(RNAi) is a gene silencing phenomenon first identified in the
nematode, C. elegans, but was subsequently found to occur in
higher organisms including humans. It probably evolved as an ancient
defense mechanism for cells to fend off mobile genetic elements, such
as RNA viruses and transposons, but today it has been implicated in a
growing number of cellular processes.
As discussed by
Stevenson, RNAi involves sequence specific degradation of target RNAs
triggered by the formation of double stranded RNA (dsRNA). When it
occurs naturally, long dsRNA is processed to short interfering RNAs
(siRNAs) 21-24 bases in length by a dsRNA-specific endonuclease named
Dicer (Figure). They are incorporated into a nuclease complex
referred to as the RNA-induced silencing complex or RISC. Unwinding
of the siRNAs activates and directs RISC to the target RNAs, which
are cleaved and degraded. The complementarity between the siRNA and
the target RNA determines the sequence specificity of RNAi.
Mechanism of Gene Silencing by RNA Interference. The pathway of RNA interference can be broken down into two main phases. In the first phase, long double-stranded RNA (dsRNA) is recognized and processed by Dicer, an RNase III enzyme, into duplexes of short interfering RNA (siRNA) of 21 to 24 nucleotides (nt) in length. Exogenous synthetic siRNAs or endogenous expressed siRNAs can also be incorporated into the RNA-induced silencing complex (RISC), thereby bypassing the requirement for dsRNA processing by Dicer. In the second phase, siRNAs are incorporated into the multiprotein RISC.
A helicase in RISC unwinds the duplex siRNA, which then pairs by means of its unwound antisense strand to messenger RNAs (mRNAs) that bear a high degree of sequence complementarity to the siRNA. An as yet unidentified RNase (Slicer) within RISC proceeds to degrade the mRNA at sites not bound by the siRNA. Cleavage of the target mRNA begins at a single site 10 nucleotides upstream of the 5'-most residue of the siRNA–target mRNA duplex. Although the composition of RISC is not completely known, it includes members of the Argonaute family that have been implicated in processes directing post-transcriptional silencing. ADP denotes adenosine diphosphate, Pi inorganic phosphate, P phosphate, and OH hydroxyl. The figure was adapted from Stevenson. Nat Rev Immunol 2003;3:851-8.
with permission. Stevenson, M. N Engl J Med 2004;351:1772-7. Copyright
© 2004 Massachusetts Medical Society. All rights reserved.
An important advance
in the RNAi field was the discovery that exogenous synthetic siRNAs
or endogenously synthesized siRNAs driven by viral vectors could be
incorporated into RISC and induce sequence-specific degradation of
target RNAs. This created an extremely powerful tool for scientists
to “knock down” expression of genes of interest simply by
adding synthetic RNA duplexes to the medium of cultured cells,
introducing viral vectors that express siRNAs into cells or even
generating transgenic animals that synthesize siRNAs.
RNAi is much more
complex than outlined here, and there are many technical difficulties
that complicate the use of RNAi to knock-down gene expression in
experimental systems. Nevertheless, RNAi has stimulated considerable
interest in the pharmaceutical/biotech industry as a potential
therapeutic agent for human disease. The best examples to date have
to do with treatment of infectious diseases, such as those caused by
HIV, hepatitis viruses and poliovirus, as well as cancers that are
mediated in part by overactive oncogenes. In the case of viral
infections, interfering RNAs could be targeted to viral transcripts
required for viral replication or survival. In the second case, using
RNAi to silence expression of BCR-ABL, the fusion gene that results
from the Philadelphia chromosome translocation in chronic myelogenous
leukemia or mutated RAS oncogenes that drive several types of cancer
would be appealing.
attention to date, but of probably at least as much interest to
readers of GGH, is the potential use of RNAi to knock down
expression of mutant alleles in dominantly inherited genetic disease.
In concept, siRNAs could be tailored to distinguish mutant from
normal (wild type) alleles and block only mutant allele expression.
This could convert a dominant negative disorder, ie, a disorder in
which the product of the mutant allele interferes with the function
of the normal (wild type) allele product, to a disorder that results
from haploinsufficiency or functional loss of one allele. For
families in which both forms occur, manifestations are usually milder
in the form resulting from haploinsufficiency, ie, osteogenesis
imperfecta type I – haploinsufficiency vs osteogenesis type II
– dominant negative. Thus, there is potential benefit from this
excitement and promise of therapeutic RNAi, there are many obstacles,
the greatest of which is delivery. Systemically delivered siRNAs face
degradation by nucleases, and the use of viral vectors to target
organs of interest is still in its infancy. A recent publication by
Soutscheck and colleagues provides evidence that chemically modified
siRNAs can successfully knock down endogenous genes in living mice.
More specifically, they targeted expression of the gene encoding
apoprotein B (apoB) in the mouse liver and jejunum where it is
known to be expressed at high levels with 2 siRNAs known to silence
apoB in cultured cells. They modified the apoB siRNAs
by chemically stabilizing their backbone and also by adding
cholesterol to their 3’ end. The modified siRNAs were then
compared to unmodified apoB siRNAs and other controls.
The results showed
that the cholesterol-conjugated apoB siRNAs were significantly
more stable in serum than their unconjugated counterparts. When
administered intravenously, one of the conjugated apoB siRNAs
was very effective at lowering apoB mRNA and apoB
protein levels, as well as total cholesterol and LDL cholesterol.
They observed no evidence of “off-target” effects, that
is, effects attributed to silencing of genes other than apoB
or other obvious complications from the injections. The authors
concluded that exogenously administered chemically modified siRNAs
can potentially be used to silence expression of endogenous genes
involved in human disease.
M: Therapeutic potential of RNA interference. N Eng J Med
J,Akinc A, Bramlage B, et al. Therapeutic silencing of an endogenous
gene by systemic administration of modified siRNAs. Nature
Comment: RNAi has had a major impact on science since its
relatively recent discovery. It is still not entirely clear how it
works and there remain concerns about specificity and the so-called
off target effects on genes other than specifically targeted genes.
Nevertheless, it has great promise as a means to treat not only
cancer and infectious diseases, but genetic diseases in which mutant
alleles differing from their normal alleles by only a single base can
be specifically targeted. It will probably be years before such
treatment becomes realistic for humans, but the success of
substantially knocking down apoB expression by systemically
administering chemically modified apoB siRNAs in mice is very
encouraging. One note of caution is that the growing skeleton may be
difficult to target because the cartilaginous growth plate is
relatively avascular compared to most tissues such as liver and gut.
William A. Horton, MD