Viral Vector Training

1. Viral Vector Training

2. Viral Vectors • Can be used as gene delivery systems • Can also be used for human gene therapy • All work with viral vectors mustbe 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

3. 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

4. Common Eukaryotic Viral Vectors • Adeno-associated Virus • Adenovirus • Retrovirus • Includes Lentivirus, MMLV, HIV or SIV replication, incompetent viruses • Herpes Virus • Vaccinia Virus

5. 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

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 cells • Insertional mutagenesis/oncogenic potential • Inappropriate expression of gene product • Germ-line transfer of genes • Rescue by other human pathogenic viruses

7. 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!.

8. Risk Assessment Summary

9. Risks Associated with Viral Vectors: Rescue of Replication Deficient Viruses by Superinfection with Wild Viruses Viral DNA Gene of Interest Wild Virus Virus Cell’s DNA Target Cell

10. Virus Wild Virus Risks Associated with Viral Vectors: Rescue of Replication Deficient Viruses by Superinfection with Wild Viruses Viral DNA Gene of Interest Target Cell Cell’s DNA

11. Risks Associated with Viral Vectors: Insertional Mutagenesis Virus Viral DNA Gene of Interest Target Cell Host Cell DNA Proto-Oncogene

12. 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

13. Viral Pseudotyping: A Double-Edged Sword Special care should be used when working with pantropic or amphotropic viruses which can infect humans!

14. 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

15. 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

16. Specific Risks for AAV Vectors • Insertional mutagenesis • Increased risk when using helper virus • Increased risk when gene of interest is an oncogene • Latent infection

17. 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

18. 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 vitroandin vivo use http://cshprotocols.cshlp.org/content/2009/5/pdb.prot5011.full

19. 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

20. 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

21. 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)

22. 2 Plasmid System 2 plasmid systems present safety concerns due to theincreased risk of recombination from homologous recombination resulting in a replication competent virus

23. 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

24. 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

25. 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 http://www.sciencephoto.com/media/200779/enlarge#

26. 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

27. 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

28. 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

29. 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

30. Ways to Minimize Exposure

31. Ways to Minimize Exposure

32. 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

33. Engineering ControlsBiosafety Cabinets (akaBSC, 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

34. 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

35. 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 Work Clean to Dirty

36. 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 The pressure readings on the sticker MUST match the gauge!

37. 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 The pressure readings on the sticker MUST match the gauge!

38. More on BSCs

39. 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

40. Centrifuge Safety Cups Centrifuge safety cups or sealed rotors must be used when working with viral vectorsThey are to be loaded and unloaded in the biological safety cabinet

41. 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

42. 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

43. 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!!

44. 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

45. 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

46. 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

47. 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

48. Disinfection & Waste Disposal Disinfection • Most effective germicides for viral vectors are: • 1% sodium hypochlorite (bleach) • 2% glutaraldehyde • 5% phenol • All waste generated MUSTbe 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 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