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Working with ribonucleic acid
(RNA)-based biotechnologies)
Advances in the laboratory now indicate that ribonucleic acid (RNA)-based
biotechnologies are fast approaching application in animals. The most
likely area of use will be in disease prevention or mitigation. There
are several RNA-based biotechnologies but, in general, they all aim
to reduce the production of protein form a given gene. At present,
RNA-based biotechnologies have most promise in combating virus
infection in animals through reducing viral gene activity. RNA-based
biotechnologies are likely to impact on animal health within the next
ten years.
RNA-based approaches to regulate gene activity have been practiced in the lab for some
time. Significant technical advances and validation of strategies now mean they are likely to
see application in animals in the field soon.
There are a variety of RNA-based biotechnologies differing in technical aspects of the
mode of action. However, they all aim to reduce the production of protein from a given
gene. This article will introduce the different types of RNA–based biotechnologies, briefly
indicate how they might be delivered to an animal and indicate possible applications.
The focus will be only impact to animal health.
RNA-interference (RNAi)
Although less than a decade has passed since RNAi was
shown to work in mammals, this term has come common
interaction leads to the destruction of the messenger RNA,
place in discussion about regulating gene expression. But
preventing production of protein which should have been
what is RNAi? In its simplest definition it is the use of RNA
produced by that messenger RNA. This activity is often
(ribonucleic acid) molecules to modulate the activity of a
called ‘gene knockdown’. To give an example, if an RNAi
target gene. When you delve deeper a multitude of terms
molecule was designed to target a viral gene, then its
appear – siRNA, shRNA, miRNA to name a few. These terms
activity would prevent normal virus production
will be expanded more later; but first how are genes
activated.
Genes within an animals’ genome are transcribed when
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can bind directly to a target messenger RNA. This
RNAi molecules come in different forms. As
independent small double stranded RNA molecules called
short-interfering RNA (siRNA) that can only survive
the appropriate activation signals are present in a given cell.
transiently in an animal. For prolonged an activity the
The action of transcription results in the production of
siRNA must be incorporated into an expression vector that
messenger RNA. An RNA molecule comprises of a string of
functions like a gene. In this case they are called short-
nucleotides; the string of nucleotides in messenger RNAs
hairpin RNA (shRNA), due to their physical structure, or
encode for the proteins that make up an animals cell. It is
microRNA (miRNA). The latter form are found naturally in
against this messenger RNA that RNAi acts. The important
animals and are now believed to play a major role in
functional component of RNAi is a short single-stranded
normal cell growth and survival. Synthetic miRNA can also
RNA molecule of about 20 nucleotide bases in length which
be constructed in the lab for delivery to an animal.
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Other RNA-based biotechnologies
For both gene therapy delivery and transgenic strategies,
Although currently most optimism for successful
more elaborate RNAi molecule designs that incorporate
application of RNA-based biotechnologies revolves around
inducible gene knockdown activity are
the use of RNAi, other types of RNA molecule exist that
possible. In this case the RNAi molecule is
could be applied to animals. These include RNA decoys to
only produced in the presence of the
block activity of an enzyme. A potential application would
appropriate inducing factor.
be to block the activity of a viral polymerase and thus
The practicalities of
prevent viral replication. For some time the prospect of
delivery of RNA molecules to
using antisense molecules or other oligonucleotide-type
animals are likely to be
molecules.
common to those addressed
Both antisense and RNAi molecules evoke normal
during development of drug
cellular enzymes to destroy the target RNA. There are
treatment strategies and
some RNA molecules that contain their own enzymatic
vaccination.
activity. Ribozymes, that specifically bind to and
subsequently catalyze the cleavage of other target RNA
molecules, and represent an interesting alternative to the
RNAi. Unfortunately ribozyme design has proved difficult.
It is likely that further RNA molecules will be identified.
Applications
of RNA-based
biotechnologies
in animal health
For example, recently RNA molecules that can destroy
Most applications that could be
miRNA called antagomirs. To date, all RNA-based
applied to animals are still in the
biotechnologies function to reduce target gene activity.
experimental phase. There are,
however, several preclinical studies
Delivering RNA-based molecules
for applications in humans and it is
How can RNA-based molecules be delivered to animals?
reasonable to expect that these will
Two different strategies are possible. First they could be
accelerate the use of RNA-
delivered in a gene-therapy or vaccination-type of
based biotechnologies in
approach. Depending on what type of RNA-based
animals. A few current studies
molecule was delivered the effect could be either a
will be described to illustrate
transient of stable change in gene activity. For example the
the potential of RNA-based
use of siRNA would result in a transient alteration in gene
biotechnologies.
activity somewhat similar to vaccination, except that the
Perhaps the most likely
timescale would most likely be considerably shorter that
application of RNA-based
the intended protection period offered by vaccination. This
biotechnologies will be to interfere with
timescale will impact on what you could use siRNA for.
virus infection. Several projects are
With a gene therapy delivery strategy the aim would be to
focussing on the use of RNAi to prevent virus
enable the change in gene activity to be present for as
replication and thus combat virus induced
long as cells carrying the RNA-based molecule survive in
disease. These studies are most
the animal. For most applications, actual delivery vehicles
advanced for FMDV, EIAV and flu.
remain to be evaluated.
Commonly the target is the viral
Alternatively, shRNA or miRNA could be delivered as a
polymerase. Antisense-type
transgene to generate genetically modified animals. This
approaches are also being targeted
approach would result in the stable inheritance of the
against viruses. In one project the RNA molecule
RNAi transgene by offspring and subsequent generations
functions as a decoy, again aiming to prevent influenza
of animals derived from the original founder animal.
viral polymerase enzyme activity to intervene in virus replication.
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Alternative uses of RNA-based biotechnologies
Another widely discussed concern relates to possible
that can be envisaged for the future include
inefficient eaffects on non-target genes. This is possible
modulation of some aspect of the immune system
through weak interaction of the RNA molecule to
In this scenario the variation in gene activity
seen in natural animal populations could be
messenger RNAs transcribed form non-target genes.
Obviously if this occurred in a living animal the effect
mimicked through the knockdown afforded by
could be quite severe. This is an area of intense
RNAi. In a similar vein, some other aspect of animal
investigation at the moment and the imminent human
function, for example, growth could be modulated.
clinical trials with RNA-based biotechnologies will be
informative in this regard.
Potential risks associated
with RNA-based biotechnologies
It is now widely acknowledged that RNAi can be an
efficient way to reduce gene activity. However, as
with most emerging technologies, some concerns
remain to be overcome. The use of RNA-based
technologies can provoke a evoke the internal
immune response within an animal’s cell. In
extreme situations this can lead to cell death. For
those applications that aim to combat virus infection
this may be an advantage.
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2007 • 4
Bruce Whitelaw