Biomedical Engineering Reference
In-Depth Information
number of cell-produced mRNA copies, thereby overcoming the endogenously administered
AS-ODN.
Because of this, antisense as a treatment has not proven to be a killer of cells and,
therefore, not a revolutionary anti-cancer agent, as it was originally hoped. However, it
has played a pivotal role in leading to the next advance in cellular gene inhibition - small
interfering RNA.
12.1.1.4 siRNA
Fire and Mello [12] were using antisense to study behavioral effects on the primitive worm
Caenorhabditis elegans
. They tested sense RNA and antisense RNA on the worms but
there was no effect. They then tested a combination of sense and antisense RNA. The
effect was dramatic. The worms started to twitch spontaneously. The gene that was hold-
ing back the twitching had been silenced. Fire and Mello had discovered gene silencing
by double-stranded (ds) RNA which acted as small interfering RNA (siRNA). They were
rewarded for the discovery with the Nobel Prize in 2006. RNAi has become widely rec-
ognized as a biological mechanism for the regulation of gene expression and used for
intracellular inhibition. dsRNA is produced in the nucleus. In the cytoplasm it binds to an
enzyme, Dicer. Dicer literally dices up the dsRNA into short strands (15-20 nucleotides).
One of the strands is loaded into a protein complex, RNA-induced silencing complex
(RISC). The RISC now has the single strand of short RNA as a binding site to bind to a
complementary sequence on the cell's mRNA. This binding leads to cleavage of mRNA,
degrading the message and stopping it from translating a specific protein. And hence it is
silenced.
RNAi is a fundamental cellular process of gene regulation in the cells of animals and
plants. Since both animals and plants are subject to diseases induced by viruses, RNAi may
have evolved to protect cells from invasion by viruses. The genome of retroviruses is in
double strands of RNA. A retrovirus, lacking cellular mechanisms and DNA, injects its
genomic dsRNA into a cell to reproduce itself using the DNA of the invaded cell. RNAi
protects the cell by destroying the viral RNA through the RISC mechanism.
siRNA is more powerful than antisense in silencing genes, but it has its difficulties. It
is not long lasting, it may silence off-target sites and it has been not been easy to inject
systemically as a therapy. We have directly compared siRNA with AS-ODN to inhibit
the Beta-1 adrenergic receptor gene [13]. The effect was measured on blood pressure in
hypertensive rats and on measures of heart performance, because beta blockers have long
been used for hypertension and heart failure treatments. The siRNA and AS-ODN were
injected systemically in a Lipofectamine vehicle. The result was a significantly better effect
on lowering blood pressure and improving heart performance with the siRNA than with the
AS-ODN. Both approaches lasted about 1 week with a single injection [13].
12.1.1.5 MicroRNA
MicroRNA (miRNA) offers completely new possibilities for gene modification, cell therapy
and drug development. It is involved in almost every biological process regulated by genes
and its absence or mutations could be the cause of many disease states from birth defects
to cancer.
Although miRNAs were discovered over 20 years ago in
C. elegan
s [14] and later
found in mammals, we are still in an early stage of discovering how many there are,
