Biological and Genetic Modification Safety Course October 2013 Medical School: Post Graduate Students

1. Biological and Genetic ModificationSafety Course October 2013 Medical School: Post Graduate Students Prof Ron Croy, Biosafety Consulting – University Biological Safety Consultant

2. Genetic ModificationSafety Additional safety considerations for research with GM organisms

3. Biosafety Course Covered - • Biosafety Laws and regulations • Risk assessments - BioCOSHH • Laboratory risks • Schedule 5 – bioterrorism agents • Working with microorganisms, animals, plants • Hazard groups of biological agents • Containment and control • Microbiological Safety Cabinets – class 1-3 • Emergency spillage • Sharps • Storage • Transport • Containment levels 1-3 • PPE • Disinfection

4. What 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 centres • Newcastle University's GM Centre Reference number is GM540 • Note: 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.

5. GM Safety Law • The 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

6. DEFINITIONS: Genetically Modified Organisms? 3 components are required to generate a GMM / GMO: Host GM organisms (GMO’s) Genetic material Gene / cDNA GM cells GM microorganisms (GMM’s) Vector

7. DEFINITIONS: GM Exemptions – but still subject to BioCOSHH • Mutagenesis (eg x-rays, chemicals) • Synthetic nucleotides • Self cloning – genes into same organism • “Natural” transformations • Hybridoma’s (mab’s) • Humans and human embryos - IVF

8. Genetically Modified Organisms

9. RISKS: GM Cell lines • Plasmid/siRNAtranfection: transient or stable transfection – antibiotic resistance! • Cancer cell lines – GM may effect • Cell phenotype or functions, Increased tumourigenicity • Immune evasion ? • Some cell lines already contain viral components: HPV-E6, SV40, adenoviruses • Know your cell line before transfecting in recombinant components! • Effects of GM modification could be unknown

10. Risks With Viral Vectors • Vectors encoding for cDNA or shRNA of choice can be inserted • The major risks to be considered • Potential 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 later • Caution when handling mutant TSG’s*/oncogenes, with upstream c.a. promoters *TSG=Tumorsuppressor gene • Working with Viral DNA - replication? • Clean lab area, pipettes, minimise aerosol, avoid sharps, wear PPE at all times • DNA can be destroyed by: • UV - transluminator crosslinks DNA • specific chemicals (hypochlorite, Exitus-Plus)

12. Plasmid + Expression Vectors • Supercoiled DNA plasmid containing cDNA/shRNA • UV light – viewing/cutting out DNA - sunburn • DNA Gels – acrylamide, ethidium bromide, Gel Red • potential carcinogen/mutagen action • What protein is expressed and how much? • Wear PPE – UV opaque face mask • Clean benches and pipettes regularly • DNA can be destroyed by UV light (UV crosslinker) and specific chemicals • Pipettes, plates, racks, gloves

13. Severe 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 vaccine • Mousepox does not normally infect humans • GM mousepox infection of workers or escape from lab? • Could achieve same for smallpox in humans? • Smallpox was responsible for 3-500 million deaths in the 20th Century alone!

14. Severe Unknown Risks Interleukin-4 (IL-4) mousepoxvirus “This was the first example of a virus overcoming vaccination, and this was very worrying” http://www.nature.com/embor/journal/v11/n1/full/embor2009270.html 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, 18 - 24 Published online: 11 December (2009) An interview with Ronald Jackson and Ian Ramshaw

15. Severe Unknown Risks recombinant CD28-SuperMAB 2) Catastrophic failure of recombinant antibody In 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 humans The 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.

16. Examples of “dual-use dilemma” Synthetic virulent viruses and immune evasion 2) 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: 1016-8 and Pollack A. Scientists create a live polio virus. New York Times, 2 July 2002 3) 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. ProcNatlAcadSci USA 2002; 99: 8808-13 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 1918-19.TumpeyTM, 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

17. Unknown Risks • GM often induces unexpected changes in transgenic cells and organisms • it is important that as far as possible the changes are envisioned and controlled or in the worst possible scenario the GMO/GMM is controlled What this means is that You need to try to predict what properties will be conferred on each of your GMO’s and assess how this might affect the Risk Where there is little evidence of the effects of a genetic modification may need to consider a ‘worst case scenario’ and implement appropriate controls

18. Things 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 modification • Toxins • Affects to cell signalling • Mutated genes • Oncogenes and tumour supressor genes • Phenotype and stability of GMM/GMO • How will you contain and control these risks?

19. GM Activity Class ? • As well as Hazard group rating of host we now also need to consider GM Class • CL1 – HG1 – GM Class 1 • CL2 – HG2 – GM Class 2 • CL3 – HG3 – GM Class 3 • BUT an HG1 organism could become a GM class 2 depending on how it’s modified - what it’s expressing • Hazard group rating sets the “base level” then depending on the modification the organism may be elevated to a higher risk group based on the modification • Oncogenes / Tumour Supressor genes • Pathogenic genes • Toxin genes • Increased survival, spread, resistance etc

20. GM Activity Class ?

21. Additional Considerations for Genetically Modified Organisms (GMO)

22. GM Regulations • Health and Safety Executive is the competent authority regulating all aspects of GM • Genetically Modified Organisms (Contained Use) Regulations (2000) modified 2002, 2005, 2010 • HSE plans to consolidate GMO(CU) legislation by October 2014 • Genetically Modified Organisms (Deliberate Release) Regulations 2002. 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

23. Containing GMO Animals/Plants • Possible increased risk to environment…. • Escape of animals – ease of recapture • Sheep > Mice > Insects > Pollen! • Ability to breed with native population • Rate of breeding? • Transfer of stable GM genes? • However unlikely, Risk Assessment needs to consider animal escape and consequences! • Crop plants a major consideration

24. Biological containment egEscherichia coli K12 • E.coli K-12 was originally isolated from a convalescent diphtheria patient in 1922 – lacks pathogenicity • E. coli K-12 is defective in at least three cell wall characteristics • Lipopolysaccharide core • Glycocalyx • Capsular (K) antigens • K12 strains (and others!) are therefore debilitated and do not colonise the human intestine and survive poorly in the environment • K12 is used routinely for plasmid transformation for “bulking up” of DNA and for expressing encoded proteins

25. Biological containment 3rd Generation Lentiviral Vectors • The packaging vector – minimal set of lentiviral genes required to generate the structural proteins and packaging • The pCMV-VSV-G envelope vector – provides the heterologous envelope • The shRNA transfer vector – contains the sequence of interest and cis acting sequences (RNA production) • Particles are replication-incompetent • Deletion in the U3 portion of the 3’ LTR eliminates the promoter-enhancer region • Similar systems for other viral vectors • Lentivirus used for studying mammalian gene expression in cultured target cells

26. Inactivation of GMM’s/GMO’s • 100% kill of GMM / GMO is required before disposing of waste • Autoclaving is the most effective method for inactivating GM waste – essential requirement • Standard 121°C or 134 °C for 15-30 minutes • Validation of effectiveness using annual thermocouple testing is required; cycle verification for GM at CL2 and CL3 • Do not autoclave GMM / GMO containing radioactive or hazardous chemical substances

27. Summary • All GM work must be in registered facilities and all GM projects properly risk assessed and CL2, CL3, CL4 projects notified to HSE • All GM work must be risk assessed taking into account the effects of the GM on the agents and organisms used; products expressed • GMM’s / GMO’s must be contained • GMM / GMO waste must be 100% inactivated • Ensure HG of host and final activity class are considered and respected • Take additional care with mammalian viral vectors with harmful inserts • Transport of GMO / GMM must be done according to transport and associated regulations(secure packaging, certified carriers, authorisation of receiving lab/BSO)

28. GM Risk Assessment

29. GM Risk Assessments http://safety.ncl.ac.uk/gmriskassessment.aspx Comprehensive Guidelines also available http://safety.ncl.ac.uk/uploads/GM_Risk_Assessment_Guidelines.pdf GM Project Application procedures (http://safety.ncl.ac.uk/activityclass.aspx)

30. GM Risk assessment forms

31. GM Risk Assessments You are here: Safety Office » Safety Topics » Biological Safety » Example Risk Assessments http://safety.ncl.ac.uk/gmriskassessment.aspx example RA available Example GM application for Transduction of mammalian cells using lentiviral vectors

32. HSE Forms Notifications and Fees • Genetically modified organisms • CU1 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 forms • CU1 - Notification of intention to use premises for genetic modification • CU2 - Notification of intention to conduct individual contained use activities involving genetic modification • CU3 - Notification of accidents involving Genetically Modified Organisms (Regulation 21) • CU4 - Transfer of notified activity form (eg GM research transferred from another institute) • Biological Agents • CBA1 - Notification of use and consignment of biological agents

33. WHAT IF?

34. Emergency Situations In 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

35. Emergency Situations What are realistic emergencies? • Fire (real or a false alarm) • Spillage • Injury with a SHARP (HG2 / 3!) • Flood • Power / instrument failure (MSC, centrifuge) • Bio-terrorism ? (mousepox lesson)

36. Emergency Situations • BEFORE an emergency occurs…….consider what might happen • adopt working procedures which minimise the risk of an accident happening • the most likely emergency is spillage of a liquid culture or loose samples; leakage from a container • know what to do in the event of a spillage of your materials • the problem is greater with larger scale experiments - so be prepared accordingly! • special situation in centrifuges due to bottle leakage

37. Emergency Situations An example spillage SoP • don’t panic - but act quickly • leave the lab to allow aerosols to settle (~hours) • put on protective clothing • minimise aerosol risk ASAP - cover spillage with layers of paper towels (absorbent granules) • extreme care if broken glass present but don’t remove pieces • cover whole area with disinfectant (conc ?) • leave to disinfect - notify other lab users • seek advice - supervisor / BSO • cleanup (24h) – autoclave waste emergency? what emergency? don’t panic!

38. Emergency Situations Video: Emergency Procedures Dealing with Spillages

39. Risk Assessments • remember 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 done • this applies to risk assessments, standard operating procedures,testing of facilities/equipment and training records/schedules for staff and students

40. Training 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

41. Emergency procedures - expect the unexpected !

42. 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 certificate