Presentation on theme: "Medical School: Post Graduate Students"— Presentation transcript:
1Medical School: Post Graduate Students Biological and Genetic Modification Safety Course October 2013Medical School: Post Graduate StudentsProf Ron Croy, Biosafety Consulting – University Biological Safety Consultant
2Additional safety considerations for research with GM organisms Genetic Modification SafetyAdditional safety considerations forresearch with GM organisms
3Biosafety Course Covered - Biosafety Laws and regulationsRisk assessments - BioCOSHHLaboratory risksSchedule 5 – bioterrorism agentsWorking with microorganisms, animals, plantsHazard groups of biological agentsContainment and controlMicrobiological Safety Cabinets – class 1-3Emergency spillageSharpsStorageTransportContainment levels 1-3PPEDisinfection
4What is different in GM?The GM process produces a NEW organism with different, potentially unknown properties.Thus the route of exposure and virulence may have changed requiring altered containment.Therefore it is a legal requirement for all GM work to be i) notified to HSE and ii) can only be carried out in registered GM centresNewcastle University's GM Centre Reference number is GM540Note: HSE tend not to accept that the new (GM) organism may be less viable or less infective even if it is a deletion transgenic organism.
5GM Safety LawThe project manager/PI has ultimate legal responsibility for adequately risk assessing the GM project and is liable for all their projects.All of the regulations from Biological Agents cover GM work also (including COSHH)plus:Genetically Modified Organisms (Contained Use) Act and Regulations
6DEFINITIONS: Genetically Modified Organisms? 3 components are required to generate a GMM / GMO:Gene / cDNAHostVectorGeneticmaterialGM cellsGM organisms (GMO’s)GM microorganisms (GMM’s)
7DEFINITIONS: GM Exemptions – but still subject to BioCOSHH Mutagenesis (eg x-rays, chemicals)Synthetic nucleotidesSelf cloning – genes into same organism“Natural” transformationsHybridoma’s (mab’s)Humans and human embryos - IVF
9RISKS: GM Cell linesPlasmid/siRNA tranfection: transient or stable transfection – antibiotic resistance!Cancer cell lines – GM may effectCell phenotype or functions, Increased tumourigenicityImmune evasion ?Some cell lines already contain viral components: HPV-E6, SV40, adenovirusesKnow your cell line before transfecting in recombinant components!Effects of GM modification could be unknown
10Risks With Viral Vectors Vectors encoding for cDNA or shRNA of choice can be insertedThe major risks to be consideredPotential for generation of replication-competent lentivirus (RCL)User infection - potential for oncogenesis, (oncogenes, TSGs)“Some” liver tumours have been observed in neo-natal animals following Lentiviral administration(source SACGM)
11“Naked” DNA Safety Naked DNA used safely in gene therapy Sub cutaneous injection show plasmid DNA presence in organs, lymph, skin ~1month laterCaution when handling mutant TSG’s*/oncogenes, with upstream c.a. promoters *TSG=Tumor suppressor geneWorking with Viral DNA - replication?Clean lab area, pipettes, minimise aerosol, avoid sharps, wear PPE at all timesDNA can be destroyed by:UV - transluminator crosslinks DNAspecific chemicals (hypochlorite, Exitus-Plus)
12Plasmid + Expression Vectors Supercoiled DNA plasmid containing cDNA/shRNAUV light – viewing/cutting out DNA - sunburnDNA Gels – acrylamide, ethidium bromide, Gel Redpotential carcinogen/mutagen actionWhat protein is expressed and how much?Wear PPE – UV opaque face maskClean benches and pipettes regularlyDNA can be destroyed by UV light (UV crosslinker) and specific chemicalsPipettes, plates, racks, gloves
13Severe Unknown Risks e.g. GM Mousepox-IL4 Virus Scenarios where the results of well- intentioned scientific research can be used for both good and harmful purposes give rise to what is now widely known as the “dual-use dilemma”Hypervirulent strain of highly pathogenic GM virus – with no vaccineMousepox does not normally infect humansGM mousepox infection of workers or escape from lab?Could achieve same for smallpox in humans?Smallpox was responsible for million deaths in the 20th Century alone!
14Interleukin-4 (IL-4) mousepox virus Severe Unknown RisksInterleukin-4 (IL-4) mousepox virus“This was the first example of a virus overcoming vaccination, and this was very worrying”1) One of the most cited examples of dual-use research is that of Australian researchers who inadvertently developed a lethal mouse virus. In this now-famous study, the researchers used standard genetic engineering techniques to insert the gene for interleukin-4 (IL-4) into the mousepox virus. They hoped that the altered virus would induce infertility in mice—which are a major pest in Australia—and would thus serve as an infectious contraceptive for pest control. To their surprise, they discovered that the altered virus could kill both mice that were naturally resistant to, and mice that had been vaccinated against ordinary mousepox. When they published their findings, along with a description of the materials and methods, in the Journal of Virology in 2001 ( Jackson et al, 2001), critics complained that they had thereby alerted would-be terrorists to new ways of making biological weapons and had provided them with explicit instructions.Of particular concern was the possibility that the same techniques used to engineer the mousepox virus could be applied to create more virulent forms of poxviruses that afflict humans, including a vaccine-resistant strain of smallpox; one of the most devastating diseases in human history. Although it was eradicated in the 1980s, fears remain that former Soviet stockpiles—or genetically reconstituted forms of the virus—could be put to use by nefarious agents.“Boy this is scary—this is the kind of thing that science fiction is made of.” This research was the first example of a virus overcoming vaccination, and this was very worrying. And I suppose there was a little bit of excitement about it as well—it wasn't all doom and gloom. This is exciting stuff, no matter how evil or bad it may turn out to be. We went away wondering what to do about it. In those times there was no pathway in the structure of scientific institutions for resolving a case like this.EMBO reports (2010) 11, Published online: 11 December (2009)An interview with Ronald Jackson and Ian Ramshaw
15recombinant CD28-SuperMAB Severe Unknown Risksrecombinant CD28-SuperMAB2) Catastrophic failure of recombinant antibodyIn its first human clinical trials, recombinant CD28-SuperMAB an immunomodulatory drug being developed for treatment of leukemia and rheumatoid arthritis, caused catastrophic systemic organ failure in the subjects, despite being administered at a sub-clinical dose 500 times lower than the dose found safe in animals. Six volunteers were hospitalized, at least four of these suffering from multiple organ dysfunction. trial subjects was an unpredicted biological action of the drug in humansThe molecule was genetically engineered by transfer of the complementarity determining regions (CDRs) from heavy and light chain variable region sequences of a monoclonal mouse anti-human C28 antibody into human heavy and light chain variable frameworks. Humanised variable regions were subsequently recombined with a human gene coding for the IgG4 gamma chain and with a human gene coding for a human kappa chain, respectively. The recombinant genes were transfected into Chinese hamster ovary cells and the recombinant antibody harvested from culture supernatant.
16Examples of “dual-use dilemma” Synthetic virulent viruses and immune evasion2) Synthetic Virulent Polio Virus: In a second study, researchers at the State University of New York at Stony Brook artificially synthesized a “live” polio virus from scratch.6 Following the map of the polio virus RNA genome, which is published on the Internet, they stitched together corresponding strands of DNA, which they purchased via mail-order. The addition of protein resulted in the creation of a virus that paralysed and killed mice. Upon publication of results in Science in 2002, the researchers said they “made the virus to send a warning that terrorists might be able to make biological weapons without obtaining a natural virus”.7 Similar techniques might enable production of smallpox or Ebola. Cello J, Paul AV, Wimmer E. Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template. Science 2002; 297:and Pollack A. Scientists create a live polio virus. New York Times, 2 July 20023) Smallpox Immune Evasion: In a third study, published in the Proceedings of the National Academy of Sciences in 2002, researchers used published DNA sequences to engineer a protein – known as SPICE – produced by the smallpox virus.8 The study revealed the ways in which, and the extent to which, this protein defeats the human immune system. Though the findings may facilitate development of protective medicines, they may also reveal ways to increase the virulence of the closely-related vaccinia virus (which is used in the smallpox vaccine). Rosengard AM, Liu Y, Nie YZ, Jimenez R. Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement. Proc Natl Acad Sci USA 2002; 99:4) Spanish Flu Virus: A more recent study, published in Science in 2005, employed techniques of synthetic genomics (similar to those used in the polio study) to “reconstruct” the Spanish Flu virus, which killed between 20 and 100 million people in Tumpey TM, Basler CF, Aguilar PV, Zeng H, Solorzano A, Swayne DE, et al., et al. Characterization of the reconstructed 1918 Spanish Influenza pandemic virus. Science 2005; 310: 77-80
17Unknown Risks What this means is that GM often induces unexpected changes in transgenic cells and organismsit is important that as far as possible the changes are envisioned and controlled or in the worst possible scenario the GMO/GMM is controlledWhat this means is thatYou need to try to predict what properties will be conferred on each of your GMO’s and assess how this might affect the RiskWhere there is little evidence of the effects of a genetic modification may need to consider a ‘worst case scenario’ and implement appropriate controls
18Things to Consider when reading or preparing a GM Risk Assessment Infectious Vectors – viruses?Potential to transfer genetic material to other organisms – mobilisable vectors!Products of GM modificationToxinsAffects to cell signallingMutated genesOncogenes and tumour supressor genesPhenotype and stability of GMM/GMOHow will you contain and control these risks?
19GM Activity Class ?As well as Hazard group rating of host we now also need to consider GM ClassCL1 – HG1 – GM Class 1CL2 – HG2 – GM Class 2CL3 – HG3 – GM Class 3BUT an HG1 organism could become a GM class 2 depending on how it’s modified - what it’s expressingHazard group rating sets the “base level” then depending on the modification the organism may be elevated to a higher risk group based on the modificationOncogenes / Tumour Supressor genesPathogenic genesToxin genesIncreased survival, spread, resistance etc
20GM Activity Class ? Class Description Examples 1 2 3 Unlikely to cause human disease or environmental damageHG1 Biological agents (Minimum for host)- E. coli K12 with harmless genes- Replication defective virus vectors with harmless genes2May cause human disease but unlikely to cause significant environmental damageHG2 Biological agents (Minimum for host)- E. coli K12 with harmful genes- Replication defective vectors or competent HG2 viruses with harmless or harmful genes3May cause severe human disease or significant environmental damageHG3 Biological agents (Minimum for host)Competent HG3 viruses with harmless or harmful genes
21Additional Considerations for Genetically Modified Organisms (GMO)
22GM RegulationsHealth and Safety Executive is the competent authority regulating all aspects of GMGenetically Modified Organisms (Contained Use) Regulations (2000) modified 2002, 2005, 2010HSE plans to consolidate GMO(CU) legislation by October 2014Genetically Modified Organisms (Deliberate Release) Regulations Defra governs the release of any GMO’s - mainly crop plants.Defra also involved with import of GM animal pathogens and numerous other animal products; CITES
23Containing GMO Animals/Plants Possible increased risk to environment….Escape of animals – ease of recaptureSheep > Mice > Insects > Pollen!Ability to breed with native populationRate of breeding?Transfer of stable GM genes?However unlikely, Risk Assessmentneeds to consider animal escape andconsequences!Crop plants a major consideration
24Biological containment eg Escherichia coli K12 E. coli K-12 was originally isolated from a convalescent diphtheria patient in 1922 – lacks pathogenicityE. coli K-12 is defective in at least three cell wall characteristicsLipopolysaccharide coreGlycocalyxCapsular (K) antigensK12 strains (and others!) are therefore debilitated and do not colonise the human intestine and survive poorly in the environmentK12 is used routinely for plasmid transformation for “bulking up” of DNA and for expressing encoded proteins
25Biological containment 3rd Generation Lentiviral Vectors The packaging vector – minimal set of lentiviral genes required to generate the structural proteins and packagingThe pCMV-VSV-G envelope vector – provides the heterologous envelopeThe shRNA transfer vector – contains the sequence of interest and cis acting sequences (RNA production)Particles are replication-incompetentDeletion in the U3 portion of the 3’ LTR eliminates the promoter-enhancer regionSimilar systems for other viral vectorsLentivirus used for studying mammalian gene expression in cultured target cells
26Inactivation of GMM’s/GMO’s 100% kill of GMM / GMO is required before disposing of wasteAutoclaving is the most effective method for inactivating GM waste – essential requirementStandard 121°C or 134 °C for minutesValidation of effectiveness using annual thermocouple testing is required; cycle verification for GM at CL2 and CL3Do not autoclave GMM / GMO containing radioactive or hazardous chemical substances
27SummaryAll GM work must be in registered facilities and all GM projects properly risk assessed and CL2, CL3, CL4 projects notified to HSEAll GM work must be risk assessed taking into account the effects of the GM on the agents and organisms used; products expressedGMM’s / GMO’s must be containedGMM / GMO waste must be 100% inactivatedEnsure HG of host and final activity class are considered and respectedTake additional care with mammalian viral vectors with harmful insertsTransport of GMO / GMM must be done according to transport and associated regulations (secure packaging, certified carriers, authorisation of receiving lab/BSO)
31GM Risk Assessments example RA available You are here: Safety Office » Safety Topics » Biological Safety » Example Risk Assessmentsexample RA availableExample GM application for Transduction of mammalian cells using lentiviral vectors
32HSE Forms Notifications and Fees Genetically modified organismsCU1 forms and CU2 forms must be provided before work begins. Please also send your risk assessment and appropriate fee with forms CU1 and CU2. No fee or risk assessment is required with an accident notification, CU3.Downloadable formsCU1 - Notification of intention to use premises for genetic modificationCU2 - Notification of intention to conduct individual contained use activities involving genetic modificationCU3 - Notification of accidents involving Genetically Modified Organisms (Regulation 21)CU4 - Transfer of notified activity form (eg GM research transferred from another institute)Biological AgentsCBA1 - Notification of use and consignment of biological agents
34Emergency SituationsIn Section 5 of your RA you need to consider what emergencies could happen during the handling of the GM biological agent that might affect its containment and then detail the procedures to be used to deal with this situation
35What are realistic emergencies? Emergency SituationsWhat are realistic emergencies?Fire (real or a false alarm)SpillageInjury with a SHARP (HG2 / 3!)FloodPower / instrument failure (MSC, centrifuge)Bio-terrorism ? (mousepox lesson)
36Emergency SituationsBEFORE an emergency occurs…….consider what might happenadopt working procedures which minimise the risk of an accident happeningthe most likely emergency is spillage of a liquid culture or loose samples; leakage from a containerknow what to do in the event of a spillage of your materialsthe problem is greater with larger scale experiments - so be prepared accordingly!special situation in centrifuges due to bottle leakage
37Emergency Situations An example spillage SoP don’t panic - but act quicklyemergency? what emergency?leave the lab to allow aerosols to settle (~hours)put on protective clothingminimise aerosol risk ASAP - cover spillage with layers of paper towels (absorbent granules)extreme care if broken glass present but don’t remove piecescover whole area with disinfectant (conc ?)leave to disinfect - notify other lab usersseek advice - supervisor / BSOcleanup (24h) – autoclave wastedon’t panic!
38Emergency Situations Video: Emergency Procedures Dealing with Spillages
39“ If it’s not documented – it’s not done! ” Risk Assessmentsremember when it comes to the enforcing authorities, such as HSE, Defra/Fera ……….“ If it’s not documented – it’s not done! ”So if you have no evidence (usually a printed document) that your work has been properly and adequately risk assessed it is assumed (by HSE) that this has NOT been donethis applies to risk assessments, standard operating procedures, testing of facilities/equipment and training records/schedules for staff and students
40Training Records Important advice! Keep a well documented portfolio of all your safety training (date, title, course content, signatures)As well as verifying your competency to perform specific tasks it forms part of your CV for your future career / appointments
42Any questions about GM or emergencies Any questions about GM or emergencies? Please make sure that you sign the attendance sheets for this course and the GM course (this afternoon) to make sure that you are registered as having attended these courses and that you receive your GM certificatequestions please?Issue handouts