Infection followed by disease will depend on the microorganisms ability to
multiply in the host and on the host's ability to resist or control the infection. It
has proved useful to categorise all microorganisms into 4 groups which define
their pathogenicity to humans; with only the first group containing
non-pathogens. This categorisation applies only to the infectivity towards
humans, and is of significance only, therefore, for the contained use of
organisms:
| Hazard Group 1 |
Organisms that are most unlikely to cause human disease |
| Hazard Group 2 |
Organisms capable of causing human disease and which
may be a hazard to laboratory workers, but are unlikely to
spread to the community. Laboratory exposure rarely
produces infection and effective prophylaxis or effective
treatment is usually available |
| Hazard Group 3 |
Organisms that may cause severe human disease and
present a serious hazard to laboratory workers. They may
present a risk of spread to the community, but there is
usually effective prophylaxis or treatment available |
| Hazard Group 4 |
Organisms that cause severe human disease and are a
serious hazard to laboratory workers. They may present a
high risk of spread to the community, and there is usually
no effective prophylaxis or treatment |
The intention of this categorisation, which applies to non-modified organisms
as well, is to identify appropriate containment which would be required to
protect those working with the organisms. The higher the hazard group, the
greater the containment required to control the organism and ensure that it does
not infect those working with it.
Pathogenicity is not a simple characteristic. Many genes must interact
appropriately for a microbe to cause disease. the pathogen must possess and
express characteristics such as recognition factors, adhesion ability,
toxigenicity and resistance to host defence systems. Single gene modifications
of organisms with no pathogenic potential or history, or even the introduction
of multiple genes unlikely to confer pathogenicity are unlikely to result in
unanticipated pathogenicity. For example, E. coli K12 has been disabled to
remove some of the factors that might be associated with pathogenicity (wild
type E. coli is a group 2 pathogen). The factors which have been lost include
the cell-surface K antigen, part of the LPS side chain, the adherence factor
(fimbriae) that enable adherence to epithelial cells of human gut, resistance to
lysis by complement and some resistance to phagocytosis. This variant of E.
coli is a common host organism for genetic modifications within the laboratory.
The starting point for the risk assessment is, therefore, an assumption that the
level of risk associated with the modified organism is at least as great as that of
the host organism (until proved otherwise, either by direct observation, or by
argument where the factors which are likely to enhance or decrease
pathogenicity are considered as in the case of K12 above). Whether in the
laboratory or in industry the capacity to choose a host means that in all but a
few cases the host organism will have been chosen to be in hazard category 1. It
is assumed that the modified organism will be used under the same containment
as the host wild-type organism unless the modification inserts information
which would alter the pathogenicity. The vector has also to be considered, both
for its own potential for pathogenicity and for its ability to transfer the insert to
other than the intended organism -- horizontal transfer of the information. Most
vectors used for E. coli contain no sequences which might result in pathogenic
behaviour. The presence of genes coding for antibiotic resistance might be of
concern, but for most of these the antibiotic resistance is already so common in
the environment that it may be discounted.
Most common E. coli vectors are transfer deficient, but the ability to transfer
information either directly or with the assistance of other plasmids and the host
range of the vector must be taken into account when considering the safety of
the mechanism of insertion of the required genes into the host organism.
The properties of the insert are again of importance in considering the risk
assessment for the modified organism. Clearly if the information encodes a
toxic gene product, or one which is known to be likely to modify the
pathogenicity of the organism into which it is inserted, the great the risk. If the
gene product is non-toxic and is not one which may pose a risk to the people
working with the organism in containment, the risk management will largely be
based on the pathogenicity of the host organism.
In most instances the characteristics of the donor organisms are of less
relevance to the risk assessment than those of the host. If the donor organism is
merely used as a source of well characterised DNA for a selectable phenotype
or a promoter or other control sequence, the characteristics of the donor are
unimportant to the risk assessment. If however, the insert contains genes which
are biologically active, toxins or virulence factors, then information from the
donor organism are of consequence. The construction of cDNA or genomic
libraries make it essential to consider all the possible hazards associated with
the donor organism, and in this instance, the hazard group may well have to be
the higher or the two within which the host and donor fall.
It is now possible to examine the modified organism and consider the likely
risk. During the 1970's Dr. Sidney Brenner and others in the United Kingdom
attempted to systematise the approach by considering three factors -- Access,
Damage, and Expression. The approach was incorporated in the United
Kingdom's approach to risk assessment for contained use of bacteria, and is
discussed in detail in a document produced by the Advisory Committee on
Genetic Modification in the United Kingdom. The latest version of the
guidance was published in 1999 and provides clear guidance as to the risk
assessment for the contained use of genetically modified microorganisms
(including any cells in culture). The guidance note is free and may be obtained
from the Health & Safety Executive in Britain. More information is available
by looking at the newsletters published by the ACGM which are available on
the internet on http://www.shef.ac.uk/~doe.
Access is a measure of the probability that a modified micro-organism, or the
DNA contained within it, will be able to enter the human body and survive
there. It is a function of both host and vector. Depending on the organism being
used, there are a number of routes of entry which allow access. The properties
of the vector, particularly mobilisation functions need to be taken into account.
In general if the organism is capable of colonising humans then access is high,
whereas if the host is disabled so as to require the addition of specific nutrients
not available in humans or outside of the culture media and is also sensitive to
physical conditions or chemical agents present in humans, then the access
factor is likely to be low.
Expression and Damage are usually associated with the insert and the gene
product.
Expression is a measure of the anticipated or known level of expression of the
inserted DNA; if the 'gene' inserted is intended to be expressed at a high level,
for example, by deliberate in-frame insertion down-stream of a strong
promoter, expression is likely to be high. If the insert is simply there to allow
probes to detect the DNA, and is non-expressible DNA, i.e. with no foreseeable
biological effect or gene containing introns which the host is incapable of
processing, then the expression factor will be low. Examination of the final
product, the modified organism itself, will determine the actual expression,
which may be higher or lower than expected.
Damage is a measure of the likelihood of harm being caused to a person by
exposure to the genetically modified micro-organism, and is
independent of either expression or access. It is associated with the known or
suspected biological activity of the DNA or of the gene product. The activity of
the organism which results in any toxic, allergenic or pathogenic effect need be
taken into account within this parameter. It may be that the biological activity
of a protein is dependent on the host cell system in which it is expressed. An
oncogene expressed in a bacterium will have no discernible effect, when
present in a human cell, problems may arise. The full biological function of
many gene products require post-translational modification which will not
occur within a bacterial cell normally. The potential biological activity of the
gene product should be considered in the context of where and how it has been
expressed and the effect on its structure and activity of the mode of
manufacture. The range products with 'damage' potential might include:
- a toxic substance or pathogenic determinant that is likely to have a
significant biological effect - damage is high
- a biologically active substance which might have a deleterious effect if
delivered to a target tissue
- a biologically active substance which is very unlikely to have a deleterious
effect or where it could not approach the normal body level. When cloning in
E. coli the 'worst case' would be if all the E. coli in a person were replaced by
the modified organism expressing a foreign polypeptide in an active form at
a high rate. If all of these are absorbed in an active form and arrive at a site
where they might have their maximum effect, what would be the damage?
- a gene sequence where any biological effect is unlikely because of known
properties of the protein or because of the high levels encountered in nature
Once an estimate of each of these parameters has been made (in the United
Kingdom this is numerical in steps of 10-3), they may be combined. The result
provides a qualitative measure of the risk, and allows a containment level to be
assigned for the use of the organism in order to protect those working with the
genetically modified micro-organism.
Unfortunately, this Brenner scheme is only easily applicable to a small class of
experimental uses of modified micro-organisms, but the number of experiments
in research laboratory environments which fit the requirements for the
application of this scheme make its retention useful.
Modified organisms may be used in containment in laboratories (or pilot plants)
or may be used in an industrial setting. It may be that the primary distinction
here is not the size of plant or type of organism, but rather the skill and training
of those working in the facility.
It is likely that a research or development laboratory will be working with
organisms which pose a greater threat to either the individuals working therein
or to the environment than do those organisms developed for large scale factory
use. The great majority of organisms used in industrial production are
well-characterised, 'familiar' organisms capable of being used under conditions
of 'Good Industrial Large Scale Practice' or GILSP. Given that it is usually
possible to 'choose' the parental organism into which a gene is inserted for a
particular 'industrial' purpose, there would be no good reason to choose an
organism likely to pose problems to either those working in the facility, or to
the environment in the event of an escape.
The same logic would apply to the development stage where 'industrial' use of
the modified organisms is being planned. There is a possible extra hazard in
that it is at this stage that the modified genes may be inserted into the organism,
and the unpredictability of insertion site may, arguably, require slightly greater
care than that taken at the production facility.
In the research laboratory, organisms may be pathogenic to humans and/or to
the environment, as it is here that fundamental research would be conducted.
Experiments will involve organisms and /or inserts which may be injurious to
the health of the workers or to those who are incidentally on site in the
laboratory.
