The scientists used the nematode
(roundworm) Caenorhabditis elegans to study this. Subsequently, however,
considerable problems arose in the attempt to transfer the strategy used
by Mello and Fire to vertebrates. In particular the administration of
small RNAs, known as siRNAs (small interfering RNAs), in animals proved
difficult. Although it was possible to administer siRNAs successfully by
using various methods such as high-pressure injections or in conjunction
with cholesterol, the underlying mechanisms remained obscure. Markus
Stoffel, ETH Zurich Professor at the Institute for Molecular Systems
Biology, together with chemists from the Alnylam Company, has now
succeeded in elucidating the mechanism for the uptake of siRNA in
combination with fatty acids in mammals. The corresponding paper, which
has been published in the scientific journal �Nature Biotechnology� and
will also adorn the title page of the printed version in October,
represents the basis for possible siRNA therapies, among other things.
This is because Stoffel showed that siRNA can be coupled effectively to
various fatty acids.
Cholesterol transporters also play a part
Stoffel and his team turned to chemically modified
siRNAs in combination with cholesterol, not because the method based
on this compound was particularly efficient but because it had the
least side-effects. First of all the researchers wanted to know
whether siRNA was capable of being bonded to other lipophilic
substances in addition to cholesterol, and caused a reduction in the
activity of a target gene in the liver at the same time. It turned out
that there are several such fatty acids. But what is it in the blood
to which all these RNAs conjugated with so-called lipophilic
substances bond" The ETH Zurich researchers discovered that, depending
on the fatty acid used, the binding partners are the well-known
cholesterol transporters High Density Lipoproteins (HDL) and Low
Density Lipoproteins (LDL) as well as the albumin (protein) present
everywhere in the blood. Without these lipoprotein particles there is
no uptake of siRNAs into the tissues, as became apparent from further
experiments. In an additional experiment the scientists demonstrated
that the uptake can be made considerably more efficient if the
siRNA-fatty acid molecules are already firmly bonded to HDL and LDL
before being administered. Stoffel�s team also discovered that there
is preferential uptake into different tissues depending on whether an
siRNA-fatty acid molecule is bonded to HDL or LDL: all LDL compounds
trigger responses in the liver, but HDL compounds also do so in the
intestine or kidneys.
An irritating finding
The latter finding indicated that the uptake
proceeds via HDL and LDL receptors. The researchers proved this
assumption by inactivating the receptors, with the result that uptake
no longer occurred. Despite the clarity of the finding, it irritated
Stoffel slightly. He found it hard to imagine that the siRNAs were
able to enter the cell via the normal absorption route like HDL,
because this route leads into the cell�s own digestive system with
lysosomes that would degrade the siRNAs. So how would the siRNAs be
able to avoid this degradation" Stoffel concluded that they simply use
a different doorway into the cell. Thus the HDL and LDL receptors
would only act as docking stations but not as an entry portal.
But what might the alternative doors be" The ETH
Zurich researchers remembered that a gene product Sid1, which is
necessary for the cellular uptake of siRNA, occurs in the worm
Caenorhabditis elegans. The corresponding gene also has a homologue in
mammals. By inactivating it, the scientists showed that it is also
necessary in mammals. The overall result from all the discoveries is a
mechanism for siRNA administration that starts with the bonding of
siRNAs to particular fatty acids. This combination is linked to
lipophilic proteins that bring about docking onto the tissue cells.
The doors that allow the siRNA-fatty acid molecules to enter are then
situated close to the docking station.
Prospects for new therapies and research
Stoffel thinks that through their work they were
able to determine the elements that are most important for the uptake
mechanism. However, he says it is very likely that yet more molecules
play a part. But since an insight into the mechanism now exists for
the first time, it will be possible to make specific improvements in
the technique. For example Stoffel�s group wants to find out whether
HDL and LDL can be replaced by synthetic proteins or lipid-rich
particles. He says that basically the technique has the potential to
be used in gene therapy. However, the determination of the siRNA doors
also opens up new approaches to fundamental research. Instead of siRNA
it might also be possible to smuggle in miRNAs, another group of small
RNAs, in the same way. The same mechanism ought to work for miRNA
inhibitors as well. Since miRNA is increasingly �suspected� of
occupying a decisive role in gene regulation in nature, its targeted
administration or inhibition could yield completely new insights. |