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

2. Additional safety considerations for research with GM organisms
Genetic Modification Safety
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:
Gene / cDNA
Host
Vector
Genetic
material
GM cells
GM organisms (GMO’s)
GM microorganisms (GMM’s)

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/siRNA tranfection: 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=Tumor suppressor 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 million deaths in the 20th Century alone!

14. Interleukin-4 (IL-4) mousepox virus
Severe Unknown Risks
Interleukin-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

15. recombinant CD28-SuperMAB
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:
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. 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

17. Unknown Risks What this means is that
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 ? Class Description Examples 1 2 3
Unlikely to cause human disease or environmental damage
HG1 Biological agents (Minimum for host)
- E. coli K12 with harmless genes
- Replication defective virus vectors with harmless genes 2
May cause human disease but unlikely to cause significant environmental damage
HG2 Biological agents (Minimum for host)
- E. coli K12 with harmful genes
- Replication defective vectors or competent HG2 viruses with harmless or harmful genes 3
May cause severe human disease or significant environmental damage
HG3 Biological agents (Minimum for host)
Competent HG3 viruses with harmless or harmful genes

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 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 eg Escherichia 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 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
GM Project Application procedures (

30. GM Risk assessment forms

31. GM Risk Assessments example RA available
You are here: Safety Office » Safety Topics » Biological Safety » Example Risk Assessments
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. What are realistic emergencies?
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
emergency? what emergency?
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
don’t panic!

38. Emergency Situations Video: Emergency Procedures
Dealing with Spillages

39. “ If it’s not documented – it’s not done! ”
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
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
questions please?
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