Genetic Modification Proposal Form :
Risk Assessment template for Genetic Modification involving Lentiviral constructs – v.MESB1.4
To be allocated by GM safety officer:-
Final Risk Class : ___________________
HSE Ref (where appropriate): _____________________
Title of Project:
Insert overall title here…
Sub-title
Leave blank unless the project is an amendment or extension to an
existing project…
Proposal Ref No:
GM37/______________
Please refer to the specific and general guidance notes below before completing this form. Please remove the notes from
the submitted document
1. Name of Principal Investigator (PI):
2. Department, building and room location of PI:
3. Telephone number and email address of PI:
If this proposal is an for an umbrella project, please give names, positions and brief
contact details for the other academic staff whose work is included:
4. Summary of risks considered (specify only those risks deriving from the existence and use of the GM
construct or infected/integrated organism, not general health, safety and COSHH issues):
Place a ‘’ in the
relevant box(es)
Risk being
considered for which
organism?
Likelihood of event
Nature of GM risk
Negligible
v. low
but
finite
low but
signif.
insertional mutation leading
to oncogenesis or other
health effect
infection by replicationThe experimenter
competent retrovirus
effects of infection and/or
The experimenter
gene / fRNA* expression at
infection site
insertional mutation leading
Other humans locally
to oncogenesis or other
e.g. lab workers
health effect
Other humans locally
infection by replicatione.g. lab workers
competent retrovirus
effects of infection and/or
Other humans locally
gene / fRNA* expression at
e.g. lab workers
infection site
*fRNA – functional RNA e.g. shRNA used for knockdown of endogenous gene product
The experimenter
1
Consequences
of event
quite
likely
None (of
significance)/
mild/medium/
severe
Risk Assessment for Genetic Modification involving Lentiviral constructs (cont.)
4. Summary of risks considered (cont.)
Place ‘’ in the
relevant box(es)
Risk being
considered for
which organism?
Likelihood of event
Nature of GM risk
Host species in
lab/ containment
facility
effects on behaviour (e.g.
aggression, biting risk) or
pathogenicity of host
Host species,
external
breeding of host with wild
counterpart leading to persistence
of integrated GM construct and
effects on wild popn.
Other species,
external
escape of GMO with ecol. impact
on other spp.
Other species,
external
horizontal transmission of GM
construct/gene to other spp. with
ecol. impact.
Other species,
external
horizontal transmission to human
pathogen increasing pathogenicity
Negligible
v. low
but
finite
low but
signif.
Consequences
of event
quite
likely
None (of
significance)/
mild/medium/
severe
Other? – specify…
5. Summary of the proposed work:
Place a ‘’ in the relevant box(es)
Nature/subtitle of work
Will it involve any of the following?
in vitro / cell
in vivo /
manufacture of
culture
animal
viral particles
infection
infection
Manufacture of lentiviral particles containing construct
encoding biological/marker fusion protein
Delivery via lentiviral construct of biological/marker fusion
protein to mouse and rat primary cell cultures, cell lines and
organotypic culture
Delivery via lentiviral construct of biological/marker fusion
protein to mouse and rat tissues in vivo
6. Short summary description of aims of the overall project and intention behind the GM
work proposed:
e.g. The protein product of gene Y is thought to participate in process Z in mammals and we are examining
its function in relation to its presumed downstream partner, protein Q. We propose to knock down the
expression of gene Y in MMM cells using lentiviral shRNA constructs incorporating a marker gene (eGFP) to
highlight infected cells.
Where the planned activities involve elements related to gene therapy, please give these extra visibility here.
e.g. We plan to use lentiviral constructs to restore activity of gene Y in haematopoietic progenitor cells from
disease YY patients deficient for gene Y and reinject the modified cells into patients etc. etc.
2
Risk Assessment for Genetic Modification involving Lentiviral constructs (cont.)
7. Primary location(s) of proposed laboratory work (building and room location):
7a. Nature of work in primary location:
8. Secondary locations where GMO may be handled (e.g. culture suites, Central Research
Facility etc):
8a. Nature of work in secondary location(s):
If the answer to 7 or 8 above is a Biological Services facility, please  here [ ]
Please read the Generic Animal Containment Appendix (dated 19/05/10) downloaded from
http://www.gla.ac.uk/colleges/mvls/campus/healthsafety/gmcommittees/gm-37/
By ticking this form, you confirm that the guidelines are sufficient to safely handle and house the proposed
genetically modified strain.
9. Brief description of the biology of the system under investigation:
e.g. Gene Y is a GeneYfunctionalpathwaymember that is expressed [state where and when] throughout both
adult and foetal human tissues with highest levels found in the heart and brain. GeneY [state functional
attributes] binds a range of growth factors including GrowthFactorsX, Y &Z. Upon ligand binding GeneY
forms a homodimer or a heterodimer (with a GeneZ receptor), the latter of which potentiates GeneY’s
signalling capability. Following dimerisation, GeneY undergoes autophosphorylation, activating the receptor
and initiating several intracellular signalling pathways such as the GenesM,N&P pathway.
10. Summary description of the constructs to be used/made:
a) Overall system name/description
(e.g. 4th generation lentivirus construct based on XYZ vector, containing shRNA vs Gene Y, and marker gene
eGFP).
b) Summary of Lentiviral Coat Protein and Tropism to be employed
(e.g. VSV-G; can infect mammals and insects). The important element to focus on here is whether
the construct will be packaged into particles with enhanced dispersal/infection capability of
relevance to human or ecosystem health, not just the breadth of the tropism as such.
In parts c) – h) below – if you are bringing ready-made infectious lentiviral particles into the
University, give details just for the construct(s) in those particles and the packaging system used to
create the particles; if you are manufacturing the particles, give, in addition to this, details of the
shuttle and other vectors used to create the construct itself. See notes for further info.
c) Vector backbone and origins of replication etc.
- Lentiviral contruct(s):
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
d) Resistance genes
3
Risk Assessment for Genetic Modification involving Lentiviral constructs (cont.)
- Lentiviral contruct(s):
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
e) Marker genes and tags
- Lentiviral contruct(s):
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
f) Biological insert (i.e. the ‘functional’ part, designed to have an effect on the biology of the host
cells / organism)
- Lentiviral contruct(s):
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
g) Expression modulators (e.g. promoter, WPRE, stating the version and its functional
constituent parts where applicable)
- Lentiviral contruct(s):
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
h) Other constituents (only where significant to function and not covered in c)-g))
- Lentiviral contruct(s):
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
i) Summary of the differences between the experimental/test and control constructs
Please include a diagram of the vector/construct showing the features referred to in c) – h)
wherever possible.
11. Justification for the use of each component of the construct:
use as much space as you need here
12. If the lentivirus particles will be manufactured on campus (in any part of the University
or associated hospitals), summarise the facilities available and the manufacture system,
with consideration of GM safety issues:
13. Summary of procedures to be employed:
use as much space as you need here
13a Laboratory Staff working on this project
Provide a list of the personnel initially intended to be working with the GM materials in this
proposal. If the list changes, an addendum can be provided to the Committee Chair on an annual
basis.
14. Detailed risk assessment (this should illustrate why each box in the summary table on
page 1 has been ticked, and should include consideration of appropriate containment):
Special note for items 13 and 14:
i) needles – if hypodermic syringe needles or glass capillary needles containing infectious GM organism or
virus or construct are used at any point in the procedures, the project will inevitably be considered Class 2 or
above.
ii) safety cabinets/hoods – where a Class II safety cabinet is used to protect the worker from significant harm
involved in the use of the GM organism, virus or construct, the project will inevitably be considered Class 2 or
above.
4
Risk Assessment for Genetic Modification involving Lentiviral constructs (cont.)
15. How mature is the field? Cite evidence supporting the use of the GM technology
proposed for the purpose proposed:
use as much space as you need here
16. What classification do you wish this project and its activities to be considered under?:
The answer you give here should be commensurate with a) the requirements laid down in the HSE
Compendium of Guidance and its addenda, b) the risks explained in Section 14 above, and c) the
proposed containment procedures.
Place a ‘’ against the relevant classification:
Class 1
Class 2
Class 3
Note – All except Class 1 will require notification to the HSE and payment of a fee.
Signature of Proposer:
Date of Proposal:
Signature of GM Biological Safety Officer:
Date approved and confirmed classification:
Final classification:
Date to HSE (if applicable):
5
Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
If you have any questions about filling in or submitting this form, please contact:
Jane Paton,
Clerk to GM37 Committee,
Room 140, West Medical Building, University Avenue, Glasgow G12 8QQ
Tel: 0141 330 6151
Jane.Paton@glasgow.ac.uk
LEGAL NOTES:
In completing this submission, researchers are confirming they have consulted and are following the
SACGM Compendium of Guidance (CoG) [Revised 2007], issued by the Scientific Advisory Committee for
Genetic Modification Contained Use (//www.hse.gov.uk/) and the guidance to the regulations "A guide to
the Genetically Modified Organisms (Contained-Use) Regulations - 2000" (HSE Books L29) (guide-L29).
Copies of Notes and Regulations are available from Jane Paton
REMIT NOTES:
This form should be used whenever work using whole, or components of, lentiviruses is proposed
that does not come within the remit of the general microbiological cloning (in e.g. E. coli) template
form. Manipulation of single plasmids of 4th generation lentivirus vectors with no more than one
component of a lentivirus (e.g. GAG, or POL, or ENV etc.), might come under the E. coli template,
but handling of more than one component plasmid in a single project/lab or of complete infectious
lentivirus particles does not.
Guide to filling in each section:
1-3. One member of academic staff should be nominated here as PI for the GM proposal. Where
two or more project leaders are involved collaboratively, or separately, in the work being proposed
in a single GM submission, their names, affiliations and contact details should be given in an
addendum to the submitted form.
Please note – HSE practices have changed recently. It is still possible to notify to the HSE a
‘Connected Programme of Work’ that covers several types of activity at more than one Class level.
In addition, where several groups want to do a similar activity/programme of activities, it is possible
to submit a single ‘umbrella’ notification to HSE. On this document one person needs to be
specified as P.I., but this person will not be held uniquely responsible for everything that happens
under that notified/approved set of activities – each sub-P.I. will still be responsible for their part of
the notified work exactly as they would be in all safety procedures within the University.
4. Please summarise the identified risks associated with the GM aspect of the work proposed.
Consideration should be given to the experimenter, other third-party humans (e.g. lab co-workers,
other employees, people outside the University), other individuals of the GM host species in the lab
or outside, organisms of other species in the University or wild in the environment. Specify, using
the summary table provided, the nature of the risk, the likelihood of the risk event(s) happening and
the severity of the consequences of such occurrences. This section should tie in with the risk
assessment details given in Section 14. The template form specifies a number of hazards that
typically need to be considered in work using lentiviruses – alter this to suit if other risks need to be
6
Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
specified or risks can be removed due to the nature of the work. In terms of risk likelihood,
‘negligible’ indicates effectively no risk (but we can’t specify a risk of exactly 0), v. low but finite
indicates that some researchers or government officials have considered there to be a risk associated
with that element of the GM work, and that risk has not been ruled out by further experimental
studies, but it is not thought to be likely to occur other than in exceptional circumstances. In terms
of Consequences and severity, ‘None (of significance)’ indicates that any effects would be
localised, very mild, short term, highly treatable and extremely unlikely to cause future health or
other problems in the organism being considered.
It should be noted that, under ‘Host species in lab/containment facility’, mice kept in Biological
Services facilities would never be mixed with non-infected mice after exposure to the infective
material rendering them genetically modified (i.e. after lentivirus infection). Thus, only risks
associated with unusual circumstances, such as escape of GM animals within the unit, need be
considered here.
5. Each aspect or element of the GM work proposed should be summarised here (typical examples
are given – again, add or remove rows to suit the proposed work). If viral particles are to be
manufactured in the University of Glasgow or its associated hospitals, please state. Other work can
be divided into ex vivo (in vitro, cell culture, tissue slices or organotypic culture) and in vivo
(injection or other transfer method to live animals/plants).
Please note – under some circumstances, manufacture of lentiviral particles can itself take the
project into a situation where Class 2 notification to HSE is necessary. If you include this row and
tick the box indicating that manufacture will take place on campus, you will be expected to justify
carefully your procedures if you want the project to be approved at Class 1.
6. This should cover broad aims only, and not be a full introduction and discussion of issues (refer
to Section 14 below).
8. The ‘GMO’ will include packaged, infectious lentiviral particles, where relevant.
9. This should consist of a paragraph or so introducing the biology of the system being manipulated
- please avoid unexplained jargon. This section is to help the committee see where the issues
highlighted in the risk assessment come from and assess the completeness of the risk assessment.
10. In this section, please describe the overall system being used, and split the construct(s) up into
their component parts. See General Guidelines Sections F and G below for examples. Again, this
will help the committee to ascertain whether best GM practice is being employed in each aspect of
the design and experimental plan.
11. Where a component is non-controversial, this can be brief, but the committee need to decide,
from their own expertise and knowledge, whether there are issues that have not been fully covered
in the risk assessment.
13. This should focus exclusively on methods/methodology to be employed, in simple, clear
language.
14. The risk assessment should cover all possible aspects of risk, from the potential affected entity,
through the nature of the risk, the construct components, to the methodological issues relating to
risk. It should draw on, but not repeat in detail, items covered in any/all of the other sections. When
considering the effect on other organisms, ‘harm’ is defined as effects on numbers or functioning of
7
Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
organisms in an ecosytem. Factors to consider will include: shedding of viable virus in fluids from
infected organisms; capacity of GM(M)O to survive, establish, disseminate and or displace other
GM(M)Os; potential for gene transfer to other organisms; pathogenicity to animals/plants; gene
expression products that are toxic to other organisms; phenotypic and genetic stability; no. of
organisms released; and the characteristics of the environment into which GM(M)Os may escape.
15. Use this section to describe and cite work supporting your statements in the preceding sections
and risk assessment. Highlight any aspects of the proposed work that are recent innovations or
novel aspects of lentiviral construct work.
16. To summarise the current opinion about classification, if any of the following are the case in
your proposed project and activities, then it is likely or inevitable that the project will be considered
as Class 2 or above and will require notification to the HSE and payment of a fee:
- use of a vector system earlier than 3rd/4th gen., particularly those without SIN
(self-inactivating) LTRs
- having a WPRE segment in the construct that contains sequence encoding full length
WPV X protein in an expressible form (even if there is no promoter specifically driving its
expression)
- use of sharps (metal or glass needles, including small-diameter, blunt-ended instruments
such as Hamilton syringes) to deliver viral particles to the host organism/tissue/cells or to
harvest/purify infectious viral particles – risk here is skin puncture with transfer of virus
particles to the experimenter or other worker
- use of inherently dangerous inserts, such as oncogenes
- use of larger volumes of fluid containing high-titre viral particles in such a way that risk of
spillage is greater than ‘very low but finite’
- use of protocols that are likely to generate lots of viral particle-containing fluid aerosol or
fluid splashes
- use of Class 2 safety cabinets to protect the worker or the wider environment
8
Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
General guidelines:
A) Comments from HSE – the GM issues surrounding use of lentiviruses in biological and
clinical research have been discussed several times by the SACGM committee. Below, extracts
from several communications from the SACGM are given.
Comments in a letter in Nov. 2007 (now superseded by more recent HSE statements) suggested that “Class
2 [was recommended] for all activities involving gene delivery via human pseudo-typed retrovirus/lentivirus
constructs (including third generation replication defective pseudo particles). This is based on the inherent
risks of insertional mutagenesis from these genetically modified constructs. This approach means that
unless the users can justify why the work should be undertaken at lower containment, then
containment level 2 should be should be the default position.’. This seems to have been intended to
apply both to cell/tissue culture work and in vivo delivery. The minutes of SACGM meeting 10/07/2008 stated
that “The working group discriminated between activities that involved, or did not involve, the use of sharps
as their use was seen to significantly increase the potential for worker exposure. Consequently, the revised
guidance bases classification, to a large degree, on whether control measures are required to minimise
exposure to contaminated sharps….. Similarly some distinction in terms of risk was recommended for work
with low titre/small volume work as opposed to high titre/large volume activities….. percutaneous injury
should remain the focus of the guidance, but that duty holder should also be made aware of additional routes
of exposure that may affect classification of the work…” [this latter referring to the risk of fluid
splashes/aerosols infecting the worker via mucous membranes].
In a meeting of SACGM on 20/11/08 there was specific discussion of the guidelines for lenti work to be
incorporated into the draft final revision of the Retrovirus section of the Compendium of Guidance (segments
of which reproduced here):
- The retrovirology section of the SACGM(CU) Compendium of Guidance has been revised in light of:
members’ comments and discussion at preceding SACGM(CU) meetings (13 th & 14th meetings); and
deliberations at the virology working group meeting in June. SACGM(CU)’s intention is to provide clarification
on classification of activities involving retroviral/lentiviral vectors to permit researchers to make a sufficient
and proportional assessment of the risks associated with such work. A final draft of the guidance is provided
for consideration by the committee. A revised version of the section of the Compendium of Guidance
dealing with lentivirus work was issued on December 2008.
- Risk assessment for work with these vectors, should not only consider the properties of the transgene but
also the inherent hazards of the vectors i.e. replication competence; and insertional mutagenesis (e.g.
activation of adjacent proto-oncogenes or insertional inactivation of tumour suppressor genes) and whether
these are relevant to the type of activities undertaken in the laboratory and clinical setting.
- The current (i.e. pre-Nov. 2008) advice in the Compendium of Guidance is that work involving replication
defective pseudotyped lentiviral or retroviral vectors should be undertaken as a Class 2 activity as a
minimum (unless justification can be provided why this is not appropriate), because of the inherent
hazardous properties of the viral vectors. The revised guidance (i.e. post-Nov. 2008) changes this advice
such that certain activities including small scale, laboratory studies (e.g. transduction of cells in vitro), which
involve the use of multiply disabled viral constructs containing a non-harmful transgene and do not involve
the direct use of sharps or aerosol/splash generation, can be undertaken as a Class 1 activity. The revised
guidance retains extensive advice on the key aspects of vector design that need to be built into the risk
assessment.
- In terms of likelihood of exposure, the use of sharps is relatively clear cut. However, the guidance provides
advice that where mucosal or inhalation routes of exposure are deemed significant then the work should also
be classified as Class 2. ‘Significance’ is described in terms of titre, volume and the likelihood of generating
aerosols or splash. Comments from dutyholders (i.e. Safety officers / GM committees) relate to how they
might decide on whether this exposure route is deemed significant.
- Applications of these vectors in the clinic may involve the use of sharps for delivery of vectors into patients
and indeed the potential for splashes or aerosol generation is perceived as greater than in the laboratory.
Consequently, the revised guidance is likely to result in clinical applications being considered as Class 2.
However, in many cases, the use of lentiviral/retroviral vectors involves transduction of cells ex-vivo, with
subsequent transduced cells used for patient treatment. In such circumstances, the hazards associated with
9
Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
the viral vectors are not deemed relevant to the risk assessment and a lower classification of the work may
be appropriate.
- Hazards associated with these vectors are summarised as: stable expression of transgenes, insertional
mutagenesis and potential for generation of replication competent virus. Replication defective vectors that
cannot infect human cells can generally be considered class 1. For replication defective retroviruses and
lentiviruses capable of infecting human cells, if the risk assessment demonstrates they are adequately
attenuated, it is possible to designate the activity as class 1 :- Factors supporting this classification will
include: low risk of generation of RCV (e.g. a third generation packaging system), self inactivating (SIN) LTR
and non-harmful insert. However, contaminated sharps represent a significant hazard…and their use should
be excluded for vectors that can infect human cells, if the activity is to be designated class 1.
- The most likely route of accidental infection with a retrovirus will be via inadvertent percutaneous
inoculation. Consequently, the direct use of sharps (e.g. needles, blades, glass Pasteur pipettes, Hamiulton
syringes, glass capillaries, broken glass in recent contact with viral particles) for virus manipulation would be
incompatible with Class 1 designation, for retroviruses and lentiviruses that are both replication defective and
capable of infecting human cells….. where the use of sharps for work with these viruses cannot be avoided,
it is incumbent upon the user to formulate a policy for the control of sharps to minimise inadvertent exposure.
This should include documented training of staff in their safe use and disposal and the work classified as
Class 2. Hollow needles and other sharps should be used with extreme care and only when necessary.
Needles should never be re-sheathed, but rather disposed of directly into an appropriate container for
autoclaving or incineration.
- Worker exposure can occur via other routes, which become significant where the laboratory work involves
large volumes/titres of viral vectors and/or the use of aerosol generating procedures; in which case exposure
via a mucosal or inhalation route needs to be carefully assessed. Where exposure via this route is deemed
significant, use of a microbiological safety cabinet is appropriate and the work should be classified as Class
2. Where product sterility is the sole purpose for necessitating the use of a microbiological safety cabinet
(and indeed gloves), then this should not be used as the basis for classification. Where gloves and/or Class
2 cabinet are used to minimise a real risk of exposure of the worker handling the viral particles, then the
inherent risk of the activity will almost certainly be deemed to require Class 2 notification or above.
- The risk of exposure to lentiviral/retroviral vectors in transduced cells subsequently used for other activities
(e.g. non-fixed flow cytometry; inoculation of animals; in vitro propagation; clinical gene therapy applications)
is negligible, where the cells do not incorporate any helper function and where residual virus titres have been
reduced by replacing the potentially infectious cell supernatant medium. In those circumstances, no
additional containment measures beyond those needed for the non-transduced cells are required. For
experimental animals that have been inoculated with non-replicating virus, the risk of operator exposure from
the infected animals is minimal as is the potential for virus shedding. It is therefore appropriate to house
these animals in a containment level 1 facility. Where the animals are used for post mortem procedures
classification should be assessed on a case-by-case basis.
- classification into a GM activity does not necessarily mean that all the measures from the associated
containment-level will need to be applied. If it is adequately justified by the risk assessment, derogation may
be sought from the Competent Authority to exclude unwarranted measures.
B) Summary description of the origin, development and current state of the art of lentiviral
vectors.
Viral vectors have become a widely used tool for the delivery of foreign genes into cells, either in
vivo or in vitro. Lentiviral vectors are increasingly popular choices for this purpose, as they can be
easily and safely manipulated to allow the insertion of foreign genes into the genome for stable
expression. Lentivirus vectors have several key advantages including the ability to cause long-term
gene expression changes and the capability to infect almost all dividing and non-dividing cells (refs
1-4). These vectors have proven highly efficient in vivo and have achieved stable long term gene
expression in target tissues, such as the brain, retina, liver and muscle.
The complexity of the lentivirus genome has been exploited to build novel biosafety features in the
design of these vectors. In addition to the structural gag, pol and env genes common to all
10
Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
retroviruses, HIV contains two regulatory genes, tat and rev, essential for viral replication, and four
accessory genes, vif, vpr, vpu and nef, that are not crucial for viral growth in vitro but are critical
for in vivo replication and pathogenesis. The first two generations of lentivirus vector carried the
HIV-derived cis-acting sequences necessary for transcription, encapsidation, reverse transcription,
and integration. They thus encompassed, from the 5' to 3' end, the HIV 5' LTR, the leader sequence
and the 5' splice donor site, approximately 360 bp of the gag gene (with the gag reading frame
closed by a synthetic stop codon), 700 bp of the env gene containing the rev-responsive element
(RRE) and a splice acceptor site, an internal promoter (typically the immediate-early
enhancer/promoter of human cytomegalovirus (CMV) or that of the phosphoglycerokinase gene
(PGK)), the transgene, and the HIV 3' LTR. Vector particles were produced by cotransfection of the
three constructs in 293T cells (4). In this design, significant levels of transcription from the vector
LTR and of accumulation of unspliced genomic RNA occur only in the presence of Tat and Rev.
Diagram of retroviral genomes and structure of a retrovirus particle
In 3rd and 4th generation vectors, a 4-plasmid viral particle production system is employed: i) HIVbased vector plasmid designed to accommodate the desired biological insert and marker genes, as
well as harbouring the packaging signal, , and 5’ LTR/promoter and 3’ self-inactivating LTR (SIN
LTR); ii) Packaging construct containing HIV gag and pol genes; iii) Construct expressing HIV rev;
iv) Construct expressing envelope protein. Tat was eliminated from the system as its job could be
performed by constitutively active promoter sequences in the 5’ LTR (7). This design makes the
expression of the packaging functions conditional on complementation available only in producer
cells and incorporates only three of the nine genes of HIV1 in the construct that infects target cells
and becomes integrated into the genome. Only 6-7% of the original vector sequence is left in the
recombinant virus and recombination with integrated wild-type HIV virus or endogenous
retroviruses is more or less impossible. Furthermore, many nucleotide changes have been made to
sequences used in these vectors to render sequence similarity to wild strains of the parent viruses
even lower. All lentivirus vector systems can be tested if need be for production of replication
competent lentivirus, RCL (usually occurring via recombination). Previous experience in large11
Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
scale production facilities has shown that RCL is typically not detected when using 3rd and 4th
generation vector systems in the production of lentiviral constructs packaged into infective
particles. Assays for RCL can include p24 ELISA and qRT-PCR type assays. Please see Sastry et
al. (2005) for details of how to perform such assays.
The final GMMs therefore harbour replication- and recombination-disabled lentivirus vectors
capable of expressing user-specified genes at high level.
References.
1) Gallay, P., S. Swingler, C. Aiken, and D. Trono. 1995. HIV-1 infection of nondividing cells:
C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator. Cell 80:
379-388.
2) Lewis, P. F., M. Hensel, and M. Emerman. 1992. Human immunodeficiency virus infection
of cell arrested in the cell cycle. EMBO J. 11: 3053-3058.
3) Lewis, P. F., and M. Emerman. 1994. Passage through mitosis is required for
oncoretroviruses but not for the human immunodeficiency virus. J. Virol. 68: 510-516.
4) Naldini, L., U. Blömer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D.
Trono. 1996. In vivo gene delivery and stable transduction of nondividing cells by a
lentiviral vector. Science 272: 263-267.
5) Burns, J. C., T. Friedmann, W. Driever, M. Burrascano, and J.-K. Yee. 1993. Vesicular
stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high
titer and efficient gene transfer into mammalian and non-mammalian cells. Proc. Natl. Acad.
Sci. USA 90: 8033-803.
6) Zufferey, R., D. Nagy, R. J. Mandel, L. Naldini, and D. Trono. 1997. Multiply attenuated
lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15: 871-875.
7) Dull, T. Zuffery, R. Kelly, M. Mandel, RJ. Nguyen, M. Trono, D., and Naldini, L. 1998. A
third generation lentivirus vector with a conditional packaging system. J. Virol. 72: 84638471.
8) Sinn P.L., Sauter S.L. and McCray Jr P.B. 2005. Gene therapy progress and prospects:
development of improved lentiviral and retroviral vectors – design, biosafety and
production. Gene Therapy 12: 1089-1098.
9) Sastry L., Xu Y., Duffy L., Koop S., Jasti A., Roehl H., Jolly D., and Cornetta K. 2005.
Product-enhanced reverse transcriptase assay for replication-competent retrovirus and
lentivirus detection. Human Gene Therapy 16: 1227-1236
C) Pseudotyped particles
Lentiviral constructs are often packaged into VSV-G pseudotyped lentiviral particles. VSV-G
pseudotyping involves packaging the genome and other contents of the virus into the VSV
(vesicular stomatitis virus) glycoprotein envelope, which will then exhibit infection behaviour (and
antigenicity) characteristic of the VSV virus, providing the constructed viral particles with a broad
tropism and the ability to deliver genes to many different cell types. The VSV genome does not
contain the same functional packaging elements as those employed in the creation of the lentiviral
constructs. Thus, no recombination can occur with wild-type strains of VSV virus leading to
creation of novel VSV virus with unintended genes in the environment. The VSV is an arbovirus
and transmission of wild strains normally occurs via mechanical events (splashing onto wounds
etc.) rather than via inhalation of aerosol. There is thus a small but finite risk of infection via the
splashing route. Wild strains of VSV are considered high risk and come under anti-terrorism
legislation.
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
Other envelope proteins can be utilised to provide advantages for transducing other defined cell
types. These may include Ebola virus, Hantavirus, Rabies virus, Ross River virus, murine leukemia
virus and baculovirus. The envelope to be used is a consideration for the risk assessment and details
of proposed envelopes to be used should be included.
D) 3rd and 4th generation lentiviral particles used to deliver the construct by one-time
infection usually consist of:
- VSV-G glycoprotein envelope
- HIV-1 or -2 Gag-encoded capsid proteins
- HIV-1 or -2 Pol-encoded reverse transcriptase and integrase enzymes
- two RNA copies of the expression constructs.
These particles are replication-incompetent and cannot be produced in infected cells during the
experiment. There is no risk of inadvertent production of replication-competent retrovirus by
recombination as the potentially recombining stages have been designed in such a way as to make
recombination impossible.
E) The lentiviral particles are assembled using vectors with the following features:
- Rous Sarcoma virus/HIV-1 5’LTR hybrid promoter
- HIV-1  packaging signal
- Rev Response Element
- HIV-1 cPPT - central polypurine tract
- Promoter – ubiquitous or cell/tissue/stage restricted, to direct expression of biological and marker
genes in the construct
- WPRE
- U3/HIV-1 truncated, self-inactivating 3’LTR.
The pseudotyped lentiviral particles contain no viral genes but only viral sequences to facilitate the
packaging of the expression construct into virions (5’ and 3’ LTRs and  packaging signal). The
U3/HIV-1 truncated 3’LTR self-inactivates the 5’LTR in transduced cells.
The gag gene encoding the capsid and matrix proteins, pol gene encoding proteins required for
reverse transcription and integration of the viral genome, the env gene encoding the VSV-G
envelope protein and the Rev protein are encoded by genes in three accessory plasmids that are only
used in the generation of the pseudotyped lentiviral particles. These genes are not present in the
genome of the ‘host’ virus that produces the VSV-G pseudotyped lentiviral particles.
The central polypurine tract (cPPT) serves to enhance the efficiency of integration and insert gene
expression by facilitating nuclear translocation of pre-integration complexes.
The viral vectors may contain the WPRE (Woodchuck Hepatitis Virus Post-transcriptional
Regulatory Element) to enhance expression of the genes harboured in the expression construct. Its
mechanism of action is unclear, but may involve the facilitation of nuclear export and/or stability of
viral mRNA, or enhanced transcriptional termination and avoidance of readthrough beyond the
construct. This element was first described in Donello et al., 1998 and in Zufferey et al., 1999 and
there has been a good deal of work on it since. Kingsman et al., 2005 wrote a summary highlighting
the concerns about the presence of promoter and partial (60 a.a.) coding sequence of the WHV X
protein within the first generation WPRE element. Protein X has been reported to be a major factor
in the increased incidence of liver cancer after infection by natural routes with Hepatitis B-type
hepadnaviruses. It is unclear whether there is any real evidence that this 60 a.a. N-terminal fragment
of protein X is directly oncogenic, although it may act as a cofactor for oncogenicity with weak
effect. It also seems that partial-Protein X cannot be expressed under the control of the promoter in
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
the WPRE element, even in lentiviral constructs, except in the presence of a second enhancer,
examples of which are not present in the WPRE construct. It could potentially be expressed as a
result of readthrough from endogenous genes adjacent to the viral construct integration site,
although there is no direct evidence that this happens. Such readthrough effects would be reduced
by the deliberate inclusion of insulator elements flanking the expressed portions of the integrated
construct. It is also possible to abrogate the potential expression from the included protein X
promoter using newer generation WPRE elements lacking all or part of this promoter, and/or by
site-directed mutagenesis to remove the start codon or other portions of the coding sequence. These
modifications have been shown not to affect the efficiency of the WPRE element in enhancing
expression of genes contained within the lentiviral construct.
F) Description of components of the lentiviral vectors to be used (see Q10 parts a) to g) on the
form)
Below are examples of the kind of description you should use, to make it easy for the committee to
evaluate each of the components in the GM system:
a) Overall system name/description
(e.g. 4th generation lentivirus construct based on XYZ vector, containing shRNA vs Gene Y, and marker gene
eGFP).
Entries in this section might look something like the following:
4th generation lentivirus system based on pLentiLox pLL4.0 and pLL4.2 vectors, containing a
variety of promoters, GeneX/FLAG/RFP fusion protein, insulator sequences and GeneX binding
target element
System constitutes:
- Main expression construct vector pLL4.0 or pLL4.2
- packaging system on 3 plasmids (2 separate systems will be tried):
pLP1 (Invitrogen) or pMDLg/pRRE – expresses Gag and Pol proteins in Rev-dependent manner
pLP2 (Invitrogen) or pRSV.rev – expresses Rev protein
pLP/VSVG (Invitrogen) or pMD2.VSVG – expresses VSV-G envelope protein
The main expression construct has been made elsewhere, and is based on the insertion of the
biological and marker sequences described below into the multicloning site of pcDNA3.1 Zeo+
(Invitrogen), based on pBR322 and the pUC plasmid series.
b) Summary of Lentiviral Coat Protein and Tropism to be employed
Entries in this section might look something like the following:
Coat protein of the packaged infectious particles will be VSV-G. This has a wide tropism and can
infect all mammal species tested so far, as well as a range of insect vectors.
c) Vector backbone and origins of replication etc.
- Lentiviral contruct(s):
Entries in this section might look something like the following:
Main construct – pUC6 backbone and E. coli ColE1 origin, multiple cloning site with TOPO cloning
site, V5 epitope tag coding sequence and transcriptional stop sequence
Accessory plasmids:
pLP1, pLP2, pLP/VSVG, and pMDLg/pRRE, pRSV.rev, pMD2.VSVG – all based on pUC sequence
and E. coli ColE1 origin
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
pcDNA3.1 clones: f1 origin, ColE1 origin, SV40 origin
d) Resistance genes
Entries in this section might look something like the following:
- Lentiviral contruct(s):
Main construct: - AmpR; Blasticidin resistance gene
Accessory plasmids: pLP1, pLP2, pLP/VSVG, and pMDLg/pRRE, pRSVrev, pMD2.VSVG – AmpR
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
pcDNA3.1 clones: AmpR; Neomycin resistance
Note – these sections (and see below) on ‘Other plasmids…’ will be needed wherever the inserts
to be included in the lenti constructs are cloned in other vectors initially and are not covered by a
relevant E. coli GM RA.
e) Marker genes and tags
Entries in this section might look something like the following:
- Lentiviral contruct(s):
C-terminal V5 tag for convenient detection; FLAG tag within fusion protein
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
pcDNA3.1 clones: FLAG tag within fusion protein
f) Biological insert (i.e. the ‘functional’ part, designed to have an effect on the biology of the host
cells / organism)
Entries in this section might look something like the following:
- Lentiviral contruct(s) and pcDNA3.1 clones:
Fusion protein with the following components:
Full length mouse GeneX ORF – FLAG – RFP (Red fluorescent protein). This insert will be cloned
into the vector in such a way as to remove the eGFP coding sequence but leave the LoxP sites
flanking the expression insert.
g) Expression modulators (e.g. promoter, WPRE, stating the version and its functional
constituent parts where applicable)
Entries in this section might look something like the following:
- Lentiviral contruct(s):
- CMV promoter for high-level expression of biological insert
- Other promoters instead of or alongside CMV - including UbiC, U6, CamKII, PGK, synapsin1, tau.
- HS4 insulator and/or CTCF binding site sequences from chicken -globin locus
- RRE – rev response element
- WPRE – modified to remove potential for expression of N-term. fragment of WHV protein X
- Mecp2 binding target elements – a) Mecp2 binding target oligonucleotide from Bdnf promoter IV;
b) 500bp from Bdnf promoter IV; c) fragment from Mecp2 promoter thought to be a binding target
for Mecp2; synthetic binding target oligonuc. based on known binding properties and selectivity
- Polyadenylation signals / transcriptional termination sequences
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
pcDNA3.1:
- CMV promoter (for biological insert)
- T7 promoter (for biological insert)
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
- bGH polyadenylation signal / transcriptional terminator (pA; for biological insert)
- SV40 origin / promoter (for Neomycin resistance gene expression)
- SV40 pA (for Neomycin resistance gene expression)
h) Other constituents (only where significant to function and not covered in b)-f))
Entries in this section might look something like the following:
- Lentiviral contruct(s):
- Other plasmids e.g. those initially containing the biological insert / fRNA etc.
Ψ packaging sequence
U3 3’ LTR
5’ LTR – these LTRs together function in packaging to create lentiviral particles, but are selfinactivating (SIN) after integration into the target cell genome and thus render the integrated
lentiviral construct incapable of being mobilised into infectious particles even in the presence of
active endogenous or exogenous retrovirus.
LoxP sites flanking the biological insert – these will not be used in this series of experiments.
G) Controls for lentiviral delivery experiments (see Q10 part h) on the form)
Experiments utilising lentiviral constructs to deliver genes or gene knockout reagents to
cells/organisms usually involve several controls. Each application should describe both the main
experimental constructs and the control constructs, as appropriate to the experiment, explaining
what each is for. The descriptions of the constructs should include the vector-based constituents
from which the final construct is derived, the gene(s) to be inserted, marker genes deployed after
infection, and resistance genes deployed during construct creation and packaging or after infection,
plus any other components.
Entries in this section might look something like the following:
e.g. for an experiment to test the efficacy of GeneX overexpression in changing the phenotype of
Cell types Y, Z and W, with additional tests to find the optimum conditions for such an effect:
A series of test lentiviral constructs will be made, variably containing:
- GeneX/FLAG/RFP fusion protein
- insulator sequences flanking expression cassette, as above
- variety of GeneX binding target elements, as above
- variety of promoters, as above
Control constructs will be made with the same regulatory features, but from which only RFP is
expressed, or no exogenous protein at all.
OR
e.g. for an experiment to test the effect of RNAi-based knockdown of gene Y:
Main experimental construct:
-
pRRLU6shGeneY(R)sincpptCMV.GFP.p ( GeneY shRNA hairpin + eGFP)
Control construct for testing effect of scrambled sequence RNAi-based reagent for gene Y – tests
non-specific effect of a insert with that base composition but no targeted functional purpose:
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
-
pRRLU6shGeneYscr(R)sincpptCMV.GFP.p (scrambled GeneY shRNA hairpin + eGFP)
Construct for testing effect of marker gene alone:
-
pRRLCMV.GFP.p (eGFP)
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
Diagrams of vectors and constructs – please use clear diagrams made to illustrate the features
of most relevance to the risk assessment. Please remember that the committee may not have
seen the particular vectors to be employed or have come across the construct design before, so
these diagrams are of considerable use in familiarising them with the proposed elements of
the work. Examples are given below:
pLentiLox4.0
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
H) Safety procedures and justification
Where mammalian cell lines and organotypic cultures are used for transduction using lentiviral
constructs, they should be manipulated in sterile conditions using standard tissue culture practice.
This will involve the use of Class II Microbiological Safety Cabinets (‘hoods’) to protect the work.
Where any further safety concerns require the use of Class II cabinets to protect the worker, this
should be given enhanced visibility in the risk assessment and the concerns clearly stated. Usually,
any work requiring this level of containment for the protection of the worker will require
classification of the GM activity at Class 2 or above and HSE notification.
For primary cells and cell lines, viral transduction is normally carried out using a range of MOI
(multiplicity of infection) dependent on each cell line and each viral particle titre. After
transduction, media are replaced and cells allowed to express genes from the construct for periods
of 24 to 96 hours. Viral particles can be pipetted onto the cells, reducing the risk of accidental
infection of the experimenter and lab co-workers.
To inactivate remaining packaged virus particles in the medium, all plasticware and reagents that
come into contact with the viral particles and transduced cells should be soaked in 3% Virkon for
24 hours prior to disposal either by autoclaving or by incineration.
Again, if safety and escape issues arise in relation to virus inactivation and disposal beyond those
normally associated with Class 1 GM work, these should be given prominence in the risk
assessment and may require Class 2 classification.
Experiments using unfixed cells and/or media supernatant should only be carried out in containment
level 1 facilities if the work is classified at Class 1, or if the work is done in sealed containers and
the risk of the containers being compromised with consequent release of infectious lentiviral
particles is judged to be negligible or very low. Any procedures involving puncture of sealed
vessels containing viral particles should receive enhanced visibility in the risk assessment.
Any procedures involving hypodermic syringe needles will need to be classified at Class 2 or
above, as needlestick injury becomes a significant risk (see section I) below).
I) Routes of escape for lentiviral constructs packaged into viral particles
Potential routes of escape are:
Aerosol
Incorrect disposal of culture materials
Needlestick injury to experimenter or third party
Escape of recently infected animal/plant outside of the Biological Services or other
containment facility
The potential for escape by aerosol is low if all work with unsealed virus particle-containing fluids
is done within Class II hoods. Aerosol could also possibly arise during application of viral particles
to cells/tissues if this has to be carried out outside a hood. Aerosol could also possibly arise during
waste disposal if fluids are released before autoclaving is complete.
Contact with fluids containing viral particles would only result in infection of the experimenter or
lab co-workers if contact was oral, or via skin lesions, cuts etc., and then only if unprotected by
gloves etc.
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
Where infectious lentiviral particles are to be transported between labs, the protocol should involve
double-sealed containment e.g. the use of screw-top tubes packed securely into a container with a
sealing lid.
If lentiviral particles are being delivered to live animals by injection, there is a risk of needlestick
injury to the experimenter or to third parties present during delivery (e.g. Biol. Services personnel,
other lab workers). The likelihood of such an injury taking place should be considered. The risk
would seem to be greatest to the experimenter unless the organism to which the particles are being
delivered needs to be handled during delivery by other staff.
J) Other routes of escape for lentiviral components and inserts
Lentiviral particles are often packaged using producer cell lines such as HEK293-FT. Many of these
lines are certified free of adventitious agents. Where cell lines not so certified are used, there exists
a risk that parts of the lentiviral system or biological insert could be picked up by a resident
adventitious agent (e.g. HIV, EBV etc.) and incorporated into its genome in such as way as to
enhance its transmissibility or ability to persist in the environment. These risks should be
considered in the risk assessment.
K) Consequences of escape
If the experimenter or third party worker are exposed to virus or viral infected cells, the likelihood
of disease is very low or negligible with 3rd and 4th generation lentiviral constructs. Infection via
inhalation or injection of virus would be limited to mucosal cells or cells at the site of injection (in
vivo experiments have shown diffusion of infectious particles no more than millimetres from the
injection site). Even if the infected worker had a pre-existing HIV infection, the chances of
recombination are extremely small due to the design of the vector. There is no available evidence
for transfer of infectious viral particles to other vulnerable cells via the bloodstream or lymphatic
system except in cases where the intention was to transduce cells of the immune system in the
bloodstream/bone marrow. Viral particles that do not infect cells are estimated to become inactive
within hours and no viable virus should remain after 48 hours. Experiments to look for viral
particles in urine and faeces of animals infected with lentiviral constructs by injection into nervous
system tissues have found none. Virus is not incorporated into the germline and will not be passed
to offspring.
Contact with viral particles and infection of cells at the contact site could result in a transient
immune response with local swelling and inflammation. This is unlikely to be more than a minor
effect unless the nature of the biological insert(s)/markers make it more likely or more severe.
Any effects of expression of the functional parts of the construct are likely to be negligible, but the
following should be considered:
- gene expression in cells local to the injury (intended biological constituents of construct and other
genes in the construct, including WHV protein X and marker genes)
- gene knockdown in cells local to the injury (due to intended biological constituents of construct)
- other effects of the construct on endogenous gene expression
- insertional mutagenesis in the infected individual’s genome leading to enhanced expression of an
oncogene or oncogenic cofactor or repression of a tumour suppressor gene
- insertional mutagenesis in the infected individual’s genome leading to disruption of other
endogenous gene function
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Guidance Notes for Risk Assessment for Genetic Modification involving Lentiviral constructs
Since 3rd and 4th generation lentiviral constructs cannot replicate after infection, there is no risk of
continued infectivity of the needlestuck individual, so no virus could be passed on to other people.
The design of 4th generation lentiviral constructs is such that there is essentially no risk of
recombination with other extant human or animal/plant viruses to create new strains with altered
properties.
Insertional mutagenesis effects have been considered to be the main element of risk with newer
generation lentivirus constructs. The construct is designed to become integrated into the genome.
Sites of integration have been studied and the current consensus is that, with current generations of
lentivirus construct in common use in non-therapeutic experiments, there is little selectivity. Thus,
the construct can integrate anywhere within or between genes, with no preference for genic regions,
coding regions or promoter regions. Little risk is thought to be associated with integration into
introns, although sense-orientation integration within a gene might disrupt splicing and expression
of that gene. No enhancer/repressor sequences are present, so direct transcriptional effects on such
integration events are unlikely, although effects acting via disruption of chromatin structure cannot
be ruled out. The a priori probability of integration into an exon or promoter of an endogenous gene
is thus ~3.5%, the proportion of the genome occupied by these kinds of sequence. Any integration
event that disrupts endogenous gene function would only do so if dominant effects on gene
expression manifest for the gene in question, as typically only one integration event will happen per
cell, and that will occur monoallelically. The oncogenesis observed in clinical trials for treatment of
X-linked SCID has been shown to be strongly associated with multiple features of the design of that
experiment, and is not thought likely to occur in cases where the biological gene components of the
construct do not give cells a growth advantage. Several other trials have monitored oncogenesis and
have reported no adverse events of this nature over many patient-months of follow-up.
22