do not use elevators. Walk don t rush or crowd. Use handrails in stairways. Assist people with disabilities. Move away from the building quickly watch for falling glass and other hazards. Move to your emergency meeting location and stay there so that all personnel may be accounted for. Never re-enter the building until notified by emergency personnel that it is safe to do so Employee Right to-know It is important that all lab personnel (even those not directly working with the virus) be informed and aware that Adenovirus is being used in the lab. Page 9 of 11 Date: 13 June 2014

10 Return this form to the Neuroscience Coordinator Perry pavilion, Room E3205 with all appropriate signatures including laboratory staffs declaration Page 10 of 11 Date: 13 June 2014

11 As the principal Investigator your signature below indicates that you agree to comply with these requirements and will acknowledge and accept responsibility for ensuring compliance including all individuals who enter or work in your laboratory or collaborate in carrying out your research. Although you may choose to delegate aspects of the biosafety program in your laboratory to other laboratory staff, you are ultimately responsible for all activities occurring in your laboratory. Contact the DHRC Institutional Biosafety Committee with questions related these responsibilities PI Name: Signature: Date: I have attended laboratory specific safety training for this standard operation procedure. I have read and understood this standard operating procedure and I have had my questions answered. Date Laboratory Staff Name (Print) Signature Page 11 of 11 Date: 13 June 2014