REVIEWS

Knocking down barriers:

advances in siRNA delivery
Kathryn A. Whitehead*, Robert Langer*‡ and Daniel G. Anderson‡
Abstract | In the 10 years that have passed since the Nobel prize-winning discovery of RNA
interference (RNAi), billions of dollars have been invested in the therapeutic application
of gene silencing in humans. Today, there are promising data from ongoing clinical trials
for the treatment of age-related macular degeneration and respiratory syncytial virus.
Despite these early successes, however, the widespread use of RNAi therapeutics for
disease prevention and treatment requires the development of clinically suitable,
safe and effective drug delivery vehicles. Here, we provide an update on the progress
of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and
intracellular delivery of nucleic acids.

RNA interference
RNA interference (RNAi) gained international attention of the siRNA is cleaved12. The activated RISC, which
(RNAi). A fundamental in 1998 when Fire, Mello and colleagues discovered the contains the antisense strand (or guide strand) of the
pathway in eukaryotic cells by ability of double-stranded RNA to silence gene expres- siRNA, selectively seeks out and degrades mRNA that is
which a short piece of RNA is sion in the nematode worm Caenorhabditis elegans 1. complementary to the antisense strand13 (FIG. 1). The cleav-
able to induce the destruction
Three years later, Tuschl and co-workers published their age of mRNA occurs at a position between nucleotides 10
of mRNA containing a
complementary sequence. celebrated proof-of-principle experiment demonstrating and 11 on the complementary antisense strand, relative to
that synthetic small interfering RNA (siRNA) could achieve the 5′-end14. The activated RISC complex can then move
Small interfering RNA sequence-specific gene knockdown in a mammalian cell on to destroy additional mRNA targets, which further
(siRNA). RNA fragments line2. The first successful use of siRNA for gene silencing propagates gene silencing 15. This extra potency ensures a
approximately 21–23
nucleotides long that are
in mice was achieved for a hepatitis C target shortly therapeutic effect for 3–7 days in rapidly dividing cells,
capable of inducing the thereafter 3. Since that time, the biotechnology sector has and for several weeks in non-dividing cells16. Eventually,
sequence-specific destruction made considerable efforts in the advancement of siRNA siRNAs are diluted below a certain therapeutic threshold
of complementary mRNA. therapeutics for the treatment of various disease targets, or degraded within the cell, and so repeated administra-
including viral infections4,5 and cancer 6–8. tion is necessary to achieve a persistent effect.
RNA-induced silencing
complex RNAi is a fundamental pathway in eukaryotic cells Theoretically, when using appropriately designed
(RISC). The protein complex by which sequence-specific siRNA is able to target and siRNA, the RNAi machinery can be exploited to silence
responsible for the binding cleave complementary mRNA2. RNAi is triggered by the nearly any gene in the body, giving it a broader thera-
and unwinding of the presence of long pieces of double-stranded RNA, which peutic potential than typical small-molecule drugs.
double-stranded siRNA
within the cytoplasm,
are cleaved into the fragments known as siRNA (21–23 Indeed, it has already been reported that synthetic
and for the subsequent nucleotides long) by the enzyme Dicer 9. In practice, siRNAs are capable of knocking down targets in various
identification and destruction siRNA can be synthetically produced and then directly diseases in vivo, including hypercholesterolaemia17, liver
of the target mRNA. introduced into the cell, thus circumventing Dicer cirrhosis18, hepatitis B virus (HBV)4,19, human papilloma-
mechanics (FIG. 1). This shortcut reduces the potential for virus20, ovarian cancer 21 and bone cancer 22. In order for
*Department of an innate immune interferon response and the shutdown these advances to be implemented in a clinical setting, safe
Chemical Engineering,
of cellular protein expression that can occur following the and effective delivery systems must be developed. While
‡
The David H. Koch Institute
for Integrated Cancer interaction of long pieces (>30 nucleotides) of double- ‘naked’, chemically modified siRNA has shown efficacy
Research, Massachusetts stranded RNA with intracellular RNA receptors10. in certain physiological settings such as the brain23 and
Institute of Technology, Once siRNA is present in the cytoplasm of the cell, the lung24, there are many tissues in the body that require
Cambridge, Massachusetts it is incorporated into a protein complex called the RNA- an additional delivery system to facilitate transfection.
02142, USA.
induced silencing complex (RISC)11. Argonaute 2, a multi- This is because naked siRNA is subject to degradation
Correspondence to D.G.A.
e-mail: dgander@mit.edu functional protein contained within RISC, unwinds the by endogenous enzymes, and is too large (~13 kDa)
doi:10.1038/nrd2742 siRNA, after which the sense strand (or passenger strand) and too negatively charged to cross cellular membranes.

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REVIEWS

There are several tissues that are amenable to topical

or localized therapy, including the eye, skin, mucus
membranes, and local tumours 25–28 (TABLE 1) . local
siRNA delivery is particularly well-suited for the treat-
ment of lung diseases and infections. The direct instillation
of siRNA into the lung through intranasal or intra-
tracheal routes enables direct contact with lung epithelial
cells. These cells play a part in a myriad of lung con-
ditions and infections, including cystic fibrosis, asthma,
influenza and the common cold24. It has been reported
that respiratory syncytial virus (RSV) replication can
be inhibited by nasally administered siRNA formu-
lated with or without transfection agents in mice29,30.
Progress in the treatment of RSV continues with Phase II
clinical trials using an aerosolized siRNA delivery
system31. Intratracheal administration of siRNA has
also been reported to offer prophylactic and therapeu-
tic effects in the treatment of severe acute respiratory
syndrome32.
Another example of local delivery is direct intra-
tumoral injection of siRNA delivery complexes into
various mouse xenograft models. siRNA complexed with
the delivery agent polyethyleneimine (PEI) was shown
to inhibit tumour growth upon intratumoral injection
in mice bearing glioblastoma xenographs28. Niu and
co-workers have also reported naked siRNA efficacy
upon direct injection into a subcutaneous cervical cancer
model in mice20.

Barriers to systemic siRNA delivery in vivo

In contrast to the direct accessibility of localized targets,
many tissues can only be reached through the systemic
administration of delivery agents in the bloodstream.
siRNA formulations for systemic application face a series
Figure 1 | The mechanism of rNA interference. Long double-stranded RNA (dsRNA) of hurdles in vivo before reaching the cytoplasm of the
is introduced into the cytoplasm, where it is cleaved into small interfering RNA (siRNA) by target cell (FIG. 2). Post-injection, the siRNA complex
the enzyme Dicer. Alternatively, siRNA can be introduced directly into the cell. The siRNA must navigate the circulatory system of the body while
is then incorporated into the RNA-induced silencing complex (RISC), resulting in the avoiding kidney filtration, uptake by phagocytes, aggre-
cleavage of the sense strand of RNA by argonaute 2 (AGO2). The activated RISC–siRNA gation with serum proteins, and enzymatic degradation
complex seeks out, binds to and degrades complementary mRNA, which leads to the by endogenous nucleases33.
silencing of the target gene. The activated RISC–siRNA complex can then be recycled for
Phagocytosis serves as a significant immunological
the destruction of identical mRNA targets.
barrier, not only in the bloodstream but also in the
extracellular matrix of tissues. Phagocytic cells such as
macrophages and monocytes remove foreign material
The issue of effective and non-toxic delivery is a key chal- from the body to protect against infection by viruses,
lenge and serves as the most significant barrier between bacteria and fungi. unfortunately, phagocytes are also
siRNA technology and its therapeutic application. highly efficient at removing certain therapeutic nano-
complexes and macromolecules from the body, and steps
Modes of siRNA administration must be taken to avoid opsonization when designing
The ease of siRNA delivery is partly dependent on the drug delivery vehicles33.
Antisense strand accessibility of the target organ or tissue within the Egress from the bloodstream and across the vascu-
The strand of the siRNA body. localized siRNA delivery — that is, application lar endothelial barrier poses a significant challenge for
molecule that is of siRNA therapy directly to the target tissue — offers delivery of siRNA to many tissues within the body. In
complementary to the target
several benefits, including the potential for both higher general, molecules larger than 5 nm in diameter do not
mRNA, which activates RISC
and has an important role in bioavailability given the proximity to the target tissue, readily cross the capillary endothelium, and therefore
target mRNA identification and reduced adverse effects typically associated with will remain in the circulation until they are cleared from
and destruction. systemic administration. By contrast, systemic delivery, the body. There are certain tissues, however, that allow the
meaning the intravenous injection of delivery particles entry of larger molecules, including the liver, spleen, and
Transfection
The process of delivering
that then travel throughout the body to the target organ some tumours. These organs allow the passage of mol-
nucleic acid material into or tissue, requires that particles have the ability to avoid ecules up to 200 nm in diameter, which can accommodate
the cell. uptake and clearance by non-target tissues (FIG. 2). a typical drug delivery nanocarrier 34.

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siRNAs can induce nonspecific activation of the immune

system through the Toll-like receptor 7 (TlR7) path-
way 38,39. This effect can be reduced by the incorporation
of 2′-O-methyl modifications into the sugar structure of
selected nucleotides within both the sense and antisense
strands38,40 (FIG. 3a). 2′-O-methyl modifications have also
been shown to confer resistance to endonuclease activity 41
and to abrogate off-target effects when incorporated
into the seed region, which corresponds to nucleo-
tides 2–8 on the antisense strand42. Other common
modification approaches to mitigate enzymatic deg-
radation include the introduction of phosphorothioate
backbone linkages at the 3′-end of the RNA strands to
reduce susceptibility to exonucleases. It is also possible
to incorporate alternative 2′ sugar modifications (for
example, a fluorine substitution) to increase resistance
to endonucleases43.
Another strategy to improve the therapeutic efficacy
of siRNA involves the conjugation of small molecules or
peptides to the sense strand of the siRNA. Several small
molecules have been reported to increase target-gene
knockdown in vitro, including membrane-permeant
peptides44 and polyethylene glycol (PEG)45. Of particu-
lar note are cholesterol-modified siRNAs, which have
demonstrated increased binding to serum albumin,
resulting in improved biodistribution to certain tar-
gets including the liver (FIG. 3b). Cholesterol-modified
siRNA were capable of silencing apolipoprotein B
(ApoB) targets in mouse liver and jejunum, and of
ultimately reducing total cholesterol levels46. Another
study by DiFiglia and co-workers details the ability of a
Figure 2 | Physiological barriers to the systemic delivery of small interfering rNA
cholesterol-modified siRNA to knockdown a gene asso-
(sirNA) nanoparticles. An injected nanoparticle must avoid filtration, phagocytosis
and degradation in the bloodstream (a); be transported across the vascular ciated with Huntington’s disease. A single intrastriatal
endothelial barrier (b); diffuse through the extracellular matrix (c); be taken up into injection was able to delay the abnormal behavioural
the cell (d); escape the endosome (e); and unpackage and release the siRNA to the phenotype observed in a rapid-onset mouse model of
RNA interference (RNAi) machinery (f). this disease23.
Given the success of cholesterol-modified siRNA
in vivo, Wolfrum and co-workers attempted to identify
alternative lipid-like molecules to serve as RNA con-
After an siRNA complex leaves the bloodstream, it jugates for improved delivery of siRNA47. Specifically,
must diffuse through the extracellular matrix, which is fatty acids and bile-salt derivatives were conjugated to
a dense network of polysaccharides and fibrous proteins siRNA and injected into mice and hamsters in order to
that can create resistance to the transport of macro- elucidate how modified siRNA conjugates interact with
molecules and nanoparticles35. This can slow or even the high-density lipoprotein (HDl) and low-density
halt the drug delivery process and create an additional lipoprotein (lDl) receptors that enable delivery to the
opportunity for nanoparticles to be taken up by resident liver. It was found that shorter fatty-acid chain lengths
macrophages. Having been taken up by the target cell, (<C18) did not induce gene knockdown, whereas bile-
particles must then escape the endosome to reach the salt derivatives and fatty-acid conjugates with longer
cytoplasm36. If the siRNA nanocomplex is unable to exit chain lengths enabled potent silencing in hepatocytes
the endosome, it will be trafficked through endomem- via the HDl receptor 47.
brane compartments of decreasing pH and be subject to Another example of the possibility of introducing
degradative conditions in the lysosome37. Finally, if for- beneficial modifications to nucleic acid therapeutics
mulated with delivery agents, siRNA must be released comes from the antisense drug mipomersen. Mipo-
from the carrier to the cellular machinery. mersen is a 2′-O-(2-methoxyethyl)-modified single-
stranded RNA molecule that is targeted to ApoB48,
Modified siRNA for improved delivery a protein that has been implicated in cardiovascular
Humans have evolved a number of host-defence mecha- disease. Isis Pharmaceuticals has reported promising
nisms against siRNA, as it is a feature of certain viral Phase II safety and efficacy results, and a Phase III trial
infections. However, chemical modifications can be to assess efficacy in patients with a familial history of
introduced into the siRNA molecule to evade immune hypercholesterolaemia is currently underway in a joint
defences in vivo. For example, many non-modified venture with Genzyme49.

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Table 1 | Modes of siRNA delivery and potential targets Biodistribution. The biodistribution of siRNA can be
significantly influenced through formulation with a
Mode of Potential organ target Potential disease target refs delivery vehicle. Systemic administration of synthetic
administration
delivery nanoparticles often results in accumulation in
Topical Eye Macular degeneration 102 the organs of the reticuloendothelial system, including
Skin Atopic dermatitis 25 the liver, spleen, kidneys and lungs16,55,56. It is no coin-
Vagina Herpes simplex virus 27 cidence that much of the successful siRNA delivery
seen in recent years has targeted disease within these
Rectum Inflammatory bowel disease 105
organs4,29,30,57,58.
Local/direct Lung SARS 32 Excretion through the kidney typically occurs for
Brain Huntington’s disease 23 molecules less than 50 kDa in size59. As such, naked
Spinal cord Chronic pain 90 siRNA experiences rapid renal clearance upon systemic
administration43. Several studies monitored the biodis-
Isolated tumour Glioblastoma multiforme 28
tribution of siRNA in mice after an intravenous injection
Systemic Liver Hypercholesterolaemia 57 and observed naked siRNA accumulation in the kidney
Heart Myocardial infarction 106 and urine within 5 minutes of administration60,61. By
Kidney Kidney disease 61 complexing siRNA with synthetic materials, the size
of the delivery nanoparticle can be increased to avoid
Metastasized tumours Ewing’s sarcoma 97 glomerular filtration through the kidneys and reserve
SARS, severe acute respiratory syndrome; siRNA, small interfering RNA. the siRNA for alternative organ targets59.
Additionally, it has been reported that certain siRNA
formulations are capable of accumulation in subcutan-
Properties of synthetic delivery nanoparticles eous tumours22,62. This phenomenon has been attributed
For tissues and cells that are not amenable to the delivery to the enhanced permeability and retention (EPR) effect.
of naked or chemically modified siRNA, delivery of It has therefore become a common approach to exploit
nanoparticles that incorporate siRNA are used. In gen- the leaky vasculature of tumours for the purposes of
eral, delivery vehicles are designed to both facilitate directed delivery 7,62,63. Others have also reported success
uptake into the target tissue of interest and, when used in targeting tumours through conjugation to ligands
for systemic delivery, to protect siRNA payloads and such as antibodies64,65.
inhibit nonspecific delivery. Below, we highlight sev-
eral important characteristics of delivery nanoparticles Toxicity. Even the most efficacious siRNA delivery
and provide specific examples of their construction agents are rendered useless if they provoke unaccept-
and use. able toxicity on either a cellular or systemic level. Viral
vectors, which were among the first vehicles to be studied
Surface properties. The surface charge of a delivery for siRNA delivery, can induce unacceptable levels of
nanoparticle can significantly influence the way it toxicity through the activation of immune responses66.
interacts with the target cell and other physiological Therefore, synthetic lipids and polymers have been
molecules. In the simplified in vitro setting, a positively developed to offer alternatives to viral vectors for nucleic
charged delivery vehicle can facilitate uptake by asso- acid delivery applications, and are carefully formulated
ciating with the negatively charged cellular membrane50. to avoid stimulation of the immune system39. Clearance
A positive charge also promotes complex formation of larger molecular mass materials typically requires
and compression with the polyanionic nucleic acids them to be biodegradable. The use of biodegradable,
Enhanced permeability of the siRNA. The situation, however, becomes more high molecular mass polycations and polymers contain-
and retention (EPR) effect complicated in vivo as negatively charged serum pro- ing linkages that can be cleaved inside the cell can help
The effect by which
teins in the bloodstream will often bind to a positively reduce cytotoxicity 67.
macromolecules undergo
increased accumulation in charged nanocomplex, therefore rendering it ineffective.
tumours. This is attributable The addition of PEG or other hydrophilic conjugates to Synthetic materials for siRNA delivery
to the quickly growing the surface of a delivery vehicle can assist in mitigating Synthetic materials have demonstrated potential as effec-
tumour vasculature, which this problem51. Additionally, PEG conjugation can con- tive non-viral siRNA delivery carriers. Although many
is improperly formed and
subsequently more permeable
trol particle size and prevent particle aggregation in the types of compound have been investigated as potential
to large molecules. presence of serum51. candidates, this Review focuses on synthetic materials
Coating of the nanoparticle with hydrophilic molecules that have successfully delivered nucleic acids in vivo.
Liposome such as PEG can also play an important role in the ability An overview of the delivery agents presented here can
A type of drug delivery
of the siRNA delivery carrier to evade the immune sys- be found in TABLE 2.
vehicle made of lipids.
These nanocomplexes can tem and associated phagocytes. PEG forms a barrier
be unilamellar (one set of around nanoparticles that provides steric stabilization Liposomes and lipid-like materials. unilamellar and
head-groups) or multilamellar and protection from the physiological surroundings52. multilamellar liposomes are commonly used as pharma-
(two or more sets of The length of the PEG chain can have a significant influ- ceutical delivery vehicles68. In an aqueous environment,
head-groups). They can be
used to deliver hydrophilic
ence on its stabilization and protective properties, and certain materials have the ability to form liposomes, in
or hydrophobic payloads, chain length is typically optimized for each individual which a lipid bilayer forms a sphere with an aqueous core.
depending on their structure. delivery system53,54. For example, one set of polar head-groups can create the

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Figure 3 | Two common small interfering rNA (sirNA) modifications used for therapeutic applications.
a | The 2′-O-methyl sugar modification prevents activation of the Toll-like receptor 7 immune response and confers
enzymatic resistance to the siRNA molecule. b | The conjugation of cholesterol to the sense strand of an siRNA duplex
improves delivery of naked siRNA to certain cellular targets, including hepatocytes. The cholesterol conjugate is
shown in red, and the linker is shown in green.

outer surface of the nanocomplex, while another set of in mice have indicated an activation of interferon α and
polar head-groups faces the interior hydrophilic core, β, suggesting that stimulation of the immune system
which houses the nucleic acid payload69 (FIG. 4). It is also may also have a role in efficacy 72.
possible for liposomes to be amorphous in structure, More recently, Sato and co-workers used vitamin-
with the lipids and nucleic acids interspersed through- A-coupled lipotrust liposomes to deliver anti-gp46
out. liposomes can be created using single or multiple siRNA to fibrogenic hepatic cells for the treatment of liver
types of lipid, which allows for additional flexibility cirrhosis. Five administrations of the siRNA lipocomplexes
when optimizing the physical and chemical properties were reported to resolve liver fibrosis and prolong survival
of the nanoparticle68. in rats that had otherwise lethal liver cirrhosis, in a dose-
liposomes have been used for the delivery of nucleic and duration-dependent manner. It was also shown that
acids for over 20 years, originating with studies by Felgner rescue was not related to off-target effects or associated
and colleagues detailing the ability of the cationic with recruitment of the innate immune system18.
lipid DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N- The use of lipid complexes for localized siRNA admin-
trimethlylammonium chloride) to deliver both DNA istration has also proved beneficial. Vaginal instillation
and RNA into mouse, rat and human cell lines70,71. More of cationic liposomes comprised of Oligofectamine and
recently, stable nucleic acid–lipid particle (SNAlP) for- siRNA targeting herpes simplex virus (HSV)-2 reportedly
mulations have demonstrated efficacy in several models led to uptake by the epithelial and lamina propria cells
in vivo. A study by Morrissey and co-workers indicated in the vagina in mice and protected against lethal infec-
that HBV replication was inhibited through the delivery tion for up to 9 days. The siRNA complexes protected
of an siRNA–SNAlP complex that targeted HBV RNA. mice when administered before and/or after (otherwise)
Three daily intravenous injections of 3 mg per kg per lethal HSV-2 challenge27. In another study, intrathecal
day reduced serum HBV levels by at least one order of administration of complexes formulated by i-FECT and
magnitude, and the effect was specific, dose-dependent siRNA protected mice from fatal Japanese encephalitis
and lasted for up to 7 days after dosing 4. In addition, virus and West Nile virus after intracranial administra-
Zimmerman and colleagues demonstrated the ability tion, and reduced pain receptor expression following
of SNAlPs to enable knockdown of ApoB in the liver of intrathecal administration in rats73.
cynomolgus monkeys 57. A single siRNA injection Also of note are several synthetic lipid-based materi-
resulted in dose-dependent silencing of ApoB mRNA als that have demonstrated DNA transfection efficiency.
expression in the liver 48 hours after administration, Similar materials may also potentially serve as candi-
with maximal silencing of more than 90%. Knockdown dates for siRNA delivery, given the similarities involved
was confirmed to be caused by ApoB mRNA cleavage at in the two types of nucleic acid delivery 74. For example,
precisely the site predicted for the RNAi mechanism, and Wheeler et al. used liposomes comprising a combination
persisted for 11 days at the highest administered dose of of the cationic lipid GAP-DlRIE (N-(3-aminopropyl)-
2.5 mg per kg 57. N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium
An additional study details the use of SNAlP formu- bromide) and the neutral co-lipid DOPE (dioleoylphos-
lations to combat the Zaire strain of Ebola virus, which phatidylethanolamine) to transfect mouse lung with a
proves fatal to 90% of its victims via haemorrhagic gene that confers chloramphenicol resistance. After a
fever 72. When contained within the SNAlP formula- single intranasal administration, gene expression was
tion, siRNA targeting of the polymerase gene of the virus enhanced by more than 100-fold relative to plasmid DNA
completely protected guinea pigs against viraemia and alone, followed by a gradual return to baseline levels
death when administered shortly after an Ebola virus by 21 days post-administration75. Also of interest are
challenge. It is important to note that preliminary studies transactivating transcriptional activator (TAT)-modified

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Table 2 | Selected synthetic materials for in vivo siRNA delivery

Material Model Target route Animal refs
Liposomes and lipids
i-FECT Japanese encephalitis JEV and WNV Intracranial Mouse 70
virus (JEV) and West envelope
Nile virus (WNV)
Lipidoids Dyslipidaemia FVII/ApoB Intravenous Mouse, rat, 30
monkey
Dyslipidaemia FVII/ApoB Intravenous Mouse, 79
hamster
Malaria Haem oxygenase 1 Intravenous Mouse 80
Hypercholesterolaemia PCSK9 Intravenous Mouse, rat 17
LipoTrust Liver cirrhosis gp46 Intravenous Rat 18
Oligofectamine Herpes simplex virus 2 HSV-2-associated Intravaginal Mouse 27
(HSV-2) viral proteins UL27
and UL29
SNALP Hepatitis B virus (HBV) HBV Intravenous Mouse 4
Dyslipidaemia ApoB Intravenous Monkey 56
Ebola (Zaire) Polymerase L Intravenous Guinea pig 69
Cationic polymers
Cyclodextrin Ewing’s sarcoma tumour EWS–FLI1 Intravenous Mouse 94
xenograft
Healthy monkey model RRM2 Intravenous Monkey 95
Dynamic Dyslipidaemia ApoB/PPARα Intravenous Mouse 98
PolyConjugate
Poly- Glioblastoma xenograft PTN Intratumoral Mouse 28
ethyleneimine
Formalin-induced pain NMDAR2B Intrathecal Rat 87
Cervical tumour xenograft HPV E6/E7 Intratumoral Mouse 20
Ovarian tumour xenograft HER2 Intraperitoneal Mouse 88
Small interfering RNA (siRNA) conjugates
Cholesterol Dyslipidaemia ApoB Intravenous Mouse 46
Huntington’s disease Huntingtin gene Intrastriatal Mouse 23
Fatty acids/ Dyslipidaemia ApoB Intravenous Mouse, 47
bile salts hamster
ApoB, apolipoprotein B; EWS–FLI1, Ewing’s sarcoma–friend leukaemia virus integration 1; FVII, factor VII blood protein; gp46,
a glycoprotein gene; HER2, human epidermal growth factor receptor 2; HPV E6/E7, human papillomavirus oncogenes; NMDAR2B,
N-methyl-d-aspartate receptor type 2B; PCSK9, proprotein convertase subtilisin/kexin type 9; PPARα, peroxisome proliferator-
activated receptor α; PTN, pleiotrophin (a secreted growth factor); RRM2, ribonucleoside-diphosphate reductase subunit M2;
SNALP, stable nucleic acid–lipid particles.

liposomes, which, when complexed with a gene encoding In another study, a combinatorial library of lipid-like
green fluorescence protein (GFP) and injected locally, materials was developed for use in siRNA delivery 30.
induced the expression of GFP in lewis lung carcinoma These ‘lipidoids’ were synthesized through the conju-
tumour cells in mice50. gate addition of alkyl-acrylates and -amides to primary
Although liposomes are among the most popular and secondary amines and then studied in cell culture.
nucleic acid delivery agents, some concerns regarding One leading candidate for in vivo gene knockdown was
their safety for therapeutic use remain. Toxicity of certain identified as 98N12-5(1), which comprises five 12-carbon
cationic lipid particles has been reported both in vitro alkyl-acrylamide chains attached to an amine core (FIG. 4d).
and in vivo76–78, and certain synthetic agents have been Formulations of this material with siRNA were capable of
found to induce a gene signature of their own that might achieving potent and persistent silencing of various lung and
increase the off-target effects of siRNA79,80. Despite these liver targets in mice, rats and cynomolgus monkeys30.
issues, liposomes show promise for future clinical use, as In a more recent primate study, 98N12-5(1) was also
evidenced by the approval of PEGylated liposomes for used to deliver siRNA against PCSK9, a protein that
doxorubicin and amphotericin B delivery by the uS Food regulates lDl receptor protein levels and function17.
and Drug Administration79,81. liver-specific siRNA silencing of PCSK9 reduced PCSK9

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Figure 4 | synthetic materials for small interfering rNA (sirNA) delivery. a | A representation of stable nucleic
acid–lipid particles (SNALPs), which are liposomes comprising cationic lipids, non-ionic lipids and polyethylene glycol
(PEG). siRNA is contained in the hydrophilic interior of the particle. b | Polyethyleneimine is used to fabricate both
linear and branched polymeric delivery agents. c | A cyclodextrin-based delivery agent. d | The lipidoid 98N12-5(1).

mRNA levels by 50–70% in mice and rats, as well as It has also been frequently used for various local siRNA
reducing human PCSK9 transcript levels in transgenic delivery applications. Intrathecal administration of PEI–
mice by more than 70%. Silencing persisted for up to siRNA complexes was reported to selectively knockdown
3 weeks after a single intravenous dose, indicating that a pain receptor in rats. Maximal effect occurred on day 3
anti-PCSK9–lipidoid complexes may serve as a potent for mRNA levels and day 7 for associated protein levels
and effective treatment for hypercholesterolaemia17. following an injection of 5 mg of siRNA targeting a
Importantly, lipidoid materials were shown to facilitate subunit of the NMDA (N-methyl-d-aspartate) receptor
siRNA delivery without disrupting endogenous micro- NR2B90. PEI has also shown efficacy in a subcutaneous
RNA processing 82. Finally, Hmox1, a gene expressed in mouse tumour model. The intraperitoneal administra-
the liver encoding the protein haem oxygenase 1, has tion of complexed siRNA led to the delivery of the intact
also been silenced in mice by siRNA nanoparticles formu- siRNA into the tumours and a marked reduction of
lated with lipidoids. Knockdown of Hmox1 may represent tumour growth through siRNA-mediated downregulation
a potential approach for the treatment of malaria infection of human epidermal growth factor 2 (HER2; also known
and disease progression83. as ERBB2)91.There has been significant concern regarding
the toxicity of PEI at higher molecular masses and high
Polymers. Cationic polymers with a linear or branched doses92,93. However, strategies to modify the structure of
structure can serve as efficient transfection agents because PEI to reduce toxicity while retaining its potent ability
of their ability to bind and condense large nucleic acids to transfect cells are in development94–96.
into stabilized nanoparticles84,85. Such materials have also Cyclodextrin polymers have also been developed
been shown to stimulate nonspecific endocytosis as well as siRNA delivery agents. Tumour growth in a mouse
as endosomal escape79. A proposed mechanism for this model of metastatic Ewing’s sarcoma was shown to
is the ‘proton-sponge’ effect 84, whereby buffering of the be inhibited by the systemic delivery of nanoparticles
endosome leads to an accumulation of ions within this formed by cyclodextrin, the targeting ligand transfer-
compartment and an osmotic pressure that eventually rin, and siRNA specific for the EWS–FLI1 fusion gene
bursts the endosome86,87. commonly associated with the condition. Knockdown
PEI is a broadly investigated delivery carrier for the was not observed upon removal of the targeting ligand,
administration of a wide range of nucleotide-based ther- nor was there any evidence of immune stimulation or
apies, including DNA, siRNA and oligonucleotides88,89. toxicity 97. In another study, Heidel and co-workers used

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Table 3 | Current clinical trials for siRNA therapeutics (VEGFR), has shown therapeutic potential in its inhibi-
tion of the excessive vascularization of the eye that leads
company Disease Mode of status to AMD26.
administration
In 2004, the first clinical trial involving siRNA was
Allergan Age-related macular Topical Phase II carried out by Acuity Pharmaceuticals for the treatment
degeneration
of AMD. The completed Phase II trials reported that all
Alnylam Respiratory syncytial Local/direct Phase II doses were well tolerated with a lack of adverse systemic
virus effects. Testing has now moved into Phase III trials, which
Nucleonics Hepatitis B virus Systemic Phase I have been taken over by Opko Health. Allergan is cur-
Quark Pharmaceuticals/ Acute renal failure Systemic Phase I rently conducting a Phase II clinical trial on an siRNA for
Pfizer AMD, with completed Phase I results indicating minimal
Opko Health Age-related macular Topical Phase III side effects and improved vision in some of the patients.
degeneration Although Silence Therapeutics also had an siRNA product
Silence/Quark/Pfizer Diabetic macular Topical Phase II for AMD in the pipeline, they have now refocused their
oedema Phase II clinical study on the treatment of diabetic mac-
Transderm Pachyonychia Topical Phase Ia/b
ular oedema, which is another condition caused by leaky
congenita vasculature within the eye. Importantly, a recent study
siRNA, small interfering RNA.
has reported that anti-VEGF siRNA efficacy in the eye
is not due to specific gene silencing, but is instead caused
by nonspecific stimulation of the TlR3 pathway, which
cyclodextrin–transferrin nanoparticles (FIG. 4c) to study can reduce angiogenesis102. Although this study calls into
the effects of siRNA delivery on the immune system of question the nature of the anti-angiogenic effect reported
cynomolgus monkeys98. Specifically, an siRNA targeting in AMD clinical trials, it does not explain the therapeutic
the M2 subunit of ribonucleotide reductase was delivered effects observed in other applications of siRNA in which
intravenously in escalating doses, and it was established appropriate controls have been performed.
that multiple, systemic doses of targeted nanoparticles Also of note is Alnylam’s RSV01 formulation, which
containing non-chemically modified siRNA can safely targets the nucleocapsid N gene of RSV — a major cause
be administered to non-human primates98. In addition, of respiratory illness in infants and young children. The
cyclodextrin–transferrin nanoparticles demonstrated RSV01 formulation completed Phase I trials and was
efficacy in knocking down luciferase and ribonucleotide found to be well tolerated in healthy adults. Since then,
reductase genes in mice99,100. Phase II trials have demonstrated significant antiviral
Polymer–siRNA conjugates have also shown potential efficacy of this formulation in adults103.
for applications in systemic siRNA delivery. Rozema et al.
have developed a polymer-conjugated delivery system Future directions and conclusions
called Dynamic PolyConjugates to facilitate delivery of Moving forward, we expect that synthetic nanoparticles
siRNA to hepatocytes101. Key features of the Dynamic composed of polymers, lipids, lipidoids or conjugates
PolyConjugate technology include a membrane-active will have a key role in the systemic application of siRNA
cationic polymer, the ability to reversibly mask the activity in the clinic. The incorporation of tissue-specific ligands
of this polymer until it reaches the acidic environment into these particles may enable targeting, which will
of the endosome, and the ability to target this modified assist with in vivo biodistribution and delivery. We also
polymer and its siRNA cargo specifically to hepatocytes anticipate that future developments will require atten-
after intravenous injection. Dynamic PolyConjugates tion to nonspecific activation of the immune system by
were capable of inducing knockdown of two mouse liver siRNA, including TlR3 and TlR7 pathways102,104. Given
genes. Analyses of serum liver enzyme and cytokine levels the potential for nonspecific effects, it is important that
in treated mice indicated that siRNA complexes formed the therapeutic mode of action be validated when possible,
with this synthetic polymer were well tolerated101. such as through direct measurement of target mRNA
levels in vivo. Chemical modification of siRNA — such
Clinical trials as 2′-O-methyl substitutions — can minimize nonspecific
Today, siRNA therapeutics are progressing in the clinic effects40, and we expect that additional improvements
(TABLE 3). Many of the most advanced trials rely on forms in both activity and delivery will be mediated by direct
of localized delivery, although several ongoing clinical chemical modification of the siRNA sequence.
Age-related macular trials involve the use of delivery agents. Not included In summary, the field of RNAi therapeutics has made
degeneration in TABLE 3 are many formulations that are in preclinical significant progress since the first demonstration of gene
(AMD). This disease of the
or animal-study phases at the major pharmaceutical knockdown in mammalian cells. siRNA-based formulations
eye is caused by excessive
growth and rupture of blood companies developing siRNA therapeutics. offer significant potential as therapeutic agents to induce
vessels within the cornea Several of the most advanced clinical trials focus on the potent, persistent and specific silencing of a broad
and is a leading cause of the treatment of age-related macular degeneration (AMD), range of genetic targets. Delivery remains the most signifi-
blindness. Several early siRNA which is a leading cause of blindness. AMD arises from cant barrier to the widespread use of RNAi therapeutics in
clinical trials have targeted
this disease, primarily due
excessive blood-vessel growth and rupture within the a clinical setting, and future work focusing on the develop-
to the ease of local delivery cornea. Naked siRNA targeted to genes for vascular ment of safe and effective delivery materials is needed to
to the eye. endothelial growth factor (VEGF) and its receptor ensure the broadest application of RNAi in the clinic.

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CoRRigENDuM

Knocking down barriers: advances in siRNA delivery

Kathryn A. Whitehead, Robert Langer & Daniel G. Anderson
Nature Reviews Drug Discovery 8, 129–138 (2009) | doi:10.1038/nrd2742
On page 136 in Table 3, the clinical trial for acute renal failure has been attributed to Quark/Pfizer when it should be
attributed to Quark alone. In addition, the authors would like to clarify that, following a Phase I/II trial in patients with wet
age-related macular degeneration, the siRNA drug candidate PF-4523655 (RTP801i-14) is now being studied in a Phase II
trial for diabetic macular oedema conducted by Pfizer in collaboration with Quark.