Download IN CONFIDENCE Ref. No.: H2016-003 Risk Assessment for Genetic

Ref. No.: H2016-003
IN CONFIDENCE
Risk Assessment for Genetic Modification Project to be considered by the
Biological Safety Committee of the Hutchison-MRC Research Centre, Cambridge.
1.
Name of applicant:
Prof. Rebecca Fitzgerald
Division or Unit:
MRC Cancer Unit
Group Leader responsible (if not
applicant):
Prof. Rebecca Fitzgerald
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2.
Functional Analysis of Oesophageal Adenocarcinoma
Genomic variants
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Title of project:
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3.
Overview, including details of
insert(s):
This project aims to better understand the functional
effects of genomic aberrations in oesophageal
adenocarcinoma by permanently or transiently altering
the genomic and transcriptomic state of in vitro models
including oesophageal cell lines and organoids derived
from primary tumour tissue. This requires the use of
lentiviral RNAi and cDNA over-expression vectors as
well as expression of targeted nucleases that will
permanently alter cancer cell genomes.
Inserts will encode reporters (e.g. EGFP, DsRed,
luciferase); transcriptional regulators (e.g. Tet-On);
normal and modified transcription factors or epigenetic
regulators and other cancer-associated transcripts,
including known oncogenes; shRNAi constructs, or
targeted nucleases and their associated RNA molecules
(CRISPR/Cas9), including constructs that functionally
ablate known tumour suppressor genes or activate known
oncogenes.
Genes will be expressed under the control of viral and
mammalian promoters, both constitutive (e.g. CMV or
E2F) and/or inducible (e.g. regulated by Tet-On transactivator protein).
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4.
Plasmid production
Host/ vector system(s):
K12-derived E. coli strains (e.g. DH5alpha) of bacteria are
used for transformations and amplification of plasmids. No
constructs will contain mammalian extra-cellular protein
encoding genes driven by bacterial promoters as these
could potentially enhance E.coli pathogenicity.
lentiviral production
Stable gene transduction will be achieved by HIV1-derived
2nd generation lentiviral vectors (e.g. psPAX2, psMD2.G).
Later generation systems with further safety mechanisms
and higher efficacy may be adopted as they become
established. 293T human embryonic kidney cells and
derivatives will be used as packaging/producer cells. Viral
particles will be filtered to remove any possible
contamination by 293T cells, which could allow continued
viral production.
Targeted nuclease-mediated genome engineering
The CRISPR/Cas9 system will be used for targeted genome
editing in somatic cells both by transient transfection and
stable integration. For transient transfection, plasmids such
as but not limited to pX330, pX260, and pX335 will be used.
Cas9 nuclease and guide RNAs will also be stably
integrated in the target cells using lentiviral gene delivery
(e.g. but not limited to using pCW57.1 and pLX304-based
vectors). Genetically manipulated cell lines will be
propagated as regular cancer cell lines in containment level
1 once confirmed lentiviral negative using reverse
transcriptase activity assays. Genetically manipulated
organoid cultures will be maintained in containment level
2.
Target cells
1. Established human tumour cell lines and 2. Oesophageal
organoid cultures derived directly from patient samples.
These can be passaged >30 times in many cases, without
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transformation. The patient samples are not in general
screened for blood-borne viruses, and we are not permitted
to screen them, for ethical/consent reasons. There is a
separate risk assessment for these cultures.
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5.
E. coli
Hazard identification in respect of
human health and environmental
safety.
(Consider host, vector, insert and
final GMM.)
Estimation of the severity or
consequence of the harmful effect
were it to occur.
Laboratory strains of E. coli K12 are recognised as
non-colonising and may be considered to be ACDP hazard
group 1.
Viral vectors
We will use 2nd generation lentiviral vectors containing
several safety mechanisms:
• Reduced number of genes from HIV-1 in lenti-viral
vectors (i.e. gag, pol, tat and rev are absent).
• Separation of genes encoding the structural and other
components required for packaging the virus to
substantially reduce the risk of undesirable recombination
events that could lead to the generation of a replicationcompetent virus (Dull et al., J Virology 72 8463-8471 1998).
• None of the HIV-1 structural genes are present in the
packaged viral genome and are therefore never expressed
in the transduced target cell.
• The lentiviral particles produced are replicationincompetent and only carry the gene of interest. No other
viral species are produced.
• In some cases expression of the gene insert of interest
will be dependent on a tet-responsive promoter, and
regulated by a co-transduced lentivirus doxycyclinresponsive Tet regulator, giving an added level of safety
for insert expression.
These vectors contain the WPRE (Woodchuck hepatitis B
virus post-transcriptional regulatory element), which may
have oncogenic properties, so, whether or not the inserts are
oncogenic, all these lentiviruses are potentially oncogenic.
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Viral vectors in unscreened human oesophageal organoid
cultures
In principle, there is a small risk that organoid cultures
might carry HIV, which, by acting as a helper virus, would
make the lentivirus inserts replication-competent.
However, to our knowledge HIV does not replicate in cells
such as oesophageal epithelium, so, provided the organoids
have been passaged by disaggregation so that they do not
also carry macrophages or lymphocytes, the risk of HIV is
low.
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After infected organoids have been passaged further, the
risk of active virus will be very low.
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6.
Provisional Class/containment
level (in particular taking account
of the biological agents hazard
group and other classification
scheme for pathogens).
E. coli
This step will often involve
considering the containment level
necessary to control the risk of the
host and making a judgement
about whether the modification
will result in a GMM which is more
hazardous, less hazardous or about
the same.
Sometimes it might help to
compare the GMM with the
relative hazard presented by other
organisms.
Viral vectors and infection
The E. coli and tissue culture cells are disabled and belong
to Hazard Group 1.
The risk of Lentiviral infection of a worker during the
packaging/infection step is small, but still not negligible. As
the vector contains the complete WPRE element as well as
tumorigenic inserts this part of the work is Class 2.
However any theoretical hazard is only during initial
contact since such viruses could not propagate.
Given these non-negligible hazards, particular attention
will be paid to staff awareness when working with genes
with a potential growth-promoting function (virus
production, labelling, storage). No sharps will be used
when working with lentiviruses. Also, appropriate personal
protective equipment will be used (gloves, lab coats).
DNA grown up from clones should also be handled with
care as it is potentially oncogenic because of the WRPE
element and oncogenic inserts; i.e. gloves should be worn,
sharps avoided and all wastes be rendered harmless before
disposal.
Virus storage
For some experiments, virus will be stored outside the Class
2 lab. For this, it will be in a designated section of a clearly
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Ref. No.: H2016-003
designated freezer, in double containers, according to the
Hutchison-MRC Research Centre Class 2 Code of Practice.
Moving Infected Cell Lines to CL1
After infection, selection and passaging at least once, for cell
line cultures only, NOT organoids, cells that are virus
negative may be transferred to CL1. To show that cells are
negative for replicating lentivirus, we will use well
characterised methods such as the Molecular Probes'
EnzChek® Reverse Transcriptase Assay.
All organoids (prior to and after infection) will remain in
CL2.Cell storage
To cryopreserve cells in CL2, storage will be at -80 outside
the CL2 lab as specified above for virus storage. When it is
really necessary for long-term preservation to store cells in
a vapour phase liquid nitrogen refrigerator this will be in a
clearly designated location, in a double container, according
to an agreed Hutchison-MRC Research Centre Class 2 Code
of Practice and with explicit permission from the Hutchison
Lab Manager responsible.
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7.
The prokaryotic cells are highly disabled and the plasmid
vectors are non-mobilisable, therefore neither has any
possibility of further spread.
Environment and activity
considerations.
This includes an estimation of the
likelihood that hazards will be
realised. Given that the provisional
Class (and hence containment
level) has already been decided it
helps to bear this in mind when
deciding how likely a harmful
event is.
Use these considerations of
likelihood to revise the provisional
containment so that all risks are
controlled to low or effectively
zero.
Double check that all hazards are
properly controlled by the
proposed containment.
The principle hazard in the human cell work is the low risk
of blood-borne virus and particularly HIV. Neither are
expected to survive passage of cultures and reverse
transcriptase assay should confirm the absence of HIV in
cultures maintained after infection.
We are not going to produce aerosols, which contain viral
particles. Also, the amounts of virus produced are modest.
The lentiviruses have unstable infectivity and infection is
only obtained by co-cultivation of the packaging cell
supernatant with the recipient cells. There will be no
animals present in the tissue culture facilities.
The risk to the environment is therefore effectively zero.
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8.
Class 1, with no additional precautions required, for the
work with E. coli.
Assign final activity Class.
This is done by comparing the
containment and control measures
identified as necessary to control
the risk with the table of
containment in the Regulations.
Class 2, with no additional precautions required for the
packaging/infection steps with the retro and lentivirus.
Potentially growth-promoting DNA will be handled
appropriately (see above).
Class 1, with no additional precautions required, for the
work with stably infected or genetically engineered cells.
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Final Class/containment level:
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2 and 1
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9.
ALL persons involved in GM work
(give experience or name of trainer
if not already experienced):
Xiaodun Li
Post-Doctoral Researcher. More
than 8 years of experience in
molecular biology.
Gianmarco Contino
Post-Doctoral Researcher. More than
8 years of experience in molecular
biology.
Alex Frankell
of those above)
PhD student (under the direction
Nuria Galeano-Dalmau Research Assistant (under the
direction of those above)
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10. Notes (including any abbreviations No animals are involved in this work.
used):
If work involves transgenic animals
and is not exempt, give Licence
number:
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11.
Ref. No.: H2016-003
Signed:
Date:
(Group Leader)
__________________________________________________________
12.
Comments of Biological Safety Committee:
This is principally a project to use lentivirus to manipulate cancer-relevant
genes in human cells. The non-standard element of the proposal is that
organoid cultures of human epithelium from unscreened patients are among
the target cells to be used. Screening is not permissible for ethical reasons. Since
such cultures might rarely be contaminated with HIV, the general principle that
modern lentivirus constructs cannot propagate could break down, because the
HIV could package the lentivirus vector. Self-inactivating constructs would be
preferable in this case, since they are less likely to transcribe packageable
RNAs. An SOP for cryopreserving CL2 cells needs to be developed.
Clearance given to start work/Notified to HSE
13.
Signed:
Date:
(Chairman of BSC)
__________________________________________________________
14.
Agreement of person responsible for supervision and safety:
Signed:
Date:
(Competent person under MHSW Regs 1999/Biological Safety Officer)