siRNA

Over the past 20 years the idea of targeting the molecular level of
diseases by modifying gene expression patterns has attracted great
interest. Based on the sequencing of the human genome and our
burgeoning understanding of the molecular causes of diseases, the
possibility of turning off pathogenic genes at will appears to be an
appealing approach for treatment of a wide variety of clinical
pathologies. Rationale-based drug design for development of
compounds suited to specifically switch-off target genes holds promise
in finding therapies for complex diseases, such as diabetes or cancer,
with fewer side effects.

In 2002, RNAi (RNA interference) as a further mRNA-transcript-

targeting strategy came to the attention of the general public by the
technique’s designation as the ‘breakthrough of the year’ by Science.
Since then, the number of publications utilizing this novel technology
has increased exponentially, which has led to a more profound
understanding of the underlying mechanism of RNAi and has provided
indications for potential therapeutic applications.

RNAi: The Mechanism

The first observations of gene silencing by dsRNA (double-stranded

RNA) derived from experiments with plants. In the late 1980s and early
1990s, genetic experiments conducted on petunias yielded bizarre
results. Introducing numerous copies of a gene coding for a deep
purple colour led to plants with white or patchy blossoms and not to a
darker hue, as would have been expected. Somehow, the introduced
genes had silenced both themselves and the plant’s intrinsic colour-
coding genes.

The puzzling findings of colour alterations in petunias after transfection

remained unexplained for nearly a decade, until, in 1998, Fire and co-
workers reported that injection of dsRNA into Caenorhabditis elegans
resulted in potent gene silencing. dsRNA several hundred bases in
length not only caused significant gene silencing, but was clearly more
active than the corresponding singlestranded antisense molecules, a
revolutionary finding in the field of molecular biology. Since the
introduced dsRNA molecules interfered with the function of the
targeted gene, the process was coined ‘RNA interference’.
The unprecedented potency of gene silencing by RNAi prompted
intensive research efforts over the next couple of years, leading to our
current understanding of the RNAi machinery. These first mechanistic
clues for the RNAi machinery were obtained from experiments using
Drosophila extracts. In essence, RNAi is initiated by long stretches of
dsRNA that undergo processing by an enzyme referred to as Dicer.
Dicer cuts the long stretches of dsRNA into duplexes with 19 paired
nucleotides and two nucleotide overhangs at both 3’-ends. These
duplexes are called siRNA (short interfering RNA). The double-stranded
siRNA then associates with RISC (RNA-induced silencing complex), a
fairly large (approx. 160 kDa) protein complex comprising Argonaute
proteins as the catalytic core of this complex. Within RISC, the siRNA is
unwound and the sense strand removed for degradation by cellular
nucleases. The antisense strand of the siRNA directs RISC to the target
mRNA sequence, where it anneals complementarily by Watson–Crick
base pairing. Finally, the target mRNA is degraded by RISC
endonuclease activity.
Although gene silencing by ASOs was known to be a reliable method
for targeted gene down-regulation in human cell culture, RNAi was first
studied in C. elegans and plants and it was unclear whether it would
work in mammalian cells too. In 2001, Tuschl and co-workers provided
the first evidence that siRNAs can mediate sequence-specific gene
silencing in mammalian cells and that the dicing step can be bypassed
by the transfection of siRNA molecules into cells. From that time on it
was evident that RNAi could be used to study, and maybe even
influence, the molecular basis of human disease.

RNAi represents an outstanding strategy for modulating gene

expression. In contrast with other mRNA targeting strategies, RNAi
takes advantage of the physiological gene-silencing machinery, which
might explain the excellent potency of RNAi. The finding that long
dsRNAwas processed to siRNA prompted a search for interfering RNA
encoded by the cellular genome itself. To date, a still increasing
number of similar, yet different, short RNA species have been
identified called miRNA (microRNA), tiny non-coding RNA,
heterochromatic RNA or small modulatory RNA. This classification may
be somewhat artificial and many processes involving short RNA are still
incompletely understood. miRNA, as the best characterized short RNA
species, are approx. 70 nucleotides in length and transcribed from
their coding genes to form a double-stranded stem–loop hairpin
molecule by self-complementary binding. They have been found in all
multicellular organisms investigated so far, from Drosophila to man.
Approx. 250 different miRNA sequences are predicted in humans from
genomic screening, with some 175 miRNAs already confirmed
experimentally. The miRNA hairpin is processed by Dicer and
incorporated into RISC. Although even a single base mismatch may
completely abrogate siRNA silencing, translational inhibition by
miRNAs tolerates a mismatch to the mRNA target sequence. As a
consequence, a single miRNA is able to regulate many, maybe even
hundreds of, different genes, which may contribute significantly to
siRNA off-target effects.
The long dsRNA cannot be used in mammalian cells for gene silencing
due to the fact that introduction of long dsRNA elicits a strong antiviral
response that obscures any gene-specific silencing effect. This makes
the process of selecting sequences for generation of siRNA a very
tricky process.
siRNA Designing Strategies:
Default parameters: AA(19mer)

The default criteria selects target sequences of 21 nucleotides that

begin with AA and are located within a region of the coding sequence
that is within 50-100 nucleotides of the AUG start codon and within 50-
100 nucleotides from the termination codon. The presence of AA at the
start of the sequence allows for the use of dTdT at the 3’-end of the
antisense sequence. The sense strand can also be synthesized with
dTdT at the 3’ end, because only the antisense strand is involved in
target recognition. The use of dTdT makes the siRNA duplex more
resistant to exonuclease activity. Because a number of reports have
demonstrated that the presence of AA at the beginning of the target
sequence is not an absolute requirement, the selection program
includes the option to search for sequences that begin with other
nucleotide pairs. Beginning with the AUG start codon of your transcript,
scan for AA dinucleotide sequences. Record each AA and the 3'
adjacent 19 nucleotides as potential siRNA target sites.

This strategy for choosing siRNA target sites is based on the

observation by Elbashir et al. that siRNAs with 3' overhanging UU
dinucleotides are the most effective. This is also compatible with using
RNA pol III to transcribe hairpin siRNAs because RNA pol III terminates
transcription at 4-6 nucleotide poly(T) tracts creating RNA molecules
with a short poly(U) tail.

GC Content

The G-C content of the sequence is also used as a condition for

selecting target sequences. Ideally the GC content will be less than
50%, although successful gene silencing has been reported with
siRNAs that have G-C contents between 50 and 60%. The default
parameter selects for a G-C content in the 40-50% range, however,
options are available that allow for selection over wider ranges.

Stretches of Nucleotide Repeats

The default mode avoids sequences with repeats of three or more G’s
or C’s, as their presence initiates intra-molecular secondary structures
preventing effective siRNA silencing hybridization. As an option, repeat
stretches of A’s and T’s can also be eliminated, as they tend to reduce
the specificity of the target sequence.
Blast Search

Once a target sequence has been chosen, a BLAST search is initiated

to ensure that your target sequence is not homologous to other gene
sequences. Target sequences that have more than 15 contiguous
bases pairs of homology to other genes in the NCBI database are
eliminated.

siRNA delivery options

Delivery of siRNA directly in cells can be achieved by using
microinjection or electroporation. Another popular option is the use of
transfection reagent. Several companies offer specialized siRNA-
delivery reagents. Usually RNAi effect is seen within 4 hours and the
maximum down regulation observed in 24-48 hrs. The effect lasts
several cell generations and from 4-10 days depending on cell culture
type.

Methods for generating siRNAs

Short siRNA molecules can be prepared by direct chemical synthesis or
transcription driven by RNA polymerase promoters. In term of high-
throughput applications, vector- based strategies are favoured
because such strategies enjoy the advantages of much lower cost and
ability to regenerate. Transfection efficiency of siRNA into cells
depends on cell types and the RNAi effect of synthetic siRNA only
sustain for a period of time. The advantage of the vector-based siRNA
is the capability of removing those cells that are not transfected with
the plasmids by selecting the transfected cells with antibiotic resistant
genes. Virus vectors also enable the delivery of siRNA expression
cassettes into cells with higher transfection efficiency, and in case of
lentivirus and retrovirus, it is easy to make stable knockdown cells by
integration into the genome.

siRNA production by pol III promoters

The use of pol III promoters in vectors for encoding siRNA was
pioneered by a number of groups in early 2002. The vectors contain a
single pol III promoter followed by a segment of DNA that encodes a
short hairpin RNA (shRNA). The shRNA closely resembles the structure
of microRNAs and will be integrated into the RISC complex in the same
way as the normal siRNA does. The backbones of these constructs
were initially plasmids, but the grafting the shRNA expressing
cassettes into different viral vectors such as lentivirus, retrovirus and
even adenovirus also explored successfully to facilitate the
transfection of cells that were hard to penetrate, such as primary cells
and suspension cells. Another strategy of encoding siRNA that has gain
more popularity is a vector that contains dual pol III promoters
arranged in a convergent manner that drive the expression of two
short complementary RNA strand from a single 19 bp DNA region.
While being as effective as the shRNA encoding vectors, these dual-
promoter vectors are firstof all more robust for cloning both because of
the lack of secondary structure in the siRNA coding region, and much
lower level of DNA synthesis errors due to the shorter sequence.

A third method is a technique using two tandem pol III promoters to

drive the expression of siRNA from separate 19 mer DNA fragments.
This method might not be widely used due to the fact that construction
of such a plasmid is considerably more cumbersome than the other
two methods. This method as well as the shRNA method, however,
offers the possibility to integrate mismatches into the resulting siRNA,
thus possibly enhance the efficacy of the siRNA or increase the
frequency of effective siRNA.

H1 and U6 promoters from human and mouse are the most widely
used pol III promoters in the siRNA vector construction. Other
promoters that have been used include promoters of tRNAVal and
tRNAMet. All these pol III promoters are active in a large variety of
cells, and appear to perform similarly in different species. This makes it
very easy to use the same vector in different experimental systems.
One interesting feature of the pol III promoters is that they can be
easily transformed into inducible promoters. This feature is very
important for some functional studies and screening.

siRNA production by other promoters

Other promoters that have been used for siRNA production include T7
promoter and CMV promoter. Since T7 promoter does not work in
mammalian cells, strategies employing T7 or similar promoters
normally can only support siRNA production in test tubes, but not
within cells. People have overcome this shortcoming by either pre-load
the promoter with CMV promoter is a much stronger promoter
compared to other pol III promoters, and this can allow more siRNA
molecules to be transcribed from a given amount of DNA vector. But
since CMV promoter is an RNA polymerase II
(pol II) promoter, the resulting transcripts are normally capped at the
5’-end and tailed at the 3’-end with a long poly (A) sequence. It
appears that such modifications are well tolerated as shown in the
efficacy assays. Due to the potentially higher degree of flexibility in
enabling different spatial and temporal expression patterns, these
types of vector-based siRNA strategies might be much more useful for
in vivo research purposes and for siRNA based gene therapy at a later
stage although pol III based vectors seem to be able to satisfy most of
the gene knock-down needs in cultured cells.
Delivering RNAi therapeutics
The ~14 kDa mass and polyanionic charge of a typical 21 bp siRNA
duplex ensure that achieving good tissue bioavailability is often a
greater challenge than lead selection. Despite this, both local and
systemic delivery to various tissue and cellular compartments have
been demonstrated preclinically. For local delivery, administration of
unmodified siRNA in simple formulations such as saline has resulted in
target mRNA knockdown in a wide variety of tissues, including the
respiratory and urogenital epithelia, central nervous system and the
eye. Most of the local delivery studies have been in mice, and involve
substantial substantial doses, presumably associated with high local
concentrations that drive cellular uptake. Although the exact
mechanism of uptake in most of these cases remains unknown, some
notable effects have been observed, including decreasing respiratory
syncitial virus (RSV) or parainfluenza virus (PIV) infection in respiratory
epithelium, herpes simplex virus-2 infection in the vaginal epithelium
[11], 2',3'-cyclic nucleotide 3'- phosphodiesterase expression in
oligodendrocytes, huntingtin expression in neurons, and vascular
endothelial growth factor (VEGF)-A [14] and VEGF receptor (VEGFR)I
expression in ocular tissues.

Systemic delivery of RNAi therapeutics offers both the greatest

opportunities and challenges. An unmodified saline-formulated siRNA
injected intravenously is subject to simultaneous RNase-mediated
degradation and rapid renal excretion. Hence, any attempts at
systemic delivery must involve mechanisms for increasing the
circulation half-life (t1/2) of the siRNA, its distribution to an appropriate
tissue compartment and then its uptake, followed by intracytoplasmic
release and activity. In this regard, the greatest success to date has
been achieved with respect to hepatic delivery, with three distinct
approaches, each involving a conjugated or formulated siRNA,
warranting review.

Soutschek et al. [16] were the first to report a delivery strategy with
significant translational potential. In a mouse system, they
demonstrated that cholesterol conjugation to the sense or 'passenger'
strand of an ApoB-specific siRNA administered intravenously resulted
in a significant reduction in clearance and an associated 16- fold
increase in t1/2, relative to the unconjugated control Subsequent work
elucidated that the change in pharmacokinetic (PK) characteristics was
secondary to loading of the conjugated siRNA into circulating
lipoprotein particles via the appended cholesterol moiety. This also
facilitated receptor mediated uptake of the siRNA into hepatocytes,
and resulted in ~60% knockdown of ApoB mRNA in the mouse liver.
Notably, intravenous administration of cholesterol-conjugated siRNAs
targeting ApoB also resulted in efficient knockdown of ApoB mRNA in
the jejunum.

More recently, conjugation-based approaches with other ligands have

been used, such as prostate-specific membrane antigen aptamer-
siRNA conjugates to deliver drug to tumor cells in vivo.

Two groups have reported liposomal nanoparticle (LNP)-mediated

delivery of siRNA to the liver, demonstrating knockdown in the mouse
and the nonhuman primate. In both studies, the LNP formulation, also
known as a 'stable nucleic acid lipid particle' (SNALP), was composed
of several non-covalently associated components (an ionizable lipid,
polyethylene glycol
(PEG)-lipid for prolonging t1/2, cholesterol, and a neutral lipid) which
self-assembled and encapsulated the siRNA were able to show that
relative to unencapsulated siRNA, LNPs that encapsulated a variety of
anti-hepatitis B virus (HBV) siRNAs showed reduced plasma clearance
and efficient hepatic uptake with dose-dependent, potent (>1 log10)
and durable knockdown of circulating HBV DNA levels in a mouse
model of infection. Zimmermann et al. also demonstrated hepatic
uptake of LNPs with specific knockdown of ApoB in the non-human
primate. The pharmacodynamic (PD) profile in this study was notable
not only for the extent of hepatic ApoB mRNA knockdown (>80%), but
also for its translation to systemic lowering of lowdensity lipoprotein
cholesterol (LDLc) (by 82%) which persisted for several weeks after a
single dose. The latter observation was particularly interesting because
the drug was undetectable in the liver of animals beyond 48 hrs,
suggesting an apparent PK-PD hysteresis. Recent work with more
sensitive quantitative PCR assays has clarified that in fact the drug is
present (and presumably stabilized in RISC) at all time points when PD
is observed after LNP-mediated siRNA delivery (Alnylam
Pharmaceuticals Inc., unpublished observations, Renta Hutabarat The
mechanism of ionizable LNP-mediated delivery has been dissected,
and in vivo it involves the opsonization of the LNP by ApoE in the
circulation, LDL receptor-mediated uptake of the opsonized particles by
hepatocytes, and then endosomal release of siRNA into the cytoplasm
(Alnylam Pharmaceuticals Inc.).
The third systemic delivery approach of note involves multicomponent
complexes. Rozema et al. studied particles composed of siRNA
conjugated with a dynamic polyconjugate and complexed with PEG to
extend t1/2, plus a liver targeting ligand, N-acetyl galactosamine
(NAG). This strategy was successfully used in rodents and non-human
primates to show potent and durable hepatic ApoB knockdown with
associated reduction in circulating LDLc. Mechanistically, the role of
NAG was crucial, as its replacement with mannose abrogated uptake
by hepatocytes and instead directed delivery to other hepatic cellular
compartments such as Kupffer cells. Bartlett et al. [24] used a similar
multicomponent concept but complexed a ribonucleotide reductase
(RRM2) siRNA to cationic cyclodextrin, along with PEG and a targeting
ligand, transferrin. In this system, the transferrin allowed delivery to
extrahepatic sites, and target mRNA knockdown was achieved in a
subcutaneous tumor xenograft.

Of the three hepatic delivery concepts above, the LNP mediated

approach has received the most attention. Recently, optimization of
LNP structure and function has resulted in potent in vivo knockdown at
doses as low as
0.01 mg/kg (whereas in local delivery (see above) doses are typically
>1 mg/kg). LNP-mediated delivery of RNAi therapeutics has now been
applied to several different target mRNAs other than ApoB and has
been described in five species including the mouse, rat, hamster,
guinea pig and non-human primate (Alnylam Pharmaceuticals Inc.,
unpublished observations).

In terms of pharmacology, the LNP experiments have fully validated

the drug-like behavior of siRNAs. For all the LNP studies cited, there is
a consistency of observations with respect to PD onset, which
generally occurs within 24 hrs with peak effects at 48-72 hrs; duration,
which lasts several weeks depending on the potency (and perhaps
stability) of the siRNA concerned; strict dose dependency; and return
of PD to baseline. Akinc et al. also elegantly showed reproducibility of
effects with multiple treatment cycles by giving a LNP-formulated
Factor VII siRNA once monthly. Many studies have also confirmed the
RNAi mechanism of action in vivo by demonstrating (via 5" rapid
amplification of cDNA ends assay) the anticipated cleavage site in the
target mRNA and the lack of effect by identical LNP-mediated delivery
of an irrelevant siRNA. Overall, the potency, predictable and
reproducible pharmacology across different targets and species (as
anticipated for an endogenous mechanism), the selectivity and the
clear mechanism of action suggest a robust translational potential for
LNPbased hepatic delivery of RNAi therapeutics. To fully exploit the
therapeutic potential of RNAi, systemic delivery beyond the liver will
need to be accomplished, and indeed some creative approaches have
been reported. With optimization to further increase the circulation
t1/2 relative to LNPs delivering to the liver, LNP mediated extrahepatic
delivery to subcutaneous tumors in mice has been achieved.
Antibodies facilitating siRNA delivery offer several advantages,
including an intrinsic targeting mechanism and good PK properties.

Antibody-protamine fusion proteins have been shown to complex with

siRNAs and reported to deliver specifically to subcutaneous tissue and
to lung tumors in mice. By bringing together the advantages of LNPs
and antibodies, Peer et al. devised sophisticated neutral LNPs with a
covalently attached antibody against β7 integrin and carrying an
anticyclin D1 siRNA complexed with protamine. Using this approach,
they were able to show specific knockdown of cyclin D1 in gut
mononuclear leucocytes with translation to therapeutic effects in a
mouse model of colitis. Oral delivery of RNAi therapeutics would offer
the greatest convenience if translatable clinically. Recently, glucan-
encapsulated siRNA particles were reported to be efficient oral delivery
vehicles to target gut macrophages [33]. Although the extrahepatic
delivery concepts above demonstrate progress, further work is
required to reproduce the observations and demonstrate a clear RNAi-
dependent mechanism of action, much as described for extrahepatic
delivery to subcutaneous tumors .

Clinical RNAi pipeline

A total of 14 RNAi therapeutic programs have entered clinical practice
in the past decade. Of the 14 programs, seven involve local/topical
delivery to the eye (four), respiratory tract (two) and skin (one). The
remaining seven are systemic programs targeting liver (two), hepatic
and extrahepatic cancer (three), leukocytes (one) and kidney (one). Six
of the 14 programs (ALN-RSV, ALN-VSP, ALN-TTR, TD101, bevasiranib,
Bcr-abl) have clear target validation, and the other eight programs
address targets of significant interest (for example, p53 for kidney
injury or RTP801 for age-related macular degeneration (AMD), but lack
robust prior validation. Equally, only a few programs (ALN-TTR, ApoB-
SNALP) offer the possibility of early PD demonstration of target
knockdown, whereas the rest will require further development into
phase II before adequate proof of concept is achieved. ALN-RSV has
achieved initial proof of concept.

Several have targets (RSV (N) nucleocapsid protein, p53, TTR, ApoB,
K6a) that would be 'undruggable' by small molecules or protein
moieties. The attempt to target Bcrabl and mutant K6a shows the
potential for allele-specific knockdown. Finally, ALN-VSP builds on data
from animal models in which five transcripts were suppressed in
parallel by simultaneously targeting two transcripts, VEGF and kinesin
spindle protein (KSP), a clear advantage in complex indications such as
cancer. From an overall safety perspective, almost 1500 patients and
healthy volunteers will soon have been enrolled onto RNAi clinical
programs. In total, 1065 have already been studied across all phases of
clinical development, including 522 in the bevasiranib phase III studies.

These numbers are important because a unique siRNA related adverse

event occurring at a high incidence (for example >10%), would
probably already have been identified.
Equally, no data have emerged to suggest serious adverse events
linked to siRNA exposure, and none of the programs have been placed
on 'clinical hold' by regulatory agencies, a probable outcome if
untoward safety events were being uncovered. The largest fraction of
the safety experience (1284 subjects) relates to the three ocular
programs (bevasiranib, PF-04523655 and AGN211745) and to the
respiratory program (ALNRSV), which suggests good local tolerability
to RNAi therapeutics. ALN-RSV (Alnylam Pharmaceuticals Inc.,
Cambridge, MA, USA) in phase 2b is currently the most advanced
program in the global RNAi pipeline. It uses an unmodified siRNA
formulated in saline targeting the RSV N gene transcript. Three phase I
studies (two intranasal [59] and one inhalational [60]) showed safety
and tolerability at doses up to 3 mg/kg. In 2008, this program reported
the first initial human proof of concept for an RNAi program.

Conclusions
With barely a decade since its initial characterization, the translation of
RNAi biology toward RNAi therapeutics has progressed at a rapid pace.
Moreover, the following years promise to be crucial for demonstration
of the safety and efficacy of this new method in controlled, randomized
studies. Certainly, much remains to be accomplished. In delivery
research and preclinical studies, further advancement of the field will
require continued progress in conjugation and formulation strategies. It
would be naive to believe that any one single technology will provide
all the solutions, so it is gratifying to see such a broad-based effort
across multiple delivery technologies under investigation in academia
and industry. In development efforts, many milestones remain to be
achieved, including the conduct of large phase III studies and
regulatory approvals. Of course, it is likely that there will be product
failures, which could be accounted for by a number of factors including
target selection, delivery technologies, clinical trial design and even
commercial considerations. Nevertheless, the steadfast commitment in
the field is apparent in the advancement of this critically needed
innovation to patients.