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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.

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Presentation on theme: "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."— Presentation transcript:

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 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

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 3

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

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. 5 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 R eplication C ompetent V iruses (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

7 Risk Assessment 7 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 Biosafety Considerations Higher RiskLower Risk Vector ReplicationReplication competentReplication incompetent Vector Design Vector packaging functions on two plasmids Viral genes present & expressed Vector and packaging functions separated onto multiple (3+) plasmids Viral genes deleted Transgene Oncogene, Toxin encoding, Tumor Suppressor Non – oncogene, structural gene Vector GenerationLarge ScaleLaboratory Scale Animal Host Permissive 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) 8

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

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

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

12 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 12

13 Viral Pseudotyping: A Double-Edged Sword TropismHost RangeViral Envelope Protein Receptor for Viral Envelope Ecotropic Mouse/Rat (narrow host range) Gap70mCAT-1 Amphotropic / Dualtropic Mammals (wider host range) 4070A / 10A1Ram-1 / GALV PantropicAll AnimalsVSV-G Phosphotidyl serine Phosphotidyl inositol GM3 ganglioside Special care should be used when working with pantropic or amphotropic viruses which can infect humans! 13

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 14

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 when working with human cells 15

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 16

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 17

18 Adenovirus Vector Characteristics Vector capacity 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 18

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 19

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 20

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 ) 21

22 2 Plasmid System 22 2 plasmid systems present safety concerns due to the increased 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 23 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 24

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 in post-mitotic neurons Useful for nervous system applications 25

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 26

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 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

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 28

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 29

30 Ways to Minimize Exposure 30

31 Ways to Minimize Exposure 31

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 32

33 Engineering Controls Biosafety Cabinets ( aka BSC, Tissue Culture Hood) 33 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

34 Working Inside a BSC Allow cabinet to run for 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 34 Work Clean to Dirty

35 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 35 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 36 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 37 The pressure readings on the sticker MUST match the gauge!

38 38 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 More on BSCs

39 Filtered Vacuum Lines for Liquid Waste 39 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 vectors They are to be loaded and unloaded in the biological safety cabinet 40

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 41

42 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

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!! 43

44 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

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 45

46 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

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 47

48 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

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 49

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 50

51 Work Practices – Autoclaves (Contd) 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 autoclave is capable of killing microorganisms – A load test using Geobacillus stearothermophilus should be performed monthly 51

52 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

53 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 53 Testing for Replication Competent Viruses (RCV) (Contd) Hehir, 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:

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 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 10 6 virus particles compared to known positive control 54 Zhang WW, Kock, PE, Roth, JA Detection of wild-type contamination in a recombinant adenoviral preparation by PCR. Biotechniques. 18:

55 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 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): 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 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 55 Potential for Replication Competent Viruses

56 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 56 Strathdee CA, McLeod, MR A modular set of helper dependent simplex virus expression vectors. Mol Ther. 5: Potential for Replication Competent Viruses

57 Vaccinia virus Testing is not required since replicating viruses are used 57 Potential for Replication Competent Viruses

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 vector – Potential pathogenic effects of expressed transgene 58

59 It is the responsibility of the protocol applicant 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 59

60 Standard Operating Procedures Adenoassociated Viruses Adenovirus Retroviruses Herpes Virus Vaccinia Virus 60 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

61 EOHSS Contact Information 61 Director: Marta Figueroa, MS, CIH Newark/ Scotch Plains Biosafety: Jessica McCormick, Ph.D. RBP Tamara McNair, MS Brian Eggert, MPH Piscataway/ New Brunswick Biosafety: Tracy Pfromm, MPH Camden/ Stratford Biosafety: Tom Boyle, MS, RBP

62 Were on the Web! 62 ready.umdnj.edu

63 63 /umdnjalerts rss.xml /umdnjoem

64 Braun, A Biosafety in Handling Gene Transfer Vectors. Current Protocols in Human Genetics CDC-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.pdfhttps://research.uiowa.edu/ehs/files/documents/biosafety/AAV.pdf Forestell, 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 Resources Hehir, 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 64 References

65 NCI-Fredrick Safetygram (ISM-193, April 2001): 193.pdfhttp://web.ncifcrf.gov/Campus/safety/safetygram/ism- 193.pdf 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: References

66 Completed Training! 66 Lets take the Quiz! Thank you for taking the time to complete the: UMDNJ Viral Vector Online Training


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