Biosafety of Recombinant Viral Agents · • Biology of wild-type virus – Most vectors based on...
Transcript of Biosafety of Recombinant Viral Agents · • Biology of wild-type virus – Most vectors based on...
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Biosafety of Recombinant Viral Agents
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Viral Vectors for Gene Therapy
• Introduction• Biosafety Principles • Biosafety Risk Categories• Biosafety Risk Assessment
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Viral Vectors for Gene Therapy
• Classes of Viral Vectors– Methods– Applications– Biosafety Considerations
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Gene Therapy
• The transfer of genetic material to living organisms:– Correct a genetic defect– Exert a phenotypic change– Drug delivery– Vaccines
• Viral and non-viral vectors
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Biosafety – Basic Principles• Primary containment
– protection of lab personnel and the immediate laboratory environment
– laboratory practice and technique• knowledge of infectious agents and risks • strict adherence to standard microbiologic
techniques• develop protocols and procedures to be
followed by all lab personnel working with biologic hazards
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Standard Operational Practices
1. Access to the laboratory is limited or restricted at thediscretion of the laboratory director when experiments orwork with cultures and specimens are in progress.
2. Persons wash their hands after they handle viable materials,after removing gloves, and before leaving the laboratory.
3. Eating, drinking, smoking, handling contact lenses,applying cosmetics, and storing food for human use arenot permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or aface shield .
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Standard Operational Practices
4. Mouth pipetting is prohibited
5. Policies for the safe handling of sharps are instituted.
6. All procedures are performed carefully to minimize thecreation of splashes or aerosols.
7. Work surfaces are decontaminated at least once a dayand after any spill of viable material.
See Health Canada “Laboratory Biosafety Guidelines” for details
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Standard Microbiological Practices
8. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving.
9. A biohazard sign may be posted at the entrance to thelaboratory whenever infectious agents are present. Thesign may include the name of the agent(s) in use and thename and phone number of the investigator.
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Biosafety – Basic Principles• Primary containment
– safety equipment• bio-safety cabinets (class 2)• centrifuge covers• personal safety devices - gloves, goggles,
protective clothing, etc
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Biosafety – Basic Principles
• Secondary containment– protects personnel in the laboratory and
persons outside the laboratory– facility design
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Bio-Safety Levels• Bio-Safety Level 1
– suitable for organisms not known to cause disease in humans
– primary containment is standard operational practices
– personal protection - eyeware, gloves,labcoat, etc.
– other primary or secondary barriers are not required
– sink for handwashing
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Bio-Safety Levels
• Bio-Safety Level 2– suitable for organisms known to cause
moderate disease in humans – suitable for work involving human-derived
tissues and fluids– hazard relates to accidental percutaneous
or mucous membrane exposure or ingestion
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Bio-Safety Levels• Bio-Safety Level 2
– other primary or secondary barriers are not required if the potential for aerosolization is low
– bio-safety cabinets should be utilized if there is a risk of aerosols or large volumes/concentrations of infectious agent are used
– Restricted access to infectious agents (locked up)
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Bio-Safety Levels• Bio-Safety Level 2
– primary containment is standard operational practices
– personal protection - eyeware, gloves, labcoat, etc.
– waste decontamination– sink for handwashing
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Bio-Safety Levels• Bio-Safety Level 3
– required for agents where respiratory transmission is possible and cause serious or potentially lethal disease in humans
– hazard to lab personnel relate to autoinocluation and exposure to infectious aerosols
– greater emphasis on primary and secondary containment to protect lab personnel
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Bio-Safety Levels• Bio-Safety Level 3
– all work must be performed in biosafety cabinets or other enclosed equipment
– secondary containment includes restricted lab access and special ventilation to minimize release of infectious aerosols into the surrounding environment
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Bio-Safety Levels• Bio-Safety Level 4
– suitable for rare and exotic agents that are transmissible via aerosols, pose a high risk of life-threatening disease, and for which there is no known treatment or cure
– complete barrier between lab personnel and the infectious agent is required
– special secondary containment facilities
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Risk Groups• Group 1
– Agents that are not associated with disease in adult humans
• Group 2– Agents that are associated with human disease which is
rarely serious and for which there are preventative or therapeutic measures often available
• Group 3– Agents that are associated with serious or lethal disease in
humans for which preventive or therapeutic measures may be available
• Group 4– Agents that are associated with serious or lethal disease in
humans for which preventive or therapeutic measures are not usually available
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• Refer to Laboratory Biosafety Guidelines manual from Health Canada
http://www.hc-sc.gc.ca/pphb-dgspsp/publicat/lbg-ldmbl-6/index.html
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Biosafety – Basic Principles
• Risk assessment– biology of wild-type virus– prediction of biology of recombinant virus
vectors• Principle investigator in conjunction with
the institution biosafety office is responsible for assessing risk and setting biosafety level
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Biosafety - Risk Assessment
• Biology of wild-type virus– Most vectors based on ubiquitous viruses, most of
which cause mild or no significant disease in humans
– Route of transmission (potential for aerosol)– Agent stability (capsid vs envelope)– Animal viruses - some are controlled independently
by governmental regulations
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Biosafety - Risk Assessment
• Recombinant vectors– Human viruses adapted for gene therapy are
generally modified to reduce pathogenicity• understand the origin and mechanism of attenuation
– Some recombinant vectors are still replication competent
– Risk of generating replication-competent virus• in vitro or in vivo through homologous sequences• changes in selective pressure can produce viral mutants
– genetic variability
– Integrating vectors
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Biosafety - Risk Assessment• Recombinant vectors
– Genetic alteration of viruses • tropism or host range • route of transmission• stability • pathogenicity or virulence
– Effect of the transgene• toxins• oncogenes• suicide genes• cell cycle genes• genes that might increase the replicative capability of the
virus• immune-modulating genes
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Biosafety - Risk Assessment
• Recombinant vectors– Consideration of the nature of the work
• Volume or concentration of vector• Potential to produce aerosols
– Previous toxicity or animal studies– Availability of prophylaxis or therapy– Consideration of the likelihood of “rare” events– Animal transmission
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Biosafety
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Refer to “NIH Guidelines on Recombinant DNA”
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Viral Vectors
• Retrovirus• Adenovirus• Adeno-Associated Virus• Lentivirus• Other viruses
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Retrovirus Vectors• Moloney murine leukemia virus based
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Retrovirus Vectors
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Retrovirus Vectors
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Retrovirus Vectors
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Retrovirus Vectors• Advantages:
– Broad tropism that can be altered via the envelope protein
– Easy to generate (especially with VSV pseudotype).
– Packaging cell lines available– Accommodate up to 8 kb insert– Integrates into host genome– Non-immunogenic
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Retrovirus Vectors• Disadvantages:
– Unable to transduce non-dividing cells– Integration into host is random, and thus
the potential for insertional mutagenesis– LTR can affect activity of downstream
promoters– Transgene expression unstable
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Retrovirus Vectors• Biosafety
– murine based vectors are BSL-1• especially if produced in ecotropic cell lines
– risk of RCR depends on the packaging cell line used
• viral genes separated into different transcription units (env gene)
• amphotropic cell lines (lack endogenous retroviral sequences)
– effects of transgene
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Lentivirus Vectors• Subgroup of the retrovirus family
– includes HIV
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Lentivirus Vectors
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Lentivirus Vectors
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Lentivirus Vectors
• Advantages– Infects dividing and non-dividing cells
• due to nuclear localization signals on the pre-integration complex
– Risk of RC virus low• absence of most HIV genes including env
– Integrates– Silent LTR – Non-immunogenic
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Lentivirus Vectors
• Disadvantages– RC virus production is not completely
eliminated– Vectors can be rescued by wild type
infection. Creates the possibility of novel infectious agents
– Insertional mutagenesis
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Lentivirus Vectors - Biosafety
• Based on HIV (Group 3 agent)• Major risk is production of RCR• Effect of transgene• BSL-2+ or BSL-3 should be employed
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Adenovirus Type 5 Genome
0 mu 100 mu
E1AE1B
L1-4 L5
E3
E2B E2A E4
ITR ITR
regulatory proteins
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Adenovirus Vectors
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Adenovirus Vectors
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Adenovirus Vectors
• Disadvantages– Induce host inflammatory and immune
responses– transient gene expression– HD systems not completely devoid of
helper virus
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Adenovirus Vectors
• Advantages:– Can be grown in high titer– Very efficient gene transfer– Infects a variety of replicating and non-
replicating cells– Epichromosomal, thus insertional
mutagenesis does not occur– Helper-dependent systems can
accommodate over 30 kb of DNA
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Adenovirus Vectors
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Adenovirus Vectors
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Adenovirus Vectors - Biosafety
• Group - 2 agent• Persists on surfaces for long periods• Transmitted via numerous pathways• Risk of aerosol• Effect of transgene• BSL-2 recommended
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Adeno-Associated Virus
• Non-enveloped, single-stranded DNA parvovirus
• Needs helper virus for replication– adenovirus E2, E4
• Integrates into host genome to produce latent infection
• No known disease in humans
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Adeno-Associated Virus
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Adeno-Associated Virus
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Adeno-Associated Virus
• Advantages– Broad tropism, infects dividing and non-
dividing cells– low immunogenicity– stable, long term gene transfer– improved production methods has
eliminated helper virus contamination– Neglible risk of RCR during production
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Adeno-Associated Virus
• Disadvantages– conversion to double stranded DNA rate
limiting step– small, accomodates only 4.7 kb DNA– does not integrate in the absence of rep– can be rescued in vivo– difficult to work with in vitro
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Adeno-Associated Virus
• Biosafety– AAV is a group 1 agent– can be handled in BSL-1 conditions if not
produced with helper virus– risk of aerosol– prudent to use BSL-2 – effect of transgene
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Other Virus-Based Vectors
• Herpesvirus• Vaccinia Virus• Alphavirus
– Semliki Forest and Sindbis• Baculovirus• Influenza• Hybrids
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The End