Transcript GeneticModificationSafety2013x.0001.8f61.bak

Occupational Health
& Safety Service
Biological and Genetic Modification Safety
Course October 2013
Medical School: Post Graduate Students
Prof Ron Croy, Biosafety Consulting – University Biological Safety Consultant
Occupational Health
& Safety Service
Genetic Modification Safety
Additional safety considerations for
research with GM organisms
Biosafety Course Covered •
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Occupational Health
& Safety Service
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
3
What is different in GM?
Occupational Health
& Safety Service
•
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.
4
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
Occupational Health
& Safety Service
DEFINITIONS: Genetically Modified
Organisms?
Occupational Health
& Safety Service
3 components are required to generate a GMM / GMO:
Host
Genetic
material
GM organisms (GMO’s)
Gene / cDNA
GM cells
GM microorganisms (GMM’s)
Vector
6
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
Occupational Health
& Safety Service
Occupational Health
& Safety Service
Genetically Modified Organisms
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
Occupational Health
& Safety Service
Risks With Viral Vectors
Occupational Health
& Safety Service
• 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)
10
“Naked” DNA Safety
Occupational Health
& Safety Service
• 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)
11
Plasmid + Expression Vectors
Occupational Health
& Safety Service
• 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
12
Severe Unknown Risks
e.g. GM Mousepox-IL4 Virus
Occupational Health
& Safety Service
• Scenarios where the results of wellintentioned 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
13
million deaths in the 20th Century alone!
Severe Unknown Risks
Interleukin-4 (IL-4) mousepox virus
Occupational Health
& Safety Service
“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
14
Severe Unknown Risks
recombinant CD28-SuperMAB
Occupational Health
& Safety Service
2) Catastrophic failure of recombinant antibody
In its first human clinical trials, recombinant CD28SuperMAB 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.
15
Examples of “dual-use dilemma”
Synthetic virulent viruses and immune evasion
Occupational Health
& Safety Service
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. Proc Natl Acad Sci 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. 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
16
Unknown Risks
Occupational Health
& Safety Service
• 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
17
Things to Consider when reading or
preparing a GM Risk Assessment
Occupational Health
& Safety Service
• 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?
18
GM Activity Class ?
Occupational Health
& Safety Service
• 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
19
GM Activity Class ?
Class
Occupational Health
& Safety Service
Description
Examples
1
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
20
Occupational Health
& Safety Service
Additional Considerations for Genetically
Modified Organisms (GMO)
GM Regulations
Occupational Health
& Safety Service
• 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
22
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
Occupational Health
& Safety Service
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
Occupational Health
& Safety Service
Biological containment
3rd Generation Lentiviral Vectors
Occupational Health
& Safety Service
• 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
25
Inactivation of GMM’s/GMO’s
Occupational Health
& Safety Service
• 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
Summary
Occupational Health
& Safety Service
• 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)
27
Occupational Health
& Safety Service
GM Risk Assessment
28
GM Risk Assessments
Occupational Health
& Safety Service
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)
29
GM Risk assessment forms
Occupational Health
& Safety Service
30
GM Risk Assessments
You are here: Safety Office » Safety Topics » Biological Safety » Example Risk Assessments
Occupational Health
& Safety Service
http://safety.ncl.ac.uk/gmriskassessment.aspx
example RA available
Example GM application for Transduction of mammalian cells using lentiviral vectors
31
HSE Forms Notifications and FeesOccupational Health
& Safety Service
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
32
Occupational Health
& Safety Service
WHAT IF?
Emergency Situations
Occupational Health
& Safety Service
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
34
Emergency Situations
Occupational Health
& Safety Service
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)
•
35
Emergency Situations
•
Occupational Health
& Safety Service
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
•
36
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
Occupational Health
& Safety Service
emergency? what emergency?
don’t panic!
37
Emergency Situations
Occupational Health
& Safety Service
Video: Emergency Procedures
Dealing with Spillages
38
Risk Assessments
•
Occupational Health
& Safety Service
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
39
Training Records
Occupational Health
& Safety Service
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
40
Emergency procedures
- expect the unexpected !
Occupational Health
& Safety Service
Occupational Health
& Safety Service
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